An efficient synthesis of a 6″-BODIPY-α-Galactosylceramide probe for monitoring α-Galactosylceramide uptake by cells

Article abstract of DOI:10.1016/j.carres.2019.107840

Herein, an efficient synthesis of BODIPY-α-Galactosylceramide 3, which can be used to study the cellular uptake of the potent immunostimulatory parent compound α-Galactosylceramide, is reported. Key in our synthetic strategy is the six-step synthesis of the core BODIPY scaffold (64% yield overall) and its quantitative conversion to an N-hydroxysuccinimidyl ester to facilitate conjugation and purification of the target glycolipid. For the preparation of the core of the glycolipid, the solubility of the lipid acceptor proved to be critical. The ability of BODIPY-αGalCer 3 to activate invariant natural killer cells was then demonstrated in vitro.

Full text of DOI:10.1016/j.carres.2019.107840

Journal Pre-proof
An efficient synthesis of a 6″-BODIPY-α-Galactosylceramide probe for monitoring α-
Galactosylceramide uptake by cells
Janice M.H. Cheng, Stephanie H. Chee, Yusuf Dölen, Martijn Verdoes, Mattie S.M.
Timmer, Bridget L. Stocker
PII:
S0008-6215(19)30471-9
DOI:
https://doi.org/10.1016/j.carres.2019.107840
CAR 107840
Reference:
To appear in: Carbohydrate Research
Received Date: 8 August 2019
Revised Date: 14 October 2019
Accepted Date: 15 October 2019
Please cite this article as: J.M.H. Cheng, S.H. Chee, Y. Dölen, M. Verdoes, M.S.M. Timmer, B.L.
Stocker, An efficient synthesis of a 6″-BODIPY-α-Galactosylceramide probe for monitoring α-
Galactosylceramide uptake by cells, Carbohydrate Research (2019), doi: https://doi.org/10.1016/
j.carres.2019.107840.
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Graphical Abstract

An efficient synthesis of a 6″-BODIPY-α-Galactosylceramide probe for
monitoring α-Galactosylceramide uptake by cells
Janice M. H. Cheng,a Stephanie H. Chee,a Yusuf Dölen,b Martijn Verdoes,b Mattie S.
M. Timmer,*,a Bridget L. Stocker*,a
a School of Chemical and Physical Sciences, Victoria University of Wellington, PO
Box 600, 6140, Wellington, New Zealand
b Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences,
Radboud University Medical Center, Geert Grooteplein 26, 6525 GA Nijmegen, The
Netherlands.
Abstract
Herein, an efficient synthesis of BODIPY-α-Galactosylceramide 3, which can be used
to study the cellular uptake of the potent immunostimulatory parent compound α-
galactosylceramide, is reported. Key in our synthetic strategy is the six-step synthesis
of the core BODIPY scaffold (64% yield overall) and its quantitative conversion to an
N-hydroxysuccinimidyl ester to facilitate conjugation and purification of the target
glycolipid. For the preparation of the core of the glycolipid, the solubility of the lipid
acceptor proved to be critical. The ability of BODIPY-αGalCer 3 to activate invariant
natural killer cells was then demonstrated in vitro.
1

Introduction
Over the years there has been much interest in the potential of α-galactosylceramide
(αGalCer, 1, Figure 1) as an immunostimulant.1 αGalCer was the first ligand shown
to bind to CD1d [a member of the CD1 family of proteins on the surface of antigen-
presenting cells (APCs)] and activate a subset of T cells known as invariant natural
killer T (iNKT) cells, which ultimately leads to the production of inflammatory (Th1-
type) cytokines, including interferon-γ (IFN-γ), and regulatory (Th2-type) cytokines
such as interleukin-4 (IL-4) and interleukin-17.2,3,4 Since this seminal discovery,
iNKT cells have been reported to have important roles in infectious diseases,5
autoimmune disorders (e.g., lupus and diabetes),6 and in anti-tumour immunity,7 and
the myriad of potential medical applications of αGalCer has resulted in many groups
preparing fluorescently labeled αGalCer probes8,9,10,11,12 with potential applications
including studies into lipid trafficking and the quantification of αGalCer loading onto
APCs.
Although αGalCer activates iNKT cells via CD1d-mediated presentation to the iNKT
cell TCR, derivatising the lipid portion of αGalCer with a fluorescent group8,9,12 may
interfere with αGalCer-CD1d interactions13 and influence intracellular
trafficking.14,15,16,17 Notwithstanding, it is generally accepted that the CD1d-
glycolipid-NKT cell receptor interaction tolerates the appendage of small molecules
at C6 of αGalCer.18 19,20 To this end, C6″-dansylated αGalCer 2 was synthesised, first
’
by Savage and co-workers9 and later by our group using an improved synthetic
route,11 with the dansyl-analogue 2 being found to activate iNKT cells in a manner
similar to the parent compound.11 However, we found that the low fluorescent
quantum yield (Φ = 0.20) of the danysl group limited the use of the C6″-dansylated
2

αGalCer 2 in vivo, thus necessitating the need for the preparation of a C6″-substituted
αGalCer derivative with enhanced fluorescent properties.
Figure 1. αGalCer (1) and 6″-labeled probe Dansyl-αGalCer 2.
In an effort to synthesise a C6″-fluorescent αGalCer probe with improved
fluorescence, we focused on the use of the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
(BODIPY) as a fluorescent reporter. The BODIPY scaffold has found wide
application as a fluorescent marker due to its small size, high fluorescent quantum
yield (Φf = 0.40-0.60), and large extinction coefficient (log ε0 = 4-5)21 (cf. Dansyl: Φf
= 0.065 [H2O] and log ε0 = 3).22 To this end, we envisioned developing BODIPY-
αGalCer 3 (Figure 2), which can be made via amide bond formation between the
carboxyl-modified BODIPY 4 and amine-functionalised αGalCer 5. It was then
proposed that the BODIPY scaffold could be obtained via the condensation of
terephthalic acid mono-methyl ester 6 and two equivalents of dimethylpyrrole 7,
hydrolysis of the corresponding methylester and treatment with boron trifluoride
diethyl etherate.21,23 6′-Amino-αGalCer 5 was envisioned to be made by coupling
galactosyl imidate 8, itself obtained from D
-galactose,11,24 with lipid acceptor 9, which
3

can also be prepared from
D-galactose accordingly to modified literature
procedures.11,25 26
’
Scheme 1. Retrosynthesis for the preparation of BODIPY-αGalCer 3.
Results and Discussion
The synthesis of the BODIPY fluorophore started with the methylation and partial
hydrolysis of terephtalic acid 11 to give monoester 6 in 85% yield over two steps
(Scheme 2). Acid chloride 12 was then generated using thionyl chloride, followed by
immediate condensation with dimethylpyrrole 7 to form dipyrrole 13. Here, the
addition of excess thionyl chloride (2.3 equivalents per equivalent of dimethylpyrrole
7) to remove the water formed during the condensation reaction, rather than the use of
a Dean-Stark apparatus, was critical for obtaining a good yield (77%) of the desired
dipyrrole 13. Initial attempts to hydrolyse ester 13 with subsequent extraction of the
carboxy-product and treatment with boron trifluoride diethyl etherate to give
carboxyl-modified BODIPY 4 proved unfruitful in our hands,23 with compound
degradation and poor product yield being observed. However, Inoue et al.27 and
Bolydev et al.28 demonstrated that ester groups on BODIPY scaffolds can be
4

hydrolysed after the addition of boron into the BODIPY framework since the
inclusion of boron stabilises the bora-diaza-s-indacene core. Accordingly, we treated
dipyrrole 13 with boron trifluoride diethyl etherate to give indacene 14 in excellent
yield (98%). Crystallisation from methanol then provided red crystals suitable for X-
ray diffraction analysis, which confirmed the structure of difluoro-bora-diaza-
indacene 14 (Scheme 2).29 Hydrolysis of the methyl ester in 14 under optimised
conditions (0.1 M KOH in iPrOH at room temperature) and purification by silica gel
flash column chromatography then afforded carboxy-fuctionalized BODIPY 4 in
quantitative yield. BODIPY 4 could also be crystallised from MeOH to give red
crystals with a melting point of 182.5-182.8 °C.
Scheme 2. Synthesis of carboxy-fuctionalised BODIPY 4
5

