Synthesis of potential allosteric modulators of Hsp90 by chemical glycosylation of Eupomatenoid-6

Article abstract of DOI:10.1016/j.carres.2014.03.006

Hsp90 (Heat shock protein-90) is a chaperone protein and an established anti-apoptotic target in cancer therapy. Most of the known small-molecule inhibitors that have shown potent antitumor activity target the Hsp90 N-terminal domain and directly inhibit its ATP-ase activity. Many of these molecules display important secondary effects. A different approach to Hsp90 inhibition consists of targeting the protein C-terminal domain (CTD) and modulating its chaperone activity through allosteric effects. Using an original computational approach, allosteric hot-spots in the CTD have been recently identified that control interdomain communication. A combination of virtual and experimental screening enabled identification of a rhamnosylated benzofuran (Eupomatenoid-2) as a lead for further development. In this paper we describe glycodiversification of Eupomatenoid-2 using chemical glycosylation of the 2-(4′-hydroxyphenyl)benzofuran aglycon (a.k.a. Eupomatenoid-6). Glycosylation of the phenol by glycosyl bromides under basic conditions afforded the desired products in the gluco-, galacto-, and fuco-series. This approach failed in the manno- and rhamno-series. However, mannosylation and rhamnosylation of Eupomatenoid-6 could be obtained under carefully controlled acidic conditions, using O-benzoxazolyl imidate (OBox) donors. The glycosides obtained are currently under investigation as modulators of Hsp90 chaperone activity.

Full text of DOI:10.1016/j.carres.2014.03.006

Accepted Manuscript
Synthesis of potential allosteric modulators of Hsp90 by chemical glycosylation
of Eupomatenoid-6
Laura Morelli, Anna Bernardi, Sara Sattin
PII:
S0008-6215(14)00094-9
DOI:
http://dx.doi.org/10.1016/j.carres.2014.03.006
CAR 6701
Reference:
Carbohydrate Research
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 January 2014
4 March 2014
5 March 2014
Please cite this article as: Morelli, L., Bernardi, A., Sattin, S., Synthesis of potential allosteric modulators of Hsp90
by chemical glycosylation of Eupomatenoid-6, Carbohydrate Research (2014), doi: http://dx.doi.org/10.1016/
j.carres.2014.03.006
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Synthesis of potential allosteric modulators of Hsp90 by chemical
glycosylation of Eupomatenoid-6
Laura Morelli, Anna Bernardi, Sara Sattin*
Università degli Studi di Milano, Dipartimento di Chimica, via Golgi 19, 20133 Milano, Italy.
*corresponding author: E-mail address: sara.sattin@unimi.it, Phone: +39 0250314096
Abstract
Hsp90 (Heat shock protein-90) is a chaperone protein and an established anti-apoptotic target in cancer
therapy. Most of the known small-molecule inhibitors that have shown potent antitumor activity target
the Hsp90 N-terminal domain and directly inhibit its ATP-ase activity. Many of these molecules display
important secondary effects. A different approach to Hsp90 inhibition consists of targeting the protein
C-terminal domain (CTD) and modulating its chaperone activity through allosteric effects. Using an
original computational approach, allosteric hot-spots in the CTD have been recently identified that
control interdomain communication. A combination of virtual and experimental screening enabled
identification of a rhamnosylated benzofuran (Eupomatenoid-2) as a lead for further development. In
this paper we describe glycodiversification of Eupomatenoid-2 using chemical glycosylation of the 2-(4’-
hydroxyphenyl)benzofuran aglycon (a.k.a. Eupomatenoid-6). Glycosylation of the phenol by glycosyl
bromides under basic conditions afforded the desired products in the gluco-, galacto- and fuco- series.
This approach failed in the manno- and rhamno- series. However, mannosylation and rhamnosylation of
Eupomatenoid-6 could be obtained under carefully controlled acidic conditions, using O-benzoxazolyl
imidate (OBox) donors. The glycosides obtained are currently under investigation as modulators of
Hsp90 chaperone activity.
Keywords : Eupomatenoid, phenol glycosylation, Hsp90, O-benzoxazolyl imidate (OBox),
Glycodiversification, Phase Transfer Catalysis
1. Introduction
Heat Shock Proteins (HSPs) are a class of functionally related chaperone proteins, which are
overexpressed as a protective mechanism in cells exposed to a variety of stressful events. They have
been shown to possess a pivotal role in cell cycle progression and cell death (apoptosis) and to be
involved in many diseases. In particular, Hsp90 is established as an anti-apoptotic target in cancer
therapy.1,2 Hsp90 consists of four domains, an N-terminal ATP binding site domain, a middle domain
that regulates ATP hydrolysis, a charged region, and a C-terminal homodimerization domain.3 These
domains are involved in complex internal dynamics processes that control the chaperone activity of the
Page 1 of 20

protein and, with it, the signaling pathways regulated by Hsp90, which controls a number of client
proteins.1,4
Numerous small-molecule inhibitors of Hsp90 have been identified and have shown potent
antitumor activity in a wide-range of malignancies.5 Most of these molecules, such as Geldanamycin or
Radicicol,6 target the Hsp90 N-terminal domain and directly inhibit its ATP-ase activity. The
aminocoumarin Novobiocin and its analogues7,8 have been shown to bind the C-terminal domain (CTD)
of Hsp90 and to cause proteosomal degradation of the clients by inhibiting correct folding. A recent
study has shown that the activity and selectivity of Novobiocin can be tuned by glycosylation at the 4’
position.9
Using an original computational approach,10,11 allosteric hot-spots in Hsp90 CTD that control
interdomain communication have been recently identified by Colombo and co-workers. Virtual
screening enabled the identification of a group of 14 molecules targeted to these hot spots, six of which
were experimentally shown to bind the CTD and to control Hsp90 function in cellular studies.11
Allosteric modulation of Hsp90 may allow adjustments to the internal dynamics of the protein, leading
to fine tuning of the signalling pathways it regulates. Thus, allosteric inhibitors or activators of Hsp90
activity may become useful tools for system biology studies.
Among the hits identified,11 we focused our attention on the rhamnosylated 2-(4’-hydroxyphenyl)-5-
propenyl-benzofuran scaffold 1 (Eupomatenoid-2,12 Figure 1). The aglyconic part is also known as
Eupomatenoid-6 (2, Figure 1), a natural product extracted from the leaves of Piper fulvescens that has
been the subject of several syntheses.13-16 Glycodiversification17-19 of 1 has the potential to generate a
set of diverse modulators of Hsp90 activity.9,20,21
In this paper, we report our studies on the chemical glycosylation of Eupomatenoid-6 (2).
Figure 1. The rhamnosylated 2-phenyl-5-propenyl-benzofuran scaffold 1 and its aglycon 2.
2. Results and discussion
Eupomatenoid-6 (2) was prepared according to a reported procedure13 shown in Scheme 1, that starts
from 2-bromo-4-chlorophenol 3 and leads to the intermediate 5-chlorobenzofuran 4, which is
Page 2 of 20

transformed in 2 using a Stille coupling. Due to the diasteroemeric purity of the Stille reagent used, the
target 2 was obtained as a 3:1 E:Z mixture.
Scheme 1: Synthesis of Eupomatenoid-6 (2) and the glycosylation model aglycon 6. i. Pd(OAc)2, rac-2-(di-tert-
butylphosphino)-1,1′-binaphthyl (rac-DTBPB), NaOtBu, toluene, MW 100°C, 1h; ii. TFA/DCM, r.t., 45% over two
steps; iii. Tri-N-butyl(1-propenyl)tin, [HPtBu3][BF4]Pd2dba3, CsF, 85%, E:Z 3:1; iv. NaSEt, DMF, 145°C, >95%.
For the exploration of different glycosylation strategies the 5-chlorobenzofuran 6 (Scheme 1) was
chosen as a model aglycon, because it is synthetically more accessible than 2 and avoids the analysis of
E:Z mixtures. Compound 6 was obtained in good yield by demethylation of 4 using sodium
ethanethiolate.13
Glycosylation of phenols is associated with several specific problems.22,23 First, under acidic conditions,
phenols are weaker nucleophiles compared to alcohols, because the aromatic ring is electron
withdrawing. Moreover, under classical Lewis acid catalyzed glycosylation conditions, C-glycosylation
competes with, or often prevails over, O-glycosylation. Nonetheless, acceptable yields can usually be
obtained even with not very active glycosyl donors (such as O-acetates) using phenols carrying electron-
donating groups (e.g., p-methoxyphenol).24
We initially tested the glycosylation of 6 with β-D-glucose penta-O-acetate 7 in the presence of boron
trifluoride etherate24 and with α-D-glucose tetra-O-acetyl-trichloroacetimidate 825 in the presence of
TMSOTf at -20°C (Scheme 2). In both cases, 9 was isolated in low yields (24% and 27%) as a 6:1 (β:α)
anomeric mixture and extensive decomposition of the aglycon was observed. Upon suggestion by a
referee, the glycosylation reaction was also performed in the presence of boron trifluoride and TEA.26
slight improvement in the diastereoselectivity was indeed observed (β:α, 10:1) but yields remained
disappointingly low (30% ca.).
A
Page 3 of 20

