Synthesis of α-1,2- and α-1,3-linked di-rhamnolipids for biological studies

Article abstract of DOI:10.1016/j.carres.2020.108102

For a detailed examination of the interaction of rhamnose containing derivatives with recombinant horseshoe crab plasma lectin (rHPL), two di-rhamno-di-lipids (an α-1,2- and an α-1,3-linked) were synthesized via a new simple method. The N-iodosuccinimide/triflic acid mediated glycosylation of the methyl (R)-3-hydroxydecanoate with phenyl-1-thio-rhamnobioside donors afforded the mono-lipid disaccharides. Removal of the methyl ester group followed by esterification of the mono-lipids with a second (R)-3-hydroxydecanoate unit resulted in fully protected di-lipid derivatives, transformation of which into the target compounds was accomplished in two steps. This method allows the synthesis of both regioisomers in only 6 steps starting from the corresponding free disaccharides. Both synthetic di-rhamnolipids were biologically active for lectin binding differential binding preference between two isomeric di-rhamno-di-lipids. The rHPL lectin favours the α-1,3-linked di-rhamno-di-lipids over its α-1,2-linked regioisomer.

Full text of DOI:10.1016/j.carres.2020.108102

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Synthesis of α-1,2- and α-1,3-linked di-rhamnolipids for biological studies
Fruzsina Demeter, Margaret Dah-Tsyr Chang, Yuan-Chuan Lee, Tse-Kai Fu, Mihály
Herczeg, Anikó Borbás
PII:
S0008-6215(20)30349-9
DOI:
https://doi.org/10.1016/j.carres.2020.108102
CAR 108102
Reference:
To appear in: Carbohydrate Research
Received Date: 5 June 2020
Revised Date: 10 July 2020
Accepted Date: 13 July 2020
Please cite this article as: F. Demeter, M. Dah-Tsyr Chang, Y.-C. Lee, T.-K. Fu, Mihá. Herczeg, Anikó.
Borbás, Synthesis of α-1,2- and α-1,3-linked di-rhamnolipids for biological studies, Carbohydrate
Research (2020), doi: https://doi.org/10.1016/j.carres.2020.108102.
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Synthesis of α-1,2- and α-1,3-linked di-rhamnolipids for biological studies
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Fruzsina Demetera,b,c, Margaret Dah-Tsyr Changd, Yuan-Chuan Leed,e, Tse-Kai Fud, Mihály
Herczeg*,a,f, Anikó Borbás*,a
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aDepartment of Pharmaceutical Chemistry, University of Debrecen, H-4032, Debrecen,
Egyetem tér 1, Hungary, bMTA-DE Molecular Recognition and Interaction Research Group,
UD, Egyetem tér 1, H-4032 Debrecen, Hungary, cDoctoral School of Chemistry, University of
Debrecen, dInstitute of Molecular and Cellular Biology, National Tsing Hua University,
Hsinchu, Taiwan, Republic of China, eDepartment of Biology, Johns Hopkins University,
Baltimore, Maryland, MD 21218, USA, fResearch Group for Oligosaccharide Chemistry of
HAS, UD, H-4032, Debrecen, Egyetem tér 1, Hungary
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herczeg.mihaly@science.unideb.hu, borbas.aniko@pharm.unideb.hu
Highlights
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Di-lipid derivatives of α-1,2- and α-1,3-linked rhamnobiosides were prepared.
Methyl (R)-3-hydroxydecanoate was glycosylated with benzyl-protected phenyl-1-
thio-rhamnobiosides.
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The di-rhamnosyl mono-lipids obtained were esterified with a second 3-
hydroxydecanoate at the disaccharide level.
The linking position of the rhamnobiosyl donors greatly affected the glycosylation
stereoselectivity and esterification efficacy.
The α-1,3-linked di-rhamnolipid showed stronger binding affinity to horseshoe crab
plasma lectin than that of α-1,2-linked.
Abstract
For a detailed examination of the interaction of rhamnose containing derivatives with
recombinant horseshoe crab plasma lectin (rHPL), two di-rhamno-di-lipids (an α-1,2- and an
α-1,3-linked) were synthesized via a new simple method. The N-iodosuccinimide/triflic acid
mediated glycosylation of the methyl (R)-3-hydroxydecanoate with phenyl-1-thio-
rhamnobioside donors afforded the mono-lipid disaccharides. Removal of the methyl ester
group followed by esterification of the mono-lipids with a second (R)-3-hydroxydecanoate
unit resulted in fully protected di-lipid derivatives, transformation of which into the target
compounds was accomplished in two steps. This method allows the synthesis of both
regioisomers in only 6 steps starting from the corresponding free disaccharides. Both
synthetic di-rhamnolipids were biologically active for lectin binding differential binding
preference between two isomeric di-rhamno-di-lipids. The rHPL lectin favours the α-1,3-
linked di-rhamno-di-lipids over its α-1,2-linked regioisomer.
Keywords
glycolipids, L-rhamnose, stereoselective glycosylation, decanoic acid, lectin

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1. Introduction
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Appearance of pathogenic bacterial strains which are resistant to currently available
antibiotics is a serious problem worldwide [1]. Most of these antibiotic-resistant organisms
form biofilm composed of secreted proteins, glycolipids, polysaccharides and small
molecules. These bacteria may cause life-threatening infection that is highly difficult to
manage even with combinations of available methods [2]. To solve this problem one possible
approach may be the use of lectins. Lectins are carbohydrate-binding proteins and known to
have high specificity for properly arranged sugar moieties [3]. Lectins can recognize
carbohydrates on bacterial cell-wall and this specific recognition may provide an alternative
route for detection and inhibition of pathogens [4]. A soluble and functional recombinant
horseshoe crab plasma lectin (rHPL) possessing specific L-rhamnose-binding capability has
recently been expressed in an Escherichia coli system [5]. It was shown that rHPL selectively
bound to pathogens isolated from clinical samples, such as Pseudomonas aeruginosa,
Klebsiella pneumoniae, and Streptococcus pneumoniae serotypes. The binding was
established to be a specific molecular interaction between rHPL and rhamnose moiety of
pathogen-associated molecular patterns on the bacterial surface. Moreover, rHPL is capable of
inhibiting the growth of P. aeruginosa PAO1 in a concentration-dependent manner [4,5]. The
results show that a specific protein-glycan interaction between rHPL and rhamnosyl moieties
may facilitate the development of new diagnostic and therapeutic strategies for the treatment
of microbial pathogens-associated diseases. Preliminary results suggested that rHPL could
bind to rhamnose-containing components (lipopolysaccharides, di-rhamnolipid, structural
polysaccharides, eg. Psl) in bacterial biofilm and interrupt biofilm development [5-8]. From
these results, it can be stated that di-rhamnolipids are glycolipidic biosurfactants produced by
various bacterial species. The naturally produced rhamnolipids (RLs) are always found as a
mixture of different RL congeners, because the variations and distribution of these
compounds is highly depend on the diverse cultivation conditions and the type of the bacterial
strain. Their glycon part is composed of one (mono-RL) or two (di-RLs) rhamnose moieties.
The sugar units are linked to each other through an α-1,2-O-glycosidic linkage. The aglycon
part is mainly one or two (rarely three) β-hydroxy fatty acid chains which can be saturated,
mono-, or polyunsaturated and the chain length may be from C8 to C16. The fatty acid units
are linked to each other through an ester bond which formed between the β-OH group of the
distal (relative to the glycosidic bond) chain with the carboxyl group of the proximal chain.
The carboxyl group of the distal β-hydroxy fatty acid part mostly remains free, but few
congeners contain a short alkyl ester group. The 2-hydroxyl group of the distal (relative to the
glycosidic bond) rhamnose group remains mostly free similar to the distal β-hydroxy fatty
acid residue. RLs known to promote the uptake and biodegradation of poorly soluble
substrates, have antibacterial and antiviral activities, act as immune modulators and are
involved in surface motility respectively in bacterial biofilm development. Although several
studies have been performed in relation to RLs, unfortunately the exact physiological function
of these molecules is still not clear, but many of these functions came from the
physicochemical properties of these compounds such as surface activity, wetting ability,
detergency, and other properties related the amphipathic nature [9,10].

