Synthesis of the Fungal Metabolite YWA1 and Related Constructs as Tools to Study MelLec-Mediated Immune Response to Aspergillus Infections ?

Article abstract of DOI:10.1021/acs.joc.0c02324

We describe the chemical synthesis of the fungal naphthopyrones YWA1 and fonsecin B, as well as their functionalization with an amine-spacer arm and the conjugation of the resulting molecules to three different functional tags (i.e., biotin, Oregon green, 1-[3-(succinimidyloxycarbonyl)benzyl]-4-[5-(4-methoxyphenyl)-2-oxazolyl]pyridinium bromide (PyMPO)). The naphthopyrone-biotin and -PyMPO constructs maintained the ability to bind the C-type lectin receptor MelLec, whose interaction with immunologically active fungal metabolites (i.e., 1,8-dihydroxynaphthalene-(DHN)-melanin and YWA1) is a key step in host recognition and induction of protective immune responses against Aspergillus fumigatus. The fluorescent Fonsecin B-PyMPO construct 21 was used to selectively visualize MelLec-expressing cells, thus validating the potential of this strategy for studying the role and functions of MelLec in immunity.

Full text of DOI:10.1021/acs.joc.0c02324

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Article
Synthesis of the Fungal Metabolite YWA1 and Related Constructs as
Tools to Study MelLec-Mediated Immune Response to Aspergillus
Infections†
Monica Piras,* Ilaria Patruno, Christina Nikolakopoulou, Janet A. Willment, Nikki L. Sloan,
Chiara Zanato, Gordon D. Brown,* and Matteo Zanda*
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ABSTRACT: We describe the chemical synthesis of the fungal naphthopyrones YWA1 and fonsecin B, as well as their
functionalization with an amine-spacer arm and the conjugation of the resulting molecules to three different functional tags (i.e.,
biotin, Oregon green, 1-[3-(succinimidyloxycarbonyl)benzyl]-4-[5-(4-methoxyphenyl)-2-oxazolyl]pyridinium bromide (PyMPO)).
The naphthopyrone-biotin and -PyMPO constructs maintained the ability to bind the C-type lectin receptor MelLec, whose
interaction with immunologically active fungal metabolites (i.e., 1,8-dihydroxynaphthalene-(DHN)-melanin and YWA1) is a key step
in host recognition and induction of protective immune responses against Aspergillus fumigatus. The fluorescent Fonsecin B-PyMPO
construct 21 was used to selectively visualize MelLec-expressing cells, thus validating the potential of this strategy for studying the
role and functions of MelLec in immunity.
genesis, we recently demonstrated that recognition of DHN-
melanin components by the host immune system has a crucial
role in the control of A. fumigatus infections in both mice and
humans.9 Fungal DHN-melanin and its metabolic precursors
(YWA1, THN, and DHN) are sensed by the host immune
system through the C-type lectin receptor (MelLec).9 Our
findings indicate that the interaction of MelLec with
immunologically active components of DHN-melanin is a
key step in host recognition and induction of protective
immune responses against A. fumigatus. To investigate the
interaction of the melanin-sensing receptor MelLec with
YWA1 and evaluate how structural modifications of the
naphthopyrone scaffold could affect the ligand−receptor
binding properties, we have now developed the first synthesis
of YWA1. To date, YWA1whose structure offers very limited
options for chemical modificationcould only be obtained in
INTRODUCTION
■
Naphthopyrones are important aromatic polyketides displaying
a characteristic polyhydroxynaphthalene system fused to a
pyran-4-one ring. These fungal metabolites have attracted a
great deal of interest due to their broad range of biological
activities, encompassing immunomodulatory,1 antiprolifera-
tive,2 cytotoxic,3 and antibacterial4 properties (Figure 1a).
The hemiketal naphthopyrone YWA1, whose structure was
unambiguously determined by spectroscopic methods two
decades ago,5 was isolated from the human opportunistic
pathogen Aspergillus fumigatus and other fungal species
expressing specific types of nonreducing polyketide synthases
(NR-PKS). These multidomain enzymes assemble acyl and
malonyl units in a polyketide chain and successively catalyze
regiospecific cyclization/aromatization reactions to convert the
polyketide chain into the YWA1 aromatic scaffold.6,7 YWA1 is
the first intermediate in the biosynthetic pathway leading to
1,8-dihydroxynaphthalene-(DHN)-melanin (Figure 1b), a
highly insoluble conidial pigment formed by polymerization
of 1,8-DHN monomers that confers fungal cell resistance
against the host immune response, oxidative stress, and
ultraviolet light.8 Although melanin represents an important
protective factor contributing to fungal virulence and patho-
Received: September 30, 2020
Published: April 22, 2021
https://doi.org/10.1021/acs.joc.0c02324
© 2021 American Chemical Society
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Figure 1. (a) Structures of YWA1 and other biologically active fungal metabolites incorporating a naphthopyrone system: fonsecin B (stabilizing
ligand of c-myc G-quadruplex DNA),2 nigerone (cytotoxic agent),3 and fonsecinone C (antibiotic).4 (b) Biosynthetic pathway to DHN-melanin.
Scheme 1. Examples of Syntheses of Polyketide Naphthopyrones
a
Scheme 2. Syntheses of YWA1 and Fonsecin B
a
Reagents and conditions: (i) Ac2O, pyridine, r.t., overnight (70%); (ii) AlCl3, 150 °C, 1.5 h, and then MeOH, H2SO4, r.t., 4 h (60%); (iii) BnBr,
K2CO3, acetone, 60 °C, overnight (70%); (iv) lithium bis(trimethylsilyl)amide (LiHMDS), tetrahydrofuran (THF), 0 °C, 2 h (90%); (v) (a)
BnOH, PPh3, diisopropyl azodicarboxylate (DIAD), Et2O, r.t., 4 h for compound 13a (17%); (b) BOMCl, N,N-diisopropylethylamine (DIPEA),
CH2Cl2, r.t., overnight for compound 13b (50%); (c) Me2SO4, K2CO3, acetone, 60 °C, overnight for compound 13c (70%); (vi) LiHMDS, THF,
−40 °C, acetaldehyde, 15 min (90%); (vii) LiHMDS, THF, 0 °C, Ac2O, 15 min (50%); (viii) Dess−Martin periodinane (DMP), CHCl3, 40 °C;
(ix) H2, Pd(OH)2 20 wt % on C, AcOH, MeOH, r.t., 3−12 h (90%).
tiny amounts by extraction from Aspergillus conidia. Despite
the biological importance of polyketide naphthopyrones, very
few methods have been described for their preparation,10−13
and none of these turned out to be suitable for our aims, which
were (i) to have ready access to sufficient quantities of YWA1
for biological studies and (ii) to generate YWA1-based
functional chemical probes for studying the activity of MelLec
in cells, with the view of enabling the study of MelLec-
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mediated immune responses in vivo and the biosynthesis of
DHN-melanin in fungi.
product 11a (Scheme 3). The nature of the solvent was found
to have a remarkable effect on the chemoselectivity of the
Herein, we report (1) a concise synthesis of YWA1 and
some of its structural analogues, which are recognized by
MelLec protein;9 (2) the functionalization of the YWA1
scaffold with an amine-spacer arm and its conjugation with
Oregon green (19), biotin (20), and 1-[3-(succinimidyloxy-
carbonyl)benzyl]-4-[5-(4-methoxyphenyl)-2-oxazolyl]-
pyridinium bromide (PyMPO) (21), without significantly
altering its ability to bind MelLec; and (3) the capacity of the
fluorescent conjugate 21 to specifically visualize MelLec in cell
cultures.
a
Scheme 3. Synthesis of 12a
a
Reagents and conditions: (i) BnBr, K2CO3, DMF, r.t., 4 h (95%);
(ii) LiHMDS, THF, −40 °C, acetaldehyde, 2 h (90%).
RESULTS AND DISCUSSION
■
Syntheses of YWA1 and Fonsecin B. Existing synthetic
procedures for the preparation of tricyclic oxygen-heterocycles,
similar to YWA1, generally start with the formation of a
naphthalene unit followed by condensation of a pyran-4-one
ring.10−12 In their work on the total synthesis of nigerone,
Kozlowski et al. used a 2-naphthoate precursor (2) to generate
the naphthopyrone heterocycle 4 (Scheme 1a).10 According to
the authors, the formation of the pyrone ring was the most
challenging part of the synthesis, where the obvious
disconnection involving addition of acetone to the methyl
ester of 2 failed to supply the requisite pyrone ring and an
alternative approachbased on the addition of a dimesyl
anion and condensation of acetaldehydewas instead
required. Our attempts to adapt this strategy to prepare
YWA1 from the naphthol intermediate 2 were unsuccessful,
mainly because the pyrone ring could not be obtained in its
hemiketal form, characteristic of YWA1. Additionally, this
route seemed to lack the synthetic flexibility we required to
develop YWA1 analogues suitable for bioconjugation to
chemical handles and functional probes. In earlier studies
based on the preparation of naphthopyrones, Arima and co-
workers used a carboxy-diketone precursor (5) to access the 2-
acetyl naphthol 6, which was then readily converted into
Fonsecin B by Claisen condensation with EtOAc (Scheme
1b).11 Although this latter strategy seemed particularly well-
suited for the synthesis of our target molecule, numerous
attempts to convert 5 into the desired 6 turned out to be
unsuccessful in our hands. In a more recent publication,6 a
concise approach to the 2-acetyl naphthol unit 12 was
described (Scheme 2), comprising (i) the Fries rearrangement
of phenyl diacetate 9, (ii) benzylation of the key intermediate
10, and (iii) Dieckmann’s cyclization of the nonsymmetrical
penta-substituted phenyl ester 11. Following this procedure
and optimizing the reaction conditions for the Fries rearrange-
ment, we successfully obtained 12 in satisfactory yields. When
the Fries rearrangement was conducted using AlCl3 in refluxing
dichloroethane (DCE) for 72 h, as previously reported, we
could only obtain small quantities of the desired product 10
(15% yield). A considerable amount of the unreacted starting
material was recovered along with a complex mixture of
byproducts resulting from the hydrolysis of the O-acetyl/
methyl ester groups. Significant improvements were made by
treating neat 9 with AlCl3 at 150 °C for 1 h and then
performing a Fisher esterification to repristinate the methyl
ester partially hydrolyzed during the acylation step. O-
Benzylation of 10 followed by intramolecular Dieckmann
condensation of 11 afforded the desired naphthol 12. During
the O-benzylation step, the acidic character of the methylene
protons of 10 led to the formation of the overalkylated side-
process, with O-benzylation being favored in acetone, while C-
alkylation waspredominant in dimethylformamide (DMF).