Scheme 2: Glycosylation of 6 under acidic conditions. i. 7 (1.5 eq), BF3.Et2O (3 eq), r.t. ii. 8 (1.2 eq), TMSOTf (0.1
eq), -20 °C.
The aglycon instability and the scarce reactivity under acidic conditions associated with poor
stereoselectivity prompted us to examine glycosylation by glycosyl halide donors under basic conditions,
where phenols are easily deprotonated.23
Glycosylation of model acceptor 6 with α-D-glucosyl bromide tetra-O-acetate donor 10 was screened
under different experimental conditions (Table 1). Reaction of 10 with 6 in the presence of excess silver
carbonate afforded only traces of the expected product 9 (β anomer) and the acetylated acceptor 11
was recovered as the major product (Table 1, entry 1). Reaction of the Cs salt of 6 with excess 10 in
DMF at 60°C led to recovery of unreacted starting material (Table 1, entry 2). Phase Transfer Catalysis
(PTC) conditions were explored under various experimental setups. Tetrabutylammonium iodide (TBAI)
was initially examined, using an excess (3 eq) of donor in CHCl3 as a solvent and aq. K2CO3 as the base,
following a reported procedure27 (Table 1, entry 3). Under these conditions the β-glycosylation product
9 was formed in good yields (60%), but the conversion was incomplete. Similar results were obtained
using the more efficient catalyst tetrabutylammonium hydrogensulfate (TBAHSO4) (entry 4, 65% yield).
Lowering the amount of donor (0.66 eq, entry 5) was beneficial for the chromatographic isolation of 9,
but worsened considerably the yields of the reaction. Indeed, it was more convenient to isolate the
glycosylation product after deprotection (MeONa) to yield 9a (Table 1, entry 6, 60% yield over the two
steps).
The anomeric configuration of 9 was assigned as β after deacetylation to 9a, which showed a J1,2
coupling constant of 7.3 Hz (DMSO-d6, Supplementary Figure 3).
Page 4 of 20

Table 1: Initial screening for optimal glycosylation conditions of acceptor 6 with donor 10
Entry Eq. donor Reagents
Solvent
T
Time 9 Yield
Other products
1
2
3
3
Ag2CO3 (3.65 eq)
Pyridine
r.t.
24 h
11 (50%)
Traces
-
6 Cs salta
DMF
60°C 24 h
starting materials
traces of 11
3
4
5
6
3
TBAI (0.2 eq)
K2CO3 (6 eq)
CHCl3-H2Ob r.t.
24 h 60%
starting materials
traces of 11
3
TBAHSO4 (0.5 eq)
K2CO3 (6 eq)
CHCl3-H2Oc
CHCl3-H2Oc
CHCl3-H2Oc
r.t.
r.t.
r.t.
8 h
65%
starting materials
traces of 11
0.66
3
TBAHSO4 (0.5 eq)
K2CO3 (6 eq)
24 h 30%
24 h 60%d
starting materials
11 (20 %)
TBAHSO4 (0.5 eq)
K2CO3 (6 eq)
starting materials
11 (10 %)
a Generated with Cs2CO3 b 0.04M, H2O: 5% w/wK2CO3; c 0.1M, H2O: 5% w/wK2CO3; d Yield calculated over 2 steps after
deprotection to afford 9a.
The reaction of different glycosyl bromide donors (L-Glc, D- and L-Gal and L-Fuc configuration) with 6
under the optimized conditions led to the desired products in modest to excellent yields (Table 2). An
excess of donor or acceptor was used depending on the availability of the bromide. The fucosylated
product was obtained in almost quantitative yield using an excess of donor (Table 2, entry 4). The
anomeric configuration of all glycosylated products was confirmed to be β only (18a-21a, Table 2) by
experimental NMR JH1,H2 coupling constants of the deacetylated products.
Page 5 of 20

Table 2: Glycosylation of acceptor 6 with different donors.
Isolated yielda
36% (43%)b
41%b
Entry
Donor Eq donor
Product
By-products
1
2
3
4
5
6
16
12
15
14
13
17
0.83
3
18a
19a
20
21a
-
11 (30%)
-
0.83
3
62% (77%)
traces of 11
>95%b
-
-
-
c
0.66
0.66
c
-
a
i. CHCl3 (0.1-0.2M)/ water (5% w/wK2CO3), TBAHSO4 (0.5 eq), K2CO3 (6 eq) at room temperature for 24h. Yields in
brackets are calculated based on the limiting agent. b Yield calculated over 2 steps after deprotection. c No
reaction occurred.
Disappointingly, under these conditions glycosides of manno- configuration (D-Man and L-Rha) were not
accessible; attempted reaction gave only complete recovery of starting material (Table 2, entries 5-6).
Mitsunobu conditions were explored whereby 6 was treated with tetra-O-benzoyl-rhamnose and Ph3P,28
but again only unreacted starting material was recovered.
Attempts to glycosylate 6 with rhamnose penta-O-acetate in the presence of boron trifluoride
etherate24 or with mannose tetra-O-benzoyl-trichloroacetimidate in the presence of TMSOTf were not
successful. No reaction was observed in the first case, while C-glycosylation and aglycon decomposition
were detected in the second case. Demchenko and co-workers recently reported a new class of glycosyl
donors, the O-benzoxazolyl (OBox) imidates,29 that possess intermediate reactivity properties relative to
O-trichloroacetimidates and thioglycosides. In particular OBox glycosides react with nucleophiles under
Lewis acid activation at very low temperature, which may prevent degradation of our acid-sensitive
Page 6 of 20

acceptor. Following Demchenko procedure, a mixture of acceptor (6) and the known donor α-D-
Man(OBz)4-OBox 2229 (1.2eq) was dissolved in 1,2-dichloroethane (1,2-DCE) and cooled to -78°C, with
the solvent solidifying at -35°C. After addition of a 0.1M TMSOTf solution in 1,2-DCE (0.1 eq) the
reaction mixture was slowly warmed to -30°C and quenched by water addition after 2 min stirring.
Work-up and chromatographic purification afforded product 23 as a single (α) isomer in 33% yield
(Table 3, entry 1). Similarly, the unprecedented rhamnosyl donor 24 was prepared in 65% yield from tri-
O-benzoyl-α-L-rhamnosyl bromide 26 and AgOBox 27 (Scheme 3) and reacted with 6, providing product
25 in high yield (87%, Table 3, entry 2). Zemplén deprotection of the O-benzoyl glycosides followed by
reverse phase automated chromatography afforded the corresponding glycosides 23a and 25a.
Table 3: TMSOTf-promoted glycosylation reaction of acceptors 6 with D-Man-OBox (22) and L-Rha-OBox (24).
Entry Acceptor Donor Product Isolated yield
1
2
6
6
22
24
23
25
33%
87%
i. 6 (1 eq, 0.2M in 1,2-DCE), 22 or 24 (1.2 eq), TMSOTf (0.1 eq), -78°C to m.p., 5 min; ii. MeONa, MeOH, r.t.
Scheme 3: Preparation of α-L-Rha(OBz)3-OBox 24 by reaction of tri-O-benzoyl-α-L-rhamnosyl bromide 26 with
AgOBox 27.
Page 7 of 20