With BODIPY 4 successful prepared, we then embarked on the synthesis of the core
6″-amino functionalised αGalCer (Scheme 3). First, imidate donor 8 was prepared
from
D-galactose (10) via formation of methyl azidoglycoside 15 (5 steps, 47%
overall yield),11 hydrolysis of the anomeric methoxy, and subsequent installation of
the imidate. The isopropylidene-protected lipid 9, as first reported by Schmidt and co-
workers,25,26 and which could be readily prepared from intermediates that we used en
route to the synthesis of dansyl-αGalCer 2,11 was then prepared. Unfortunately, all
attempted glycosylation reactions between 8 and 9 proved unsuccessful despite
31
investigations into the use of either TMSOTf30 or BF3 OEt2 as an activator in a
myriad of solvent systems and with reactions being undertaken at temperatures
ranging from -20 – 30°C. At cooler temperatures, acceptor 9 proved too insoluble, as
evidenced by a cloudy suspension being observed in the reaction flask, while warmer
temperatures led to donor decomposition. Herein, it should be noted that Schmidt and
co-workers did not directly use 9 in a glycosylation reaction, but converted it to a 1-
phosphite derivative for the synthesis of glycophosphatidylinositols.25,26
Scheme 3. Attempted glycosylation reaction of imidate donor 8 with acceptor 9
6

Due to the poor reactivity of isopropylidene protected acceptor 9, the use of
alternative protecting groups on the phytosphingosine backbone was evaluated. Xia et
al. reported on the successful coupling of a per-benzylated imidate donor with a
phytosphingosine lipid acceptor with the 3- and 4-hydroxyls protected as benzoate
esters.32 With this in mind, commercially available phytosphingosine 17 was N-
acylated with hexacosanoic (C26) acid (Scheme 4).32,33 The lipophilicity and hence
poor solubility of the C26 acyl chain has resulted in various reagents being used for
this transformation, 34,35,36,37 however, we found that the use of PyBOP in the presence
of DiPEA and DMF gave a quantitative yield of the target trihydroxy-ceramide,
which is the highest reported yield to date. The primary hydroxyl of the ceramide was
then protected as a tritylether to give diol 18 in 79% yield. The secondary hydroxyls
in ceramide 18 were then benzoylated, and the trityl group removed to afford the
ceramide acceptor 19 in 67% over two steps.32,38
Scheme 4. First attempted synthesis of BODIPY-αGalCer 3
7

A TMSOTf-mediated glycosylation reaction between acceptor 19 and the galactose
imidate donor 8 was then performed. Through optimisation of the glycosylation
reaction, it was observed that 0 °C was the lowest temperature that could be used for
optimal activation of the imidate donor 8 while keeping the highly lipophilic acceptor
19 dissolved. In this way, the protected 6″-azido glycolipid 20 was obtained in
excellent (91%) yield with satisfactory α-selectivity (α:β = 3:1), with the
stereoselectivity of the glycosylation reaction being readily determined by 1H and 13C
1
NMR spectral analysis of 20 [α-20: J1′,2′ = 3.6 Hz, JCH = 168.8 Hz; β-20: J1′,2′ = 7.8
1
Hz, JCH = 159.4 Hz]. The α- and β-anomers could be isolated by silica gel flash
column chromatography in 68% and 23% yields, respectively. This result was
encouraging as α-selective glycosylation reactions using the hexacosanoyl ceramide
acceptor are difficult and typically result in modest-good yields.32,35
With the fully protected glycolipid α-20 in hand, the protecting groups were then
removed. This was initially attempted with a Birch reduction and quenching of the
reaction with methanol, which would remove the benzyl groups and benzoyl groups.
However, difficulty in isolating deprotected product 5 following neutralisation of the
reaction with Dowex H+ necessitated a stepwise approach. Accordingly, the 3-O- and
4-O-benzoyl groups were first removed under basic conditions to afford the diol
glycolipid in excellent (93%) yield. Subsequent hydrogenolysis of the three benzyl
ethers and reduction of the azide to the amine, using Pearlman’s catalyst in the
presence of 5% formic acid, then afforded 6″-amino-αGalCer 5 in 33% yield.
Although difficulties in the isolation and purification of 5 resulted in a low
deprotection yield, the overall yield for the synthesis of 6″-amino-αGalCer 5 (21%
over 3 steps from the ceramide lipid acceptor) is comparable to the route developed
8

by Savage and co-workers for the synthesis of dansylated αGalCer 2 (19% over 4
steps from the ceramide acceptor).9 Next, amine 5 was coupled to BODIPY-COOH 4
using HBTU and DiPEA, but, although the reaction appeared promising based on
TLC analysis, BODIPY-α-GalCer 3 could not be isolated free from coupling reagents
and by-products in acceptable yields.
To develop a BODIPY reagent that would eliminate the need for coupling reagents in
the final step, BODIPY-COOH 4 was first converted into N-hydroxysuccimidyl ester
21 (Scheme 5). This reaction occurred uneventfully to give BODIPY-NHS ester 21 as
a crystalline material29 in quantitative yield (Scheme 5). Coupling of NHS ester 21 to
amino-αGalCer 5 then proceeded smoothly, and following purification by silica gel
flash column chromatography BODIPY-αGalCer 3 was isolated as a red solid in 44%
yield. Amide coupling yields on the 6″-position of αGalCer typically range from 33-
53% because of the poor solubility and reactivity of the amphiphilic glycolipid.9,39
Characteristic three-bond correlations (HMBCs) between CH2-6′ and the carbonyl
carbon of the BODIPY amide were observed, which confirmed conjugation of the
BODIPY scaffold to the glycolipid.
Scheme 5. Synthesis of BODIPY-αGalCer 3
9

With BODIPY-αGalCer 3 in hand, the ability of the compound to activate iNKT cells
was then investigated using an iNKT cell hybridoma cell line DN32.D3, with IL‐2
production being used as a measure of iNKT cell activation. Cell line JAWS II was
used as a source of murine DCs (mDCs). Accordingly, mDCs were combined with
DN32.D3 cells and cultured for 24 h with titrated amounts of αGalCer 1 or BODIPY-
αGalCer 3 (Figure 2). Here, both αGalCer 1 and BODIPY-αGalCer 3 stimulated the
release of IL-2 in a dose-dependent manner. Although the response to BODIPY-
αGalCer 3 was approximately 1 log less than that of the parent compound, BODIPY-
αGalCer 3 is capable of activating iNKT cells, which is consistent with reported
activities of fluorescently labeled αGalCer derivatives.9,12,33 Overall, this observation
confirms that the C6″-position of αGalCer is well-suited for the incorporation of a
fluorescent label. Moreover, incorporating a fluorescent group at the C6″-position
leaves native ceramide lipid, which is important in the intracellular and extracellular
trafficking of glycolipids,15,16,17 in CD1d loading and antigen exchange,40 and CD1d-
antigen presentation,14,41 unaltered.
10

Figure 2. In vitro activation of iNKT cells by BODIPY-αGalCer 3. iNKT cell
hybridoma cell line DN32.D3 and JAWS II cells were cultured in the presence of
various concentrations of either unlabeled or BODIPY labeled α-GalCer for 48 h.
The supernatants were then analyzed for the presence of IL-2 ( ELISA). Data are
means of triplicate wells and are representative of two independent experiments.
Conclusion
In conclusion, BODIPY-αGalCer 3 was successfully synthesised from the fully
deprotected amine 5 and BODIPY-NHS 21. To this end, highly efficient syntheses of
BODIPY-NHS 21 and its carboxy-precursor 4 were achieved (64% overall yield for
each), which not only demonstrates the value of this BODIPY scaffold as a
fluorescent group in this work, but also, provides a ready synthesis of a fluorophore
that can be readily incorporated into other chemical probes. With regard to the
synthesis of BODIPY-αGalCer 3, the solubility of the ceramide lipid acceptor proved
crucial for its reactivity in subsequently glycosylation reactions. The potential of
BODIPY-αGalCer 3 to activate iNKT was then demonstrated, thus providing a C6″-
functionalised fluorescent αGalCer probe which should find wide application in a
number of biological settings.
Experimental:
11

General procedure: Unless otherwise stated all reactions were performed under N2.
Prior to use, THF (Pancreac) was distilled from sodium and benzophenone, pyridine
was distilled and dried over 4Å molecular sieves (4Å MS), DCM (Pancreac) was
distilled from P2O5, and H2O and benzene (Fisher Scientific) were distilled. Trityl
chloride (Acros), anhydrous Et2O (Pancreac), PPh3 (Merck), Pd(OH)2/C (Aldrich, 20
wt%), anhydrous DMF (Acros), TFA (Aldrich), trichloroacetonitrile (Aldrich),
triethylsilane (Fluka), TMSOTf (Aldrich), H2SO4 (Lab-Scan), Formic acid (Aldrich),
AcCl (B&M), BnBr (Fluka), PMe3 (Aldrich, 1m in THF), AcOH (Ajax Finechem),
Ac2O (Peking Reagent), TMSOTf (Aldrich), DiPEA (Aldrich), NaOMe (Janssen
Chimica), phytosphingosine (TCI), C25H51COOH (Acros), BzCl (Aldrich, distilled an
stored under argon), HBTU (Acros), PyBOP (Aldrich), EDCI (Aldrich), DMAP
(Merck), sodium (Aldrich), Pd(OH)2/C (Aldrich, 20 wt%), EtOAc (Pancreac),
hexanes (Fisher Scientific), petroleum ether (Pure Science), MeOH (Pure Science),
CHCl3 (Pancreac), EtOH (absolute, Pure Science), NaOH (Pure Science), NaHCO3
(Pure Science), NaCl (Pancreac) were used as received. All solvents were removed by
evaporation under reduced pressure. Reactions were monitored by TLC-analysis on
Macherey-Nagel silica gel coated plastic sheets (0.20 mm, with fluorescent indicator
UV254) with detection by UV-absorption (short wave UV – 254 nm; long wave UV –
366 nm), by dipping in 10% H2SO4 in EtOH followed by charring at ~150 °C, by
dipping in I2 in silica, or by dipping into a solution of ninhydrin in EtOH followed by
charring at ~150 °C. Column chromatography was performed on Pure Science silica
gel (40-63 micron). AccuBOND II ODS-C18 (Agilent) was used for reverse phase
chromatography. Infrared spectra were recorded as thin films using a Bruker Tensor
27 FTIR spectrometer equipped with an Attenuated Total Reflectance (ATR)
sampling accessory and are reported in wave numbers (cm-1). Nuclear magnetic
12