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Earlier our research group designed and performed the synthesis of several different
rhamnose-containing derivatives such as rhamnobiosides, multivalent rhamnobiosides and the
pentasaccharide part of the Psl exopolysaccharide of Pseudomonas aeruginosa as potential
ligands of rHPL [6-8,11]. In our previous studies on multivalent rhamnobiosides, we have
found that the rHPL lectin favours the α-1,3-linked di-rhamnosides over α-1,2- and α-1,4-
linked derivatives [6]. To more accurately study the rHPL-biofilm interaction, we have
decided the synthesis of two RLs, composed of two (R)-3-hydroxdecanoic acid residues and
either an α-1,2-linked (1) or an α-1,3-linked (2) rhamnobiosyl unit.
2. Results and discussion
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2.1. Chemistry
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There are two examples in the literature [12,13] for the synthesis of the α-1,2-linked
(Figure 1., 1) derivative, but the α-1,3-bonded di-rhamnolipid (2) has not been reported. For
the preparation of the final products a straightforward synthetic pathway was developed.
Which is different from the methods presented in the literature, and can be used for
production of both regioisomers. The optimization of the synthesis of free disaccharide donors
(3, 9) used for the synthesis of the planned α-1,2- (1) and α-1,3-linked (2) di-rhamnolipids has
been presented in a previous publication [8].
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Figure 1. Structure of the α-1,2- (1) and α-1,3-linked (2) di-rhamnolipids.
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The hydroxyl groups of the α-1,2 and α-1,3-linked disaccharide donors were uniformly
protected with benzyl groups, the use of which allows the selective removal of the ester group
of the (R)-3-hydroxydecanoic acid methyl ester. Van Boom’s group demonstrated that both
the pre-synthesis of dilipid and its glycosylation proceeded with very low yields [12].
Therefore, our synthetic sequence includes preparation of the di-rhamnosyl mono-lipids
followed by introduction of the second lipid unit at the disaccharide levels.

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Scheme 1. Glycosylation of methyl (R)-3-hydroxydecanoic acid with the α-(1,2)-linked
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rhamnobioside.
First, the α-1,2-linked derivative was synthesized (Scheme 1.). In this reaction, the free
hydroxyl groups of the starting disaccharide 3 [8] were benzylated under alkaline conditions,
then the fully protected derivative 4 obtained was used to glycosylate methyl (R)-3-
hydroxydecanoate with by means of the NIS/TfOH promoter system. In the glycosylation
reaction, in addition to the expected α-linked derivative 6 (50%), the β-linked stereoisomer 5
was isolated in 46% yield. This is presumably due to the fact that the rhamnosyl unit at the C-
2 position functioned not only as a nonparticipating group but also could exert a shielding
effect on the α-side due to its bulk and proximity to the anomeric centre. As a result, the two
anomers were formed nearly in a 1:1 ratio. Fortunately, the two stereoisomers could be
successfully separated by column chromatography and we could continue the synthesis with
the pure α-linked di-rhamnolipid 6.
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Scheme 2. Esterification of the mono-decanoic derivative (7) and removal of the protecting
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groups.

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For the preparation of the fully protected di-rhamno-di-lipid (8), firstly the methyl ester
group of the decanoic acid part of 6 was removed by alkaline hydrolysis (Scheme 2.) to
produce derivative 7 in an excellent yield. For the esterification of compound 7 with the
second lipid moiety, the active carboxylic acid derivative was generated in situ using N,N′-
dicyclohexylcarbodiimide (DCC) reagent in the presence of 4-(dimethylamino)pyridine
(DMAP) and then reacted with the second decanoic acid methyl ester to result in the expected
compound 8 in a modest yield. The α-1,2-bonded target compound was finally obtained by
the selective removal of the methyl ester group using LiI reagent followed by the cleavage of
the benzyl groups of the disaccharide moiety by catalytic hydrogenolysis. The reactions
yielded the required rhamnobiosyl dilipid 1 in good yield.
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Scheme 3. Glycosylation of methyl (R)-3-hydroxydecanoic acid with the α-(1,3)-linked
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rhamnobioside.
Next, the α-1,3-linked derivative (2) was prepared from the disaccharide 9 [8] (Scheme 3.).
In the same pathway as the α-1,2-linked derivative, the free -OH groups of the carbohydrate
moiety were first benzylated using BnBr and NaH reagents to yield 10. Subsequently, under
the previously used glycosylation conditions, the methyl (R)-3-hydroxydecanoate and the
protected disaccharide 10 were coupled to produce the expected α-linked compound 11 with
excellent yield and stereoselectivity. In this case the bulky rhamnosyl unit at position 3
shielded exclusively the β-side (bottom side) of the glycosidic centre of the first rhamnose
unit. This steric hindrance, the anomeric effect and the presence of the non-participating
benzyl group at position 2 jointly ensured the exclusive α-selectivity of the glycosylation
reaction.

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Scheme 4. Esterification of the mono decanoic derivative (11) and cleavage of the protecting
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groups.
Next the methyl ester group of the decanoic acid unit was removed with 0.5 M NaOH
solution (Scheme 4.) affording the free carboxylic acid derivative 12 in good yield.
Esterification of 12 with the second methyl (R)-3-hydroxydecanoate using the previously
described DCC/DMAP reagent combination provided the protected di-rhamno-di-lipid (13) in
acceptable yield. Finally, the free α-1,3-linked end-product 2 was obtained in good yield after
selective removal of the methyl ester group and subsequent catalytic hydrogenolysis of the
benzyl groups.
2.2. Biological Evaluation
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The binding of rHPL to two di-rhamno dilipid derivatives (1, 2) in comparison to di-
rhamnolipid extracted from bacteria was evaluated by direct ELISA (Figure 2.). The binding
of rHPL to the α-1,3-linked di-rhamnolipid (2) was significantly stronger than that of α-1,2-
linked di-rhamnolipid (1) or bacterial α-1,2-linked di-rhamnolipid, being their relative binding
119.7 ± 3.9%, 102.3± 4.5% and 100 ± 6.3%, respectively. The A590nm value of rHPL to α-1,3-
linked di-rhamnolipid, α-1,2-linked di-rhamnolipid and bacterial α-1,2-linked di-rhamnolipid
was 0.98± 0.03, 0.84± 0.04 and 0.82± 0.05, respectively. There was no significant difference
between the binding strength of rHPL to the bacterial or synthetic α-1,2-linked di-
rhamnolipid. This result confirmed that rHPL preferred α-1,3-linked di-rhamnoside over α-
1,2-linked di-rhamnoside.