Thus, with these two strategies in hands, scaffold 2 was glycosylated with different glycosyl donors. The
OBox strategy was successfully applied for the preparation of the D-Man and L-Rha derivatives (28 and
29, respectively), which were obtained in moderate to good yields (Table 4, entries 1-2).
When performing the reaction on the pure E isomer of 2 (E-2, Eupomatenoid-6), product E-29 was
obtained in 70% yield (Table 4, entry 3). Benzoate hydrolysis of E-29 under Zemplén conditions afforded
α-L-rhamnosyl 2-(4’-hydroxy-phenyl)-5-prop-1-E-enyl-benzofuran 1 (Eupomatenoid-2) which was found
to be identical to an authentic sample obtained from the National Cancer Institute Collection (NCI). The
α configuration of Eupomatenoid-2, previously assigned to the natural product without unequivocal
experimental evidence,12 was confirmed by analysis of 1H coupling constants and of the 1H NOESY
spectrum. The signal of H4 of 1 appears as a t with J3,4 = J4,5 = 9.5 Hz, which is consistent with a chair
(1C4) conformation. The anomeric coupling constant (J1,2) of 1.8 Hz is typical of α-rhamno glycosides and
strong nOe contacts for H1 are observed only with H2 and Hortho of the phenol moiety, as expected for an
α rhamnoside (see Supplementary Figure 13). Glycosylation of 2 under PTC conditions afforded the D-
/L-Glc, D-/L-Gal and L-Fuc β glycosides in moderate yields (Table 4). As shown in the model system,
higher yields of the desired products were obtained using an excess of donor (Table 4, compare entries
4 and 5). Nonetheless, in some cases, an excess of acceptor was employed to simplify the purification
procedure, since the glycosylation products often co-elute with the donor. For the same reason in some
cases products were purified after deacetylation and yields are given over the two steps (Table 4, entries
6-8 and 10).
Page 8 of 20

Table 4: Glycosylation of acceptor 2 with different glycosyl donors.
Entry
Donor Eq donor
Product
Isolated yielda
1
2
22
24
24
10
10
16
12
12
15
14
1.2
1.2
1.2
0.66
3
28
29
54%
55%
3
E-29
30
70%
4
25% (39%)
50%
5
30b
31ac
32a
32a
33
6
0.83
0.66
3
32%d (38%)
47%d (70%)
50%d
7
8
9
0.83
0.66
61% (71%)
49%d (73%)
10
34a
i. TMSOTf (0.1 eq), 1,2-DCE (0.2M), -78°C to m.p., 5 min; ii. CHCl3 (0.1-0.2M)/water (5% w/wK2CO3), TBAHSO4 (0.5
a
eq), K2CO3 (6 eq) r.t., 24h; iii. MeONa, MeOH, r.t. Yields in brackets are calculated based on the limiting agent;
b 35 (20%); c 35 (30%); d Yield calculated over 2 steps after deacetylation.
3. Conclusions
Chemical glycosylation of the phenol functionality of Eupomatenoid-6 (2) and of its synthetic precursor 6
was achieved using glycosyl halides under basic phase transfer conditions for the D- and L-gluco-, D- and
L-galacto- and L-fuco- glycosides. Products of manno- configuration (L-Rha and D-Man) were obtained
using the corresponding OBox-imidate donor under Lewis acid catalysis at low temperature. In all cases
6-deoxy sugars (L-Rha and L-Fuc) afforded the corresponding glycosides in considerably higher yields;
this is in accord with previous reports for chemical glycosylation of phenols.23 The anomeric
configuration of the products was determined by NMR JH1-H2 coupling constants or, when this was not
Page 9 of 20

distinctive, by analysis of the nOe contacts. β-Glycosides were obtained in the gluco-, galacto- and fuco-
series, α-glycosides in the manno- and rhamno- series.
The compounds synthesized have undergone preliminary testing for binding to the Hsp90 C-terminal
domain.30 Several have been found to bind as well as Eupomatenoid-2. These molecules are currently
being analyzed for allosteric modulation of Hsp90 function in cellular models.
4. Experimental
4.1 General Methods
All chemical reagents were of analytical grade, and used as supplied. Organic solvents were dried
according to common literature protocols. All non-hydrolytic reactions were conducted under a positive
pressure of nitrogen. Analytical thin layer chromatography (TLC) was performed on silica gel 60-F254
(Merck). Plates were visualized by ultraviolet (UV) light, or treatment with molibdic reagent solution,
followed by heating. Compounds were purified by flash chromatography using Silica gel 60 (0.040-0.063
mm, 230-400 mesh particle size) by Merck. Automated flash chromatography by Biotage instrument
(Biotage Isolera™ Prime) was used to purify final compounds. KP-SilTM (40–65 µm, average 50 micron)
Biotage SNAP cartridges were used for direct-phase purifications (for reverse-phase: KP-C18-HSTM,
1
average 25 micron); solvents were of reagent grade and were used as supplied. H-NMR spectra were
recorded at 400 MHz and 13C-NMR spectra were recorded at 100 MHz, using a Bruker Avance 400
instrument. Chemical shifts (δ) are reported in ppm using residual solvent signals from deuterated
solvents as references.31 Signals in 1H and 13C NMR spectra were assigned with the aid of two
dimensional COSY and HSQC spectra. For some signals in 1H NMR spectra, the coupling patterns were
reported as multiplets due to high order coupling or signal overlap. Mass spectrometry was performed
under positive/negative-mode electrospray ionization (ESI) on a ThermoFinnigan LCQ™ Classic or
Waters® Micromass® Q-Tof micro™ Mass (ESI-HRMS) and for high resolution on Bruker Daltonics APEX II
(FT-ICR). Optical rotations were measured in a 10 cm cell with a Perkin-Elmer 241 polarimeter at 25°C.
Compounds 3, 7, 10 and 12 are commercially available (Sigma-Aldrich). Compounds 1,12 2, 4 and 5,13
13,32 14,33 15,34 16,35 1736 and 1829 are known. The characterization of protected glycosides 9, 18-21, 23,
25, 28-34 is detailed in the supplementary information.
4.2 General procedures
4.2.1 Glycosylation under phase transfer catalysis
Peracetylated glycosyl bromide (3 eq) and 2-(4’-hydroxyphenyl)benzofuran (2 or 6, 1 eq) were dissolved
in chloroform (10 mL/mmol). Tetrabutylammonium hydrogen sulfate (0.5 eq), potassium carbonate (6
Page 10 of 20

eq) and water (5% w/w of potassium carbonate) were added to the solution. The reaction mixture was
stirred for 24h at room temperature. The reaction mixture was diluted with chloroform, the organic
layer was washed with 0.1M HCl, sat. NaHCO3 and brine, dried over anhydrous sodium sulfate and
concentrated under vacuum. The crude product was purified by automated flash chromatography
(hexane/ethyl acetate gradient elution).
4.2.2 TMSOTf-promoted glycosylation with O-Benzoxazolyl imidates
A mixture of glycosyl donor (1.2 eq) and aglycon (1 eq) in dry 1,2-dichloroethane (5 mL/mmol) was
stirred under nitrogen atmosphere at room temperature for 5 min and then cooled to −78 °C. A 0.1M
solution of TMSOTf (0.1 eq) in dry 1,2-dichloroethane was added to the frozen mixture. Since 1,2-
dichloroethane melts at -35°C, the reaction mixture was let warm to -30/-20°C and stirred for 2 min at
this temperature. After the reaction was finished (TLC), the reaction mixture was diluted with CH2Cl2
and washed with 1% aq. NaOH and water. The organic phase was dried over anhydrous Na2SO4, and
concentrated in vacuo. The residue was purified by column chromatography on silica gel (hexane/ethyl
acetate gradient elution).
4.2.3 General procedure for Zemplen hydrolysis of acetates/benzoates
A 1 M solution of sodium methoxide in dry methanol (0.5 eq / O-cleavage) was drop-wise added to a
solution of the peracetylated (or a suspension of the perbenzoylated) product in methanol (0.1 M). The
reaction mixture was stirred for 1 h (2 h for O-Bz) at room temperature and then neutralized with IR-120
H+ (Amberlite) resin. The resin was filtered off and the combined filtrate was concentrated in vacuo.
The product was purified by reverse-phase automated flash chromatography (water/methanol gradient
elution).
4.3 Synthesized compounds
4.3.1 4-(5-Chloro-3-methyl-2-benzofuranyl)-phenol (6) Compound 413 (710 mg, 2.6 mmol) was
dissolved in dry DMF (13 mL). EtSNa (450 mg, 5.2 mmol) was added and the mixture was stirred at 145
°C for 4 h. The reaction was cooled to r.t. and sat. NH4Cl was added (10 mL). The mixture was extracted
with EtOAc and the combined organic layers were washed with H2O and brine, dried over anhydrous
Na2SO4, filtered off and concentrated in vacuum. The residue was purified by column chromatography
on silica gel (hexane/ethyl acetate gradient elution) to afford 662 mg (98 %) of the demethylated
benzofuran 6. Rf = 0.30 (85:15 Hex:EtOAc); 1H NMR (400 MHz, CDCl3) δ 7.74 – 7.64 (m, 2H, 2 x H-m-Phe-
O), 7.47 (dd, J = 2.1, 0.5 Hz, 1H, H-4 Bf), 7.36 (dd, J = 8.6, 0.5 Hz, 1H, H-7 Bf), 7.21 (dd, J = 8.6, 2.1 Hz, 1H,
H-6 Bf), 6.99 – 6.91 (m, 2H, 2 x H-o-Phe-O), 4.90 (s, 1H, OH), 2.40 (s, 3H, CH3 Bf). 13C NMR (101 MHz,
CDCl3) δ 155.7 (Cquat.-Phe-O), 152.3 (C-7a),152.1 (C-2), 132.9 (C-5),128.6 (2 x C-o-Phe-O), 128.0 (Cquat.
-
Page 11 of 20