resonance spectra were recorded at 20 °C in CD3OD, CDCl3, or pyridine-d5 using
either a Varian INOVA operating at 500 MHz or Varian VNMRS operating at 600
MHz. Chemical shifts are given in ppm (δ) relative to TMS. NMR peak assignments
were made using COSY, HSQC and HMBC 2D experiments.
4-(Methoxycarbonyl)benzoic acid (11). A solution of terephthalic acid 6 (15.0 g,
90.3 mmol, 1.0 equiv.) and methanol (375 mL) was refluxed for 30 min, after which
thionyl chloride (26 mL, 42.3 g, 3.97 equiv.) was added dropwise and the mixture was
maintained overnight at reflux. After being cooled to room temperature, the solvent
was removed under reduced pressure. The mixture was extracted twice with diethyl
ether (100 x 4) and washed with KOH (0.1 M) solution. The combined organic layers
were dried over anhydrous MgSO4, filtered and concentrated in vacuo to give a brown
solid. To a portion of the dimethyl ester (8.95 g, 43.0 mmol, 1 equiv.), methanol (150
mL) was added and the mixture was heated at 40 °C for 15 min. Potassium hydroxide
pellets (2.41 g, 43.0 mmol, 1 equiv.) were then added and the mixture was refluxed
overnight. The mixture was cooled and the alcohol evaporated. The resulting crude
product was dissolved in water and extracted with EtOAc (100 x 1 mL). The aqueous
layer was acidified with HCl solution (1 M) and the precipitate was filtered. The
precipitate was then extracted with diethyl ether, dried over anhydrous MgSO4,
concentrated under reduced pressure to give monoester 11 (7.05 g, 36.4 mmol, 85%
over two steps) as a white solid. This product was used without further purification.
1H NMR (500 MHz, CDCl3) δ 7.91 (m, 4H, HAr), 3.76 (3, 3H, OMe); 13C NMR (125
MHz, CDCl3) δ 166.1 (C=O), 165.0 (C=O), 132.9 (CAr), 132.4 (CAr), 128.4 (CAr),
128.2 (CAr), 51.2 (OMe); HRMS (ESI) m/z calcd. for [C9H7O4]-: 179.0339, obsd.:
179.0339.
13

(Z)-Methyl 4-((3,5-dimethyl-1H-pyrrol-2-yl)(3,5-dimethyl-2H-pyrrol-2-
ylidene)methyl)benzoate (13). Monomethylterephthalic acid (11) (3.00 g, 16.5
mmol) was refluxed in thionyl chloride (14.5 mL, 199.8 mmol, 12 equiv.) for 2.5h.
Excess thionyl chloride was removed in vacuo and the resulting crude product was
co-evaporated with dry toluene (2 x 20 mL) to remove residual thionyl chloride. The
solid was then dried on high vacuum to remove residual toluene to give acid methyl
4-(chlorocarbonyl)benzoate (12) (3.30 g, 16.6 mmol, quant.) as a white solid. This
product was used without further purification. To a solution of acyl chloride 12 (1.10
g, 5.55 mmol, 2.3 equiv.) in anhydrous CH2Cl2 (12 mL), under an atmosphere of
argon, 2,4-dimethylpyrrole 7 (0.25 mL, 0.229 g, 2.41 mmol, 1.0 equiv.) was added
dropwise. The reaction mixture was heated under reflux for 3.5h. After this time, the
reaction mixture was evaporated to dryness under vacuum. The resulting residue was
purified by column chromatography on silica gel using a CH2Cl2/methanol gradient
[99:1 → 98:2 → 95:5 → 90:10, (v/v)] to give the title compound 13 as a red solid
(0.310 g, 0.926 mmol, 77%). TLC Rf = 0.56 (MeOH/EtOAc, 1/4, v/v); IR (film) ν =
2952, 2924, 2854, 1721, 1565, 1520, 1435, 1274, 1108, 1019, 675 cm-1; 1H NMR
(500 MHz, CDCl3) δ 8.17 (d, J = 8.1 Hz, 2H, H-3′a, H-3′b), 7.43 (d, J = 8.25 Hz, 2H,
H-2′a, H-2′b), 6.07 (s, 2H, H-2, H-6), 3.98 (s, 3H, OMe), 2.55 (s, 6H, H-3, H-5), 1.34
(s, 6H, H-1, H-7); 13C NMR (125 MHz, CDCl3) δ 166.5 (C-5′), 130.5 (C-3′), 130.1
(C-2′), 120.8 (C-2), 52.5 (C-6′), 15.0 (CH3); HRMS(ESI) m/z calcd. for
[C21H23N2O2]+: 335.1755, obsd.: 335.1763. Spectral data matched that previously
reported.42
14

Methyl 4-(4,4-difluoro-1,3,5,7-tetramethyl-3a,4a-diaza-4-bora-s-indacen-8-
yl)benzoate (14). Compound 13 (286 mg, 0.854 mmol, 1.0 equiv.) was dissolved in
anhydrous toluene (23 mL) containing triethylamine (0.56 mL, 4.01 mmol, 4.7
equiv.). The mixture was stirred at room temperature for 15 minutes and kept under
an argon atmosphere while BF3·Et2O is added dropwise (0.71 mL, 5.81 mmol, 6.8
equiv.) The reaction mixture was then stirred at 80°C for 45 minutes until complete
conversion of 13 by TLC analysis. After cooling down to room temperature, the
reaction mixture was quenched by the addition of distilled water and ethyl acetate (25
mL). The organic phase was washed with water (2 x 30 mL), brine (1 x 30 mL), dried
over anhydrous MgSO4, filtered and concentrated in vacuo. The crude residue was
purified by column chromatography on silica gel using CH2Cl2. The obtained red-
orange solid (320 mg, 0.837 mmol, 98 %) was stored in the dark. Ester 14 was
crystallized from MeOH to obtain red-orange crystals. Mp 183.6–184.2 °C; Rf = 0.88
(MeOH/EtOAc, 1/4, v/v); IR (film) ν = 2926,1724, 1543, 1507, 1307, 1274, 1190,
1155, 1080, 974, 735 cm-1; 1H NMR (500 MHz, CDCl3) δ 8.11 (d, J = 8.3 Hz, 2H, H-
3′a, H-3′b), 7.34 (d, J = 8.3 Hz, 2H, H-2′a, H-2′b), 5.92 (s, 2H, H-2, H-6), 3.98 (s, 3H,
OMe), 2.49 (s, 6H, H-3, H-5), 1.29 (s, 6H, H-1, H-7); 13C NMR (125 MHz, CDCl3) δ
166.5 (C-5′), 156.0 (C-3), 142.9 (C-1), 140.2 (C-4′), 139.8 (C-1′), 130.8 (C-8), 130.4
(C-3′), 128.4 (C-2′), 121.5 (C-2), 52.4 (C-6′), 14.2 (CH3); HRMS(ESI) m/z calcd. for
[C21H21BF2N2O2Na]+: 405.1557, obsd.: 405.1555. Spectral data matched that
previously reported.42
4-(4,4-difluoro-1,3,5,7-tetramethyl-3a,4a-diaza-4-bora-s-indacen-8-yl)benzoic
acid (4). To a solution of the methyl ester 14 (0.148 g, 0.387 mmol) in isopropanol
(15 mL) under an argon atmosphere, an aqueous solution of 0.1m KOH (15 mL) was
15