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Figure 2. The binding of rHPL to bacterial or synthetic di-rhamnolipid (1, 2).
Di-rhamnolipid was coated on 96-well microplate. 0.1 μM rHPL was added to the microplates
and further detected by anti-His antibody. The value of rHPL-bacterial di-rhamnolipid
binding was set as 100%. The values were the mean ±SD from three experiments.
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3. Conclusion
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Two di-rhamno-di-lipid derivatives (1, 2) were successfully produced by a new,
straightforward method. In terms of the synthesis of compound 1, the synthetic route we used
is similar in the number of synthetic steps and overall efficiency to that described in the
literature,[12] with the advantages that easily accessible- monosaccharide building blocks
(phenyl-1-thio-rhamnobiosides) and the commercially available methyl (R)-3-
hydroxydecanoate were used in the synthesis. A further advantage was that the synthesized
monosaccharide units could be used to prepare both target compounds. The linking position
of the rhamnobiosyl donors greatly affected the stereoselectivity during glycosylation of the
hydroxydecanoate aglycone and the efficacy of the esterification of the mono decanoic
derivatives. The 1,2-linkage was disadvantageous in both steps, probably because the bulky
sugar unit at position 2 exerted a steric hindrance in transformations at or nearby the anomeric
centre. Nevertheless, the synthetic method established allowed the rapid preparation of both
regioisomers. The rhamnolipids obtained can be used for studying the binding preferences of
rHPL toward different components of the cell wall of biofilm producing bacteria. rHPL

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binding activity of α-1,3-linked di-rhamnolipid is 20% stronger than that of α-1,2-linked di-
rhamnolipid, which in turn may lead to strategic design of novel antibacterial agents [5,14].
4. Experimental section
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4.1. Materials and General Experimental Procedures
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Optical rotations were measured at room temperature on a Perkin–Elmer 241 automatic
polarimeter. TLC analysis was performed on Kieselgel 60 F254 (Merck) silica–gel plates with
visualization by immersing in a sulphuric-acid solution (5% in EtOH) followed by heating.
Column chromatography was performed on silica gel 60 (Merck 0.063–0.200 mm) and
Sephadex LH-20 (Sigma–Aldrich, bead size: 25–100 mm). Organic solutions were dried over
MgSO4 and concentrated under vacuum. One- (1D) and two-dimensional (2D) 1H, 13C, COSY
and HSQC spectra were recorded on Bruker Avance II 400 (1H: 400 MHz; 13C: 100.28 MHz)
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and Avance II 500 (1H: 500.13 MHz; C: 125.76 MHz) spectrometers at 25 °C. Chemical
shifts are referenced to SiMe4 or sodium 3-(trimethylsilyl)-1-propanesulfonate (DSS, d = 0.00
ppm for 1H nuclei) and to residual solvent signals (CDCl3: d = 77.00 ppm, CD3OD: d = 49.15
ppm for 13C nuclei). Matrix-assisted laser desorption/ionization-time-of-flight mass
spectrometric (MALDI-TOF) MS analyses were carried out in the positive reflectron mode
using a BIFLEX III mass 13 spectrometer (Bruker, Rheinstetten, Germany) equipped with
delayed-ion extraction. The matrix solution was a saturated 2,4,6-trihydroxy-acetophenone
(THAP) solution in MeCN. ESI-TOF MS spectra were recorded by using a micro-TOF-Q
type QTOFMS mass spectrometer (Bruker) in the positive-ion mode and with MeOH as the
solvent. HRMS measurements were carried out on a maXis II UHR ESI-QTOF MS
instrument (Bruker) in positive ionization mode. The following parameters were applied for
the electrospray ion source: capillary voltage: 3.6 kV; end plate offset: 500 V; nebulizer
pressure: 0.5 bars; dry gas temperature: 200 °C and dry gas flow rate: 4.0 L/min. Constant
background correction was applied for each spectrum; the background was recorded before
each sample by injecting the blank sample matrix (solvent). Na-formate calibrant was injected
after each sample which enabled internal calibration during data evaluation. Mass spectra
were recorded by otofControl version 4.1 (build: 3.5, Bruker) and processed by Compass
DataAnalysis version 4.4 (build: 200.55.2969).
4.1.1.
(R)-3-{(R)-3-[2-O-(a-L-rhamnopyranosyl)-a-L-
rhamnopyranosyl]oxydecanoyl}oxydecanoic acid (1) [12]
To a solution of compound 8 (27 mg, 0.024 mmol) in dry pyridine (255 μL) LiI (32 mg,
0.24 mmol, 10 equiv.) was added at room temperature and the reaction mixture was stirred for
6 h under reflux (110 °C). After stirring for 6 h, the reaction mixture was evaporated, and. the
residue was suspended with 10% aqueous citric acid and extracted with EtOAc (3 x 10 mL).
The combined organic layer was washed with saturated aqueous solution of Na2S2O3 (5 mL)
and brine (5 mL). The organic layer was dried over MgSO4 and concentrated. The crude
product was purified by silica gel chromatography (95:5 CH2Cl2/acetone) to give the free acid
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(15 mg, 56%) as a light yellow syrup. [α]D −21.4 (c 0.37, CHCl3); Rf = 0.67 (95:5
CH2Cl2/acetone); MS (UHR ESI-QTOF): m/z calcd for C67H88NaO13: 1123.6117 [M+Na]+;