Interestingly, the overalkylated byproduct 11a could also
undergo intramolecular Dieckmann cyclization, giving the 5-
benzyl substituted naphthalene analogue 12a (Scheme 3). The
newly formed hydroxyl groups of 12 were then fully protected
before testing the formation of the hemiketal pyrone ring.
While O-methylation of 12 to 13c was achieved smoothly, O-
benzylation proved to be particularly laborious. Treatment of
12 with BnBr under different reaction conditions did not afford
the desired fully protected intermediate 13a, while Mitsunobu
alkylation gave low yields (17%). In both cases, C-alkylation of
the naphthol ring in position 5 was the main side-reaction
observed.
Better results were obtained using the benzyloxymethyl
(BOM) group as an alternative to benzyl protection (13b).
With the protected 2-acetyl naphthols 13b, 13c in our hands,
we then tried to access the final product YWA1 and its
methylated analogue fonsecin B by Claisen condensation with
EtOAc and subsequent hydrogenolysis of the protecting
groups. Surprisingly, numerous attempts to install a diketone
moiety via Claisen condensation were unsuccessful. A variety
of solvents, bases, and temperatures were screened to condense
13b, 13c to EtOAc, but only the starting material was
recovered, while an enol acetate was formed (14a, see the
Supporting Information) when Ac2O and LiHMDS were used.
Applying a different strategy based on (i) aldol condensation of
13b, 13c with acetaldehyde, (ii) oxidation of β-hydroxyketones
14b, 14c, and (iii) final hydrogenolysis of the resulting 1,3-
diketones 15b, 15c, we finally accomplished the synthesis of
YWA1 (10.7% overall yield) and its O-dimethyl analogue
fonsecin B (15.0% overall yield), both in eight steps. Various
procedures were tested for oxidizing the β-hydroxyketone
group in 14b, 14c (Swern, Parikh−Doering, pyridinium
chlorochromate (PCC), 2-iodoxybenzoic acid (IBX)), but
only DMP led to the conversion of the secondary alcohol into
the desired diketone moiety.
Design and Synthesis of Bioconjugated Derivatives.
This synthetic strategy was used to functionalize YWA1 with
an amine-spacer arm and perform a chemoselective ligation of
the naphthopyrone scaffold to different tags (Scheme 4). Aldol
condensation of the fully protected intermediate 13b, 13c with
8-azido octanal 22 and subsequent oxidation of the β-
hydroxyketones 16b, 16c with DMP (or IBX for 16c) afforded
the desired diketones 17b, 17c. Hydrogenolysis of the
protecting groups of 17c, concomitantly with azide reduction,
quantitatively produced intermediate 18b, while cleavage of
the BOM protecting groups from 17b was impaired by the
presence of the newly formed amino group.
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a
Scheme 4. Synthesis of Bioconjugated Derivatives
a
Reagents and conditions: (i) (a) BOMCl, DIPEA, CH2Cl2, r.t., overnight for compound 13b (50%); (b) Me2SO4, K2CO3, acetone, 60 °C,
overnight, for compound 13c (70%); (c) tert-butyldimethylsilyl chloride (TBDMSCl), DIPEA, DMF, r.t., overnight for compound 13d (99%); (ii)
lithium diisopropylamide (LDA), THF, −78 °C, 45 min, and then 22, −78 °C, 15 min (99%); (iii) DMP, CHCl3, 40 °C or IBX, EtOAc, 80 °C
overnight (for 16c); (iv) (a) tetra-n-butylammonium fluoride (TBAF), THF, 0 °C, 3 h, and then H2, Pd(OH)2 on carbon 20%, AcOH, MeOH, r.t.,
3 h for compound 18a; (b) H2, Pd(OH)2 on carbon 20%, AcOH, MeOH, r.t., 3 h for compound 18b; (v) Oregon Green-N-hydroxysuccinimide
(NHS), DIPEA, DMF, r.t., 0.5 h (50%); (vi) biotin-NHS, DIPEA, DMF, r.t., 0.5 h (50%); (vii) PyMPO-NHS, DIPEA, DMF, r.t., 0.5 h (60%).
Attempts to force the removal of the BOM groups by adding
more catalyst or acetic acid led to the formation of a complex
mixture of byproducts. To overcome this problem, we decided
to protect the hydroxyl groups of 12 as tert-butyldimethylsilyl
(TBDMS) ethers. The protected intermediate 13d was
successfully condensed with 8-azido octanal to afford 16d,
which was then oxidized to give the diketone 17d. Treatment
of 17d with TBAF followed by hydrogenolysis of the Bn
groups afforded 18a. The final intermediates (18a, 18b)
equipped with an amine-spacer arm were successfully
conjugated to the NHS active esters of Oregon green
(commercial mixture of 5- and 6-isomers), biotin, and
PyMPO affording the constructs 19 (9.2% overall yield), 20a
(13.0% overall yield), 20b (9.2% overall yield), and 21 (11.0%
overall yield), each in nine steps. It should be noted that
fonsecin B and derivatives are more stable than YWA1
counterparts, and therefore more reliable tools for performing
biological studies, as OH-group methylation prevents oxidation
to the corresponding quinones.
It is also worth noting that the spacer’s arm length is
important for the synthetic accessibility of these constructs:
hydrogenolysis of an azido diketone analogue 17e synthesized
using ω-azido hexanal 23, instead of ω-azido octanal 22,
produced a complex mixture of unidentified products.
Biology. We have previously shown that MelLec is able to
recognize 1,8-DHN-melanin and other structural isomers in
the DHN-melanin biosynthetic pathway due to the presence of
the naphthalene-diol unit.9 To test our compounds, we used
the Fc-MelLec probe (the extracellular domain of MelLec,
fused to the Fc-portion of human Ig),9 which was pretreated
with or without YWA1, 1,8-DHN, fonsecin B, 2′,6′-
dihydroxyacetophenone (2,6-DHAP), 21, and 20b, and then
incubated with A. fumigatus ΔrodA conidia (Figure 2). Fc-
MelLec bound to the conidia was detected with allophyco-
cyanin (APC)-conjugated donkey antihuman antibody. Bind-
ing of Fc-MelLec to these compounds was determined by flow
cytometry, where lack of a signal on the A. fumigatus conidia
indicates inhibition of binding through competitive inhibition.
As we had described previously,9 YWA1 inhibited binding of
Fc-MelLec to ΔrodA conidia, whereas the structurally
unrelated 2,6-DHAP had no effect on binding. Notably,
fonsecin B and conjugates, 20b and 21, also inhibited the
recognition of A. fumigatus ΔrodA conidia by Fc-MelLec. This
shows that addition of the amine-spacer arm and subsequent
conjugation do not impair the ability to bind the receptor.
Immunofluorescence experiments showed that, compared to
the untreated cells, RAW 264.7 cells expressing hMelLec can
bind to the fluorescent conjugate 21 (Figure 3), and the
binding is concentration-dependent (data not shown). The
binding of 21 was shown to be specific by treating RAW 264.7
cells expressing hMelLec in the presence of competing
unlabeled YWA1 (Figure 3), which resulted in a strong
decrease of fluorescence at the highest concentrations, such as
25 and 50 μM (for further competition experiments and data,
see Figure S1, Supporting Information).
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Figure 2. Fc-MelLec binds to 1,8-DHN-melanin precursors and other structurally related bioconjugates. (a) Flow cytometry histogram showing
that YWA1 (1,8-DHN-melanin precursor) inhibits recognition of A. fumigatus ΔrodA conidia by Fc-MelLec (green) compared to the uninhibited
Fc-MelLec control (gray). (b) Inhibition of Fc-MelLec binding to ΔrodA conidia of A. fumigatus by 1,8-DHN-melanin precursors (YWA1, 1,8-
DHN), Fonsecin B, and the bioconjugates 21 and 20b compared to the uninhibited Fc-MelLec control. 2,6-DHAP is included as a negative control
as it lacks the naphthol unit. The experiment was repeated independently twice, with similar results. Two technical replicates were used for each
sample in each experiment. Data shown here is pooled from the two independent experiments. Fold change was calculated by dividing the mean
value of pretreated Fc-MelLec with each compound by the mean value of the uninhibited Fc-MelLec. Values show mean standard error of the
mean (SEM); ****p < 0.05 (one-way analysis of variance (ANOVA) and Bonferroni post hoc test comparing inhibition of Fc-MelLec binding to
ΔrodA conidia by the different compounds versus uninhibited Fc-MelLec). (c) Structures of the compounds tested.
1
phase silica columns. H NMR and 13C NMR spectra were recorded
CONCLUSIONS
We described an efficient synthesis of YWA1 and other
■
on a Bruker AVANCE III 400 NMR spectrometer and calibrated
1
using residual undeuterated solvent as an internal reference. H δ =
structurally related fungal naphthopyrones. The flexibility of
the synthetic approach allowed us to functionalize the YWA1
scaffold with an amine-spacer arm and conjugate the resulting
molecules to three different functional tags (i.e., biotin, Oregon
green, PyMPO) without impairing the ability to bind to
MelLec. The ability of MelLec to recognize YWA1 and its
structural analogues was determined by flow cytometry
experiments, using an Fc-MelLec probe.9 The capacity to
bind to MelLec was determined as the ability of naphthopyr-
one ligands to inhibit the interaction of Fc-MelLec with
melanin components localized on the surface of A. fumigatus
conidia. The fluorescent probe 21 was then used to selectively
visualize MelLec-expressing cells using fluorescence micros-
copy, thus validating the potential of this strategy for studying
Aspergillus infections. These compounds therefore offer
exciting new tools to study the cellular and immunological
functions of MelLec in vitro and in vivo.