Phe, C-3a), 124.1 (C-6), 118.9 (C-4), 115.8 (2 x C-m-Phe-O), 111.9 (C-7), 109.5 (C-3), 9.4 (CH3 Bf). HRMS
(ESI) calcd for C15H10O2Cl1 [M-H]- 257.03748, found 257.03747.
4.3.2 Benzoxazolyl 2,3,4-tri-O-benzoyl-α-L-rhamnopyranoside (24) AgOBox29 (324 mg, 1.34mmol),
2,6-lutidine (117 µL, 1.00 mmol) and TBAI (14 mg, 0.04 mmol) were added to a solution of 2,3,4-O-
benzoyl-α-L-rhamnopyranosyl bromide (363 mg, 0.67 mmol) in dry CH2Cl2 (4.5 mL) under nitrogen
atmosphere. The reaction mixture was stirred for 4 h at 50°C and then diluted with CH2Cl2 and washed
with 1% aq. NaOH (2 x 10 mL), water (2 x 10 mL) and brine (10mL). The organic phase was dried over
anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by column chromatography on
silica gel (hexane/ethyl acetate gradient elution) to afford 24 (257 mg, 65% yield) as a white solid. Rf =
0.44 (8:2 Hex:EtOAc);
+40.3 (c 1.0, CHCl3); 1H NMR (CDCl3): δ 8.11 (d, J = 7.6 Hz, 2H, 2 x o-CH-Bz),
7.99 (d, J = 7.8 Hz, 2H, 2 x o-CH-Bz), 7.84 (d, J = 7.9 Hz, 2H, 2 x o-CH-Bz), 7.65 (t, J = 7.3 Hz, 1H, p-CH-Bz),
7.60 – 7.50 (m, 4H, 1 x p-CH Bz, 2 x m-CH Bz, H-4Box), 7.50 – 7.40 (m, 4H, 2 x m-CH-Bz, 1 x p-CH-Bz, H-
7Box), 7.34 – 7.25 (m, 4H, 2 x m-CH-Bz, H-5Box, H-6Box), 6.57 (d, 1H, J1,2 = 1.1 Hz, H-1), 6.04 – 5.90 (m, 2H,
H-2, H-3), 5.79 (t, J= 9.6 Hz, 1H, H-4), 4.44 (dq, J = 12.3, 6.1 Hz, 1H, H-5), 1.42 (d, J = 6.2 Hz, 3H, CH3-Rha).
13C NMR (CDCl3): δ 165.8 (C=O Bz), 165.6 (C=O Bz), 165.4 (C=O Bz), 161.3 (C-2 Box), 148.7 (C-7a Box), 140.7
(C-3a Box), 133.9 (p-CH-Bz), 133.6 (p-CH-Bz), 133.4 (p-CH-Bz), 130.1 (2 x o-CH-Bz), 129.9 (4 x o-CH-Bz),
129.1 (Cquat. Bz), 129.0 (2 x Cquat. Bz), 128.8 (2 x m-CH-Bz), 128.6 (2 x m-CH-Bz), 128.5 (2 x m-CH-Bz), 124.7
(C-6Box), 123.6 (C-5Box), 118.9 (C-4Box), 110.1 (C-7Box), 97.9 (C-1), 71.1 (C-4), 69.7 (C-2), 69.4 (C-3), 69.3 (C-
5), 17.8 (CH3-Rha). HRMS calcd for C34H27N1O9Na1 [M+Na]+ 616.15780, found 616.15736.
4.3.3 4-[3-methyl-5-(E-prop-1-enyl)benzofuran-2-yl]phenyl α-L-rhamnopyranoside (1,
Eupomatenoid-2) Compound 1 (42mg, 0.06 mmol) was prepared following general procedures 4.2.2 (E-
29, 70%) and 4.2.3 (quant.) and it was isolated after automated flash chromatography purification
(chloroform/methanol gradient elution). Rf = 0.32 (9:1 CH2Cl2:CH3OH); 1H NMR (400 MHz, CD3OD) δ
7.78 – 7.67 (m, 2H, 2 x H-m-Phe-O), 7.43 (bs, 1H, H-4 Bf), 7.32 (d, J = 8.5 Hz, 1H, H-7 Bf), 7.27 (dd, J = 8.5,
1.5 Hz, 1H, H-6 Bf), 7.23 – 7.10 (m, 2H, 2 x H-o-Phe-O), 6.50 (dd, J = 15.7, 1.5 Hz, 1H, =CH-Ar), 6.23 (dq, J
= 15.7, 6.5 Hz, 1H, =CH-Me), 5.51 (bd, J = 1.5 Hz, 1H, H-1), 4.04 (dd, J = 3.3, 1.8 Hz, 1H, H-2), 3.88 (dd, J =
9.5, 3.4 Hz, 1H, H-3), 3.67 (tt, J = 6.2, 4.6 Hz, 1H, H-5), 3.49 (t, J = 9.5 Hz, 1H, H-4), 2.40 (s, 3H, CH3 Bf),
1.88 (dd, J = 6.6, 1.5 Hz, 3H, Me-CH=), 1.26 (s, 3H, CH3 Rha). ESI-MS calcd for C24H26O6Na1 [M+Na]+
433.2, found 433.3; calcd for C48H51O12 [2M-H]- 819.3, found 819.6.
4.3.4 4-[5-Chloro-3-methylbenzofuran-2-yl]phenyl β-D-glucopyranoside (9a) Compound 9a (24 mg,
0.057 mmol) was prepared following general procedures 4.2.1 and 4.2.3 (60% yield, over two steps). Rf =
0.23 (9:1 CH2Cl2:CH3OH);
-55.0 (c 0.1, Dioxane); 1H NMR (400 MHz, MeOD) δ 7.80 – 7.69 (m, 2H, 2
Page 12 of 20