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found: 1123.6117. A mixture of the free acid (15 mg, 0.013 mmol) dissolved in EtOH/AcOH
(96%, 29:1, 2.0 mL) and Pd/C (10%, 10 mg) was stirred in an autoclave under a H2
atmosphere (10 bar) for 24 h. The catalyst was filtered through a pad of Celite® and the
filtrate was concentrated. The crude product was purified by silica gel chromatography
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(8:2:0.3 CH2Cl2/MeOH/H2O) to give compound 1 (6.0 mg, 75%) as a white solid. [α]D
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−22.2 (c 0.05, MeOH), lit: [α]D
−44.6 (c 1, CDCl3)11; Rf = 0.38 (8:2:0.3
CH2Cl2/MeOH/H2O); 1H NMR (D2O, 400 MHz) δ 5.39-5.25 (m, 1H, CH decanoic acid), 4.95
(s, 2H, H-1, H-1’), 4.17-4.14 (m, 1H, CH decanoic acid), 4.08 (s, 1H, H-2), 3.85-3.82 (m, 3H,
H-3, H-3’, H-2’), 3.72 (s, 2H, H-5, H-5’), 3.47-3.44 (m, 2H, H-4, H-4’), 2.61-2.47 (m, 4H, 2 x
CH2COOH decanoic acid), 1.61-1.17 (m, 30H, 12 x CH2 decanoic acid, 2 x CH3 rhamnose),
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0.91-0.90 (m, 6H, 2 x CH3 decanoic acid) ppm; C NMR (CD3OD, 100 MHz) δ 172.6 (2C,
C=O decanoic acid), 104.3 (1C, C-1’), 99.2 (1C, C-1), 75.3, 73.9, 72.3, 72.0, 72.9 (9C, 5 x
skeleton carbons, CH decanoic acid), 41.3 (2C, 2 x CH2COOH decanoic acid), 34.2, 33.1,
33.0, 30.7, 30.5, 30.4, 25.8, 23.7 (12C 12 x CH2 decanoic acid), 18.1 (2C, 2 x CH3 rhamnose),
14.4 (2C, 2 x CH3 decanoic acid) ppm; MS (UHR ESI-QTOF): m/z calcd for C32H58NaO13:
673.3770 [M+Na]+; found: 673.3761.
4.1.2.
(R)-3-{(R)-3-[3-O-(a-L-rhamnopyranosyl)-a-L-
rhamnopyranosyl]oxydecanoyl}oxydecan-oic acid (2)
To a solution of 13 (12.0 mg, 0.011 mmol) in dry pyridine (113 μL) LiI (14.4 mg, 0.11
mmol, 10 equiv.) was added and the reaction mixture was stirred for 6 h at reflux (110 °C),
and the reaction mixture was evaporated. The residue was suspended in 10% aqueous citric
acid and extracted with EtOAc (3 x 10 mL). The combined organic layer was washed with
saturated aqueous solution of Na2S2O3 (5 mL) and brine (5 mL). The organic layer was dried
over MgSO4 and concentrated. The crude product was purified by silica gel chromatography
(9:1 CH2Cl2/acetone) to give the free acid (7.4 mg, 63%) as a colourless syrup. Rf = 0.61
(95:5 CH2Cl2/acetone). A mixture of the crude product of compound 13 (15 mg, 0.013 mmol)
dissolved in EtOH/AcOH (96%, 29:1, 2.0 mL) and Pd/C (10%, 10 mg) was stirred in an
autoclave under a H2 atmosphere (10 bar) for 24 h. The catalyst was filtered through a pad of
Celite® and the filtrate was concentrated. The crude product was purified by silica gel
chromatography (8:2:0.3 CH2Cl2/MeOH/H2O) to give compound 2 (7.2 mg, 90%) as a white
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1
foam. [α]D −57.4 (c 0.05, MeOH); Rf = 0.38 (8:2:0.3 CH2Cl2/MeOH/H2O); H NMR
(CD3OD, 400 MHz) δ 5.27-5.23 (m, 1H, CH decanoic acid), 5.02 (s, 1H, H-1’), 4.76 (s, 1H,
H-1), 4.06-4.03 (m, 1H, CH decanoic acid), 3.97 (d, J = 1.8 Hz, 1H, H-2’), 3.82 (s, 1H, H-2),
3.79-3.73 (m, 3H, H-3, H-3’, H-5’), 3.70-3.65 (m, 1H, H-5), 3.47 (t, J = 9.6 Hz, 1H, H-4),
3.38 (t, J = 9.5 Hz, 1H, H-4’), 2.66-2.46 (m, 4H, 2 x CH2COOH decanoic acid), 1.62-1.24 (m,
30H, 12 x CH2 decanoic acid, 2 x CH3 rhamnose), 0.90 (t, J = 6.6 Hz, 6H, 2 x CH3 decanoic
acid) ppm; 13C NMR (CD3OD, 100 MHz) δ 172.5 (2C, 2 x C=O decanoic acid), 104.1 (1C, C-
1’), 101.1 (1C, C-1), 79.5 (1C, C-3), 76.2 (1C, CH decanoic acid), 74.6 (1C, C-4’), 73.3 (1C,
C-4), 72.9 (1C, CH decanoic acid), 72.5 (1C, C-2), 72.2 (1C, C-3’), 72.1 (1C, C-2’), 70.5 (1C,
C-5), 70.1 (1C, C-5’), 41.5 (2C, 2 x CH2COOH decanoic acid), 35.2, 34.6, 33.0, 32.9, 30.7,
30.5, 30.4, 30.3, 26.3, 26.0, 23.7 (12C 12 x CH2 decanoic acid), 18.0, 17.9 (2C, 2 x CH3
rhamnose), 14.4 (2C, 2 x CH3 decanoic acid) ppm; MS (UHR ESI-QTOF): m/z calcd for
C32H58NaO13: 673.3770 [M+Na]+; found: 673.3767.

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4.1.3. Phenyl 3,4-di-O-benzyl-2-O-(2,3,4-tri-O-benzyl-α-
pyranoside (4)
L-rhamnopyranosyl)-α-L-rhamno-
To a solution of compound 3 [8] (200 mg, 0.497 mmol) in dry DMF (2.0 mL) at 0 °C were
successively added NaH (207 mg, 5.177 mmol, 1.25 equiv./OH). After 30 min at that
temperature BnBr (369 µL, 3.106, 1.25 equiv./OH) was added and the mixture was stirred for
24 h at room temperature. Then MeOH (2.5 mL) was added and the reaction mixture was
stirred for 15 min, and the solvents were evaporated. The residue was dissolved in CH2Cl2 (75
mL), washed with water (3 x 20 mL) until neutral pH. The organic layer was dried over
MgSO4 and evaporated. The crude product was purified by silica gel chromatography (85:15
25
n-hexane/EtOAc) to give 4 (325 mg, 76%) as a colourless syrup. [α]D −78.8 (c 0.32,
1
CHCl3); Rf = 0.38 (85:15 n-hexane/EtOAc); H NMR (CDCl3, 400 MHz) δ 7.41-7.21 (m,
30H, arom), 5.41 (d, J = 1.5 Hz, 1H, H-1), 5.08 (d, J = 1.7 Hz, 1H, H-1’), 4.92-4.87 (m, 2H, 2
x Bn-CH2a), 4.67-4.52 (m, 8H, 2 x Bn-CH2b, 3 x Bn-CH2), 4.23-4.22 (m, 1H, H-2), 4.12-4.07
(m, 1H, H-5), 3.85 (dd, J = 9.3 Hz, J = 3.2 Hz, 1H, H-3’), 3.82 (dd, J = 9.4 Hz, J = 2.8 Hz,
1H, H-3), 3.80-3.79 (m, 1H, H-2’), 3.76-3.73 (m, 1H, H-5’), 3.60 (t, J = 9.3 Hz, 1H, H-4’),
3.37 (t, J = 9.4 Hz, 1H, H-4), 1.27-1.23 (m, 6H, 2 x CH3 rhamnose) ppm; 13C NMR (CDCl3,
100 MHz) δ 138.7, 138.6, 138.5, 138.4, 138.1 (5C, 5 x Cq arom), 134.7 (1C Cq SPh), 131.3-
127.4 (30C, arom), 99.7 (1C, C-1’), 87.4 (1C, C-1), 80.7 (1C, C-4’), 80.4, 80.3 (2C, C-3, C-
4), 79.3 (1C, C-3’), 76.2 (1C, C-2), 75.5 (2C, 2 x Bn-CH2), 75.1 (1C, C-2’), 72.6, 72.4, 72.3
(3C, 3 x Bn-CH2), 69.4 (1C, C-5), 68.8 (1C, C-5’), 18.1, 18.0 (2C, 2 x CH3 rhamnose) ppm;
MS (ESI-TOF): m/z calcd for C53H56NaO8S: 875.359 [M+Na]+; found: 875.278.
4.1.4. Methyl (3R)-3-[3,4-di-O-benzyl-2-0-(2,3,4-tri-O-benzyl-α-
rhamnopyranosyloxy]decanoate (5) and methyl (3R)-3-[3,4-di-O-benzyl-2-0-(2,3,4-tri-O-
benzyl-α- -rhamnopyranosyl)-α- -rhamnopyranosyloxy]decanoate (6)
L-rhamnopyranosyl)-β-L-
L
L
To a solution of disaccharide donor 4 (55 mg, 0.065 mmol, 1.25 equiv.) and methyl (R)-3-
hydroxydecanoate (11 mg, 0.052 mmol) in dry CH2Cl2 (2.5 mL) 4 Å MS (50 mg) were added
and the reaction mixture was stirred at room temperature. After 30 min the stirred mixture
was cooled to −50 °C under argon. After this temperature was reached, NIS (22 mg, 0.0975
mmol, 1.5 equiv. to the donor) was dissolved in dry THF (100 µL) and TfOH (1.7 µL, 0.0195
mmol, 0.3 equiv. to the NIS) were added. The temperature was allowed to warm up to −20 °C
and the reaction mixture was stirred for 3 h. The reaction mixture was quenched with Et3N
(50 µL), diluted with CH2Cl2 (25 mL), filtered and the mixture was washed with saturated
aqueous solution of Na2S2O3 (2 x 5 mL), saturated aqueous solution of NaHCO3 (5 mL) and
H2O (2 x 5 mL) until neutral pH. The organic layer was dried over MgSO4 and concentrated.
The crude product was purified by silica gel chromatography (8:2 n-hexane/EtOAc) to give 5
(24 mg 46%) as a colourless syrup and 6 (26 mg, 50%), ratio: 1:1 = α : β.
Data of compound 5: [α]D25 +15.7 (c 0.12, CHCl3); Rf = 0.29 (8:2 n-hexane/EtOAc); 1H NMR
(CDCl3, 400 MHz) δ 7.36-7.19 (m, 25H, arom), 5.21 (s, 1H, H-1’), 4.94 (d, J = 11.4 Hz, 1H,
Bn-CH2a), 4.88 (d, J = 11.0 Hz, 1H, Bn-CH2a), 4.69-4.42 (m, 9H, H-1, 2 x Bn-CH2b, 3 x Bn-
CH2), 4.25 (dq, J = 6.2 Hz, J = 12.5 Hz, 1H, C-5’), 4.10 (d, J = 2.1 Hz, 1H, H-2), 4.09-4.05
(m, 1H, CH decanoic acid), 3.94 (dd, J = 3.2 Hz, J = 9.4 Hz, 1H, H-3’), 3.81 (s, 1H, H-2’),
3.65 (s, 3H, OCH3), 3.58 (t, J = 9.5 Hz, 1H, H-4’), 3.49 (dd, J = 2.5 Hz, J = 8.9 Hz, 1H, H-3),

