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

Article abstract of DOI:10.1021/acsinfecdis.7b00008

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 S. 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 S. pneumoniae. These compounds contain a linear chain with a chain length from C₃ to C₉ at the 2-position (R₁) and 4′-position (R₃), as well as a 2-methyl butyryl group at the 3′-position (R₂), 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.

Full text of DOI:10.1021/acsinfecdis.7b00008

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Article
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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A Natural Lipotrisaccharide and Its Derivatives Selectively Lyse
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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,
00084, People’s Republic of China;
1
‡Center for Infectious Disease Research, School of Medicine, Tsinghua University, Beijing,
00084, People’s Republic of China;
1
Beijing Institute of Petrochemical Technology, Beijing, 102607, People’s Republic of China.
§These authors equally contribute to this study.
*Corresponding authors. E-mail: (J.R.Z.) zhanglab@tsinghua.edu.cn. Tel.: +86 10 62795892. E-
mail: (G.L.) gangliu27@tsinghua.edu.cn. 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
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compounds contain a linear chain with a chain length from C
3
to C at 2’-position and 4’-
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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
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in the last decades. 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
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pneumoniae. These pathogens not only cause devastating disease, but also represent the so-
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called “superbugs” that are extensively resistant to currently available therapeutics. 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
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clinical use in the last 40 years. Thus, novel antimicrobials are urgently needed worldwide.
Lipooligosaccharides, a type of glycolipid found in some bacteria, are truncated structures (no O-
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antigen) of bacterial lipopolysaccharide (LPS). Recent studies have suggested that both natural
and synthetic lipooligosaccharides are potential drug candidates, having demonstrated antiviral
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activity by inhibiting virus-cell binding and fusion , 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.
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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 scaffold-
anchoring 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).
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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 6 (Table 1 and Scheme 1). The
final product 1a was obtained by β-mannnosylation with acceptor 8 with donor 721, 22 (Scheme
20
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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
Donor/TfOMe/DTBMPb
c
Entry
DDQ
Solvent
DCE
Temp
45°C
Yield (%)
1
2
3
4
5
1.0/1.0/1.1
2.0/2.0/2.2
2.0/2.0/2.2
2.0/2.0/2.2
2.0/2.0/2.2
Commercial
Commercial
Commercial
Fresh Crystal
Fresh Crystal
16
25
DCE
45°C
DCM
DCE
reflux
trace
54
45°C
DCE
W, 10 min
42
6
7
2.0/2.0/2.2
2.0/2.0/2.2
Fresh Crystal
Fresh Crystal
DCE
DCE
W, 15 min
W, 20 min
52
44
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aReaction conditions: all of the reactions were performed in 3 mL of solvent in the argon
b
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atmosphere. equivalent ratio. Isolated yield.
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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
4 hours to 15 minutes (entries 5, 6, and 7).
2
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Scheme 1. Synthetic Procedures for Compound 8a
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aa) R
DCM:MeOH=2:1, 67%-85%; c) R
r.t, 77%;d) 16% CF COOH, 8% MeOH, DCM, 0°C-r.t., 70%-89%; e) Trt-Cl, DMAP, Py, 81%-
7%; f) R COOH, DMAP, EDCI, 85%-89% or R Br, NaH, DMF, 0 °C -r.t, 84%; g) FeCl .6H O,
DCM, 0.5h, 79%-86%.
COOH, DMAP, EDCI, r.t, 84%-90% or R
Br, NaH, DMF, 0 °C -r.t, 81%; b) PdCl
Br, NaH, DMF, 0 °C-
,
1
1
2
2
COOH, DMAP, EDCI, 80%-93% or R
2
3
8
3
3
3
2
Note*: While the R = 2-methylbutyl, a diastereoisomeric mixture of compounds 1d-f, 1j, 2a-d,
2
and 2g, 3 and 4 were afforded R:S=1:1, and synthetizing the compound 1h,1i,2e,2f, R represents
2
the benzyl formate.