7.26 (CDCl3), 13C δ = 77.16 (CDCl3), 1H δ = 3.31 (CD3OD), 13C δ
= 49.15 (CD3OD), 1H δ = 2.05 ((CD3)2CO), 13C δ = 29.84
((CD3)2CO), H δ = 2.50 ((CD3)2SO), 13C δ = 39.52 ((CD3)2SO).
1
19F NMR spectra were recorded on a Bruker AVANCE III 400 NMR
spectrometer and were referenced to CFCl3. 13C NMR spectra were
recorded with complete proton decoupling. Chemical shifts (δ) are
reported in parts per million (ppm), coupling constants (J) are given
in hertz (Hz), and multiplicity is described using the following
abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet;
br, broad; or combinations thereof. Mass analyses were performed
using an Agilent 1200 high-performance liquid chromatography
(HPLC) system coupled to an Agilent G6120 single quadrupole
detector equipped with an electrospray ionization (ESI) source in
direct infusion modality. ESI-MS spectra were recorded in the positive
mode. Reverse-phase (RP)-HPLC-MS analyses were performed with
an Agilent 1200 HPLC system equipped with a diode array detector
(DAD) and an ESI-MS detector. High-resolution mass spectra
(HRMS) were recorded using a Thermofisher Q-Exactive orbitrap ion
trap mass spectrometer. HPLC conditions for analytical analyses:
Phenomenex Luna C18 column, 5 μm, 100 Å, 250 × 4.6 mm2 (L ×
ID) injected volume 10 μL, flow rate 1 mL/min, unless otherwise
specified. HPLC preparative purification was performed using an
Agilent 1260 HPLC system equipped with a DAD detector. HPLC
conditions for preparative purification were as follows: Phenomenex
Luna C18 column, 5 μm, 100 Å, 250 × 21.2 mm2 (L × ID), flow rate
20 mL/min.
EXPERIMENTAL SECTION
■
General Information. Dry solvents were obtained from
commercial sources and used without further purification. The
reactions performed under a nitrogen atmosphere were carried out in
dry solvents. Reactions requiring heating were performed using an oil
bath. Reactions were monitored by thin-layer chromatography
(TLC), unless otherwise noted. TLCs were performed on Merck
silica gel glass plates (60 F254). Visualization was accomplished by UV
light (254 nm) or staining with ceric ammonium molybdate or
KMnO4 solution. Flash chromatography was performed manually
using silica gel (60 Å, particle size 40−63 μm) purchased from Merck
or automatically using Interchim PuriFlash instrument and normal-
5-(2-Methoxy-2-oxoethyl)-1,3-phenylene Diacetate (9). Com-
mercially available methyl 3,5-dihydroxyphenylacetate (12.0 g, 65.9
mmol, 1.0 equiv) was dissolved in Ac2O (31.2 mL, 0.33 mol, 5.0
equiv); then, pyridine (26.5 mL, 0.33 mol, 5.0 equiv) was added
dropwise at 0 °C. The reaction was stirred at room temperature (r.t.)
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Figure 3. Specificity control of the binding of the fluorescent conjugate 21 to hMelLec-expressing cells with or without competition by YWA1. (a)
Representative light microscopy images and immunofluorescence micrographs of hMelLec-expressing RAW 264.7 cells untreated or treated with 25
μM concentration of 21 (green) for 3 h at 37 °C 5% CO2 with or without YWA1 (50 μM) or with YWA1 (50 μM) alone. (b) Histograms showing
the binding of MelLec-expressing RAW 264.7 cells to 21 compared to untreated cells, or treated with YWA1 alone, or treated with both 21 and
YWA1, as determined by flow cytometry.
overnight, quenched with 10 mL of an ice-cold aqueous solution of
HCl (2 M), and extracted with Et2O (3 × 100 mL). The combined
organic phases were washed with 50 mL of brine and 20 mL of an
aqueous solution of HCl (1 M), dried over Na2SO4, and concentrated
under vacuum. The crude product was purified by flash chromatog-
raphy (EtOAc/n-hexane gradient from 10% EtOAc to 40% EtOAc) to
afford compound 9 as a pale-yellow oil (12.0 g, 70% yield). Rf = 0.6, n-
hexane/EtOAc, 1:1. Analytical and spectroscopic data were consistent
with those reported in the literature.14
(1.20 g, 8.40 mmol, 2.0 equiv) at room temperature in acetone (25
mL), BnBr (1.4 g, 8.4 mmol, 2.0 equiv) was added. The suspension
was heated to 60 °C and stirred overnight, quenched with 10 mL of
an ice-cold aqueous solution of HCl (1 M), and extracted with Et2O
(3 × 100 mL). The combined organic phases were washed with brine
(50 mL), dried over Na2SO4, and concentrated under vacuum. The
crude product was then purified by automated flash chromatography
(EtOAc/n-hexane gradient from 10% EtOAc to 40% EtOAc) to afford
compound 11 as a white solid (1.3 g, 70% yield). Rf = 0.3, n-hexane/
1
Methyl 2-(2,4-Diacetyl-3,5-dihydroxyphenyl)acetate (10). A
mixture of AlCl3 (5.20 g, 39.4 mmol, 3.5 equiv) and 9 (3.0 g, 11.2
mmol, 1.0 equiv) was stirred at 150 °C for 1.5 h. After cooling, the
reaction was poured into a solution of ice and aqueous HCl (2 M)
and the whole suspension was stirred for 10 min. The mixture was
extracted with EtOAc (3 x 100 mL), and the combined organic phases
were dried over Na2SO4 and concentrated under vacuum. To
repristinate the partially hydrolyzed ester, the crude mixture was
submitted to Fisher esterification using H2SO4 (0.6 mL, 11.2 mmol,
1.0 equiv) in MeOH (20 mL) and stirring at room temperature for 4
h. The product was then extracted with EtOAc (3 × 100 mL), washed
with brine (50 mL), and dried over Na2SO4. The solvent was
removed under vacuum, and the crude product was purified by
automated flash chromatography (100% DCM) to give product 10 as
EtOAc, 7:3. H NMR (400 MHz, CDCl3) δ: 7.46−7.34 (m, 10H),
6.70 (s, 1H), 5.14 (s, 2H), 4.85 (s, 2H), 3.74 (s, 2H), 3.71 (s, 3H),
2.57 (s, 3H), 2.54 (s, 3H). 13C{1H} NMR (101 MHz, CDCl3) δ:
204.0, 201.3, 171.2, 156.5, 154.3, 136.0, 135.7, 135.1, 129.6, 128.7,
128.6, 128.5, 128.3, 127.3, 125.8, 111.2, 79.5, 70.7, 52.2, 38.6, 32.5,
32.2. MS (ESI) m/z: [M + H]+ calcd for C27H27O6 447.17; found
447.2. Analytical and spectroscopic data were consistent with those
reported in the literature.14
Methyl 2-(2,4-Diacetyl-3,5-bis(benzyloxy)phenyl)-3-phenylpro-
panoate (11a). To a suspension of 10 (1.10 g, 4.20 mmol, 1.0
equiv) and K2CO3 (1.70 g, 12.6 mmol, 3.0 equiv) in DMF (25 mL),
BnBr (2.5 g, 14.7 mmol, 3.5 equiv) was added. This suspension was
stirred at room temperature for 4 h, quenched with 10 mL of an ice-
cold aqueous solution of HCl (1 M), and extracted with Et2O (3 × 30
mL). The organic phase was washed with brine (10 mL), dried over
Na2SO4, and concentrated under vacuum. The mixture was then
purified by automated flash chromatography (EtOAc/n-hexane
gradient from 10% EtOAc to 40% EtOAc) to afford compound 11a
as a white solid (2.0 g, 95% yield). Rf = 0.4, n-hexane/EtOAc, 7:3. 1H
NMR (400 MHz, CDCl3) δ: 7.34−7.03 (m, 13H), 6.98−6.93 (m,
2H), 6.85 (s, 1H), 5.06 (s, 2H), 4.72 (d, J = 10.4, 1H), 4.68 (d, J =
10.4, 1H), 3.92 (dd, J = 8.2, 6.8 Hz, 1H), 3.48 (s, 3H), 3.19 (dd, J =
13.5, 8.2 Hz, 1H), 2.87 (dd, J = 13.5, 6.8 Hz, 1H), 2.38 (s, 3H), 2.16
1
a white solid (1.9 g, 60% yield). Rf = 0.5, n-hexane/EtOAc, 1:1. H
NMR (400 MHz, CDCl3) δ: 15.33 (s, 1H), 13.94 (br, 1H), 6.35 (s,
1H), 3.92 (s, 2H), 3.76 (s, 3H), 2.77 (s, 3H), 2.64 (s, 3H). 13C{1H}
NMR (101 MHz, CDCl3) δ: 205.6, 203.6, 170.4, 169.5, 168.6, 143.3,
116.4, 114.1, 109.6, 52.6, 42.1, 33.6, 31.9. MS (ESI) m/z: [M + H]+
calcd for C13H15O6 267.08; found 267.1. Analytical and spectroscopic
data were consistent with those reported in the literature.14
Methyl 2-(2,4-Diacetyl-3,5-bis(benzyloxy)phenyl)ace-tate (11).
To a suspension of 10 (1.10 g, 4.20 mmol, 1.0 equiv) and K2CO3
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(s, 3H). 13C{1H} NMR (101 MHz, CDCl3) δ: 204.4, 201.2, 173.3,
156.3, 153.4, 138.7, 138.4, 136.0, 135.9, 129.1, 128.8, 128.61, 128.56,
128.52, 128.50, 128.3, 127.4, 126.7, 125.7, 107.8, 79.3, 70.7, 52.3,
48.8, 40.1, 32.6, 32.5. MS (ESI) m/z: [M + H]+ calcd for C34H33O6
537.22; found 537.2.
General Procedure for the Dieckman Condensation (Procedure
A). To a solution of 11 or 11a (1.0 equiv, 0.28 M in THF), LiHMDS
(1.0 M in THF, 3.0 equiv) was added dropwise at 0 °C. The resulting
orange suspension was stirred at 0 °C under a nitrogen atmosphere
for 2 h, quenched with an aqueous solution of HCl (1 M), extracted
with Et2O (50 mL x 3), washed with brine (20 mL), dried over
Na2SO4, and concentrated under vacuum to afford the title
compound 12 or 12a.