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3.35-3.26 (m, 2H, H-4, H-5), 2.53-2.42 (m, 2H, CH-CH2 decanoic acid), 1.64-1.50 (m, 2H,
CH2 decanoic acid), 1.35-1.22 (m, 16H, 2 x CH3 rhamnose, 5 x CH2 decanoic acid), 0.84 (t, J
= 6.8 Hz, 3H, CH3 decanoic acid) ppm; 13C NMR (CDCl3, 100 MHz) δ 172.4 (1C, Cq
decanoic acid), 139.5, 139.1, 138.6, 138.5, 138.1 (5C, 5 x Cq arom), 128.6-127.3 (25C, arom),
99.6 (1C, C-1), 98.3 (1C, C-1’), 83.3 (1C, C-3), 80.9 (1C, C-4’), 80.1 (1C, C-4), 79.8 (1C, C-
3’), 75.9 (1C, CH decanoic acid), 75.4 (1C, Bn-CH2), 75.3 (1C, C-2’), 74.8 (1C, Bn-CH2),
73.0 (1C, C-2), 72.8, 72.3, 72.1 (3C, 3 x Bn-CH2), 71.7 (1C, C-5), 67.8 (1C, C-5’), 51.7, (1C,
COOCH3), 39.8 (1C, CH-CH2 decanoic acid), 35.7, 32.0, 29.6, 29.3, 25.1, 22.8 (6C, 6 x CH2
decanoic acid), 18.1, 17.8 (2C, 2 x CH3 rhamnose), 14.3 (1C, CH3 decanoic acid) ppm; MS
(UHR ESI-QTOF): m/z calcd for C58H72NaO11: 967.4967 [M+Na]+; found: 967.4978.
Data of compound 6: [α]D25 −30.6 (c 0.10, CHCl3); Rf = 0.29 (8:2 n-hexane/EtOAc); 1H NMR
(CDCl3, 400 MHz) δ 7.53-7.21 (m, 25H, arom), 5.10 (d, J = 1.5 Hz, 1H, H-1’), 4.93 (d, J =
10.9 Hz, 1H, Bn-CH2a), 4.85 (d, J = 11.0 Hz, 1H, Bn-CH2a), 4.77 (d, J = 1.5 Hz, 1H, H-1),
4.66-4.47 (m, 8H, 2 x Bn-CH2b, 3 x Bn-CH2), 4.02 (dd, J = 7.3 Hz, J = 5.6 Hz, 1H, CH
decanoic acid), 3.96 (d, J = 2.5 Hz, 1H, H-2), 3.85 (dd, J = 9.2 Hz, J = 3.1 Hz, 1H, H-3’),
3.79-3.73 (m, 3H, H-2’, H-3, H-5’), 3.65 (s, 3H, OCH3), 3.63-3.61 (m, 1H, H-5), 3.60 (t, J =
7.9 Hz, 1H, H-5), 3.28 (t, J = 9.5 Hz, 1H, H-4), 2.55 (dd, J = 15.1 Hz, J = 7.5 Hz, 1H, CH-
CH2a decanoic acid), 2.42 (dd, J = 15.1 Hz, J = 5.4 Hz, 1H, CH-CH2b decanoic acid), 1.50-
1.48 (m, 2H, CH2 decanoic acid), 1.32 (d, J = 6.2 Hz, 3H, CH3 rhamnose), 1.32-1.24 (m, 10H,
5 x CH2 decanoic acid), 1.22 (d, J = 6.2 Hz, 3H, CH3’ rhamnose), 0.88 (t, J = 6.9 Hz, 3H, CH3
decanoic acid) ppm; 13C NMR (CDCl3, 100 MHz) δ 171.8 (1C, Cq decanoic acid), 138.7,
138.5 (5C, 5 x Cq arom), 128.6-127.6 (25C, arom), 99.4 (1C, C-1’), 98.0 (1C, C-1), 80.7 (1C,
C-4’), 80.3 (1C, C-4), 80.1 (1C, C-3), 79.4 (1C, C-3’), 75.4, 75.3 (2C, 2 x Bn-CH2), 75.1 (1C,
C-2’), 74.5 (2C, C-2, CH decanoic acid), 72.7, 72.3, 72.2 (3C, 3 x Bn-CH2), 68.6 (1C, C-5),
68.4 (1C, C-5’), 51.7, (1C, COOCH3), 40.4 (1C, CH-CH2 decanoic acid), 33.7, 31.9, 29.7,
29.3, 24.9, 22.8 (6C, 6 x CH2 decanoic acid), 18.1, 18.0 (2C, 2 x CH3 rhamnose), 14.2 (1C,
CH3 decanoic acid) ppm; MS (UHR ESI-QTOF): m/z calcd for C58H72NaO11: 967.4967
[M+Na]+; found: 967.4981.
4.1.5. (3R)-3-[3,4-di-O-benzyl-2-0-(2,3,4-tri-O-benzyl-α-
rhamnopyranosyloxy]decanoic acid (7)
L-rhamnopyranosyl)-α-L-
To a solution of compound 6 (125 mg, 0.132 mmol) in the mixture of MeOH (3.7 mL) and
THF (3.7 mL), 0.5 M aqueous solution of NaOH (3.7 mL) was added and the reaction mixture
was stirred at room temperature for 24 h. The reaction mixture was neutralized with acetic
acid and concentrated. The crude product was purified by silica gel chromatography (65:35 n-
25
hexane/acetone) to give 7 (112.9 mg, 92%) as a colourless syrup. [α]D −18.2 (c 0.17,
1
CHCl3); Rf = 0.46 (65:35 n-hexane/acetone); H NMR (CDCl3, 400 MHz) δ 7.32-7.20 (m,
25H, arom), 5.15 (s, 1H, H-1’), 4.93 (d, J = 10.9 Hz, 1H, Bn-CH2a), 4.84 (d, J = 10.9 Hz, 1H,
Bn-CH2a), 4.78 (s, 1H, H-1), 4.66-4.45 (m, 9H, 2 x Bn-CH2b, 3 x Bn-CH2, OH), 4.04-4.00
(m, 1H, CH decanoic acid), 3.97 (s, 1H), 3.85 (dd, J = 3.0 Hz, J = 9.2 Hz, 1H), 3.80-3.68 (m,
4H), 3.61 (t, J = 9.3 Hz, 1H), 3.29 (t, J = 9.4 Hz, 1H), 2.54 (dd, J = 7.4 Hz, J = 15.4 Hz, 1H,
CH-CH2a decanoic acid), 2.41 (dd, J = 5.3 Hz, J = 15.4 Hz, 1H, CH-CH2b decanoic acid),
1.49-1.48 (m, 2H, CH2 decanoic acid), 1.32 (d, J = 6.2 Hz, 3H, CH3 rhamnose), 1.31-1.23 (m,
10H, 5 x CH2 decanoic acid), 1.22 (d, J = 6.1 Hz, 3H, CH3’ rhamnose), 0.88 (t, J = 6.7 Hz,