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Scheme 2. Synthetic Procedures for Compound 18a
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aa) phthalic anhydride, 1.5eq DBU, 1.5eq 4Å MS; b) i, 5eq DTBMP, 2.5eq Tf
min, ii, acceptor, -78°C to 0°C, 1 h, 75%-83%; c) 3atm, H , 20% Pd(OH) , 7h, 84%-89%;
O 1.5eq -78°C, 15
2
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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 R
1
was assembled at the 2’-
position of 10 to yield 11, the 3’-position was then deprotected by removal of the allyl group
with PdCl
2
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
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acylated with R
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(compound 16), and then 8 was finally prepared after removal of the Trt group
.6H
O),
under Lewis acidic conditions (FeCl
3
2
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 β-
mannopyranosides.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) /C and
2
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)
c) undecylic acid, EDCI, DMAP, 86%; (d) PdCl
acid, EDCI, DMAP, 79%; (f) CF COOH, DCM, MeOH, 72%; (g) Trt-Cl, DMAP, Py, 84%; (h)
Heptanoic acid, EDCI, DMAP, 85%; (i) FeCl •6H O, DCM, 93%
, CSA, CH
CN, 81% (b) nBu
SnO, MeOH, reflux, then Allyl-Br, DMF, 58%,
, DCM:MeOH=2:1, 79%; (e) 2-Methylbutyric
2
3
2
(
2
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3
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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 nBu SnO in the presence of allyl
2
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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aa), phthalic anhydride, 1.5eq DBU, 1.5eq 4 Å MS; b)i, 5eq DTBMP, 2.5eq Tf
O 1.5eq -78°C,
2
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5 min; ii, acceptor, -78°C to 0°C, 1 h, 89%; c) 3atm, H
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, 20% Pd(OH) , 7 h, 85%;
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give the desired 4 under Lewis acid conditions (FeCl
3
•6H O) in DCM.
2
A stereoselective glycosylation of building block 7 with compound 4 by the procedure of β-
mannoside 18 as described above afforded the disaccharide intermediate 25. Removal of the 4,6-
O-benzal and Bn of 25 using hydrogenolysis with the help of Pd(OH) gave compound 3
2
(Scheme 4).
Collectively, all of the derivatives of 1, 2, 3 and 4 in Table 2 were prepared accordingly, where
, R , and R , respectively, represent different aliphatic alkyls anchored on either a
R
1
2
3
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.
0
1
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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 R (3’- position) exhibited
2
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 R
1
and R of 1a created compound
3
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 R
3
or C11 (1f) of R
1
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 R (1j,
1
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
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1
i, decreased potency was observed with trehalose derivatives 2e-2f (MIC=12.5~25 μg/mL),
when the 2-methylbutyl group (R at 3’ position) was omitted. Compound 4 (MIC>64 μg/mL)
2
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 R
1
, R
2
, and R substituent. This result
3
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
0
1
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9
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Compound
R
1
R
2
R
3
S. pneumoniae
S. aureus
E. faecalis
g/mL(M)
1
aa
6.25~12.5
(7.0-14.0)
>64
>64
(>70)
(>70)
1
ba
5~10
16
(18.8)
>64
>64
(>70)
>64
(5.9-11.8)
6.25~12.5
1
1
1
1
ca
da
ea
fa
(
7.0-14.0)
(>70)
(>70)
2.5~5
>64
>64
(
2.8-5.6)
2.5~5
(>70)
(>70)
>64
>64
(
2.8-5.6)
(>70)
(>70)
2.5~5
>64
>64
(
2.8-5.6)
(>70)
(>70)
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ga
2.5~5
>64
>64
4
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8
9
1
1
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1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
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4
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(2.8-5.6)
(>70)
(>70)
ha
H
H
6.25-12.5
(7.7-15.5)
>64
>64
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
(>70)
(>70)
1ia
12.5-25
>64
>64
(
15.5-31.0)
(>70)
(>70)
1
2
2
2
2
2
ja
>64
32
16
(>70)
33.3
16.6
ab
bb
cb
db
eb
6.25-12.5
(8.5-17.1)
32
32
(43.9)
(43.9)
5~10
16
32
(6.9-13.7)
(21.9)
(43.9)
5~10
16
16
(6.9-13.7)
(21.9)
(21.9)
10~20
>64
>64
(13.7-27.4)
(>70)
(>70)
H
12.5~25
>64
>64
(14.4-28.7)
(>70)
(>70)
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2
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2
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3
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2
2
fb
H
12.5~25
>64
>64
(19.4-38.7)
6.25~12.5
(>70)
(>70)
gb
64
64
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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7
8
9
0
1
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
9.7-19.3)
(>70)
(>70)
3
4
cc
6.25-12.5
>64
>64
（
8.4-16.8）
(>70)
(>70)
dd
>64
>64
>64
(>100)
(>100)
(>100)
Van
0.125-0.25
0.09-0.17)
1-2
0.5-1
(
(0.69-0.14) (0.34-0.68)
a=
, b=
, c=
, 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 Gram-
negative bacteria.