added. The reaction mixture was stirred at room temperature until complete
consumption of 14 by TLC analysis was observed. The reaction mixture was then
acidified with H+ dowex until pH=2-3, filtered and evaporated to dryness in vacuo.
The residue was purified by silica gel gradient column chromatography
(CH2Cl2/MeOH, 99:1 to 96:4, v/v) to afford the BODIPY-COOH 4 (0.142 g, 0.387
mmol, quantitative). The obtained red-brown solid is stored in the dark. Mp 182.5–
182.8 °C; Rf = 0.74 (MeOH/EtOAc, 1/4, v/v); IR (film) 2924, 1723, 1544, 1510,
1309, 1194, 1157, 983, 739 cm-1; 1H NMR (500 MHz, CDCl3) δ 8.25 (d, J14,15 = J17,18
= 8.5 Hz, 8.2 Hz, 2H, H-15, H-17), 7.44 (d, J14,15 = J17,18 = 8.2, 2H, H-14, H-18), 6.01
(s, 2H, H-2, H-6), 2.56 (s, 6H, H-9, H-10), 1.37 (s, 6H, H-11, H-12); 13C NMR (125
MHz, CDCl3) δ 130.9 (C-15, C-17), 128.5 (C-14, C-18), 121.5 (C-2), 14.6 (C-9, C-
10, C-11, C-12); HRMS(ESI) m/z calcd. for [C20H19BF2N2O2Na]+: 391.1405, obsd.:
391.1413. Spectral data matched that previously reported.42
2,5-Dioxopyrrolidin-1-yl 4-(4,4-difluoro-1,3,5,7-tetramethyl-3a,4a-diaza-4-bora-
s-indacen-8-yl)benzoate (21). To a solution of BODIPY-COOH 4 (60.4 mg, 0.164
mmol) in CH2Cl2 (6.5 ml) was added NHS (37.8 mg, 0.328 mmol) and EDCI (62.9
mg, 0.328 mmol) and the solution stirred in the dark overnight. After completion of
the reaction by TLC analysis, the mixture is diluted with distilled water, extracted
with CH2Cl2 (2 x 25 mL) and washed with brine (1 x 25 mL). The combined organic
phase was dried over anhydrous MgSO4, filtered and concentrated in vacuo.
Purification of the crude residue by silica gel gradient column chromatography
(petroleum ether to CH2Cl2) afforded BODIPY-NHS 21 (76.0 mg, 0.163 mmol,
quantitative) as a red needles. Rf = 0.83 (MeOH/EtOAc, 1/4, v/v); 1H NMR (500
MHz, CDCl3) δ 8.29 (d, J14,15 = J17,18 = 8.5 Hz, 2H, H-15, H-17), 7.51 (d, J14,15 = J17,18
16

= 8.5, 2H, H-14, H-18), 6.02 (s, 2H, H-2, H-6), 3.50 (s, 4H, CH2 NHS x 2), 2.57 (s,
6H, H-9, H-10), 1.39 (s, 6H, H-11, H-12); 13C NMR (125 MHz, CDCl3) δ 169.2
(C=O NHS), 160.3 (C=O BOD), 156.2 (C-3, C-5), 142.1 (C-1, C-7), 142.0 (C-13),
139.3 (C-16), 131.3 (C-15, C-17), 131.1 (C-7a, C-8a), 129.0 (C-14, C-18), 125.8 (C-
8), 121.7 (C-2, C-6), 53.4 (CH2 NHS x 2), 14.8 (C-9, C-10, C-11, C-12); HRMS(ESI)
m/z calcd. for [C24H23BF2N3O4]+: 466.1744, obsd.: 466.1740.
O-(6-Azido-2,3,4-tri-O-benzyl-6-deoxy-α-d-galactosyl) trichloroacetimidate (8).
Methyl 6-azido-2,3,4-tri-O-benzyl-6-deoxy-α-D
-galactoside (15)11 (360 mg, 0.74
mmol) was dissolved in aqueous acetic acid (11 mL, 80% v/v) in water followed by
the addition of 1m H2SO4 (2.72 mL, 2.72 mmol). The reaction was stirred at 100 °C
for 4.5 h, then 120 °C for a further 1.5 h. The reaction mixture was poured into ice
water and extracted with EtOAc (2 x 50 mL) and the combined organic layers were
washed with water (100 mL), saturated NaHCO3 solution (2 x 100 mL) and brine
(100 mL), dried (MgSO4), filtered and concentrated in vacuo. The residue was
purified by silica gel flash column chromatography and eluted with 5:1 (v/v)
petroleum ether/EtOAc to afford 6-azido-2,3,4-tri-O-benzyl-6-deoxy-α-D-galactose
(219 mg, 0.46 mmol, 63%) as a clear oil. Rf: 0.43 (PE/EA, 2:1, v/v); [α]D25 = +9.0 (c
= 1.0, CHCl3); IR (film) 3410, 3064, 3031, 2871, 2101, 1497, 1454, 1362, 1281,
1214, 1062, 1027, 912, 736, 697 cm-1; 1H NMR (500 MHz, CDCl3) δ 7.45–7.31 (m,
15H, Harom), 5.31 (t, J1,2 = J1,OH = 2.6 Hz, 1H, H-1), 5.03 (d, Ja,b = 11.2 Hz, 1H, CH-a,
4-O-Bn), 4.88 (d, Ja,b = 12.0 Hz, 1H, CH-a, 2-O-Bn), 4.86 (d, Ja,b = 11.8 Hz, 1H, CH-
a, 3-O-Bn), 4.81 (d, Ja,b = 12.0 Hz, 1H, CH-b, 2-O-Bn), 4.74 (d, Ja,b = 11.8 Hz, 1H,
CH-b, 3-O-Bn), 4.64 (d, Ja,b = 11.2 Hz, 1H, CH-b, 4-O-Bn), 4.08–4.06 (m, 2H, H-2,
H-5), 3.97 (dd, J2,3 = 9.9 Hz, J3,4 = 2.6 Hz, 1H, H-3), 3.86 (bs, 1H, H-4), 3.53 (dd,
17

J6a,6b = 12.5 Hz, J5,6a = 7.6 Hz, 1H, H-6a), 3.33 (d, J1,OH = 2.6 Hz, 1H, OH), 3.13 (dd,
J6a,6b = 12.5 Hz, J5,6b = 5.9 Hz, 1H, H-6b); 13C NMR (125 MHz, CDCl3) δ 138.5 (C-i,
2-O-Bn), 138.2 (C-i, 4-O-Bn), 138.1 (C-i, 3-O-Bn), 128.6, 128.53, 128.47, 128.2,
128.1, 128.0, 127.8, 127.6 (9 x Carom), 91.9 (C-1), 78.6 (C-3), 76.5 (C-2), 74.74 (C-4),
74.67 (CH2, 4-O-Bn), 73.6 (CH2, 2-O-Bn), 73.3 (CH2, 3-O-Bn), 69.7 (C-5), 51.2 (C-
6); HRMS(ESI) m/z calcd. for [C27H29N3O5+Na]+: 498.1999, obsd.: 498.2005.
Trichloroacetonitrile (448 mL, 4.47 mmol) and DBU (100 mL, 0.67 mmol) were then
added to 6-azido-2,3,4-tri-O-benzyl-6-deoxy-α-D-galactose (213 mg, 0.45 mmol) (co-
evaporated with toluene x 3) in dry CH2Cl2 (5 mL). After stirring for 1.5 h at rt, the
reaction mixture was concentrated in vacuo and purified by silica gel flash column
chromatography (silica gel was pre-washed with 1% triethylamine in 20:1 v/v
petroleum ether/EtOAc). Elution with 20:1 (v/v) petroleum ether/EtOAc afforded
imidate 8 (246 mg, 0.40 mmol, 89%) as a clear oil. Rf: 0.62 (PE/EA, 2:1, v/v); [α]D
24
= +34.0 (c = 1.0, CHCl3); IR (film) 3338, 3064, 3031, 2916, 2101, 1732, 1672, 1497,
1454, 1351, 1287, 1107, 1071, 1027, 838, 794, 736, 697 cm-1; 1H NMR (500 MHz,
CDCl3) δ 8.61 (s, 1H, NH), 7.41–7.29 (m, 15H, Harom), 6.56 (d, J1,2 = 3.3 Hz, 1H, H-
1), 5.06 (d, Ja,b = 11.3 Hz, 1H, CH-a, 4-O-Bn), 4.91 (d, Ja,b = 11.8 Hz, 1H, CH-a, 3-O-
Bn), 4.81 (d, Ja,b = 11.8 Hz, 1H, CH-b, 3-O-Bn), 4.78 (bs, 2H, CH-a, CH-b, 2-O-Bn),
4.65 (d, Ja,b = 11.3 Hz, 1H, CH-b, 4-O-Bn), 4.27 (dd, J2,3 = 10.0 Hz, J1,2 = 3.3 Hz, 1H,
H-2), 4.06–4.05 (m, 2H, H-3, H-5), 3.92 (bs, 1H, H-4), 3.53 (dd, J6a,6b = 12.6 Hz, J5,6a
= 7.4 Hz, 1H, H-6a), 3.14 (dd, J6a,6b = 12.6 Hz, J5,6b = 5.8 Hz, 1H, H-6b); 13C NMR
(125 MHz, CDCl3) δ 161.1 (C=NH), 138.4 (C-i, 3-O-Bn), 138.3 (C-i, 2-O-Bn), 138.1
(C-i, 4-O-Bn), 128.54, 128.52, 128.51 (3 x C-m, 2-O-Bn, 3-O-Bn, 4-O-Bn), 128.4 (C-
o, 4-O-Bn), 127.8 (C-m, 3-O-Bn), 127.6 (C-m, 2-O-Bn), 128.1, 127.9, 127.7 (3 x C-p,
2-O-Bn, 3-O-Bn, 4-O-Bn), 94.9 (C-1), 78.0 (C-3), 75.8 (C-2), 74.9 (CH2, 4-O-Bn),
18