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3H, CH3 decanoic acid) ppm; C NMR (CDCl3, 100 MHz) δ 176.5 (1C, Cq decanoic acid),
138.7, 138.5, 138.3 (5C, 5 x Cq arom), 128.6-127.6 (25C, arom), 99.2 (1C, C-1’), 97.9 (1C, C-
1), 80.6, 80.2, 80.0, 79.2, 74.9, 74.3, 74.1, 68.5, 68.4 (9C, 8 x skeleton carbons, CH decanoic
acid), 75.4, 75.2, 72.7, 72.2 (5C, 5 x Bn-CH2), 40.1 (1C, CH-CH2 decanoic acid), 33.5, 31.9,
29.7, 29.3, 24.8, 22.7 (6C, 6 x CH2 decanoic acid), 18.1, 17.7 (2C, 2 x CH3 rhamnose), 14.2
(1C, CH3 decanoic acid) ppm; MS (UHR ESI-QTOF): m/z calcd for C57H70NaO11: 953.4810
[M+Na]+; found: 953.4809.
4.1.6. Methyl (3R)-3-{(3R)-3-[3,4-di-O-benzyl-2-0-(2,3,4-tri-O-benzyl-α-
α- -rhamnopyranosyloxy]decanoyloxy)decanoate (8)
To a solution of compound 7 (19.6 mg, 0.02106 mmol) and methyl (R)-3-
hydroxydecanoate (5.16 mg. 0.026 mmol, 1.25 equiv.) in dry CH2Cl2 (240 μL), DCC (5.1 mg,
0.025 mmol, 1.20 equiv.) and DMAP (1.3 mg, 0.010 mmol, 0.5 equiv.) were added and the
reaction mixture was stirred at room temperature for 18 h. After stirring for 18 h the reaction
mixture was diluted with diethyl ether (800 μL) and the dicyclohexylurea was removed by
filtration. The filtrate was washed with water (2.5 mL), and the organic layer was dried over
MgSO4 and concentrated. The crude product was purified by silica gel chromatography (8:2
L-rhamnopyranosyl)-
L
25
n-hexane/acetone) to give 8 (7.2 mg, 31%) as a colourless syrup. [α]D −10.0 (c 0.06,
CHCl3); Rf = 0.46 (8:2 n-hexane/acetone); 1H NMR (CDCl3, 400 MHz) δ 7.32-7.20 (m, 25H,
arom), 5.22-5.16 (m, 1H, CH decanoic acid), 5.12 (s, 1H, H-1’), 4.93 (d, J = 10.9 Hz, 1H, Bn-
CH2a), 4.85 (d, J = 11.0 Hz, 1H, Bn-CH2a), 4.77 (s, 1H, H-1), 4.67-4.46 (m, 8H, 2 x Bn-
CH2b, 3 x Bn-CH2), 3.98-3.96 (m, 2H, skeleton proton, CH decanoic acid), 3.85 (dd, J = 3.0
Hz, J = 9.2 Hz, 1H), 3.80-3.69 (m, 4H), 3.62-3.58 (m, 4H, skeleton proton, COOCH3), 3.29 (t,
J = 9.4 Hz, 1H), 2.62-2.38 (m, 4H, 2 x CH-CH2 decanoic acid), 1.61-1.49 (m, 4H, 2 x CH2
decanoic acid), 1.36-1.22 (m, 26H, 10 x CH2 decanoic acid, 2 x CH3 rhamnose), 0.89-0.85 (m,
13
6H, 2 x CH3 decanoic acid) ppm; C NMR (CDCl3, 100 MHz) δ 170.9, 170.6 (2C, 2 x Cq
decanoic acid), 138.8, 138.5 (5C, 5 x Cq arom), 128.6-127.6 (25C, arom), 99.3 (1C, C-1’),
98.4 (1C, C-1), 80.7, 80.4, 80.2, 79.4, 75.1, 74.5, 74.3, 70.9, 68.6, 68.4 (10C, 8 x skeleton
carbons, 2 x CH decanoic acid), 75.4, 75.3, 72.7, 72.3, 72.2 (5C, 5 x Bn-CH2), 51.8 (1C,
COOCH3), 40.5, 39.1 (2C, 2 x CH-CH2 decanoic acid), 34.0, 33.5, 32.0, 31.9, 29.8, 29.4,
29.3, 25.3, 24.9, 22.7 (12C, 12 x CH2 decanoic acid), 18.1, 18.0 (2C, 2 x CH3 rhamnose), 14.2
(1C, CH3 decanoic acid) ppm; MS (UHR ESI-QTOF): m/z calcd for C68H90NaO13: 1137.6274
[M+Na]+; found: 1137.6272.
4.1.7. Phenyl 2,4-di-O-benzyl-3-O-(2,3,4-tri-O-benzyl-α-
L-rhamnopyranosyl)-α-L-rhamno-
pyranoside (10)
To a solution of compound 9 [8] (611 mg, 1.519 mmol) in dry DMF (6.11 mL) at 0 °C
were added NaH (60%) (380 mg, 15.82 mmol, 1.25 equiv./OH group) in portions. After 30
min at the same temperature BnBr (1.13 mL, 9.494 mmol, 1.25 equiv./OH group) was added,
and the mixture was stirred for 24 h at room temperature. Then MeOH (10 mL) was added
and the mixture was stirred for 15 min, and the solvents were evaporated. The residue was
dissolved in CH2Cl2 (250 mL), washed with H2O (3 x 50 mL) until neutral pH. The organic
layer was dried over MgSO4, filtered, and concentrated. The crude product was purified by
silica gel chromatography (8:2 n-hexane/EtOAc) to give 10 (977 mg, 75%) as a colourless