MIC
Species
Straina
μg/mL(M)
>64(>70)
>64(>70)
>64(>70)
>64(>70)
K. pneumoniae
P. aeruginosa
TH4079
TH4089
A. baumannii
TH4100
V. cholerae N16961
ATCC39315
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 Gram-
negative 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.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4. Antibacterial activity of compound 1f against selected Streptococcus species.
Straina
Species
MIC
μg/mL (M)
5.0-10.0
S. agalactiae
S. gordonii
ST4265
(
5.6-11.2)
ATCC49818
10.0-20
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(
11.2-22.4)
2.5-5
2.8-5.6)
S. mitis
ATCC49456
ST137
(
10
11
12
13
14
15
16
17
18
19
20
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S. oralis
2.5-5
(2.8-5.6)
2.5-5
S. pyogenes
S. sanguis
ST4285
(
2.8-5.6)
20-40
22.4-44.8)
NCTC7863
(
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
3
2
more clinical antimicrobial categories. 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
ST2784
ST2863
ST2882
ST2889
PEN, CRO, ERY, TCY, SXT, DA
ERY, TCY, SXT, DA
1.25-2.5
(1.4-2.8)
1.25-2.5
0
1
2
3
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8
9
0
1
2
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9
0
1
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
1.4-2.8)
1.25-2.5
1.4-2.8)
PEN, ERY, TCY, SXT, DA
AMX, ERY, CHL, SXT, DA
PEN, ERY, CHL, SXT, DA
(
1.25-2.5
(1.4-2.8)
1.25-2.5
(
1.4-2.8)
aAll 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
th
standards for antimicrobial susceptibility testing from the 25 informational supplement (CLSI
document M100-S25, 2015) of the Clinical and Laboratory Standards Institute (clsi.org). AMX,
amoxicillin; CHL, chloramphenicol; CRO, ceftriaxone; DA, clindamycin; ERY, erythromycin;
PEN, penicillin; SXT, sulfamethoxazole; TCY, tetracycline.
b
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.
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Figure 4. In vitro cytotoxicity (hemolysis) of compounds 1a and its representative derivatives.a
a
8
Rabbit erythrocytes were suspended in PBS to 2×10 per mL and incubated with 1% Triton X-
00 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
1
.
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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1
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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
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34
rapid autolysis of S. pneumoniae. 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
3
0 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
3
5
membrane potential sensitive dye. 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
35
membrane is depolarized by membrane-active agents. 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
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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
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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. 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,
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a, 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 Gram-
positive 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.
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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 R
1
, R
2
, and R lipid chains, reduced the selectivity to S. pneumoniae compared
3
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 (R
1
and R
3
, 1f and 1g) could improve the activity, but with a limited
acyl chain (1h and 1i) reduced
extension of the carbon number (e.g., 1j). Clearly, omitting the R
2
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
C
3
-C
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chains of R
1
and R
3
(2’-position), and the 2-methyl butyryl group of R (3’-position),
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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
1
1
0% (volume fraction) H
2
SO
4
/ethanol. Proton nuclear magnetic resonance ( H NMR) and carbon
13
nuclear magnetic resonance ( C 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
1
9
20
mmol) of 5 and (352 mg, 1 mmol) of 6 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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