74.7 (C-4), 73.6 (CH2, 3-O-Bn), 73.1 (CH2, 2-O-Bn), 72.4 (C-5), 50.9 (C-6);
HRMS(ESI) m/z calcd. for [C29H29N4O5Cl3+Na]+: 641.1096, obsd.: 641.1113.
(2S,3S,4R)-2-Hexacosanoylamido-D-ribo-octadecane-1,3,4-triol. Phytosphingosine
17 (200 mg, 0.63 mmol) and hexacosanoic acid (275 mg, 0.69 mmol) were co-
evaporated together with toluene (x 3) and dry DMF (6.5 mL) was added followed by
PyBOP (656 mg, 1.26 mmol) and DiPEA (0.28 mL, 1.58 mmol). The white
suspension was stirred at rt for 7 d, at which time the solvent was removed under
reduced pressure and the desired product crystallised from EtOAc/MeOH (30 mL,
1:1, v/v). The suspension was spun down in a centrifuge, the filtrate removed and the
white solid washed twice with EtOAc/MeOH, before it was dried in vacuo to afford
the title triol as a white solid (440 mg, 0.63 mmol, quantitative). Rf: 0.60 (15%
MeOH/CHCl3, v/v, stained with cerium ammonium molybdate); [α]D25 = -2.9 (c =
1.0, pyridine); IR (film) 3313, 2956, 2917, 2850, 1642, 1543, 1471, 1086, 1036, 981,
717, 573 cm-1; 1H NMR (600 MHz, 10% CD3OD in CDCl3) 6.90 (d, JNH,2 = 8.2 Hz,
1H, NH), 3.99 (dd, JNH,2 = 8.2 Hz, J1a,2 = J1b,2 = 4.5 Hz, 1H, H-2), 3.71 (dd, J1a,1b
=
11.4 Hz, J1a,2 = 4.5 Hz, 1H, H-1a), 3.61 (dd, J1a,1b = 11.4 Hz, J1b,2 = 4.5 Hz, 1H, H-
1b), 3.49–3.42 (m, 2H, H-3, H-4), 2.13 (t, Jα,β= 7.6 Hz, 2H, CH2-α), 1.56–1.51 (m,
2H, CH2-β), 1.36–1.30 (m, 2H, H-5), 1.61–1.56 (m, 1H, H-6a), 1.48–1.44 (m, 1H, H-
6b), 1.23–1.81 (m, 66H, H-7–H-17, H-γ–H-ω-1), 0.80 (t, J17,18 = Jω -1,ω = 7.0 Hz, 6H,
H-18, H-ω); 13C NMR (150 MHz, 10% CD3OD in CDCl3) δ 174.6 (HNC=O), 75.6
(C-3), 72.4 (C-4), 61.1 (C-1), 51.9 (C-2), 36.5 (C-α), 33.0 (C-6), 31.84, 29.64, 29.62,
29.60, 29.57, 29.56, 29.45, 29.30, 29.26, 29.23, 22.58 (C-7–C-17, C-γ–C-ω-1), 25.77,
25.72 (C-5, C-β), 13.9 (C-18, C- ω); HRMS(ESI) m/z calcd. for [C44H89NO4+H]+:
696.6864, obsd.: 696.6837.
19

(2S,3S,4R)-2-Hexacosanoylamido-1-triphenylmethoxy-octadecane-3,4-diol (18).
Trityl chloride (213 mg, 0.76 mmol) and DMAP (1.9 mg, 0.015 mmol) were added to
(2S,3S,4R)-2-hexacosanoylamido-D-ribo-octadecane-1,3,4-triol (106 mg, 0.15 mmol)
(co-evaporated with toluene x 3) dissolved in dry pyridine (1.2 mL). The reaction was
stirred for 20 h at rt, at which point further trityl chloride (128 mg, 0.46 mmol) and
pyridine (0.7 mL) were added. Stirring was continued for 4 h, then at 40 °C for 5 h to
ensure complete conversion as monitored by TLC. The reaction mixture was then
diluted with warm CHCl3 (50 mL), washed with brine (30 mL), dried (MgSO4),
filtered and concentrated in vacuo. The residue was purified by silica gel flash column
chromatography and eluted with 0.3% MeOH/CHCl3 to afford the title compound
(113 mg, 0.12 mmol, 79%) as a clear oil. Rf: 0.53 (PE/EA, 2:1, v/v); [α]D26 = +17.0 (c
= 1.0, CHCl3); IR (film) 3305, 2918, 2850, 1643, 1536, 1491, 1468, 1449, 1217,
1071, 900, 760, 704, 633 cm-1; 1H NMR (500 MHz, CDCl3) δ 7.42 (d, JCH-o,CH-m = 7.3
Hz, 6H, CPh3, CH-o), 7.32 (t, JCH-o,CH-m = JCH-m,CH-p = 7.6 Hz, 6H, CPh3, CH-m), 7.25
(t, JCH-m,CH-p = 7.2 Hz, 3H, CPh3, CH-p), 6.11 (d, JNH,2 = 8.3 Hz, 1H, NH), 4.36 (dd,
JNH,2 = 8.3 Hz, J1a,2 = J1b,2 = 4.1 Hz, 1H, H-2), 3.60–3.59 (m, 1H, H-3), 3.51 (dd, J1a,1b
= 10.0 Hz, J1a,2 = 4.1 Hz, 1H, H-1a), 3.40–3.36 (m, 1H, H-4), 3.35 (dd, J1a,1b = 10.0
Hz, J1b,2 = 4.1 Hz, 1H, H-1b), 3.23 (d, J3,3-OH = 8.6 Hz, 1H, 3-OH), 2.46 (d, J4,4-OH
=
4.1 Hz, 1H, 4-OH), 2.15 (t, Jα,β= 7.7 Hz, 2H, CH2-a), 1.90–1.64 (m, 1H, H-5a), 1.64–
1.58 (m, 2H, CH2-b), 1.48–1.38 (m, 3H, H-6, H-5b), 1.32–1.15 (m, 66H, H-7–H-17,
H-γ–H-ω-1), 0.89 (t, J17,18 = Jω-1,ω = 6.8 Hz, 6H, H-18, H-ω); 13C NMR (125 MHz,
CDCl3) δ 173.3 (HNC=O), 143.3 (C-i, CPh3), 128.6 (C-o, CPh3), 128.2 (C-m, CPh3),
127.5 (C-p, CPh3), 87.8 (Cq CPh3), 75.8 (C-3), 73.3 (C-4), 63.0 (C-1), 50.5 (C-2),
37.0 (C-α), 33.4 (C-5), 32.1, 29.84, 20.83, 29.82, 29.80, 29.79, 29.76, 29.6, 29.54,
20