x H-m-Phe-O), 7.54 (d, J = 2.1 Hz, 1H, H-4 Bf), 7.42 (d, J = 8.6 Hz, 1H, H-7 Bf), 7.27 – 7.20 (m, 3H, H-6 Bf, 2
x H-o-Phe-O), 5.00 (dd, J = 5.4, 2.1 Hz, 1H, H-1), 3.93 (dd, J = 12.1, 2.0 Hz, 1H, H-6b), 3.72 (dd, J = 12.1,
5.6 Hz, 1H, H-6a), 3.53 – 3.46 (m, 3H, H-5, H-3, H-2), 3.45 – 3.38 (m, 1H, H-4), 2.42 (s, 3H, CH3 Bf). 13C
NMR (101 MHz, MeOD) δ 159.2 (Cquat.-Phe-O), 153.5 (C-7a Bf), 153.4 (C-2 Bf), 134.0 (C-5 Bf), 129.2 (2 x C-
m-Phe-O), 126.3 (C-3a Bf, Cquat.-Phe), 125.2 (C-6 Bf), 119.8 (C-4 Bf), 117.9 (2 x C-o-Phe-O), 112.8 (C-7 Bf),
1
110.9 (C-3 Bf), 102.1 (C-1), 78.2 (C-5), 78.0 (C-3), 74.9 (C-2), 71.3 (C-4), 62.5 (C-6), 9.3 (CH3 Bf). H NMR
(400 MHz, DMSO-d6) δ 7.78 – 7.71 (m, 3H, 2 x H-m-Phe-O, H-4 Bf), 7.59 (d, J = 8.7 Hz, 1H, H-7 Bf), 7.31
(dd, J = 8.7, 2.2 Hz, 1H, H-6 Bf), 7.20 (d, J = 8.9 Hz, 2H, 2 x H-o-Phe-O), 5.36 (d, J = 4.5 Hz, 1H, OH-2), 5.13
(d, J = 4.3 Hz, 1H, OH-3), 5.06 (d, J = 5.2 Hz, 1H, OH-4), 4.96 (d, J = 7.3 Hz, 1H, H-1), 4.58 (t, J = 5.7 Hz, 1H,
OH-6), 3.76 – 3.65 (bddd, 1H, J = 11.7, 5.7, 1.5 Hz, 1H, H-6a), 3.48 (dt, J = 11.7, 5.9 Hz, 1H, H-6b), 3.42 –
3.34 (m, 1H, H-5), 3.33 – 3.11 (m, 6H, H-3, H-2, H-4), 2.41 (s, 3H, CH3 Bf). HRMS (ESI) calcd for
C21H21O7Cl1Na1 [M+Na]+ 443.08683, found 443.08705.
4.3.5 4-[5-Chloro-3-methylbenzofuran-2-yl]phenyl β-L-glucopyranoside (18a) Compound 18a (40 mg,
0.094 mmol) was prepared following general procedures 4.2.1 and 4.2.3 (43% yield, over two steps). Rf =
1
+55 (c 0.1, Dioxane). H NMR (400 MHz, CD3OD) δ 7.80 – 7.69 (m, 2H, 2
0.23 (9:1 CH2Cl2:CH3OH);
x H-m-Phe-O), 7.56 (d, J = 1.9 Hz, 1H, H-4 Bf), 7.44 (d, J = 8.6 Hz, 1H, H-7 Bf), 7.27 – 7.20 (m, 3H, H-6 Bf, 2
x H-o-Phe-O), 5.02 (dd, J = 5.4, 2.0 Hz, 1H, H-1), 3.94 (dd, J = 12.1, 2.0 Hz, 1H, H-6b), 3.74 (dd, J = 12.1,
5.6 Hz, 1H, H-6a), 3.58 – 3.38 (m, 4H, H-5, H-3, H-2, H-4), 2.43 (s, 3H, CH3 Bf). ESI-MS calcd for
C21H21O7Cl1Na1 [M+Na]+ 443.1, found 443.3; calcd for C42H42O14Cl2Na1 [2M+Na]+ 863.2, found 863.1;
calcd for C42H41O14Cl2 [2M-H]- 839.2, found 839.3.
4.3.6 4-[5-Chloro-3-methylbenzofuran-2-yl]phenyl β-D-galactopyranoside (19a) Compound 19a (29
mg, 0.07 mmol) was prepared following general procedures 4.2.1 and 4.2.3 (41% yield, over two steps).
1
Rf = 0.25 (9:1 CH2Cl2:CH3OH);
-52 (c 0.15, Dioxane). H NMR (400 MHz, CD3OD) δ 7.78 – 7.71 (m,
2H, 2 x H-m-Phe-O), 7.54 (d, J = 1.9 Hz, 1H, H-4 Bf), 7.43 (d, J = 8.9 Hz, 1H, H-7 Bf), 7.29 – 7.22 (m, 3H, H-
6 Bf, 2 x H-o-Phe-O), 4.96 (d, J = 7.7 Hz, 1H, H-1), 3.93 (d, J = 3.2 Hz, 1H, H-4), 3.87 – 3.70 (m, 4H, H-2, H-
6a, H-6b, H-5), 3.61 (dd, J = 9.7, 3.4 Hz, 1H, H-3), 2.42 (s, 3H, CH3 Bf). 13C NMR (101 MHz, CD3OD) δ
159.3 (Cquat.-Phe-O), 153.6 (C-7a Bf), 153.4 (C-2 Bf), 134.0 (C-5 Bf), 129.2 (2 x C-m-Phe-O), 126.2 (C-3a Bf,
Cquat.-Phe), 125.2 (C-6 Bf), 119.8 (C-4 Bf), 118.0 (2 x C-o-Phe-O), 112.8 (C-7 Bf), 110.9 (C-3 Bf), 102.7 (C-1),
77.1 (C-5), 74.9 (C-3), 72.2 (C-2), 70.2 (C-4), 62.5 (C-6), 9.3 (CH3 Bf). HRMS (ESI) calcd for C21H21O7Cl1Na1
[M+Na]+ 443.08680, found 443.08682.
4.3.7 4-[5-Chloro-3-methylbenzofuran-2-yl]phenyl β-L-galactopyranoside (20a) Compound 20a (55
mg, 0.13 mmol) was prepared following general procedures 4.2.1 (14, 77%) and 4.2.3 (quant. yield). Rf =
1
0.25 (9:1 CH2Cl2:CH3OH);
+52.0 (c 0.1, Dioxane). H NMR (400 MHz, CD3OD) δ 7.78 – 7.71 (bd, 2H,
Page 13 of 20

2 x H-m-Phe-O), 7.55 – 7.52 (bd, 1H, H-4 Bf), 7.45 – 7.40 (bd, 1H, H-7 Bf), 7.28 – 7.20 (bs, 3H, H-6 Bf, 2 x
H-o-Phe-O), 4.96 (d, J = 7.7 Hz, 1H, H-1), 3.93 (d, J = 3.4 Hz, 1H, H-4), 3.88 – 3.70 (m, 4H, H-2, H-6a, H-6b,
H-5), 3.66 – 3.57 (m, 1H, H-3), 2.42 (s, 1H, CH3 Bf). ESI-MS calcd for C21H21O7Cl1Na1 [M+Na]+ 443.1, found
443.3; calcd for C42H42O14Cl2Na1 [2M+Na]+ 863.2, found 863.2; calcd for C42H41O14Cl2 [2M-H]- 839.2,
found 839.1.
4.3.8 4-[5-Chloro-3-methylbenzofuran-2-yl]phenyl β-L-fucopyranoside (21a) Compound 21a (35 mg,
0.088 mmol) was prepared following general procedures 4.2.1 (21, >95%) and 4.2.3 (quant. yield). Rf =
1
+52.0 (c 0.1, Dioxane). H NMR (400 MHz, CD3OD) δ 7.79 – 7.70 (m, 2H,
0.38 (9:1 CH2Cl2:CH3OH);
2 x H-m-Phe-O), 7.54 (d, J = 2.0 Hz, 1H, H-4 Bf), 7.42 (d, J = 8.6 Hz, 1H, H-7 Bf), 7.29 – 7.13 (m, 3H, H-6 Bf,
2 x H-o-Phe-O), 4.94 (d, J = 7.7 Hz, 1H, H-1), 3.93 – 3.83 (m, 1H, H-5), 3.79 (dd, J = 9.7, 7.7 Hz, 1H, H-2),
3.69 (bd, J = 2.9 Hz, 1H, H-4), 3.61 (dd, J = 9.7, 3.4 Hz, 1H, H-3), 2.42 (s, 3H, CH3 Bf), 1.33 (d, J = 6.5 Hz,
3H, CH3 Fuc). 13C NMR (101 MHz, CD3OD) δ 159.3 (Cquat.-Phe-O), 153.6 (C-7a Bf), 153.4 (C-2 Bf), 134.0 (C-
5 Bf), 129.1 (2 x C-m-Phe-O), 126.2 (C-3a Bf, Cquat.-Phe), 125.2 (C-6 Bf), 119.8 (C-4 Bf), 117.9 (2 x C-o-Phe-
O), 112.8 (C-7 Bf), 110.9 (C-3 Bf), 102.4 (C-1), 75.0 (C-3), 72.9 (C-4), 72.3 (C-5), 72.0 (C-2), 16.8 (CH3 Fuc),
9.3 (CH3 Bf). HRMS (ESI) calcd for C21H21Cl1O6Na1 [M+Na]+ 427.09189, found 427.09228.
4.3.9 4-[5-Chloro-3-methylbenzofuran-2-yl]phenyl α-D-mannopyranoside (23a) Compound 23a (9
mg, 0.02 mmol) was prepared following general procedures 4.2.2 (23, 33%) and 4.2.3 (52%) and it was
isolated after automated flash purification (chloroform/methanol gradient elution). Rf = 0.27 (9:1
1
+85.0 (c 0.1, Dioxane). H NMR (400 MHz, CD3OD) δ 7.78 – 7.71 (m, 2H, 2 x H-m-
CH2Cl2:CH3OH);
Phe-O), 7.54 – 7.52 (m, 1H, H-4 Bf), 7.44 – 7.39 (m, 1H, H-7 Bf), 7.29 – 7.21 (m, 3H, H-6 Bf, 2 x H-o-Phe-
O), 5.57 (d, J = 1.6 Hz, 1H, H-1), 4.04 (bdd, J = 3.3, 1.8 Hz, 1H, H-2), 3.93 (dd, J = 9.4, 3.4 Hz, 1H, H-3), 3.85
– 3.68 (m, 3H, H-4, H-6a, H-6b), 3.66 – 3.55 (m, 1H, H-5), 2.41 (s, 3H, CH3 Bf). 13C NMR (101 MHz,
CD3OD) δ 158.0 (Cquat.-Phe-O), 153.5 (C-7a Bf), 153.4 (C-2 Bf), 134.0 (C-5 Bf), 129.2 (2 x C-m-Phe-O),
126.2 (C-3a Bf, Cquat.-Phe), 125.2 (C-6 Bf), 119.8 (C-4 Bf), 118.0 (2 x C-o-Phe-O), 112.8 (C-7 Bf), 111.0 (C-3
Bf), 100.1 (C-1), 75. 6 (C-5), 72.4 (C-3), 71.9 (C-2), 68.3 (C-4), 62.7 (C-6), 9.3 (CH3 Bf). HRMS (ESI) calcd
for C21H21O7Cl1Na1 [M+Na]+ 443.08680, found 443.08718.
4.3.10 4-[5-Chloro-3-methylbenzofuran-2-yl]phenyl α-L-rhamnopyranoside (25a) Compound 25a (36
mg, 0.09 mmol) was prepared following procedures 4.2.2 (25, 87%) and 4.2.3 (quant.). Rf = 0.35 (9:1
1
CH2Cl2:CH3OH);
-124.7 (c 0.1, Dioxane). H NMR (400 MHz, CD3OD) δ 7.72 – 7.66 (m, 2H, 2 x H-m-
Phe-O), 7.47 (d, J = 2.1 Hz, 1H, H-4 Bf), 7.37 (d, J = 8.6 Hz, 1H, H-7 Bf), 7.22 – 7.14 (m, 3H, H-6 Bf, 2 x H-o-
Phe-O), 5.51 (d, J = 1.6 Hz, 1H, H-1), 4.04 (dd, J = 3.4, 1.8 Hz, 1H, H-2), 3.88 (dd, J = 9.5, 3.5 Hz, 1H, H-3),
3.66 (dq, J = 9.9, 6.2 Hz, 1H, H-5), 3.49 (t, J = 9.5 Hz, 1H, H-4), 2.35 (s, 3H, CH3 Bf), 1.26 (d, J = 6.2 Hz, 4H,
CH3 Rha). 13C NMR (101 MHz, CD3OD) δ 157.9 (Cquat.-Phe-O), 153.4 (C-7a Bf), 153.3 (C-2 Bf), 134.0 (C-5
Page 14 of 20

Bf), 129.2 (2 x C-m-Phe-O), 126.2 (C-3a Bf, Cquat.-Phe), 125.1 (C-6 Bf), 119.7 (C-4 Bf), 117.7 (2 x C-o-Phe-
O), 112.7 (C-7 Bf), 111.0 (C-3 Bf), 99.7 (C-1), 73. 8 (C-4), 72.2 (C-3), 72.0 (C-2), 70.8 (C-5), 18.1 (CH3 Rha),
9.3 (CH3 Bf). HRMS (ESI) calcd for C21H21O6Cl1Na1 [M+Na]+ 427.09189, found 427.09208.
4.3.11 E,Z-4-[3-methyl-5-(prop-1-enyl)benzofuran-2-yl]phenyl α-D-mannopyranoside (28a) Compound
28a (34 mg, 0.08 mmol) was prepared following general procedures 4.2.2 (28, 54%) and 4.2.3 (quant.)
and it was isolated as 1:1 E:Z mixture after automated flash chromatography (chloroform/methanol
1
gradient elution). Rf = 0.26 (9:1 CH2Cl2:CH3OH). H NMR (400 MHz, CD3OD) δ 7.78 – 7.69 (m, 2H, 2 x H-
m-Phe-O), 7.44 (s, 0.5H, H-4 Bf E), 7.42 (s, 0.5H, H-4 Bf Z), 7.39 (d, J = 8.5 Hz, 0.5H, H-7 Bf Z), 7.33 (d, J = 8.5
Hz, 0.5H, H-7 Bf E), 7.30 – 7.15 (m, 3H, H-6 Bf E, H-6 BfZ, 2 x H-o-Phe-O), 6.61 – 6.44 (m, 1H, =CH-ArZ,
=CH-Ar E), 6.24 (dq, J = 15.6, 6.5 Hz, 0.5H, =CH-Me E), 5.77 (dq, J = 11.6, 7.1 Hz, 0.5H, =CH-MeZ), 5.56 (bs,
1H, H-1), 4.07 – 4.00 (m, 1H, H-2), 3.93 (dd, J = 9.4, 3.3 Hz, 1H, H-3), 3.84 – 3.69 (m, 3H, H-4, H-6a, H-6b),
3.62 (ddd, J = 9.7, 5.0, 2.5 Hz, 1H, H-5), 2.42 (s, 1.5H, CH3 Bf Z), 2.41 (s, 1.5H, CH3 Bf E), 1.92 (dd, J = 7.2,
1.8 Hz, 1.5H, CH3-CH= Z), 1.88 (dd, J = 6.6, 1.5 Hz, 1.5H, CH3-CH= E). 13C NMR (101 MHz, CD3OD) δ 157.7
(Cquat.-Phe-O), 157.7 (Cquat.-Phe-O), 154.3 (C-7a Bf), 153.8 (C-7a Bf), 152.2 (C-2 Bf), 152.1 (C-2 Bf), 134.3
(C-5 Bf), 133.6 (C-5 Bf), 132.6 (C-3a Bf), 132.5 (=CH-Ar E), 132.4 (C-3a Bf), 131.3 (=CH-Ar Z), 129.0 (2 x C-
m-Phe-O Z), 129.0 (2 x C-m-Phe-O E), 126.8 (2 x Cquat.-Phe), 126.5 (C-6 Bf Z), 126.3 (=CH-Me Z), 124.9 (=CH-
MeE), 123.4 (C-6 Bf E), 120.2 (C-4 Bf Z), 117.9 (4 x C-o-Phe-O), 117.3 (C-4 Bf E), 111.5 (C-7 Bf E), 111.3 (C-3
Bf), 111.2 (C-3 Bf), 111.1 (C-7 Bf Z), 100.1 (C-1), 75.5 (C-5), 72.4 (C-3), 71.9 (C-2), 68.3 (C-4), 62.7 (C-6),
18.6 (CH3-CH= E), 14.8 (CH3-CH=Z), 9.4 (2 x CH3 Bf). HRMS (ESI) calcd for C24H26O7Na1 [M+Na]+
449.15707, found 449.15713.
4.3.12 E,Z-4-[3-methyl-5-(prop-1-enyl)benzofuran-2-yl]phenyl α-L-rhamnopyranoside (29a)
Compound 29a (29 mg, 0.07 mmol) was prepared following general procedures 4.2.2 (29, 55%) and
4.2.3 (quant.) and it was isolated as 1.5:1 E:Z mixture after automated flash chromatography
(chloroform/methanol gradient elution). Rf = 0.33 (9:1 CH2Cl2:CH3OH); 1H NMR (400 MHz, CD3OD) δ
7.77 – 7.67 (m, 4H, 2 x H-m-Phe-O), 7.43 (bs, 0.6H, H-4 BfE), 7.40 (bs, 0.4H, H-4 Bf Z), 7.38 (bd, J = 8.4 Hz,
0.4H, H-7 BfZ), 7.32 (bd, J = 8.5 Hz, 0.6H, H-7 Bf E), 7.27 (dd, J = 8.6, 1.4 Hz, 0.6H, H-6 Bf E), 7.24 – 7.09 (m,
4.4H, H-6 BfZ, 2 x H-o-Phe-O), 6.62 – 6.40 (m, 2H, =CH-Ar Z, =CH-ArE), 6.23 (dq, J = 15.5, 6.5 Hz, 0.6H,
=CH-MeE), 5.77 (dq, J = 11.6, 7.1 Hz, 0.4H, =CH-MeZ), 5.51 (bs, 1H, H-1), 4.02 (dd, J = 15.9, 3.8 Hz, 1H, H-
2), 3.88 (dd, J = 9.5, 3.3 Hz, 1H, H-3), 3.76 – 3.56 (m, 1H, H-5), 3.49 (t, J = 9.5 Hz, 1H, H-4), 2.40 (s, 1.2H,
CH3 Bf Z), 2.39 (s, 1.8H, CH3 Bf E), 1.91 (dd, J = 7.2, 1.7 Hz, 1.2H, CH3-CH= Z), 1.88 (dd, J = 6.5, 1.3 Hz, 1.8H,
CH3-CH= E), 1.26 (d, J = 6.1 Hz, 3H, CH3 Rha). 13C NMR (101 MHz, CD3OD) δ 157.6 (2 x Cquat.-Phe-O), 154.3
(C-7a Bf), 153.8 (C-7a Bf), 152.1 (2 x C-2 Bf), 134.2 (C-5 Bf), 133.5 (C-5 Bf), 132.6 (=CH-Ar E), 132.6 (C-3a
Bf), 132.4 (C-3a Bf), 131.4 (=CH-Ar Z), 129.0 (4 x C-m-Phe-O), 126.7 (2 x Cquat.-Phe), 126.5 (C-6 Bf Z), 126.3
Page 15 of 20

(=CH-MeZ), 124.8 (=CH-Me E), 123.4 (C-6 Bf E), 120.2 (C-4 Bf Z), 117.7 (4 x C-o-Phe-O), 117.3 (C-4 Bf E),
111.4 (C-7 BfE), 111.1 (C-7 Bf Z), 99.8 (C-1), 73.8 (C-4), 72.2 (C-3), 72.0 (C-2), 70.8 (C-5), 18.6 (CH3-CH= E),
14.8 (CH3-CH=Z), 9.4 (2 x CH3 Bf). ESI-MS calcd for C24H26O6Na1 [M+Na]+ 433.2, found 433.5; calcd for
C48H51O12 [2M-H]- 819.3, found 819.9.
4.3.13 E,Z-4-[3-methyl-5-(prop-1-enyl)benzofuran-2-yl]phenyl β-D-glucopyranoside (30a) Compound
30a (12 mg, 0.02 mmol) was prepared following general procedures 4.2.1 (30, 39%, yield based on the
limiting agent) and 4.2.3 (>95%) and it was isolated as 3:1 E:Z mixture by a flash chromatography (CHCl3:
MeOH) followed by a reverse-phase chromatography (water/methanol) that allowed to remove donor
1
derived by-products. Rf = 0.25 (9:1 CH2Cl2:CH3OH). H NMR (400 MHz, CD3OD) δ 7.80 – 7.68 (m, 2H, 2 x
H-m-Phe-O), 7.48 – 7.44 (m, 0.75H, H-4 Bf E), 7.44 – 7.42 (m, 0.25H, H-4 Bf Z), 7.40 (d, J = 8.5 Hz, 0.25H,
H-7 BfZ), 7.34 (d, J = 8.5 Hz, 0.75H, H-7 Bf E), 7.29 (dd, J = 8.5, 1.5 Hz, 0.75H, H-6 Bf E), 7.26 – 7.19 (m,
2.25H, H-6 Bf Z, 2 x H-o-Phe-O), 6.59 – 6.47 (m, 1H, =CH-ArZ, =CH-Ar E), 6.25 (dq, J = 15.7, 6.6 Hz, 0.75H,
=CH-MeE), 5.78 (dq, J = 11.5, 7.2 Hz, 0.25H, =CH-MeZ), 4.99 (dd, J = 5.4, 2.0 Hz, 1H, H-1), 3.93 (dd, J =
12.1, 1.9 Hz, 1H, H-6a), 3.73 (dd, J = 12.1, 5.6 Hz, 1H, H-6b), 3.54 – 3.45 (m, 3H, H-5, H-2, H-3), 3.45 –
3.38 (m, 1H, H-4), 2.43 (s, J = 4.3 Hz, 0.75H, CH3 Bf Z), 2.42 (s, 2.25H, CH3 Bf E), 1.93 (dd, J = 7.2, 1.8 Hz,
0.75H, CH3-CH= Z), 1.89 (dd, J = 6.6, 1.5 Hz, 2.25H, CH3-CH= E). 13C NMR (101 MHz, CD3OD) δ 158.9
(Cquat.-Phe-O), 158.8 (Cquat.-Phe-O), 154.3 (C-7a Bf), 153.8 (C-7a Bf), 152.2 (2 x C-2 Bf), 134.3 (C-5 Bf),
133.6 (C-5 Bf), 132.6 (=CH-Ar E), 131.3 (=CH-Ar Z), 129.0 (2 x C-m-Phe-O Z), 128.9 (2 x C-m-Phe-OE), 126.8
(C-3a Bf), 126.5 (C-6 Bf Z), 126.3 (=CH-MeZ), 124.9 (=CH-Me E), 123.4 (C-6 BfE), 120.2 (C-4 Bf Z), 117.9 (4 x
C-o-Phe-O), 117.3 (C-4 Bf E), 111.4 (C-7 Bf E), 111.2 (C-3 Bf), 111.1 (C-7 BfZ), 102.1 (C-1), 78.2 (C-5), 78.0
(C-3), 74.9 (C-2), 71.4 (C-4), 62.5 (C-6), 18.6 (CH3-CH= E), 14.8 (CH3-CH= Z), 9.4 (2 x CH3 Bf). HRMS (ESI)
calcd for C24H26O7Na1 [M+Na]+ 449.15707, found 449.15714.
4.3.14 E,Z-4-[3-methyl-5-(prop-1-enyl)benzofuran-2-yl]phenyl β-L-glucopyranoside (31a) Compound
31a (35 mg, 0.08 mmol) was prepared following general procedures 4.2.1 and 4.2.3 and it was isolated
as 1.7:1 E:Z mixture in 38% yield (based on the limiting agent) over two steps. Rf = 0.25 (9:1
1
CH2Cl2:CH3OH). H NMR (400 MHz, CD3OD) δ 7.79 – 7.67 (m, 2H, 2 x H-m-Phe-O), 7.45 (s, 0.63H, H-4 Bf
E), 7.43 (s, 0.37H, H-4 Bf Z), 7.39 (d, J = 8.4 Hz, 0.37H, H-7 BfZ), 7.34 (d, J = 8.5 Hz, 0.63H, H-7 Bf E), 7.28
(dd, J = 8.5, 1.6 Hz, 0.63H, H-6 BfE), 7.26 – 7.19 (m, 2.37H, H-6 Bf Z, 2 x H-o-Phe-O), 6.61 – 6.44 (m, 1H,
=CH-Ar Z, =CH-Ar E), 6.24 (dq, J = 15.6, 6.5 Hz, 0.63H, =CH-Me E), 5.77 (dq, J = 11.6, 7.2 Hz, 0.37H, =CH-Me
Z), 5.03 – 4.95 (m, 1H, H-1), 3.93 (dd, J = 11.9, 1.7 Hz, 1H, H-6a), 3.73 (dd, J = 12.1, 5.5 Hz, 1H, H-6b), 3.57
– 3.38 (m, 4H, H-5, H-2, H-3, H-4), 2.43 (d, J = 5.4 Hz, 1.1H, CH3 Bf Z), 2.42 (s, 1.9H, CH3 Bf E), 1.92 (dd, J =
7.2, 1.8 Hz, 1.1H, CH3-CH= Z), 1.89 (dd, J = 6.6, 1.5 Hz, 1.9H, CH3-CH= E). ESI-MS calcd for C24H26O7Na1
[M+Na]+ 449.2, found 449.4; calcd for C48H52O14Na1 [2M+Na]+ 875.3, found 875.2.
Page 16 of 20

4.3.15 E,Z-4-[3-methyl-5-(prop-1-enyl)benzofuran-2-yl]phenyl β-D-galactopyranoside (32a)
Compound 32a (21 mg, 0.049 mmol) was prepared following general procedures 4.2.1 and 4.2.3 and it
was isolated as 1.7:1 E:Z mixture in 70% yield (based on the limiting agent) over two steps. Rf = 0.25 (9:1
1
CH2Cl2:CH3OH). H NMR (400 MHz, CD3OD) δ 7.78 – 7.66 (m, 2H, 2 x H-m-Phe-O), 7.44 (d, J = 11.8 Hz,
0.63H, H-4 Bf E), 7.43 (bs, 0.37H, H-4 Bf Z), 7.40 (d, J = 8.4 Hz, 0.37H, H-7 BfZ), 7.34 (d, J = 8.5 Hz, 0.63H, H-
7 BfE), 7.29 (dd, J = 8.5, 1.5 Hz, 0.63H, H-6 Bf E), 7.27 – 7.16 (m, 2.37H, H-6 Bf Z, 2 x H-o-Phe-O), 6.62 –
6.43 (m, 1H, =CH-Ar Z, =CH-Ar E), 6.25 (dq, J = 15.7, 6.5 Hz, 0.63H, =CH-Me E), 5.78 (dq, J = 11.6, 7.1 Hz,
0.37H, =CH-MeZ), 4.95 (dd, J = 7.7, 1.7 Hz, 1H, H-1), 3.93 (d, J = 3.3 Hz, 1H, H-4), 3.90 – 3.69 (m, 4H, H-2,
H-6a, H-6b, H-5), 3.61 (dd, J = 9.7, 3.3 Hz, 1H, H-3), 2.43 (s, 1.1H, CH3 Bf Z), 2.42 (s, 1.9H, CH3 Bf E), 1.92
(dd, J = 7.2, 1.8 Hz, 1.1H, CH3-CH= Z), 1.89 (dd, J = 6.5, 1.5 Hz, 1.9H, CH3-CH= E). 13C NMR (101 MHz,
CD3OD) δ 159.0 (Cquat.-Phe-O),154.3 (C-7a Bf), 152.2 (C-2 Bf), 134.3 (C-5 Bf), 132.7 (C-3a Bf), 132.6 (=CH-
Ar E), 131.3 (=CH-Ar Z), 129.0 (2 x C-m-Phe-OZ), 128.9 (2 x C-m-Phe-O E), 126.8 (2 x Cquat.-Phe), 126.5 (C-6
Bf Z), 126.3 (=CH-MeZ), 124.9 (=CH-MeE), 123.4 (C-6 Bf E), 120.2 (C-4 Bf Z), 118.0 (4 x C-o-Phe-O), 117.3
(C-4 Bf E), 111.4 (C-7 Bf E), 111.2 (2 x C-3 Bf), 111.1 (C-7 BfZ), 102.8 (C-1), 77.1 (C-5), 74.9 (C-3), 72.3 (C-2),
70.2 (C-4), 62.5 (C-6), 18.6 (CH3-CH= E), 14.8, (CH3-CH= Z), 9.4 (2 x CH3 Bf). HRMS (ESI) calcd for
C24H26O7Na1 [M+Na]+ 449.15707, found 449.15756.
4.3.16 E,Z-4-[3-methyl-5-(prop-1-enyl)benzofuran-2-yl]phenyl β-L-galactopyranoside (33a) Compound
33a (36 mg, 0.08 mmol) was prepared following general procedures 4.2.1 (33, 71%, yield based on the
limiting agent) and 4.2.3 (>95%) and it was isolated as 1.5:1 E:Z mixture . Rf = 0.25 (9:1 CH2Cl2:CH3OH).
1H NMR (400 MHz, CD3OD) δ 7.79 – 7.69 (m, 2H, 2 x H-m-Phe-O), 7.46 (bs, 0.6H, H-4 Bf E), 7.43 (bs, 0.4H,
H-4 BfZ), 7.40 (d, J = 8.4 Hz, 0.4H, H-7 BfZ), 7.34 (d, J = 8.5 Hz, 0.6H, H-7 BfE), 7.29 (dd, J = 8.5, 1.4 Hz,
0.6H, H-6 BfE), 7.27 – 7.15 (m, 2.4H, H-6 Bf Z, 2 x H-o-Phe-O), 6.62 – 6.43 (m, 1H, =CH-Ar Z, =CH-ArE), 6.25
(dq, J = 13.2, 6.5 Hz, 0.6H, =CH-MeE), 5.78 (dq, J = 11.6, 7.1 Hz, 0.4H, =CH-MeZ), 4.95 (dd, J = 7.7, 1.6 Hz,
1H, H-1), 3.93 (d, J = 3.3 Hz, 1H, H-4), 3.89 – 3.68 (m, 4H, H-2, H-6a, H-6b, H-5), 3.61 (dd, J = 9.7, 3.3 Hz,
1H, H-3), 2.43 (s, 1.2H, CH3 Bf Z), 2.42 (s, 1.8H, CH3 Bf E), 1.92 (dd, J = 7.2, 1.7 Hz, 1.2H, CH3-CH= Z), 1.89
(dd, J = 6.5, 1.4 Hz, 1.8H, CH3-CH= E). ESI-MS calcd for C24H26O7Na1 [M+Na]+ 449.2, found 449.5; calcd for
C48H52O14Na1 [2M+Na]+ 875.3, found 875.3; calcd for C48H51O14 [2M-H]- 851.3, found 851.3.
4.3.17 E,Z-4-[3-methyl-5-(prop-1-enyl)benzofuran-2-yl]phenyl β-L-fucopyranoside (34a) Compound
34a (17 mg, 0.043mmol) was prepared following general procedures 4.2.1 and 4.2.3 and it was isolated
1
as 3.5:1 E:Z mixture in 49% yield over two steps. Rf = 0.37 (9:1 CH2Cl2:CH3OH). H NMR (400 MHz,
CD3OD) δ 7.76 – 7.67 (m, 2H, 2 x H-m-Phe-O), 7.44 (bs, 0.78H, H-4 Bf E), 7.41 (bs, 0.22H, H-4 Bf Z), 7.39 (d,
J = 8.4 Hz, 0.22H, H-7 Bf Z), 7.33 (d, J = 8.5 Hz, 0.78H, H-7 Bf E), 7.30 – 7.25 (m, 0.78H, H-6 Bf E), 7.22 – 7.11
(m, 2.22H, H-6 Bf Z, 2 x H-o-Phe-O), 6.59 – 6.42 (m, 1H, =CH-Ar Z, =CH-ArE), 6.24 (dq, J = 15.6, 6.5 Hz,
Page 17 of 20

0.78H, =CH-MeE), 5.77 (dq, J = 11.6, 7.2 Hz, 0.22H, =CH-MeZ), 4.92 (dd, J = 7.7, 2.3 Hz, 1H, H-1), 3.89 –
3.74 (m, 2H, H-5, H-2), 3.68 (d, J = 3.1 Hz, 1H, H-4), 3.60 (dd, J = 9.7, 3.4 Hz, 1H, H-3), 2.41 (s, 0.66H, CH3
BfZ), 2.40 (s, 2.34H, CH3 Bf E), 1.92 (dd, J = 7.2, 1.8 Hz, 0.66H, CH3-CH= Z), 1.88 (dd, J = 6.6, 1.5 Hz, 2.34H,
CH3-CH= E), 1.32 (d, J = 6.5 Hz, 3H, CH3 Fuc). 13C NMR (101 MHz, CD3OD) δ 158.9 (Cquat.-Phe-O), 158.8
(Cquat.-Phe-O), 154.3 (C-7a Bf), 153.8 (C-7a Bf), 152.2 (C-2 Bf), 152.1 (C-2 Bf), 134.3 (C-5 Bf), 133.6 (C-5
Bf), 132.7 (C-3a Bf), , 132.6 (=CH-Ar E), 132.4 (C-4a Bf), 131.3 (=CH-Ar Z), 128.9 (2 x C-m-Phe-O Z), 128.9 (2
x C-m-Phe-O E), 126.7 (Cquat.-Phe), 126.6 (Cquat.-Phe), 126.5 (C-6 Bf Z), 126.3 (=CH-MeZ), 124.9 (=CH-Me E),
123.4 (C-6 BfE), 120.2 (C-4 Bf Z), 117.8 (4 x C-o-Phe-O), 117.3 (C-4 Bf E), 111.4 (C-7 BfE), 111.2 (C-3 Bf),
111.1 (C-3 Bf), 111.1 (C-7 BfZ), 102.4 (C-1), 75.0 (C-3), 72. 9 (C-4), 72.2 (C-5), 72.0 (C-2), 18.6 (CH3-CH= E),
16.8 (2 x CH3 Fuc), 14.8 (CH3-CH=Z), 9.4 (2 x CH3 Bf). HRMS (ESI) calcd for C24H26O6Na1 [M+Na]+
433.16216, found 433.16227.
Acknowledgments
This work was supported by Premio Fondazione Cariplo – Ricerca di frontiera 2011 (Project: Chemical
control of signalling pathways by modulation of hub proteins, CheCOSP). We thank Dr. Anna Daghetti
for technical help with MS analysis and Matteo Panza for help with the synthesis of Rha-OBox and
derivatives. The HRMS spectra have been obtained from CIGA (Centro Interdipartimentale Grandi
Apparecchiature, Università degli Studi di Milano).
Supplementary data
Supplementary data associated with this article (synthesis and characterization of all protected
glycosides, 1H-NMR and 13C-NMR spectra of all final compounds) can be found, in the online version, at
http://dx.doi.org/10.1016/
Page 18 of 20

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Page 19 of 20

*Graphical Abstract (for review)

Highlights
• Chemical glycodiversification of a complex phenol aglycon was achieved
• gluco-, galacto- and fuco-sides were prepared using bromide donors and PTC
• α-manno and -rhamno derivatives were prepared using O-benzoxazolyl imidate donors
• A library of 14 glycosylated benzofuran derivatives was obtained
Page 20 of 20