1-(1,3-Bis(benzyloxy)-6,8-dihydroxynaphthalen-2-yl)ethan-1-
one (12). From compound 11 (1.23 g, 2.76 mmol) according to
general procedure A, product 12 was obtained as a white solid (1.0 g,
90%). Rf = 0.2, n-hexane/EtOAc, 7:3. 1H NMR (400 MHz,
(CD3)2CO) δ: 9.19 (s, 1H), 8.71 (s, 1H), 7.55−7.32 (m, 10 H),
7.12 (s, 1H), 6.72 (d, J = 2.3 Hz, 1H), 6.41 (d, J = 2.3 Hz, 1H), 5.27
(s, 2H), 5.15 (s, 2H), 2.58 (s, 3H). 13C{1H} (101 MHz, (CD3)2CO)
δ: 200.8, 158.0, 155.9, 153.8, 152.1, 138.8, 136.8, 135.8, 128.9, 128.8,
128.7, 128.5, 128.0, 127.6, 122.2, 107.7, 103.2, 101.1, 100.8, 80.0,
70.2, 32.2. MS (ESI) m/z: [M + H]+ calcd for C26H23O5 415.15;
found 415.2. Analytical and spectroscopic data were consistent with
those reported in the literature.14
HRMS (ESI), m/z: [M + Na]+ calcd for C42H38O7Na 677.2510;
found 677.2510.
1-(1,3-Bis(benzyloxy)-6,8-dimethoxynaphtalen-2-yl)ethan-1-one
(13c). Me2SO4 (0.8 mL, 8.90 mmol, 10.0 equiv) and K2CO3 (616 mg,
4.46 mmol, 5.0 equiv) were added to a solution of 12 (370 mg, 0.89
mmol, 1.0 equiv) in acetone (15 mL), and the reaction was stirred
overnight at 60 °C. The mixture was quenched with 10 mL of an
aqueous solution of NaOH (1 M) and extracted with Et2O (3 × 50
mL). The solvent was removed under vacuum, and the residue was
stirred with 10 mL of an aqueous solution of NH4OH (1 M) for 2 h.
The aqueous phase was extracted with Et2O (3 × 50 mL), and the
combined organic phases were washed with H2O (2 × 30 mL) and
brine (1 × 30 mL), dried over Na2SO4, and concentrated under
vacuum. The crude product was purified by automated flash
chromatography (EtOAc/n-hexane gradient from 10% EtOAc to
20% EtOAc) to afford product 13c as a yellow oil (275 mg, 70%
yield). Rf = 0.6, n-hexane/EtOAc, 7:3. 1H NMR (400 MHz, CDCl3) δ
7.54−7.32 (m, 10H), 6.96 (s, 1H), 6.68 (d, J = 2.2 Hz, 1H), 6.46 (d, J
= 2.2 Hz, 1H), 5.21 (s, 2H), 5.02 (s, 2H), 3.92 (s, 3H), 3.88 (s, 3H),
2.58 (s, 3H). 13C{1H} NMR (101 MHz, CDCl3) δ: 202.9, 159.2,
157.6, 153.7, 152.5, 138.8, 137.6, 136.46, 128.6, 128.4, 128.2, 128.0,
127.9, 127.1, 125.2, 111.2, 103.7, 98.6, 97.4, 78.9, 70.2, 55.8, 55.3,
32.8. HRMS (ESI) m/z: [M + Na]+ calcd for C28H26O5Na 465.1672;
found 465.1672.
1-(1,3-Bis(benzyloxy)-6,8-bis((tert-butyldimethyl-silyl)oxy)-
naphtalen-2-yl)ethan-1-one (13d). TBDMSCl (85.1 mg, 0.56 mmol,
2.5 equiv) and DIPEA (0.1 mL, 0.68 mmol, 3.0 equiv) were added to
a solution of 12 (100 mg, 0.23 mmol, 1.0 equiv) in DMF (1 mL), and
the reaction was stirred at room temperature overnight under a
nitrogen atmosphere. The mixture was extracted with Et2O (3 × 10
mL), washed with 10 mL of brine, dried over Na2SO4, and
concentrated under vacuum. The crude product was purified by
automated flash chromatography (EtOAc/n-hexane gradient from 0%
EtOAc to 20% EtOAc) to afford compound 13d as a yellow oil (140
mg, 99% yield). Rf = 0.6, n-hexane/EtOAc, 9:1. 1H NMR (400 MHz,
CDCl3) δ: 7.45−7.37 (m, 10 H), 6.90 (s, 1H), 6.81 (d, J = 2.3 Hz,
1H), 6.48 (d, J = 2.3 Hz, 1H), 5.19 (s, 2H), 5.08 (s, 2H), 2.50 (s,
3H), 1.07 (s, 9H), 0.94 (s, 9H), 0.31 (s, 6H), 0.11 (s, 6H). 13C{1H}
NMR (101 MHz, CDCl3) δ: 202.4, 154.9, 153.4, 153.3, 153.0, 138.6,
137.7, 136.5, 128.6, 128.1, 128.0, 127.5, 127.5, 127.1, 125.0, 113.8,
110.8, 109.0, 103.1, 78.0, 70.1, 32.9, 26.21, 25.7, 18.9, 18.3, −4.0,
−4.2. HRMS (ESI) m/z: [M + Na]+ calcd for C38H50O5Si2Na
665.3089; found 665.3088.
1-5(Benzyl-1,3-bis(benzyloxy)-6,8-dihydroxynaphthalen-2-yl)-
ethan-1-one (12a). From compound 11a (1.00 g, 1.86 mmol)
according to general procedure A, product 12a was obtained as a
1
yellow solid (850 mg, 90% yield). Rf = 0.3, n-hexane/EtOAc, 7:3. H
NMR (400 MHz, (CD3)2CO) δ: 9.12 (s, 1H), 8.71 (s, 1H), 7.41−
6.94 (m, 16H), 6.47 (s, 1H), 5.01 (s, 2H), 4.98 (s, 2H), 4.16 (s, 2H),
2.41 (s, 3H). 13C{1H} NMR (101 MHz, (CD3)2CO) δ: 200.8, 155.3,
154.4, 153.6, 152.5, 141.7, 137.1, 136.7, 135.6, 129.0, 128.9, 128.7,
128.6, 128.3, 128.2, 128.0, 127.6, 125.5, 121.9, 110.3, 108.2, 101.2,
100.6, 80.2, 70.2, 32.3, 30.3. HRMS (ESI), m/z: [M + Na]+ calcd for
C33H28O5Na 527.1829; found 527.1828.
1-(1,3,6,8-Tetrakis(benzyloxy)naphthalen-2-yl)ethan-1-one
(13a). To a solution of 12 (230 mg, 0.56 mmol, 1.0 equiv), PPh3 (308
mg, 1.2 mmol, 2.1 equiv), and BnOH (122 μL, 1.17 mmol, 2.1 equiv)
in Et2O (10 mL) at 0 °C, DIAD (232 μL, 1.17 mmol, 2.1 equiv) was
added dropwise. The reaction was stirred for 4 h at room temperature
and then purified by automated flash chromatography (EtOAc/n-
hexane gradient from 0% EtOAc to 30% EtOAc) to afford compound
13a as a white solid (50.0 mg, 17% yield). Rf = 0.3, n-hexane/EtOAc,
1-(1,3,6,8-Tetrakis(benzyloxy)naphtalen-2-yl)vinyl Acetate
(14a). LiHMDS (33.0 μL, 0.17 mmol, 2.0 equiv) was added to a
solution of 13a (50.0 mg, 0.08 mmol, 1.0 equiv) in THF (2 mL) at 0
°C, and the reaction was stirred for 15 min. Then, acetic anhydride
(9.5 mg, 0.09 mmol, 1.1 equiv) was added dropwise and the reaction
was monitored by liquid chromatography−mass spectrometry (LC-
MS), confirming the complete conversion of the starting material into
the desired product after 2 h. The reaction was quenched with H2O
(5 mL), and the mixture was extracted with EtOAc (3 × 20 mL),
washed with 10 mL of brine, dried over Na2SO4, and concentrated
under vacuum. The crude product was then purified by flash
chromatography (n-hexane/EtOAc 8:2) to afford 14a as a white solid
1
8:2. H NMR (400 MHz, CDCl3) δ: 7.51−7.14 (m, 20H), 6.95 (s,
1H), 6.79 (d, J = 2.2 Hz, 1H), 6.62 (d, J = 2.2 Hz, 1H), 5.21 (s, 2H),
5.19 (s, 2H), 5.15 (s, 2H), 5.02 (s, 2H), 2.51 (s, 3H). 13C{1H} NMR
(101 MHz, CDCl3) δ: 203.0, 158.2, 156.9, 153.7, 152.9, 138.8, 137.4,
136.6, 136.5, 136.3, 128.7, 128.63, 128.59, 128.2, 128.0, 127.8, 127.7,
127.5, 127.1, 125.5, 111.8, 103.8, 100.2, 99.6, 78.7, 71.3, 70.2, 70.1,
32.8. HRMS (ESI), m/z: [M + Na]+ calcd for C40H34O5Na 617.2298;
found 617.2298.
1-(1,3-Bis(benzyloxy)-6,8-bis((benzyloxy)methoxy)naphtalen-2-
yl)ethan-1-one (13b). To a solution of 12 (930 mg, 2.24 mmol, 1.0
equiv) in CH2Cl2 (16 mL), DIPEA (3.8 mL, 22.4 mmol, 10.0 equiv)
and BOMCl (1.2 mL, 8.96 mmol, 4.0 equiv) were added at 0 °C. The
reaction was stirred at room temperature overnight under a nitrogen
atmosphere. The mixture was quenched with 10 mL of a saturated
aqueous solution of NH4Cl, extracted with Et2O (3 × 50 mL), washed
with brine (20 mL), dried over Na2SO4, and concentrated under
vacuum. The crude product was purified by automated flash
chromatography (EtOAc/n-hexane gradient from 0% EtOAc to 30%
EtOAc) to afford compound 13b as a yellow oil (730 mg, 50% yield).
Rf = 0.6, n-hexane/EtOAc, 8:2. 1H NMR (400 MHz, CDCl3) δ:
7.43−7.11 (m, 20H), 6.97 (d, J = 2.2 Hz, 1H), 6.88−6.83 (m, 2H),
5.29 (s, 2H), 5.21 (s, 2H), 5.10 (s, 2H), 4.98 (s, 2H), 4.68 (s, 2H),
4.54 (s, 2H), 2.46 (s, 3H). 13C{1H} NMR (101 MHz, CDCl3) δ:
202.7, 156.5, 155.2, 153.6, 152.2, 138.6, 137.8, 137.2, 137.1, 136.4,
128.6, 128.5, 128.4, 128.2, 128.0, 127.9, 127.8, 127.7, 127.1, 125.9,
112.1, 104.0, 103.9, 102.9, 93.6, 92.2, 78.5, 70.3, 70.2, 70.2, 32.8.
1
(25.0 mg, 50% yield). Rf = 0.4, n-hexane/EtOAc, 7:3. H NMR (400
MHz, CDCl3) δ: 7.51−7.26 (m, 14H), 7.23−7.13 (m, 6H), 6.90 (s,
1H), 6.71 (d, J = 2.2 Hz, 1H), 6.54 (d, J = 2.2 Hz, 1H), 5.45 (d, J =
1.3 Hz, 1H), 5.31 (d, J = 1.3 Hz, 1H), 5.20 (s, 2H), 5.12 (s, 2H), 5.11
(s, 2H) 5.05 (s, 2H), 1.94 (s, 3H). 13C{1H} NMR (101 MHz,
CDCl3) δ: 168.5, 158.1, 157.0, 155.5, 155.3, 146.3, 138.5, 138.2,
136.9, 136.7, 136.4, 128.7, 128.5, 128.4, 128.1, 128.0, 127.8, 127.71,
127.67, 127.4, 127.3, 127.1, 118.1, 112.1, 108.3, 103.6, 100.0, 99.6,
76.4, 71.2, 70.2, 70.0, 21.3. MS (ESI) m/z: [M + H]+ calcd for
C42H37O6 637.25; found 637.2.
General Procedure for Aldol Condensation with Acetaldehyde
(Procedure B1). To a solution of 13b or 13c (1.0 equiv, 0.11 M in
THF), LiHMDS (1 M in THF, 3.0 equiv) was added dropwise at −40
°C under a nitrogen atmosphere. The solution was stirred for 10 min,
and then acetaldehyde (5 M in THF, 4.0 equiv) was added. The
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reaction was stirred for 15 min keeping the temperature at −40 °C
and then quenched with a saturated aqueous solution of NH4Cl. The
mixture was extracted with Et2O, washed with brine, dried over
Na2SO4, and concentrated under vacuum. The crude product was
purified by flash chromatography eluting with the indicated solvent.
1-(1,3-Bis(benzyloxy)-6,8-bis((benzyloxy)methxy)naphtalen-2-
yl)-3-hydroxybutan-1-one (14b). Compound 13b (750 mg, 1.14
mmol) was reacted according to the general procedure B1. The crude
product was purified by flash chromatography (n-hexane/EtOAc 8:2)
to afford compound 14b as a yellow oil (700 mg, 90% yield). Rf = 0.3,
n-hexane/EtOAc, 8:2. 1H NMR (400 MHz, CDCl3) δ: 7.49−7.25 (m,
18H), 7.24−7.18 (m, 2H), 7.05 (d, J = 2.2 Hz, 1H), 6.96 (s, 1H),
6.95 (d, J = 2.2 Hz, 1H), 5.38 (s, 2H), 5.29 (s, 2H), 5.17 (s, 2H), 5.08
(d, J = 10.5 Hz, 1H), 5.04 (d, J = 10.5 Hz, 1H), 4.76 (s, 2H), 4.62 (s,
2H), 4.30−4.21 (m, 1H), 3.13 (br, 1H), 3.02 (dd, J = 17.1, 2.7 Hz,
1H), 2.86 (dd, J = 17.1, 9.0 Hz, 1H), 1.12 (d, J = 6.4 Hz, 3H).
13C{1H} NMR (101 MHz, CDCl3) δ 206.0, 156.7, 155.2, 153.4,
solution of NH4Cl, extracted with Et2O (3 × 30 mL), washed with
brine (5 mL), dried over Na2SO4, and evaporated. The crude product
was purified by flash chromatography or directly submitted to the
following step without further purification.
10-Azido-1-(1,3-bis(benzyloxy)-6,8-bis((benzyloxy)methoxy)-
naphthalen-2-yl)-3-hydroxydecan-1-one (16b). Compound 13b
(30.0 mg, 0.04 mmol) was reacted according to general procedure
B2. The crude product was purified by automated flash chromatog-
raphy (EtOAc/n-hexane gradient from 0% EtOAc to 20% EtOAc) to
afford compound 16b as a yellow oil (38.0 mg, 99%). Rf = 0.4, n-
1
hexane/EtOAc, 8:2. H NMR (400 MHz, CDCl3) δ: 7.42−7.09 (m,
20H), 6.98 (d, J = 2.2 Hz, 1H), 6.88 (s, 1H), 6.87 (d, J = 2.2 Hz, 1H),
5.30 (s, 2H), 5.21 (s, 2H), 5.09 (s, 2H), 5.02 (d, J = 10.5 Hz, 1H),
4.96 (d, J = 10.5 Hz, 1H), 4.68 (s, 2H), 4.54 (s, 2H), 3.98 (br, 1H),
3.15 (t, J = 7.0 Hz, 2H), 3.02 (d, J = 3.1 Hz, 1H), 2.97 (dd, J = 17.1,
2.5 Hz, 1H), 2.77 (dd, J = 17.1, 9.1 Hz, 1H), 1.53−1.14 (m, 12H).
13C{1H} NMR (101 MHz, CDCl3) δ: 206.2, 156.7, 155.2, 153.4,
152.3, 138.8, 137.4, 137.14, 137.07, 136.09, 128.7, 128.52, 128.48,
128.40, 128.18, 128.16, 128.0, 127.9, 127.8, 127.7, 127.2, 125.1, 112.0,
104.1, 103.9, 103.0, 93.6, 92.2, 78.78, 70.4, 70.36, 70.27, 64.4, 53.7,
22.3. MS (ESI) m/z: [M + H]+ calcd for C44H43O8 699.29; found
699.3.
152.3, 138.8, 137.5, 137.1, 137.07, 136.1, 128.7, 128.52, 128.46,
128.40, 128.2, 128.1, 128.0, 127.88, 127.85, 127.81, 127.6, 127.2,
125.1, 112.1, 104.1, 103.9, 103.0, 93.6, 92.2, 78.7, 70.4, 70.3, 70.3,
68.1, 52.2, 51.5, 36.3, 29.4, 29.1, 28.8, 26.6, 25.3. MS (ESI) m/z: [M
+ H]+ calcd for C50H54N3O8 824.38; found 824.4.
1-(1,3-Bis(benzyloxy)-6,8-dimethoxynaphtalen-2-yl)-3-hydroxy-
buta-1-one (14c). Compound 13c (275 mg, 0.62 mmol) was reacted
according to the general procedure B1. The crude product was
purified by flash chromatography (n-hexane/EtOAc 8:2) to afford
compound 14c as a yellow oil (272 mg, 90% yield). Rf = 0.5, n-
10-Azido-1-(1,3-bis(benzyloxy)-6,8-dimethoxynaphthalen-2-yl)-
3-hydroxydecan-1-one (16c). Compound 13c (450 mg, 1.0 mmol)
was reacted according to general procedure B2. The crude product
was purified by flash chromatography (n-hexane/EtOAc 8:2) to afford
compound 16c as a yellow oil (588 mg, 99%). Rf = 0.4, n-hexane/
1
1
hexane/EtOAc, 8:2. H NMR (400 MHz, CDCl3) δ: 7.42−7.21 (m,
EtOAc, 7:3. H NMR (400 MHz, CDCl3) δ: 7.49−7.31 (m, 10H),
10H), 6.86 (s, 1H), 6.57 (d, J = 2.2 Hz, 1H), 6.35 (d, J = 2.2 Hz, 1H),
5.09 (s, 2H), 4.93 (d, J = 10.5 Hz, 1H), 4.88 (d, J = 10.5 Hz, 1H) 4.19
(m, 1H), 3.81 (s, 3H), 3.77 (s, 3H), 2.96 (dd, J = 17.2, 2.7 Hz, 1H),
2.78 (dd, J = 17.2, 9.0 Hz, 1H), 1.04 (d, J = 6.3 Hz, 3H). 13C{1H}
NMR (101 MHz, CDCl3) δ: 206.2, 159.4, 157.63, 153.60, 152.7,
139.0, 137.3, 136.2, 128.7, 128.5, 128.2, 128.1, 128.0, 127.1, 124.4,
111.1, 103.9, 98.6, 97.6, 79.1, 70.3, 64.3, 55.8, 55.4, 53.6, 22.3. MS
(ESI) m/z: [M + H]+ calcd for C30H31O6 487.20; found 487.2.
General Procedure for the Synthesis of 8-Azido Octanal (22) or
8-Azido Hexanal (23). Commercially available 8-bromo-1-octanol
(100 mg, 0.48 mmol, 1.0 equiv) or 6-bromo-1-hexanol (87.0 mg, 0.48
mmol, 1.0 equiv) was dissolved in DMF (4 mL), followed by the
addition of NaN3 (31.2 mg, 0.96 mmol, 2.0 equiv). The reaction was
stirred at room temperature for 12 h. The mixture was extracted with
Et2O (3 × 50 mL), washed with brine (10 mL), dried over Na2SO4,
and evaporated. The crude product was directly submitted to the next
step without further purification. DMP (140 mg, 0.72 mmol, 1.5
equiv) was added to the solution of the azide in CHCl3 (7 mL). The
reaction was stirred at room temperature for 30 min, and the crude
product was purified by flash chromatography (n-hexane/CH2Cl2
4:6) to afford product 22 or 23 as a pale oil (99% yield for both 22
6.95 (s, 1H), 6.66 (d, J = 2.2 Hz, 1H), 6.44 (d, J = 2.2 Hz, 1H), 5.17
(s, 2H), 5.03 (d, J = 10.5 Hz, 1H), 4.97 (d, J = 10.5 Hz, 1H) 4.09 (br,
1H), 3.89 (s, 3H), 3.85 (s, 3H), 3.24 (t, J = 7.0 Hz, 2H), 3.08 (dd, J =
17.1, 2.5 Hz, 1H), 2.86 (dd, J = 17.1, 9.1 Hz, 1H), 1.62−1.11 (m,
12H). 13C{1H} NMR (101 MHz, CDCl3) δ: 206.4, 159.4, 157.6,
153.6, 152.7, 139.0, 137.4, 136.2, 128.7, 128.5, 128.2, 128.1, 128.0,
127.1, 124.5, 111.1, 103.9, 98.6, 97.6, 79.1, 70.3, 68.0, 55.8, 55.4, 52.2,
51.5, 36.3, 29.4, 29.1, 28.8, 26.6, 25.3. MS (ESI) m/z: [M + H]+ calcd
for C36H42N3O6 612.30; found 612.3.
10-Azido-1-(1,3-bis(benzyloxy)-8-((tert-butyldimethylsilyl)oxy)-6-
((isopropyldimethylsilyl)oxy)naphthalen-2-yl)-3-hydroxydecan-1-
one (16d). Compound 13d (130 mg, 0.2 mmol) was reacted
according to general procedure B2. The crude product was purified by
flash chromatography (n-hexane/EtOAc 9:1) to afford compound
16d as a yellow oil (162 mg, 99%). Rf = 0.3, n-hexane/EtOAc, 9:1. 1H
NMR (400 MHz, CDCl3) δ: 7.35−7.17 (m, 10H), 6.78 (s, 1H), 6.68
(d, J = 2.2 Hz, 1H), 6.35 (d, J = 2.2 Hz, 1H), 5.05 (s, 2H), 4.98 (d, J =
10.5 Hz, 1H), 4.93 (d, J = 10.5 Hz, 1H), 3.91 (br, 1H), 3.15 (t, J = 7.0
Hz, 2H), 2.98 (br, 1H), 2.90 (dd, J = 17.1, 2.4 Hz, 1H), 2.64 (dd, J =
17.1, 9.1 Hz, 1H), 1.54−1.44 (m, 2H), 1.30−1.13 (m, 10H), 0.94 (s,
9H), 0.82 (s, 9H), 0.18 (s, 6H), −0.00 (d, J = 2.4 Hz, 6H). 13C{1H}
NMR (101 MHz, CDCl3) δ: 205.8, 155.1, 153.5, 153.25, 153.16,
138.7, 137.3, 136.3, 128.6, 128.2, 128.1, 127.6, 127.2, 124.1, 113.7,
111.0, 109.1, 103.2, 78.3, 70.2, 67.9, 52.2, 51.5, 36.3, 29.4, 29.1, 28.8,
26.7, 26.20, 26.16, 25.7, 25.4, 18.9, 18.3, −3.89, −3.93, −4.19. MS
(ESI) m/z: [M + H]+ calcd for C46H66N3O6Si2 812.44; found 812.4.
8-Azido-1-(1,3-bis(benzyloxy)-6,8-dihydroxynaphthalen-2-yl)-3-
hydroxyoctan-1-one (16e). Compound 13c (450 mg, 1.0 mmol) was
reacted following procedure B2. The crude product was directly
submitted to the next step without further purification. Rf = 0.4, n-
1
and 23). 22: Rf = 0.5, n-hexane/CH2Cl2, 3:7. H NMR (400 MHz,
CDCl3) δ: 9.79 (t, J = 1.8 Hz, 1H), 3.28 (t, J = 6.9 Hz, 2H), 2.45 (td,
J = 7.3, 1.8 Hz, 2H), 1.71−1.58 (m, 4H), 1.44−1.34 (m, 6H).
13C{1H} NMR (101 MHz, CDCl3) δ: 202.60, 51.36, 43.77, 28.94,
28.85, 28.72, 26.47, 21.88. HRMS (ESI) m/z: [M + Na]+ calcd for
C8H15N3ONa 192.1107; found 192.1107. For 23, analytical and
spectroscopic data were consistent with those reported in the
literature.15
General Procedure for Aldol Condensation with 22 or 23
(Procedure B2). LDA (1 M in THF) was freshly prepared according
to the following procedure: 280 μL of 1,2-diisopropylamine was
added to 920 μL of THF in dry conditions under an inert atmosphere
and cooled to −78 °C, and then 800 μL of n-BuLi (2.5 M in n-
hexane) was added and the mixture was stirred at 0 °C for 40 min.
LDA (1 M in THF, 1.5 equiv) was added dropwise to a solution of
13b, 13c, or 13d (1.0 equiv, 0.04 M in THF) at −78 °C, and the
reaction was stirred for 45 min under nitrogen keeping the
temperature at −78 °C. Then, 22 or 23 (2.0 equiv), freshly prepared
and stored under an inert atmosphere until needed, was added
dropwise to the solution and the reaction was stirred for 15 min at
−78 °C. The reaction was quenched with a saturated aqueous
1
hexane/EtOAc, 7:3. H NMR (400 MHz, CDCl3) δ: 7.47−7.32 (m,
10H), 6.94 (s, 1H), 6.66 (d, J = 2.2 Hz, 1H), 6.44 (d, J = 2.2 Hz, 1H),
5.17 (s, 2H), 5.02 (d, J = 10.5 Hz, 1H), 4.96 (d, J = 10.5 Hz, 1H),
4.05 (br, 1H), 3.90 (s, 3H), 3.86 (s, 3H), 3.20 (t, J = 7.0 Hz, 2H),
3.14 (br, 1H), 3.05 (dd, J = 17.1, 2.5 Hz, 1H), 2.84 (dd, J = 17.1, 9.1
Hz, 1H), 1.57−1.11 (m, 8H). MS (ESI) m/z: [M + H]+ calcd for
C34H38N3O6 584.27; found 584.3.
General Procedure for the Dess−Martin Oxidation (Procedure
C). To a solution of β-hydroxyketone 14b, 14c or 16b−e (1.0 equiv,
0.04 M in CHCl3), DMP (1.5 equiv) was added in small portions.
The suspension was stirred at 40 °C or at room temperature until
complete consumption of the starting material (reaction monitored by
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TLC). The crude product was filtered on a silica pad to remove the
excess of DMP and directly submitted to the next step without further
purification.
(Z)-8-Azido-1-(1,3-bis(benzyloxy)-6,8-dihydroxynaphthalen-2-
yl)-3-hydroxyoct-2-en-1-one (17e). Compound 16e (255 mg, 0.44
mmol) was reacted according to general procedure C. The crude
product was filtered on a silica pad to afford 17e, which was directly
submitted to the next step without further purification. Rf = 0.5, n-
hexane/EtOAc, 7:3. Ketone 17e is in equilibrium with its enolic form.
According to the NMR, the enol is predominant. The signals of the
ketone are labeled with an asterisk when clearly distinguishable from
(Z)-1-(1,3-Bis(benzyloxy)-6,8-bis((benzyloxy)methoxy)-
naphthalen-2-yl)-3-hydroxybut-2-en-1-one (15b). Compound 14b
(700 mg, 1.0 mmol) was reacted according to general procedure C.
The crude product was filtered on a silica pad to give 15b, which was
directly submitted to the next step without further purification. Rf =
0.4, n-hexane/EtOAc, 8:2. Ketone 15b is in equilibrium with its enolic
form. According to the NMR, the enol is predominant. The signals of
the ketone are labeled with an asterisk when clearly distinguishable
from the enol. 1H NMR (400 MHz, CDCl3) δ: 15.49 (s, 1H), 7.41−
7.10 (m, 26H), 6.95 (d, J = 2.2 Hz, 1.3H), 6.87−6.83 (m, 2.6H), 5.73
(s, 1H), 5.28 (s, 2.6H), 5.20 (s, 2.6H), 5.10 (s, 2H), 5.06* (s, 0.6H),
4.98 (s, 2.6H), 4.66 (s, 2.6H), 4.55 (s, 2H), 4.51* (s, 0.6H), 3.81* (s,
0.6H), 1.96 (s, 3H) 1.95* (s, 0.9H). MS (ESI) m/z: [M + H]+ calcd
for C44H41O8 697.27; found 697.3.
(Z)-1-(1,3-Bis(benzyloxy)-6,8-dimethoxynaphthalen-2-yl)-3-hy-
droxybut-2-en-1-one (15c). Compound 14c (395 mg, 0.81 mmol)
was reacted according to general procedure C. The crude product was
filtered on a silica pad to afford 15c, which was directly submitted to
the next step without further purification. Rf = 0.4, n-hexane/EtOAc,
7:3. Ketone 15c is in equilibrium with its enolic form. According to
the NMR, the enol is predominant. The signals of the ketone are
labeled with an asterisk when clearly distinguishable from the enol. 1H
NMR (400 MHz, CDCl3) δ: 15.50 (s, 1H), 7.42−7.20 (m, 14H),
6.85 (s, 1.4H), 6.56 (d, J = 2.2 Hz, 1.4H), 6.34 (d, J = 2.2 Hz, 1.4H),
5.77 (s, 1H), 5.13 (s, 2H), 5.09* (s, 0.8 H), 4.93* (s, 0.8 H), 4.90 (s,
2H), 3.82 (s, 4.2H), 3.79 (s, 4.2H), 1.98 (s, 4.2H). MS (ESI) m/z:
[M + H]+ calcd for C30H29O6 485.19; found 485.2.
1
the enol. H NMR (400 MHz, CDCl3) δ 15.61 (br, 1H), 7.55−7.29
(m, 14H), 6.94 (s, 1.4H), 6.65 (d, J = 2.2 Hz, 1.4H), 6.43 (d, J = 2.2
Hz, 1.4H), 5.85 (s, 1H), 5.20 (s, 2H), 5.16* (s, 0.8H), 5.00 (s, 2.8H),
3.92* (s, 0.8H), 3.89 (s, 4.2H), 3.86 (s, 4.2 H) 3.21−3.15 (m, 2.8H),
2.36* (t, J = 8 Hz, 0.8H), 2.29 (t, J = 8 Hz, 2H), 1.64−1.38 (m,
8.4H). MS (ESI) m/z: [M + H]+ calcd for C34H36N3O6 582.25; found
582.2.