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syrup. [α]D −66.8 (c 0.50, CHCl3); Rf = 0.56 (8:2 n-hexane/EtOAc); H NMR (CDCl3, 400
MHz) δ 7.48-7.17 (m, 30H, arom), 5.48 (d, J = 1.2 Hz, 1H, H-1), 5.16 (s, 1H, H-1’), 4.95 (d, J
= 11.1 Hz, 1H, Bn-CH2a), 4.69-4.48 (m, 9H, Bn-CH2b, 4 x Bn-CH2), 4.16-4.11 (m, 1H, H-5),
4.06 (dd, J = 3.0 Hz, J = 9.4 Hz, 1H, H-3), 3.99-3.98 (m, 1H, H-2), 3.88 (dd, J = 3.1 Hz, J =
9.3 Hz, 1H, H-3’), 3.85-3.82 (m, 1H, H-5’), 3.79-3.75 (m, 1H, H-2’), 3.64-3.61 (m, 2H, H-4,
H-4’), 1.30-1.27 (m, 6H, 2 x CH3 rhamnose) ppm; 13C NMR (CDCl3, 100 MHz) δ 139.0,
138.7, 138.5, 138.5, 138.4, 137.8 (5C, 5 x Cq arom), 134.7 (1C Cq SPh), 131.6-127.2 (30C,
arom), 100.0 (1C, C-1’), 85.6 (1C, C-1), 80.9, 80.6 (2C, C-4, C-4’), 80.0 (1C, C-3’), 79.6 (1C,
C-2), 78.2 (1C, C-3), 75.8 (1C, C-2’), 75.2, 74.9, 72.7, 72.3 (5C, 5 x Bn-CH2), 69.6 (1C, C-5),
68.9 (1C, C-5’), 18.2, 18.0 (2C, 2 x CH3 rhamnose) ppm; MS (ESI-TOF): m/z calcd for
C53H56NaO8S: 875.359 [M+Na]+; found: 875.281.
4.1.8. Methyl (3R)-3-[2,4-di-O-benzyl-3-0-(2,3,4-tri-O-benzyl-α-
rhamnopyranosyloxy]decanoate (11)
L-rhamnopyranosyl)-α-L-
To a solution of disaccharide donor 10 (300 mg, 0.352 mmol, 1.25 equiv.) and methyl (R)-
3-hydroxydecanoate (57 mg, 0.286 mmol, 1 equiv.) in dry CH2Cl2 (13.5 mL), 4 Å MS (50
mg) were added and the reaction mixture was stirred at room temperature. After 30 min the
stirred mixture was cooled to −40 °C under argon, and NIS (119 mg, 0.528 mmol, 1.5 equiv.
to the donor) dissolved in dry THF (250.0 µL), and TfOH (9.3 µL, 0.106 mmol, 0.3 equiv. to
the donor) were added. The mixture was allowed to warm up to +5 °C and stirred for 5 h. The
reaction mixture was quenched with Et3N (100 µL), diluted with CH2Cl2 (100 mL), filtered
and the mixture was washed with saturated aqueous solution of Na2S2O3 (2 x 25 mL),
saturated aqueous solution of NaHCO3 (25 mL) and H2O (2 x 25 mL) until neutral pH. The
organic layer was dried on MgSO4 and concentrated. The crude product was purified by silica
gel chromatography (8:2 n-hexane/EtOAc) to give 11 (297 mg, 89%) as a colourless syrup.
[α]D25 −18.9 (c 0.09, CHCl3); Rf = 0.41 (8:2 n-hexane/EtOAc); 1H NMR (CDCl3, 400 MHz) δ
7.42-7.20 (m, 25H, arom), 5.14 (s, 1H, H-1’), 4.96 (d, J = 11.1 Hz, 1H, Bn-CH2a), 4.77 (s,
1H, H-1), 4.72-4.42 (m, 9H, Bn-CH2b, 4 x Bn-CH2), 4.03 (dd, J = 2.8 Hz, J = 9.5 Hz, 1H, H-
3), 3.99-3-96 (m, 1H, CH decanoic acid), 3.89 (dd, J = 2.8 Hz, J = 9.3 Hz, 1H, H-3’), 3.80-
3.73 (m, 2H, H-2’, H-5’), 3.66 (s, 3H, OCH3), 3.70-3.59 (m, 3H, H-2, H-4’, H-5), 3.53 (t, J =
9.5 Hz, 1H, H-4), 2.56 (dd, J = 7.4 Hz, J = 15.2 Hz, 1H, CH-CH2a decanoic acid), 2.40 (dd, J
= 5.4 Hz, J = 15.2 Hz, 1H, CH-CH2b decanoic acid), 1.42-1.38 (m, 2H, CH2 decanoic acid),
1.30-1.22 (m, 16H, 5 x CH2 decanoic acid, 2 x CH3 rhamnose), 0.88 (t, J = 6.9 Hz, 3H, CH3
decanoic acid) ppm; 13C NMR (CDCl3, 100 MHz) δ 171.9 (1C, Cq decanoic acid), 139.1,
138.7, 138.5, 138.2 (5C, 5 x Cq arom), 128.9-127.1 (25C, arom), 99.7 (1C, C-1’), 97.3 (1C, C-
1), 81.1 (1C, C-4), 81.0 (1C, C-4’), 80.0 (1C, C-3’), 78.1 (1C, C-2), 75.8 (1C, C-3), 75.3 (1C,
Bn-CH2), 75.2 (1C, C-2’), 74.7 (2C, CH decanoic acid, Bn-CH2), 72.8, 72.6, 72.2 (3C, 3 x
Bn-CH2), 68.8, 68.7 (1C, C-5’), 51.8, (1C, COOCH3), 40.4 (1C, CH-CH2 decanoic acid),
33.9, 31.9, 29.6, 29.3, 25.0, 22.8 (6C, 6 x CH2 decanoic acid), 18.1, 18.0 (2C, 2 x CH3
rhamnose), 14.2 (1C, CH3 decanoic acid) ppm; MS (UHR ESI-QTOF): m/z calcd for
C58H72NaO11: 967.4967 [M+Na]+; found: 967.4966.
4.1.9. (3R)-3-[2,4-di-O-benzyl-3-0-(2,3,4-tri-O-benzyl-α-
rhamnopyranosyloxy]decanoic acid (12)
L-rhamnopyranosyl)-α-L-