29.50, 29.49, 29.48, 22.8 (C-7–C-17, C-γ–C-ω-1), 26.0 (C-6), 25.9 (C-β), 14.3 (C-18,
C-ω); HRMS(ESI) m/z calcd. for [C63H103NO4+Na]+: 960.7779, obsd.: 960.7781.
(2S,3S,4R)-3,4-Dibenzoyloxy-2-hexacosanoylamido-1-triphenylmethyoxy-
octadecane. Benzoyl chloride (94 mL, 0.81 mmol) and DMAP (1.6 mg, 0.013 mmol)
were added to diol 18 (126 mg, 0.13 mmol) (co-evaporated with toluene x 3)
dissolved in dry pyridine (1.3 mL) and stirred at rt for 15 h. The pyridine was
removed in vacuo, the residue redissolved in EtOAc (70 mL) and the organic layer
then washed with water (70 mL), saturated NaHCO3 solution (70 mL), brine (70 mL),
dried (MgSO4), filtered and concentrated in vacuo. This afforded the title compound,
26
which was used without further purification. Rf: 0.49 (PE/EA, 5:1, v/v); [α]D
=
+12.0 (c = 1.0, CHCl3); IR (film) 2923, 2853, 1719, 1671, 1540, 1450, 1315, 1280,
1109, 1069, 1027, 757, 707, 632 cm-1; 1H NMR (500 MHz, CDCl3) δ 7.96 (d, JCH-
o,CH-m = 7.5 Hz, 2H, CH-o, 4-O-Bz), 7.89 (d, JCH-o,CH-m = 7.5 Hz, 2H, CH-o, 3-O-Bz),
7.59 (t, JCH-m,CH-p = 7.3 Hz, 1H, CH-p, 3-O-Bz), 7.55 (t, JCH-m,CH-p = 7.6 Hz, 1H, CH-
p, 4-O-Bz), 7.42 (t, JCH-o,CH-m = JCH-m,CH-p = 8.3 Hz, 2H, CH-m, 3-O-Bz), 7.41 (t, JCH-
o,CH-m = JCH-m,CH-p = 8.3 Hz, 2H, CH-m, 4-O-Bz), 7.31–7.30 (m, 6H, CPh3, CH-o),
7.15-7.20 (m, 9H, CPh3, CH-m, CH-p), 6.03 (d, JNH,2 = 9.3 Hz, 1H, NH), 5.81 (dd, J2,3
= 9.1 Hz, J3,4 = 2.6 Hz, 1H, H-3), 5.35 (dt, J4,5 = 10.1 Hz, J3,4 = 2.6 Hz, 1H, H-4), 4.59
(tt, J2,NH = J2,3 = 9.3 Hz, J1a,2 = J1b,2 = 3.2 Hz, 1H, H-2), 3.34 (dd, J1a,1b = 9.9 Hz, J1a,2
3.2 Hz, 1H, H-1a), 3.29 (dd, J1a,1b = 9.9 Hz, J1b,2 = 3.2 Hz, 1H, H-1b), 2.18 (dt, Ja,b =
=
14.5 Hz, Jα,β = 7.7 Hz, 2H, CH2-α), 1.93–1.81 (m, 2H, H-5), 1.68–1.58 (m, 2H, CH2-
β), 1.40–1.14 (m, 68H, H-6–H-17, H-γ–H-ω-1), 0.89 (t, J17,18 = Jω -1,ω = 7.0 Hz, 6H,
H-18, H-ω); 13C NMR (125 MHz, CDCl3) δ 172.8 (HNC=O), 166.4 (C=O, 4-O-Bz),
165.0 (C=O, 3-O-Bz), 143.3 (C-i, CPh3), 133.1 (C-p, 3-O-Bz), 132.9 (C-p, 4-O-Bz),
21

130.2 (C-i, 4-O-Bz), 129.82 (C-i, 3-O-Bz), 129.78 (C-o, 3-O-Bz), 129.75 (C-o, 4-O-
Bz), 128.5 (C-o, CPh3), 128.40 (C-m, 3-O-Bz), 128.35 (C-m, 4-O-Bz), 127.8 (C-m,
CPh3), 127.0 (C-p, CPh3), 86.8 (Cq CPh3), 74.2 (C-4), 72.7 (C-3), 61.6 (C-1), 48.5 (C-
2), 36.9 (C-α), 31.9, 29.74, 29.73, 29.72, 29.69, 29.68, 29.67, 29.66, 29.63, 29.58,
29.55, 29.53, 29.45, 29.39, 29.38, 25.72 (C-6–C-17, C-γ–C-ω-1), 28.4 (C-5), 25.70
(C-β), 14.1 (C-18, C-ω); HRMS(ESI) m/z calcd. for [C77H111NO6+Na]+: 1168.8304,
obsd.: 1168.8302.
(2S,3S,4R)-3,4-Dibenzoyloxy-2-hexacosanoylamido-octadecan-1-ol (19).
(2S,3S,4R)-3,4-Dibenzoyloxy-2-hexacosanoylamido-1-triphenylmethyoxy-octadecane
was dissolved in 1:1 (v/v) MeOH/CH2Cl2 and pTsOH.H2O (7.7 mg, 0.040 mmol) was
added. The reaction was stirred for 20 h at rt, followed by the addition of further
pTsOH.H2O (7.7 mg, 0.040 mmol), and stirring was continued for 6 h. The reaction
mixture was diluted with EtOAc (70 mL), washed with saturated NaHCO3 solution (2
x 70 mL), brine (70 mL), dried (MgSO4), filtered and concentrated in vacuo. The
crude mixture was purified by silica gel flash column chromatography and eluted with
MeOH/CH2Cl2 (3%, v/v) to afford the lipid acceptor 19 as an amorphous white solid
(81 mg, 0.090 mmol, 67% over two steps). Rf: 0.25 (PE/EA, 2:1, v/v); [α]D27 = +54.0
(c = 1.0, CHCl3); IR (film) 2918, 2850, 1721, 1649, 1541, 1468, 1315, 1280, 1177,
1111, 1070, 1027, 757, 710 cm-1; 1H NMR (500 MHz, CDCl3) δ 8.05 (d, JCH-o,CH-m
=
7.7 Hz, 2H, CH-o, 3-O-Bz), 7.94 (d, JCH-o,CH-m = 7.5 Hz, 2H, CH-o, 4-O-Bz), 7.63 (t,
JCH-m,CH-p = 7.7 Hz, 1H, CH-p, 3-O-Bz), 7.52 (t, JCH-m,CH-p = 7.5 Hz, 1H, CH-p, 4-O-
Bz), 7.49 (t, JCH-m,CH-p = JCH-o,CH-m = 7.7 Hz, 2H, CH-m, 3-O-Bz), 7.37 (t, JCH-m,CH-p
=
JCH-o,CH-m = 7.5 Hz, 2H, CH-m, 4-O-Bz), 6.48 (d, JNH,2 = 9.3 Hz, 1H, NH), 5.44 (dd,
J2,3 = 9.6 Hz, J3,4 = 2.3 Hz, 1H, H-3), 5.37 (dt, J4,5 = 9.1 Hz, J3,4 = 2.9 Hz, 1H, H-4),
22

4.40 (t, J2,3 = 9.6 Hz, 1H, H-2), 3.68–3.59 (m, 2H, H-1a, H-1b), 2.91 (dd, JOH,1a = 8.5
Hz, JOH,1b = 5.1 Hz, 1H, OH), 2.28 (t, Jα,β = 7.6 Hz, 2H, CH2-α), 2.03–2.00 (m, 2H, H-
5), 1.72–1.66 (m, 2H, CH2-β), 1.45–1.13 (m, 68H, H-6–H-17, H-γ–H-ω-1), 0.88(t,
J17,18 = Jω-1,ω = 7.0 Hz, 6H, H-18, H-ω); 13C NMR (125 MHz, CDCl3) δ 173.3
(HNC=O), 167.1 (C=O, 3-O-Bz), 166.3 (C=O, 4-O-Bz), 133.8 (C-p, 3-O-Bz), 133.1
(C-p, 4-O-Bz), 130.0 (C-o, 3-O-Bz), 129.9 (C-i, 4-O-Bz), 129.6 (C-o, 4-O-Bz), 129.1
(C-i, 3-O-Bz), 128.7 (C-m, 3-O-Bz), 128.4 (C-m, 4-O-Bz), 74.0 (C-4), 73.6 (C-3),
61.6 (C-1), 49.9 (C-2), 36.9 (C-α), 31.9, 29.73, 29.71, 29.68, 29.66, 29.65, 29.60,
29.58, 29.55, 29.40, 29.37, 29.34, 25.85, 22.7 (C-6–C-17, C-γ–C-ω-1), 28.4 (C-5),
25.75 (C-β), 14.1 (C-18, C-ω); HRMS(ESI) m/z calcd. for [C58H97NO6+Na]+:
926.7208, obsd.: 926.7220.
(2S,3S,4R)-3,4-Dibenzoyloxy-1-(2,3,4-tri-O-benzyl-6-deoxy-6-azido-α-D-
galactopyranosyloxy)-2-hexacosanoylamido-octadecane (20). Imidate donor 8 (88
mg, 0.14 mmol) and lipid acceptor 19 (64 mg, 0.071 mmol) were co-evaporated with
toluene (x 3), dissolved in dry Et2O/THF (2.7 mL, 5:1, v/v) and stirred with activated
4Å molecular sieves for 1 h. The reaction mixture was cooled to 0 °C, freshly distilled
TMSOTf (stock of 0.055m in Et2O, 76.8 mL, 0.0042 mmol) was added dropwise and
the resulting solution stirred at 0 °C for 1.5 h. The reaction was quenched with
triethylamine (50 mL, 0.36 mmol), diluted with EtOAc (20 mL), filtered through a
celite pad and concentrated in vacuo. The residue was first purified by size exclusion
column chromatography (LH20, CH2Cl2/MeOH, 1:1, v/v) to isolate a mixture of α-
and β-coupled glycolipid, which was further purified by silica gel gradient column
chromatography (petroleum ether/EtOAc, 15:1 to 5:1, v/v) to give the a-coupled
glycolipid 20α as a colourless oil (65.7 mg, 0.048 mmol, 68%) and the β-coupled
23

glycolipid 20β (22.6 mg, 0.017 mmol, 23%) also as a colourless oil. 20α: Rf: 0.58
(8% EA/Toluene); [α]D27 = –36.0 (c = 1.0, CHCl3); IR (film) 3063, 3030, 2922, 2852,
2362, 2338, 2102, 1722, 1673, 1530, 1453, 1278, 1095, 1068, 1027, 756, 711, 699
cm-1; 1H NMR (500 MHz, CDCl3) δ 8.03 (d, JCH-o,CH-m = 7.1 Hz, 2H, CH-o, 3-O-Bz),
7.96 (d, JCH-o,CH-m = 7.0 Hz, 2H, CH-o, 4-O-Bz), 7.60 (t, JCH-m,CH-p = 7.5 Hz, 1H, CH-
p, 3-O-Bz), 7.54 (t, JCH-m,CH-p = 7.5 Hz, 1H, CH-p, 4-O-Bz), 7.48-7.19 (m, 19H,
Harom), 6.66 (d, JNH,2 = 9.5 Hz, 1H, NH), 5.64 (dd, J2,3 = 9.3 Hz, J2,3 = 2.7 Hz, 1H, H-
3), 5.39–5.36 (m, 1H, H-4), 4.96 (d, Ja,b = 11.5 Hz, 1H, CH-a, 4′-O-Bn), 4.78 (d, Ja,b =
11.7 Hz, 1H, CH-a, 3′-O-Bn), 4.75 (d, J1′,2′ = 3.6 Hz, 1H, H-1′), 4.78 (d, Ja,b = 11.7 Hz,
1H, CH-a, 3′-O-Bn), 4.68 (d, Ja,b = 11.7 Hz, 1H, CH-b, 3′-O-Bn), 4.64 (s, 2H, CH-a,
CH-b, 2′-O-Bn), 4.61 (t, J1,2 = 3.2 Hz, 1H, H-2), 4.57 (d, Ja,b = 11.5 Hz, 1H, CH-b, 4′-
O-Bn), 3.98 (dd, J2′,3′ = 10.1 Hz, J1′,2′ = 3.6 Hz, 1H, H-2′), 3.93 (dd, J5′,6′a = 8.3 Hz,
J5′,6′b = 5.2 Hz, 1H, H-5′), 3.90 (dd, J1a,1b = 11.6 Hz, J1a,2 = 3.2 Hz, 1H, H-1a), 3.83
(dd, J2′,3′ = 10.1 Hz, J3′,4′ = 2.7 Hz, 1H, H-3′), 3.78 (bs, 1H, H-4′), 3.67 (dd, J1a,1b
=
11.6 Hz, J1b,2 = 3.2 Hz, 1H, H-1b), 3.55 (dd, J6′a,6′b = 12.4 Hz, J5′,6′a = 8.3 Hz, 1H, H-
6′a), 3.04 (dd, J6′a,6′b = 12.4 Hz, J5′,6′b = 5.2 Hz, 1H, H-6′a), 2.20 (t, Jα,β = 7.6 Hz, 2H,
CH2-α), 1.95-1.90 (m, 2H, H-5), 1.67-1.64 (m, 2H, CH2-β), 1.39-1.21 (m, 68H, H-6–
H-17, H-γ–H-ω-1), 0.89 (t, J17,18 = Jω -1,ω = 6.9 Hz, 6H, H-18, H-ω); 13C NMR (125
MHz, CDCl3) δ 173.1 (HNC=O), 166.2 (C=O, 4-O-Bz), 165.3 (C=O, 3-O-Bz), 138.6
(C-i, 3′-O-Bn), 138.2 (C-i, 2′-O-Bn), 138.0 (C-i, 4′-O-Bn), 133.3 (C-p, 3-O-Bz), 132.9
(C-p, 4-O-Bz), 130.1, 129.8 (C-i, 3-O-Bz, 4-O-Bz), 129.8 (C-o, 3-O-Bz), 129.8 (C-o,
4-O-Bz), 128.6, 128.5, 128.42, 128.41, 128.34, 128.32, 128.1, 128.0, 127.7, 127.62,
127.56 (21 x CHarom), 99.7 (C-1′, 1JCH = 168.5 Hz), 78.5 (C-3′), 76.4 (C-2′), 74.8 (C-
4′), 74.6 (CH2, 4′-O-Bn), 73.8 (C-4), 73.5 (CH2, 3′-O-Bn), 73.3 (CH2, 2′-O-Bn), 72.4
(C-3), 70.2 (C-5′), 69.7 (C-1), 51.3 (C-6′), 48.6 (C-2), 36.8 (C-a), 28.3 (C-5), 25.8 (C-
24

b), 31.9, 29.8, 29.72, 29.70, 29.67, 29.64, 29.61, 29.60, 29.56, 29.50, 29.4, 25.7, 22.7
(C-6–C-17, C-γ–C-ω-1), 14.1 (C-18, C-ω); HRMS(ESI) m/z calcd. for
[C85H124N4O10+H]+: 1361.9390, obsd.: 1361.9399.
(2S,3S,4R)-2-Hexacosanoylamido-(2,3,4-tri-O-benzyl-6-deoxy-6-azido-α-D-
galactopyranosyloxy)-octadecane-3,4-diol. Glycolipid 20α (66 mg, 0.048 mmol)
was dissolved in MeOH/CH2Cl2 (3 mL, 2:1 (v/v) and sodium methoxide was added
until the reaction mixture reached pH 9.0. After stirring for 12 h at rt, the reaction was
quenched with Dowex-H+, filtered and concentrated under reduced pressure. The
residue was purified by silica gel gradient column chromatography (petroleum
ether/EtOAc, 10:1 to 3:1, v/v) to afford the title compound as an amorphous white
solid (52 mg, 0.045 mmol, 93%). Rf: 0.28 (PE/EA, 2:1, v/v); [α]D24 = +6.0 (c = 1.0,
CHCl3); IR (film) 3332, 3065, 3032, 2918, 2850, 2100, 1717, 1636, 1623, 1542,
1469, 1455, 1350, 1284, 1216, 1078, 1041, 909, 757, 696 cm-1; 1H NMR (500 MHz,
CDCl3) δ 7.40-7.29 (m, 15H, Harom), 6.29 (d, JNH,2 = 8.3 Hz, 1H, NH), 4.98 (d, Ja,b =
11.3 Hz, 1H, CH-a, 4′-O-Bn), 4.884 (d, Ja,b = 11.6 Hz, 1H, CH-a, 2′-O-Bn), 4.885 (d,
J1,2 = 3.7 Hz, 1H, H-1′), 4.82 (d, Ja,b = 11.7 Hz, 1H, CH-a, 3′-O-Bn), 4.78 (d, Ja,b =
11.7 Hz, 1H, CH-b, 3′-O-Bn), 4.70 (d, Ja,b = 11.7 Hz, 1H, CH-b, 2′-O-Bn), 4.59 (d, Ja,b
= 11.3 Hz, 1H, CH-b, 4′-O-Bn), 4.27 (dd, JNH,2 = 8.3 Hz, J1a,2 = 3.4 Hz, 1H, H-2), 4.05
(dd, J2′,3′ = 10.0 Hz, J1′,2′ = 3.7 Hz, 1H, H-2′), 3.93 (dd, J1a,1b= 10.4 Hz, J1a,2 = 3.4 Hz,
1H, H-1a), 3.86 (dd, J2′,3′ = 10.0 Hz, J3′,4′ = 2.4 Hz, 1H, H-3′), 3.84-3.82 (m, 2H, H-1b,
H-4′), 3.73 (t, J5′,6′ = 6.6 Hz, 1H, H-5′), 3.52-3.46 (m, 3H, H-4, H-6′a, H-3), 3.03 (dd,
J6′a,6′b = 12.6 Hz, J5′,6′b = 5.5 Hz, 1H, H-6′b), 2.16 (t, Jα,β = 7.6 Hz, 2H, CH2-α), 1.60-
1.50 (m, 3H, CH2-α, H-5a), 1.48-1.21 (m, 68H, H-5b, H-6-H-17, H-γ–H-ω-1), 0.88 (t,
J17,18 = Jω -1,ω = 6.9 Hz, 6H, H-18, H-ω); 13C NMR (125 MHz, CDCl3) δ 173.9
25

(HNC=O), 138.2 (C-i, 3′-O-Bn), 138.0 (C-i, 4′-O-Bn), 137.8 (C-i, 2′-O-Bn), 128.69,
128.67, 128.6, 128.5, 128.3, 128.2, 128.1, 128.0, 127.7 (15 x CHarom), 99.0 (C-1′),
79.3 (C-3′), 76.3 (C-3), 75.9 (C-2′), 74.8 (CH2, 4′-O-Bn), 74.5 (C-4′), 74.4 (CH2, 2′-
O-Bn), 73.5 (C-4), 73.2 (CH2, 3′-O-Bn), 70.4 (C-5′), 69.9 (C-1), 51.2 (C-6′), 49.2 (C-
2), 37.0 (C-α), 33.5, 32.1, 29.9, 29.85, 29.81, 29.7, 29.6, 29.51, 29.46, 22.8 (C-6–C-
17, C-γ–C-ω-1), 26.0, 25.9 (C-5, C-β), 14.3 (C-18, C-ω); HRMS(ESI) m/z calcd. for
[C71H117N4O8+H]+: 1153.8866, obsd.: 1153.8871.
(2S,3S,4R)-1-(6-Amino-6-deoxy-α-D-galactopyranosyloxy)-2-
hexacosanoylamido-octadecane-3,4-diol (5). To a solution of (2S,3S,4R)-2-
hexacosanoylamido-(2,3,4-tri-O-benzyl-6-deoxy-6-azido-α-D-galactopyranosyloxy)-
octadecane-3,4-diol (25.1 mg, 0.022 mmol) dissolved in CHCl3/EtOH (2.5 ml, 3:2,
v/v) was added formic acid (0.13 mL, 0.0034 mmol) and Pd(OH)2/C (20 wt% loading,
50 wt% H2O, 36 mg, 0.033 mmol) and the reaction stirred under H2 (g) for 15 h at rt.
The reaction mixture was filtered through a celite pad and washed successively with
CHCl3/EtOH/MeOH (20 mL, 3:1:1, v/v/v) and pyridine (10 mL) and concentrated in
vacuo. The residue was semi-purified by silica gel column chromatography
(CH2Cl2→CH2Cl2/MeOH/EtOH/NH3 (35% aq)→55:2:2:1→35:2:2:1→15:2:2:1,
v/v/v/v) to afford amine 5 as an amorphous white solid (6.2 mg, 0.0072 mmol, 33%).
Rf: 0.50 (DCM/MeOH/EtOH/NH3-H2O, 5:2:2:1, v/v/v/v); 1H NMR (500 MHz, 10%
CD3OD in CDCl3) δ 4.84 (d, J1′,2′ = 3.9 Hz, 1H, H-1′), 4.12 (dd, JNH,2 = 9.1 Hz, J1a,2
=
J1b,2 = 4.9 Hz, 1H, H-2), 3.82–3.79 (m, 2H, H-1a, H-4′), 4.74–4.73 (m, 1H, H-5′), 3.71
(dd, J2′,3′ = 10.1 Hz, J1′,2′ = 3.9 Hz, 1H, H-2′), 3.63 (dd, J2′,3′ = 10.1 Hz, J3′,4′ = 3.4 Hz,
1H, H-3′), 3.53 (dd, J1a,1b = 10.6 Hz, J1b,2 = 4.9 Hz, 1H, H-1b), 3.47–3.42 (m, 2H, H-3,
H-4), 3.01 (dd, J6′a, 6′b = 13.0 Hz, J5,6′a = 7.6 Hz, 1H, H-6′a), 2.85 (dd, J6′a, 6′b = 13.0
26

Hz, J5,6′b = 3.6 Hz, 1H, H-6′b), 2.12 (t, Jα,β = 7.6 Hz, 2H, CH2-α), 1.63-1.47 (m, 4H,
CH2-α, CH2-5), 1.30–1.06 (m, 68H, H-6-H-17, H-γ–H-ω-1), 0.80 (t, J17,18 = Jω -1,ω
=
6.9 Hz, 6H, H-18, H-ω); HRMS(ESI) m/z calcd. for [C50H100N2O8+H]+: 857.7552,
obsd.: 857.7533.
(2S,3S,4R)-1-(6-Deoxy-6-[4-(4,4-difluoro-1,3,5,7-tetramethyl-3a,4a-diaza-4-bora-
s-indacen-8-yl)benzylamido])-α-D-galactopyranosyloxy)-2-hexacosanoylamido-
octadecane-3,4-diol (3). Amine 5 (4.1 mg, 0.0048 mmol) was co-evaporated with
DMF (x 3), dissolved in dry DMF (0.6 mL) and the solution was warmed to 45 °C
followed by the addition of DiPEA (3.3 µL, 0.019 mmol). BODIPY-NHS 21 (9.2 mg,
0.020 mmol) was co-evaporated with DMF (x 3) and added as a solution in DMF (0.6
mL) and stirred for 1 h at 45 °C, at which point additional BODIPY-NHS 24 (4.6 mg,
0.010 mmol) in dry CH2Cl2 (0.6 mL) and DiPEA (3.3 µL, 0.019 mmol) was added
and the reaction mixture stirred for further 14 h at 45 °C. The reaction mixture was
then concentrated in vacuo and purified immediately by silica gel gradient column
chromatography (CH2Cl2 to 3% MeOH in CH2Cl2) to afford BODIPY-αGalCer 3 as a
red solid (2.5 mg, 0.0021 mmol, 44%). Rf: 0.38 (10% MeOH/DCM, v/v); IR (film)
3346, 2851, 1642, 1631, 1608, 1547, 1513, 1468, 1450, 1409, 1308, 1192, 1157,
1071, 1044, 983, 759, 700, 635 cm-1; 1H NMR (600 MHz, pyridine-d5) δ 9.74 (t,
J6′a,NH = J6′b,NH = 5.6 Hz, 1H, NH-BOD), 8.67 (d, J2,NH = 8.5 Hz, 1H, NH-lipid), 8.46
(d, J14″,15″ = J17″,18″ = 8.2 Hz, 2H, H-15″, H-17″), 7.34 (d, J14″,15″ = J17″,18″ = 8.2 Hz, 2H,
H-14″, H-18″), 5.99 (s, 2H, H-2″, H-6″), 5.57 (d, J1′,2′ = 3.9 Hz, 1H, H-1′), 5.29–5.27
(m, 1H, H-2), 4.70 (t, J5′,6′a = J5′,6′b = 6.3 Hz, 1H, H-5′), 4.68–4.65 (2H, H-1a, H-2′),
4.49 (dd, J5′,6′a = 6.3 Hz, J6′a,6′b = 13.4 Hz, 1H, H-6′a), 4.46 (d, J3′,4′ = 1.3 Hz, 1H, H-
4′), 4.40–4.37 (m, 2H, H-1b, H-3′), 4.35–4.23 (m, 2H, H-3, H-4), 4.22–4.19 (m, 1H,
27

H-6′b), 2.71 (s, 6H, H-9″, H-10″), 2.48 (t, Jα,β = 5.6 Hz, 2H, CH2-α), 2.30–2.27 (m,
2H, CH2), 1.97–1.91 (m, 3H, H-5, CH2), 1.85–1.80 (m, 4H, CH2-β, CH2), 1.71–1.69
(m, 2H, CH2), 1.36 (s, 6H, H-11″, H-12″), 1.45–1.17 (m, 64H, H-7–H-17, H-δ–H-(ω-
1)), 0.88 (t, 6H, J17,18 = Jω -1,ω = 6.9 Hz, H-18, H-ω); 13C NMR (150 MHz, pyridine-
d5) δ 173.7 (HNC=O lipid), 168.1 (HNC=O BOD), 156.2 (C-3″, C-5″), 143.7 (C-7a″,
C-8a″), 141.9 (C-8″), 138.1 (C-13″), 136.9 (C-16″), 131.8 (C-1″, C-7″), 129.3 (C-15″,
C-17″), 128.8 (C-14″, C-18″), 122.2 (C-2″, C-6″), 77.1 (C-3/C-4), 72.9 (C-3/C-4),
71.64 (C-3′), 71.57 (C-4′), 70.8 (C-5′), 70.4 (C-2′), 69.1 (C-1), 51.8 (C-2), 72.3 (C-6′),
37.2 (C-α), 34.7, 32.5, 30.8, 30.5, 30.41, 30.40, 30.39, 30.37, 30.36, 30.34, 30.33,
30.29, 30.27, 30.26, 30.18, 30.14, 29.98, 29.97, 23.29 (C-6–C-17, C-γ–C-ω-1), 26.89
(C-5), 26.79 (C-β), 15.1, 14.9 (C-9″, C-10″, C-11″, C-12″), 14.6 (C-18, C-ω);
HRMS(ESI) m/z calcd. for [C70H117BF2N4O9+H]+: 1207.8954, obsd.: 1207.8958.
JAWS II/DN32 cultures and quantification of IL-2.
All cells were maintained in RPMI 1640 medium supplemented with 10% FBS, 2.1
mM ultra-glutamine, 100 U/mL penicilin, 100 µg/mL streptomycin, and 50 µM 2-
Mercaptoethanol. Stock α-GalCer and BODIPY α-GalCer was dissolved 1mg/ml in
DMSO (≥99.9%) and serially diluted in DMSO(≥99.9%). Diluted samples were
further diluted 1/1000 in complete RPMI medium and cultured with 5x104 JAWS II
mouse dendritic cells and DN32.D3 mouse iNKT hybridoma cells. Supernatants were
analyzed by IL-2 Mouse ELISA Kit (Invitrogen) after 48-hour incubation according
to manufacturers instructions. GraphPad Prism 8.2 was used for dose-response
analysis and curve fitting.
28