General Procedure for Hydrogenolysis (Procedure D). Glacial
AcOH (1.0 equiv) and a catalytic amount of Pd(OH)2 on carbon 20%
were added to a suspension of 1,3-diketone 15b, 15c or 17b−d (1.0
equiv, 0.014 M in MeOH) under nitrogen. The reaction was stirred
under a hydrogen atmosphere and monitored via LC-MS using a
gradient from 30% acetonitrile (ACN) (containing 0.1% trifluoro-
acetic acid (TFA)) to 100% ACN (containing 0.1% TFA) in water
over 10 min (method 1), confirming the complete conversion of the
starting material into the desired product. Pd(OH)2 was removed by
filtration on a cotton pad, and the solvent was slowly evaporated at
low temperature under vacuum. The crude product was purified by
preparative RP-HPLC using a gradient from 30% ACN (containing
0.05% TFA) to 100% ACN (containing 0.05% TFA) in water over 10
min (method 2) to obtain the final products YWA1 and fonsecin B.
Intermediates 18a and 18b instead, due to the reactivity of the amine
group, proved to be unstable in solution and therefore were used
immediately in the next conjugation step without further purification.
YWA1. Compound 15b (100 mg, 0.14 mmol) was reacted
according to general procedure D. The reaction was monitored by
LC-MS (method 1), confirming the complete conversion of the
starting material to the desired product in 12 h. The product was
submitted to preparative RP-HPLC (method 2) and isolated as a
yellow solid (36.0 mg, 90% yield), r.t. = 7.8 min. 1H NMR (400 MHz,
(CD3)2CO) δ: 15.37 (s, 1H), 9.45 (s, 1H), 9.20 (s, 1H), 6.54 (d, J =
2.2 Hz, 1H), 6.47 (s, 1H), 6.30 (d, J = 2.2 Hz, 1H), 6.05 (s, 1H), 3.19
(d, J = 17.1 Hz, 1H), 2.88 (d, J = 17.1 Hz, 1H), 1.74 (s, 3H). 13C{1H}
NMR (101 MHz, (CD3)2CO) δ: 198.8, 165.0, 162.8, 160.8, 154.1,
143.6, 105.0, 102.9, 102.8, 102.4, 101.3, 100.8, 47.8, 28.4. MS (ESI)
m/z: [M + H]+ calcd. for C14H13O6 277.06; found 277.1. Analytical
and spectroscopic data were consistent with those reported in the
literature.5
Fonsecin B. Compound 15c (100 mg, 0.21 mmol) was reacted
according to general procedure D. The reaction was monitored by
LC-MS (method 1), confirming the complete conversion of the
starting material into the desired product in 3 h. The product was
submitted to preparative RP-HPLC (method 2) and isolated as a
yellow solid (56.0 mg, 90% yield), r.t. = 8.5 min. 1H NMR (400 MHz,
CDCl3) δ: 14.21 (s, 1H), 6.45 (s, 1H), 6.39 (d, J = 2.2 Hz, 1H), 6.24
(d, J = 2.2 Hz, 1H), 3.89 (s, 3H), 3.83 (s, 3H), 2.95 (d, J = 17.0 Hz,
1H), 2.85 (d, J = 17.0 Hz, 1H), 1.68 (s, 3H). 13C{1H} NMR (101
MHz, CDCl3) δ: 196.0, 164.9, 162.5, 161.4, 153.1, 143.3, 107.4,
103.1, 102.6, 100.0, 98.6, 96.7, 56.1, 55.4, 47.0, 28.7. HRMS (ESI),
m/z: [M + H]+ calcd for C16H16O6Na 327.0839; found 327.0840.
General Procedure for the Coupling Reaction (Procedure E).
Intermediate 18a or 18b (1.0 equiv) was dissolved in DMF, and then
the desired fluorescent tag (1.5 equiv) was added in one portion. After
the addition of DIPEA (2.0 equiv), the solution was stirred for 0.5 h
under a nitrogen atmosphere. The reaction was monitored by LC-MS
using a gradient from 30% ACN (containing 0.1% TFA) to 100%
ACN (containing 0.1% TFA) in water over 10 min (method 1),
confirming the total conversion of the starting material into the
desired product. The compound was then purified by preparative RP-
HPLC with a gradient from 30% ACN (containing 0.05% TFA) to
100% ACN (containing 0.05% TFA) in water over 10 min (method
2).
(Z)-10-Azido-1-(1,3-bis(benzyloxy)-6,8-dimethoxynaphthalen-2-
yl)-3-hydroxydec-2-en-1-one (17c). Compound 16c (269 mg, 0.44
mmol) was reacted according to general procedure C. The crude
product was filtered on a silica pad to afford 17c, which was directly
submitted to the next step without further purification. Rf = 0.5, n-
hexane/EtOAc, 7:3. Ketone 17c is in equilibrium with its enolic form.
According to the NMR, the enol is predominant. The signals of the
ketone are labeled with an asterisk when clearly distinguishable from
1
the enol. H NMR (400 MHz, CDCl3) δ: 15.61 (s, 1H), 7.48−7.30
(m, 14H), 6.93 (s, 1.4H), 6.64 (d, J =2.2 Hz, 1.4H), 6.42 (d, J = 2.2
Hz, 1.4), 5.83 (s, 1H), 5.20 (s, 2H), 5.17* (s, 0.8H), 4.99* (s, 0.8H),
4.98 (s, 2H), 3.90 (s, 4.2H), 3.86 (s, 4.2H), 3.23 (t, J = 7.0, 2.8H),
2.35* (t, J = 7.4 Hz, 0.8H), 2.27 (t, J = 8.0 Hz, 2H), 1.55−1.23 (m,
14H). MS (ESI) m/z: [M + H]+ calcd for C36H40N3O6 610.28; found
610.3.
Alternative Procedure. Stabilized 2-iodoxybenzoic acid (275 mg,
0.98 mmol, 3 equiv) was added to compound 16c (200 mg, 0.33
mmol) in EtOAc (10 mL) at room temperature. The reaction mixture
was heated to 80 °C and stirred overnight. After cooling to room
temperature, the reaction was filtered on a silica pad and washed with
EtOAc. The filtrate was washed with saturated NaHCO3 (3 × 10
mL), dried (MgSO4), filtered, and concentrated under vacuum to give
17c as a yellow oil.
(Z)-10-Azido-1-(1,3-bis(benzyloxy)-8-((tert-butyldimethylsilyl)-
oxy)-6-((isopropyldimethylsilyl)oxy)naphthalen-2-yl)-3-hydroxy-
dec-2-en-1-one (17d). Compound 16d (140 mg, 0.17 mmol) was
reacted according to general procedure C (room temperature). The
crude product was filtered on a silica pad to afford 17d, which was
directly submitted to the next step without further purification. Rf =
0.4, n-hexane/EtOAc, 9:1. Ketone 17d is in equilibrium with its enolic
form. According to the NMR, the enol is predominant. The signals of
the ketone are labeled with an asterisk when clearly distinguishable
from the enol. 1H NMR (400 MHz, CDCl3) δ: 15.52 (br, 1H), 7.46−
7.27 (m, 14H), 6.88 (s, 1.4H), 6.77 (d, J = 2.2 Hz, 1.4H), 6.45 (d, J =
2.2 Hz, 1.4H), 5.73 (s, 1H), 5.19 (s, 2H), 5.15* (s, 0.8H), 5.07 (s,
2.8H), 3.84* (s, 0.8H), 3.25 (t, J = 6.9 Hz, 2.8H), 2.36* (t, J = 8 Hz,
0.8H), 2.26 (t, J = 8 Hz, 2H), 1.62−1.54 (m, 5.6H), 1.42−1.25 (m,
8.4H), 1.05 (s, 12.6H), 0.92 (s, 12.6H), 0.29 (s, 8.4H), 0.08 (s, 8.4H).
MS (ESI) m/z: [M + H]+ calcd for C46H64N3O6Si2 810.43; found
810.4.
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Oregon Green Derivative 19. Compound 17c (25.0 mg, 0.04
mmol) was reacted according to general procedure D. The reaction
was monitored by LC-MS (method 1), confirming the complete
conversion of the starting material into the desired product 18b in 3
h, r.t. = 5.7 min. MS (ESI, m/z): C22H30NO6 [M + H]+ calcd 404.20;
found 404.2. After removing Pd(OH)2, the solvent was evaporated
and the crude product was immediately reacted with Oregon green-
NHS (30.0 mg, 0.06 mmol, 1.5 equiv)previously prepared as
reported by Sun et al.16 (see the Supporting Information)and
DIPEA (15.0 μL, 0.08 mmol, 2.0 equiv) in 1 mL of DMF following
general procedure E. The reaction was monitored by LC-MS (method
1), observing the complete conversion of the starting material in 0.5 h.
The product was purified by preparative RP-HPLC (method 2) to
give 19 as a yellow solid (15.0 mg, 50% yield over two steps), r.t. =
10.1 min. 1H NMR (400 MHz, (CD3)2CO) δ: 14.13 (s, 1H), 8.22 (d,
J = 8.0, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.96 (t, J = 4 Hz, 1H), 7.76 (s,
1H), 6.96 (d, J = 7.5 Hz, 2H), 6.64−6.57 (m, 3H), 6.51 (d, J = 3.4
Hz, 1H), 6.34 (d, J = 2.4 Hz, 1H), 3.89 (s, 3H), 3.87 (s, 3H), 3.36−
3.32 (m, 2H), 3.08 (d, J = 16.9 Hz, 1H), 2.75 (d, J = 16.9 Hz, 1H),
1.58−1.51 (m, 4H) 1.40−1.23 (m, 8H). HRMS (ESI) m/z: [M +
Na]+ calcd for C43H37F2NO12Na 820.2176; found 820.2172.
(m, 2H), 1.56−1.18 (m, 14H). HRMS (ESI) m/z: [M + Na]+ calcd
for C32H43N3O8SNa 652.2663; found 652.2661.
PyMPO Derivative 21. 17c (7.0 mg, 0.01 mmol) was reacted
according to general procedure D. The reaction was monitored by
LC-MS (method 1), confirming the complete conversion of the
starting material into 18b in 3 h, r.t. = 5.7 min. MS (ESI, m/z):
C22H30NO6 [M + H]+ calcd 404.20; found 404.2. After removing
Pd(OH)2, the solvent was evaporated and the crude product was
immediately reacted with the commercially available PyMPO
succinimidyl ester (5.0 mg, 0.01 mmol, 1.5 equiv) and DIPEA (5.0
μL, 0.02 mmol, 2.0 equiv) in 0.5 mL of DMF following procedure E.
The reaction was monitored by LC-MS (method 1), confirming the
complete conversion of the starting material in 0.5 h. The product was
purified by preparative RP-HPLC (method 2) to give 21 as an orange
1
solid (5.0 mg, 60% over two steps), r.t. = 8.0 min. H NMR (400
MHz, CD3OD) δ: 9.02 (d, J = 6.4 Hz, 2H), 8.44 (d, J = 6.4 Hz, 2H),
8.01 (s, 1H), 7.92 (d, J = 7.8 Hz, 1H), 7.79 (d, J = 8.9 Hz, 2H), 7.76
(s, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.61−7.56 (m, 1H), 7.06 (d, J = 8.9
Hz, 2H), 6.45 (d, J = 2.2 Hz, 1H), 6.43 (s, 1H), 6.24 (d, J = 2.2 Hz,
1H), 5.87 (s, 2H), 3.87 (s, 3H), 3.84 (s, 3H), 3.80 (s, 3H), 3.44 (t, J =
6.6 Hz, 2H), 2.95 (d, J = 16.9 Hz, 1H), 2.64 (d, J = 16.9 Hz, 1H),
1.97−1.81 (m, 2H), 1.73−1.63 (m, 2H), 1.55−1.40 (m, 8H).
13C{1H} NMR (101 MHz, CD3OD) δ: 197.7, 167.7, 163.58, 163.56,
162.5, 161.5, 160.9, 156.0, 154.8, 153.8, 145.0, 143.4, 140.9, 135.9,
133.6, 131.8, 129.6, 128.2, 128.1, 126.6, 124.8, 122.8, 118.9, 114.4,
106.4, 103.1, 102.3, 101.6, 98.2, 96.0, 63.4, 54.8, 54.6, 54.5, 45.2, 40.3,
39.3, 28.7, 28.66, 28.1, 25.7, 22.6. HRMS (ESI) m/z: M+ calcd for
Biotin Derivative 20a. TBAF (1 M in THF, 0.1 mL, 0.13 mmol,
2.5 equiv) was added to a solution of 17d (45.0 mg, 0.05 mmol, 1.0
equiv) in THF (4 mL) at 0 °C. The reaction was stirred for 3 h under
nitrogen at 0 °C, quenched with 5 mL of a saturated aqueous solution
of NH4Cl, extracted with Et2O (3 × 5 mL), washed with brine (5
mL), and dried over Na2SO4, and the solvent was evaporated under
vacuum. The crude product was filtered on a silica pad and directly
reacted with glacial AcOH (4 μL, 0.05 mmol, 1.0 equiv) and a
catalytic amount of Pd(OH)2 on carbon 20% in 3 mL of MeOH
under hydrogen according to general procedure D. The reaction was
monitored by LC-MS (method 1) confirming the conversion of the
starting material into 18a in 3 h, r.t. = 5.0 min. MS (ESI, m/z):
C20H26NO6 [M + H]+ calcd 376.17; found 376.2. After removing
Pd(OH)2, the solvent was evaporated and the crude product was
immediately reacted with biotin-NHS (28.0 mg, 0.08 mmol, 1.5
equiv)previously prepared as reported by Susumu et al.17and
DIPEA (10.0 μL, 0.10 mmol, 2.0 equiv) in 1 mL of DMF according to
general procedure E. The reaction was monitored by LC-MS (method
1), confirming the complete conversion of the starting material in 0.5
h. The product was purified by preparative RP-HPLC (method 2) to
give 20a as a white solid (15.0 mg, 50% yield over three steps), r.t. =
+
C45H46N3O9 772.3229; found 772.3228.
Flow Cytometry. A. fumigatus ΔrodA conidia18 were incubated in
a flow cytometry buffer (1.5% (w/v) bovine serum albumin (BSA)
and 5 mM ethylenediaminetetraacetic acid (EDTA) in phosphate-
buffered saline (PBS)) on ice for 30 min. Fc-MelLec (5 μg/mL) was
pretreated with or without YWA1, Fonsecin B, 2,6-DHAP, 1,8-DHN,
20b, and 21 (all dissolved in dimethyl sulfoxide (DMSO) and used at
70 μM, resulting in a 1% DMSO solution in PBS) for 30 min at room
temperature and then incubated with ΔrodA conidia on ice for 40
min. Following incubation, fungal particles were washed in a flow
cytometry buffer and Fc-MelLec bound to the conidia was detected
with allophycocyanin (APC)-conjugated donkey antihuman IgG
antibody (Jackson ImmunoResearch) and then fixed in 1% (v/v)
formaldehyde and analyzed by flow cytometry. One-way ANOVA and
Bonferroni post hoc tests were performed to compare inhibition of
Fc-MelLec binding to ΔrodA conidia by the different compounds
versus the uninhibited Fc-MelLec control. Fold change was calculated
by dividing the mean value of the pretreated Fc-MelLec with each
compound by the mean value of the uninhibited Fc-MelLec.
Cell Culture and Growth Conditions. RAW 264.7 cells
expressing human MelLec (hMelLec)9 were maintained at 37 °C
and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% heat-inactivated fetal calf serum (FCS), 100
units/mL penicillin, 0.1 mg/mL streptomycin, 2 mM L-glutamine, and
400 μg/mL geneticin (G418) (Life Technologies, Inc.).
1
7.5 min. H NMR (400 MHz, CD3OD) δ: 6.33 (s, 1H), 6.30 (d, J =
2.1 Hz, 1H), 6.11 (d, J = 2.1 Hz, 1H), 4.37 (dd, J = 7.8, 4.7 Hz, 1H),
4.18 (dd, J = 7.8, 4.7 Hz, 1H), 3.10−3.06 (m, 3H), 2.99−2.88 (m,
1H), 2.81 (ddd, J = 12.7, 4.7, 1.3 Hz, 1H), 2.67−2.62 (m, 1H), 2.60
(d, J = 12.7 Hz, 1H), 2.10 (t, J = 7.3 Hz, 2H), 1.85−1.74 (m, 2H),
1.63−1.29 (m, 16H). HRMS (ESI) m/z: [M + Na]+ calcd for
C30H39N3O8SNa 624.2350; found 624.2350.
Biotin Derivative 20b. Compound 17c (25.0 mg, 0.04 mmol) was
reacted according to general procedure D. The reaction was
monitored by LC-MS (method 1), confirming the complete
conversion of the starting material into the desired product 18b in
3 h, r.t. = 5.7 min. MS (ESI, m/z): C22H30NO6 [M + H]+ calcd
404.20; found 404.2. After removing Pd(OH)2, the solvent was
evaporated and the crude product was immediately reacted with
biotin-NHS (21.0 mg, 0.06 mmol, 1.5 equiv)previously prepared as
reported by Susumu et al.17and DIPEA (8.0 μL, 0.08 mmol, 2.0
equiv) in 1 mL of DMF according to general procedure E. The
reaction was monitored by LC-MS (method 1), confirming the
complete conversion of the starting material in 0.5 h. The product was
purified by preparative RP-HPLC (method 2) to give 20b as a white
Immunofluorescence Microscopy. RAW 264.7 cells expressing
human MelLec (hMelLec) (1.25 × 105 cells/well) were seeded in a
48-well plate at 37 °C and 5% CO2. Next day, the cells were washed
with PBS (1×) and then incubated with 10, 20, 50, or 100 μM of 21
at 37 °C and 5% CO2 for 3 h. The cells were washed with PBS (1×)
and then were visualized under an inverted immunofluorescent
microscope (Zeiss). Cells without 21 were included as a negative
control (untreated).
ASSOCIATED CONTENT
■
sı
1
* Supporting Information
solid (13.0 mg, 50% yield over two steps), r.t. = 9.3 min. H NMR
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.joc.0c02324.
(400 MHz, (CD3)2CO) δ: 14.02 (s, 1H), 6.92 (br, 1H), 6.52 (d, J =
2.2 Hz, 1H), 6.40 (s, 1H), 6.22 (d, J = 2.2 Hz, 1H), 5.70 (br, 1H),
5.52 (br, 1H), 4.37 (dd, J = 7.4, 4.8 Hz, 1H), 4.20 (dd, J = 7.4, 4.8 Hz,
1H), 3.78 (s, 3H), 3.76 (s, 3H), 3.10−3.03 (m, 3H), 2.97 (d, J =16.8
Hz, 1H), 2.84−2.76 (m, 2H), 2.65 (d, J = 16.8 Hz, 1H), 2.57 (d, J =
12.8 Hz, 1H), 2.04 (t, J = 7.2 Hz, 2H), 1.88−1.80 (m, 2H), 1.70−1.57
Experimental procedure for the synthesis of Oregon
Green-NHS (precursor of compound 19); immuno-
fluorescence microscopy and image analysis experiments
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Article
ACKNOWLEDGMENTS
We thank Ian Fraser Flow Cytometry and Microscopy and
Histology facilities, University of Aberdeen.
on binding of 21 to human MelLec-expressing RAW
264.7 cells with competition by YWA1 at different
■
1
concentrations (1, 5, 25, and 50 μM); copies of H and
13C NMR spectra (PDF)
DEDICATION
■
†This article is dedicated to the memory of Dr. Leonardo
Manzoni.
AUTHOR INFORMATION
■
Corresponding Authors
REFERENCES
■
Monica Piras − Kosterlitz Centre for Therapeutics, Institute of
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Author Contributions
M.P., I.P., and C.Z. designed and carried out the synthesis of
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G.D.B. and M.Z. designed, supervised, and coordinated the
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Funding
Funding was provided by the Wellcome Trust (102705 and
097377) and the Medical Research Council Centre for
Medical Mycology and the University of Aberdeen (MR/
N006364/1).
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Notes
The authors declare no competing financial interest.
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J. Org. Chem. 2021, 86, 6044−6055

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Article
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J. Org. Chem. 2021, 86, 6044−6055