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To a solution of compound 11 (229 mg, 0.243 mmol) in a mixture of MeOH (6.8 mL) and
THF (6.8 mL), 0.5 M aqueous solution of NaOH (6.8 mL) was added and the reaction mixture
was stirred at room temperature for 24 h. The mixture was neutralized with acetic acid and
concentrated. The crude product was purified by silica gel chromatography (65:35 n-
hexane/acetone) to give 12 (173 mg, 77%) as a colourless syrup. [α]D25 −24.0 (c 0.20, CHCl3);
Rf = 0.57 (65:35 n-hexane/acetone); 1H NMR (CDCl3, 400 MHz) δ 7.35-7.20 (m, 25H, arom),
5.14 (s, 1H, H-1’), 4.96 (d, J = 11.0 Hz, 1H, Bn-CH2a), 4.78 (s, 1H, H-1), 4.71-4.43 (m, 10H,
Bn-CH2b, 4 x Bn-CH2, OH), 4.04 (d, J = 8.0 Hz, 1H), 3.97-3.94 (m, 1H, CH decanoic acid),
3.89 (d, J = 9.1 Hz, 1H), 3.78-3.73 (m, 3H), 3.64-3.60 (m, 2H), 3.55-3.51 (m, 1H), 2.58 (dd, J
= 7.0 Hz, J = 15.5 Hz, 1H, CH-CH2a decanoic acid), 2.40 (dd, J = 5.0 Hz, J = 15.5 Hz, 1H,
CH-CH2b decanoic acid), 1.42-1.38 (m, 2H, CH2 decanoic acid), 1.27-1.21 (m, 16H, 5 x CH2
decanoic acid, 2 x CH3 rhamnose), 0.88 (t, J = 6.6 Hz, 3H, CH3 decanoic acid) ppm; 13C
NMR (CDCl3, 100 MHz) δ 176.7 (1C, Cq decanoic acid), 139.0, 138.6, 138.5, 138.4, 138.2
(5C, 5 x Cq arom), 128.5-127.1 (25C, arom), 99.6 (1C, C-1’), 97.5 (1C, C-1), 81.0, 80.5, 79.9,
78.0, 77.3, 75.7, 75.1, 68.8, 68.7 (9C, 8 x skeleton carbons, CH decanoic acid), 75.2, 74.6,
72.8, 72.5, 72.1 (5C, 5 x Bn-CH2), 40.3 (1C, CH-CH2 decanoic acid), 34.0, 31.9, 29.5, 29.3,
24.9, 22.7 (6C, 6 x CH2 decanoic acid), 18.0, 17.3 (2C, 2 x CH3 rhamnose), 14.2 (1C, CH3
decanoic acid) ppm; MS (UHR ESI-QTOF): m/z calcd for C57H70NaO11: 953.4810 [M+Na]+;
found: 953.4808.
4.1.10. Methyl (3R)-3-{(3R)-3-[2,4-di-O-benzyl-3-0-(2,3,4-tri-O-benzyl-α-
L-
rhamnopyranosyl)-α- -rhamnopyranosyloxy]decanoyloxy)decanoate (13)
L
To a solution of compound 12 (19 mg, 0.02042 mmol) and methyl (R)-3-hydroxydecanoate
(5.16 mg. 0.025 mmol, 1.25 equiv.) in dry CH2Cl2 (240 μL), DCC (5.1 mg, 0.025 mmol, 1.20
equiv.) and DMAP (1.3 mg, 0.010 mmol, 0.5 equiv.) were added and the reaction mixture was
stirred at room temperature for 18 h. After stirring for 18 h the mixture was diluted with
diethyl ether (800 μL) and the dicyclohexylurea was removed by filtration. The filtrate was
washed with water (2.5 mL). The organic layer was dried on MgSO4 and concentrated. The
crude product was purified by silica gel chromatography (8:2 n-hexane/acetone) to give 13
25
(14 mg, 63%) as a colourless syrup. [α]D −27.0 (c 0.10, CHCl3); Rf = 0.43 (8:2 n-
hexane/acetone); 1H NMR (CDCl3, 400 MHz) δ 7.36-7.20 (m, 25H, arom), 5.21-5.18 (m, 1H,
CH decanoic acid), 5.15 (s, 1H, H-1’), 4.97 (d, J = 11.1 Hz, 1H, Bn-CH2a), 4.78 (s, 1H, H-1),
4.71-4.43 (m, 9H, Bn-CH2b, 4 x Bn-CH2), 4.04 (dd, J = 2.7 Hz, J = 8.0 Hz, 1H), 3.95-3.88
(m, 2H, skeleton proton, CH decanoic acid), 3.80-3.70 (m, 4H), 3.64-3.57 (m, 1H), 3.61 (s,
3H, COOCH3), 3.55-3.51 (m, 1H), 2.62 (dd, J = 6.6 Hz, J = 15.8 Hz, 2H, 2 x CH-CH2a
decanoic acid), 2.52 (dd, J = 6.0 Hz, J = 15.4 Hz, 1H, CH-CH2b decanoic acid), 2.40 (dd, J =
7.0 Hz, J = 15.3 Hz, 1H, CH-CH2b decanoic acid), 1.69-1.37 (m, 4H, 2 x CH2 decanoic acid),
1.27-1.22 (m, 26H, 10 x CH2 decanoic acid, 2 x CH3 rhamnose), 0.90-0.85 (m, 6H, CH3
decanoic acid) ppm; 13C NMR (CDCl3, 100 MHz) δ 170.9, 170.7 (2C, 2 x Cq decanoic acid),
139.0, 138.6, 138.5, 138.1 (5C, 5 x Cq arom), 128.5-127.0 (25C, arom), 99.7 (1C, C-1’), 97.7
(1C, C-1), 81.1, 80.5, 79.9, 78.0, 77.3, 75.8, 75.4, 70.8, 68.7, 68.6 (10C, 8 x skeleton carbons,
2 x CH decanoic acid), 75.2, 74.7, 72.8, 72.5, 72.1 (5C, 5 x Bn-CH2), 40.6, 39.0 (2C, 2 x CH-
CH2 decanoic acid), 34.0, 33.9, 31.9, 31.8, 29.6, 29.4, 29.3, 29.2, 25.2, 24.9, 22.8, 22.7 (12C,
12 x CH2 decanoic acid), 18.0, 17.9 (2C, 2 x CH3 rhamnose), 14.2 (2C, 2 x CH3 decanoic

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acid) ppm; MS (UHR ESI-QTOF): m/z calcd for C68H90NaO13: 1137.6274 [M+Na]+; found:
1137.6273.
4.2. rHPLOE binding activity
4.2.1. Binding activity of rHPLOE to synthetic di-rhamnolipid
For direct enzyme-linked immunosorbent assays (ELISA), prepared di-rhamnolipid
solution (100 μg/mL in ddH2O) was diluted in 10× the volume of the coating solution
(chloroform/ethanol= 1:9, v/v), and 100 μL of the mixture was added to each well of the 96-
well microplate (#442404, Thermo Fisher, USA) and the solvent was air-dried in chemical
hood. After blocking with a blocking reagent (#10057177103, Roche, Switzerland) at 37 °C
for 1 h, the plates were washed with PBST (PBS with 0.05% Tween-20) three times. To the
washed wells, 50 μL of 0.1 μM rHPLOE was added to the wells, which were maintained at 37
°C for 1 h. Fifty μL of 0.1 μM purified rHPLOE was added in parallel as a positive control, and
Tris-buffer (20 mM Tris-HCl and 200 mM NaCl, pH 7.4) was added in parallel as a negative
control. After washing three times with PBST, the microplates were incubated with
monoclonal anti-His-tag (1:5000; #631212, Clontech, USA) in PBST at 37 °C for 1 h.
Subsequently, horseradish peroxidase-conjugated anti-mouse IgG (1:5000; #AB2338512,
Jackson ImmunoResearch, USA) in PBST was added to the microplates, and after washing
three times with PBST, the plates were incubated at 37 °C for 1 h. After being washed three
times with PBST, 100 μL of 3,3’,5,5’-tetramethylbenzidine substrate (#5120-0077, SeraCare
KPL, USA) was added to each well and incubated at 37 °C for exactly 15 min. Finally, the
reaction was terminated by the addition of 100 μL of 2 N H2SO4. The A450nm was read using a
microplate spectrophotometer (iMark Microplate Absorbance Reader, Bio-Rad, USA). All
ELISA experiments were individually performed at least three times. The values are indicated
with the mean ± SD.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors gratefully acknowledge financial support for this research from the Premium
Postdoctoral Program of HAS (PPD 461038), the ÚNKP-19-3-I, New National Excellence
Program of the Ministry for Innovation and Technology, the EU and co-financed by the
European Regional Development Fund under the project GINOP-2.3.2-15-2016-00008 and
GINOP-2.3.3-15-2016-00004, and from the Ministry of Science and Technology (MOST 107-
0210-01-19-04, Taiwan).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.org/XXXXX
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Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: