Nonenzymatic synthesis of anomerically pure, mannosylbased molecular probes for scramblase identification studies

Article abstract of DOI:10.3762/BJOC.16.145

The chemical synthesis of molecular probes to identify and study membrane proteins involved in the biological pathway of protein glycosylation is described. Two short-chain glycolipid analogs that mimic the naturally occurring substrate mannosyl phosphoryl dolichol exhibit either photoreactive and clickable properties or allow the use of a fluorescence readout. Both probes consist of a hydrophilic mannose headgroup that is linked to a citronellol derivative via a phosphodiester bridge. Moreover, a novel phosphoramidite chemistry-based method offers a straightforward approach for the non-enzymatic incorporation of the saccharide moiety in an anomerically pure form.

Full text of DOI:10.3762/BJOC.16.145

Nonenzymatic synthesis of anomerically pure, mannosyl-
based molecular probes for scramblase identification studies
Giovanni Picca1, Markus Probst1, Simon M. Langenegger1, Oleg Khorev1,
Peter Bütikofer2, Anant K. Menon3 and Robert Häner*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2020, 16, 1732–1739.
1Department of Chemistry and Biochemistry, University of Bern,
Freiestrasse 3, 3012 Bern, Switzerland, 2Institute of Biochemistry and
Molecular Medicine, University of Bern, Bühlstrasse 28, 3012 Bern,
Switzerland and 3Department of Biochemistry, Weill Cornell Medical
College, 1300 York Avenue, 10065 New York, United States of
America
doi:10.3762/bjoc.16.145
Received: 05 May 2020
Accepted: 09 July 2020
Published: 20 July 2020
Associate Editor: S. Flitsch
Email:
Robert Häner* - robert.haener@dcb.unibe.ch
© 2020 Picca et al.; licensee Beilstein-Institut.
License and terms: see end of document.
* Corresponding author
Keywords:
carbohydrates; citronellol; phosphoramidite; photoclickable glycolipid
analogs; scramblase
Abstract
The chemical synthesis of molecular probes to identify and study membrane proteins involved in the biological pathway of protein
glycosylation is described. Two short-chain glycolipid analogs that mimic the naturally occurring substrate mannosyl phosphoryl
dolichol exhibit either photoreactive and clickable properties or allow the use of a fluorescence readout. Both probes consist of a
hydrophilic mannose headgroup that is linked to a citronellol derivative via a phosphodiester bridge. Moreover, a novel phosphor-
amidite chemistry-based method offers a straightforward approach for the non-enzymatic incorporation of the saccharide moiety in
an anomerically pure form.
Introduction
Mannosyl phosphoryl dolichol (MPD), an important, multifunc- syltransfer reactions [3,4]. A specific membrane protein – MPD
tional glycolipid, is used as a mannose donor for protein scramblase – is required to facilitate the transbilayer movement
N-glycosylation, O- and C-mannosylation, and glycosylphos- of MPD across the ER. Although the activity of MPD scram-
phatidylinositol (GPI) anchoring in the luminal leaflet of the blase has been described in microsomal vesicles and reconsti-
endoplasmic reticulum (ER) [1-8]. Interestingly, MPD is syn- tuted systems [1,2,9], the molecular identity of this protein
thesized on the cytoplasmic face of the ER and must be translo- remains unknown. To circumvent the need for traditional purifi-
cated across the ER membrane to participate in luminal glyco- cation strategies to identify the scramblase, we considered the
1732

Beilstein J. Org. Chem. 2020, 16, 1732–1739.
use of photoreactive, clickable MPD mimics. As such, an studies relied on a chemoenzymatic approach to selectively in-
attempt already showed great promise in a previous report by corporate the biologically relevant β-configured ᴅ-mannose
Rush et al. from 2015 [10]. Similarly, we envisioned that these headgroup as chemical approaches were deemed to be too
analogs could be used to capture MPD-recognizing proteins, challenging [10,13]. This method naturally has some limita-
including the scramblase, from a crude mixture of ER mem- tions regarding the quantity of the obtained compound and
brane proteins [11,12]. The captured proteins would be subse- the availability of the required mannosyltransferase. Here,
quently identified by mass spectrometry, and their function in we describe a purely chemical approach to synthesize a
MPD scrambling validated by biochemical and genetic ap- photoclickable MPD analog containing β-ᴅ-mannose (in MPC-
proaches.
1). The chemical synthesis allows for a rapid upscaling of the
reactions if necessary. In addition, having access to the indi-
A suitable molecular probe and mimic of MPD (Figure 1) can vidual building blocks (e.g., the phosphoramidites) will enable
be subdivided into three essential components: a β-ᴅ-mannose the creation of a series of molecular probes with different func-
(the α-anomer also shows biological activity, but to a lesser tional groups and varying linker lengths with relatively little
extent) [13-15], a short-chain (citronellol) mimic of dolichol effort.
[1,2,13], and a functional tag. The latter may either be a chemi-
cally reactive group (in MPC-1) or a fluorescent reporter group The crucial step in the synthetic pathway consists of the conver-
(in MPC-2). MPC-1 bears a benzophenone moiety for sion of the carbohydrate intermediates into stereodefined,
photocrosslinking to MPD-recognizing proteins. The presence anomeric phosphoramidite derivatives. The method is based on
of an additional propargyl group provides a way to further adapted procedures developed for DNA solid-phase synthesis
derivatize the probe with biotin azide via click-type chemistry [17]. Phosphoramidite chemistry allows the connection of two
[16] for the isolation of protein–lipid adducts using streptavidin molecular entities via a phosphodiester linkage and was found
resins [10]. To synthesize such molecular probes, previous to be perfectly suited for this purpose. Reports of using phos-
Figure 1: Chemical structures of MPD and the three structural analogs MPC-1, MPC-2, and MPC-3. The molecular probes MPC-1 and MPC-3 are
photoclickable derivatives, whereas the probe MPC-2 bears a fluorescent tag.
1733

Beilstein J. Org. Chem. 2020, 16, 1732–1739.
phoramidite chemistry for the preparation of carbohydrates via pholipid membrane, and the mannose and the NBD are posi-
the anomeric position are relatively rare [18-23]. Alternatively, tioned in the surrounding aqueous medium. The close prox-
the H-phosphonate approach has been used to convert carbo- imity of the NBD fluorophore to the membrane–water interface
hydrates into phosphate-linked derivatives at the anomeric [30,31] allows for fluorescence-based scramblase assays
center [24-29].
wherein the NBD can be detected with dithionite. The latter
reduces the nitro group of the NBD rapidly into an amino
The phosphoramidite approach was also applied to the synthe- group, rendering the fluorophore nonfluorescent [32-34]. While
sis of a second molecular probe, MPC-2, an anomerically pure, there are no noteworthy difficulties in the synthesis of the mo-
β-linked ᴅ-mannosephosphate derivative, which serves as a lecular probe MPC-1 to report, the same cannot be said for
fluorescent MPD analog for scramblase activity screening MPC-2. There, the NBD tag was only partially stable to the
assays. Although similar experiments have been described with conditions used to remove the protecting groups in the final step
radioactively labeled substrates, the use of fluorescently labeled (NH3 in MeOH, Figure 3). Under such alkaline conditions, a
probes offers several advantages, including the continuous mon- considerable amount of degradation of NBD was observed,
itoring of the transport and a better time resolution. For reasons making an additional purification step necessary to obtain pure
of comparison, the α-configured ᴅ-mannose probe MPC-3 was MPC-2 (see Supporting Information File 1).
synthesized in parallel. The characterization of the configura-
tion at the anomeric position was done by HSQC NMR (see As shown in Figure 3, the acetylated mannose phosphoramidite
Supporting Information File 1). Since the target enzymes are β-4Ac-Man-CEP was coupled to the free hydroxy group of the
unknown and can be expected to have stereospecific binding citronellol derivatives containing the functional tags (Cit-BZP-
sites, the α-configured ᴅ-mannose probe MPC-3 is also impor- yne or Cit-Dod-NBD). The reactions were performed in
tant as a reference for our biochemical assays (work in dichloromethane (DCM), and ETT served as the activator. The
progress). The possibility to use both probes, MPC-1 and subsequent oxidation of the unstable phosphite triester to the
MPC-3, independently may help to reduce false-positive scram- more stable phosphotriester was performed with t-BuOOH. The
blase candidates.
resulting intermediates were then treated overnight with a solu-
tion of ammonia in methanol to remove the protecting groups.
An overall yield of about 45% was achieved when no further
Results and Discussion
The synthesis of the target compounds MPC-1 and MPC-2 purification was carried out (for details see Supporting Informa-
started from commercially available (S)-citronellol (Cit), 4,4′- tion File 1).
dihydroxybenzophenone (BZP), D-mannose (Man), and 1,12-
dodecanediol (Dod, Figure 2).
The major goal of this work was the synthesis of molecular
probes with a configurationally defined mannosyl headgroup,
Both the mannose and the phosphodiester bond were intro- i.e., a pure β-anomer. In order to verify the preferred configura-
duced via phosphoramidite chemistry to yield the final com- tion at the anomeric position and to prove the working prin-
pounds, as shown in Figure 3. As opposed to the photoclick- ciple of our synthetic strategy, we used 2D NMR experiments.
able probe MPC-1, the 4-chloro-7-nitro-1,2,3-benzoxadiazole Coupled HSQC measurements revealed the 1JCH coupling con-
(NBD)-labeled analog MPC-2 carries an additional dodecanyl stants of our compounds and thereby the absolute configuration
linker between the citronellyl unit and the fluorophore. This is at the anomeric center of the carbohydrate [40-42]. For MPC-1
intended to increase the hydrophobic interactions between the and MPC-2, 1JCH values 158 Hz and 161 Hz, respectively,
probe and the lipid bilayer and also to increase the flexibility. were obtained (for comparison, for MPC-3 containing almost
More specifically, the probe should adopt a U-shaped structure exclusively the α-anomer, a value of about 171 Hz was deter-
in which the citronellyl–dodecanyl linker is located in the phos- mined, see Supporting Information File 1).
Figure 2: Chemical structures of commercially available (S)-citronellol (Cit), 4,4′-dihydroxybenzophenone (BZP), ᴅ-mannose (Man), and 1,12-dode-
canediol (Dod), the main starting materials in the synthesis of MPC-1 and MPC-2.
1734

Beilstein J. Org. Chem. 2020, 16, 1732–1739.
Figure 3: The synthetic route leading to compounds MPC-1 and MPC-2. Compound β-4Ac-Man-CEP was prepared in 4 steps from ᴅ-mannose (see
Supporting Information File 1) [35,36]. Compound Cit-BZP-yne was prepared via a Mitsunobu reaction of tetrahydropyranyl (THP)-protected
citronellol and a hydroxylated benzophenone derivative [10,37,38]. Compound Cit-Dod-NBD was obtained in 10 steps, starting from citronellol and
tert-butyldimethylsilyl (TBDMS)-protected dodecanediol [39]. The reaction of either of the alcohols with the sugar phosphoramidite using ETT led to
phosphite intermediates 1a and 1b, respectively. The intermediates were then oxidized with t-BuOOH, and finally, the protecting groups were re-
moved under basic conditions to give either MPC-1 or MPC-2 as ammonium salts. ETT = 5-(ethylthio)-1H-tetrazole.
As highlighted above, the most important step in the chemical β-ᴅ-mannopyranose. This was achieved by following literature
synthesis of the target compounds MPC-1 and MPC-2 was the procedures [35,36], which provided the pure β-anomer as a
preparation of the phosphoramidite β-4Ac-Man-CEP crystalline solid. Secondly, suitable conditions had to be identi-
(Figure 4). Two points are of particular importance for the syn- fied for the subsequent phosphitylation conditions under which
thesis of this compound. Firstly, we needed access to an no mutarotation of the carbohydrate occurs. Cooling of the reac-
anomerically pure starting material, i.e., 2,3,4,6-tetra-O-acetyl- tion mixture and performing the reaction at a temperature below
Figure 4: Preparation of mannosyl phosphoramidites. Starting from 2,3,4,6-tetra-O-acetyl-β-ᴅ-mannopyranose (β-4Ac-Man), the phosphitylation using
2-cyanoethyl N,N-diisopropylchlorophosphoramidite (CEP-Cl) and DIPEA provides β-4Ac-Man-CEP when the reaction is performed at a temperature
below −15 °C (15–30 min reaction time). When the reaction is carried out at room temperature, epimerization leads to 2,3,4,6-tetra-O-acetyl-α-ᴅ-
mannopyranose (not shown), which is subsequently converted to α-4Ac-Man-CEP.
1735

Beilstein J. Org. Chem. 2020, 16, 1732–1739.
−15 °C yielded the compound β-4Ac-Man-CEP, with no traces 114.36, 77.92, 76.10, 74.21, 61.12, 55.89, 39.83, 36.63, 29.18,
of the α-anomer. The configuration was then retained through- 25.20, 19.46, 13.85.
out the rest of the synthesis, as indicated by the NMR data of
MPC-1 and MPC-2 (Supporting Information File 1).
β-4Ac-Man-CEP
β-4Ac-Man (100 mg, 0.29 mmol, 1 equiv) was dissolved in an-
hydrous DCM (4 mL) at −60 °C. Then, DIPEA (75 μL,
Conclusion
We report herein the successful chemical synthesis of molecu- 0.43 mmol, 1.5 equiv) was added, followed by the addition of
lar probes for identifying and studying MPD scramblase, as CEP-Cl (77 μL, 0.35 mmol, 1.2 equiv). The mixture was stirred
well as proteins that use MPD as a mannosyl donor. Two types for 30 min at −50 °C under argon. The crude product was then
of probes were synthesized; both are short-chain glycolipid directly purified by flash chromatography using ethyl acetate/n-
analogs. One, MPC-1, contains a photoreactive clickable tag to hexane 1:1, v/v + 2% triethylamine. The fractions containing
capture scramblase candidates (identification by mass spectrom- the product (Rf = 0.40) were combined, and the solvents were
etry), and the other probe, MPC-2, consists of a fluorescent removed under reduced pressure on a rotary evaporator. The
label to test candidates for scramblase activity in reconstitution- product was extensively dried to give β-4Ac-Man-CEP
based assays. The molecular probes were prepared via phos- (145 mg, 0.26 mmol, 92%) as colorless foam. 1H NMR
phoramidite chemistry, which allowed the incorporation of the (300 MHz, CDCl3, δ) 5.37 (dd, J = 25.2, 3.3 Hz, 1H), 5.19 (t,
carbohydrate headgroup and simultaneously introduced the J = 9.9 Hz, 1H), 5.12–4.95 (m, 2H), 4.24–4.09 (m, 2H),
linking phosphate group. Furthermore, we demonstrate a novel, 3.91–3.48 (m, 5H), 2.66–2.53 (m, 2H), 2.14 (s, 3H), 2.05–2.00
reliable, and efficient way to synthesize the carbohydrate phos- (m, 8H), 1.95 (d, J = 1.6 Hz, 3H), 1.12 (dd, J = 10.8; 6.9 Hz,
phoramidite with a defined configuration at the anomeric posi- 12H); 13C NMR (75 MHz, CDCl3, δ): 170.58, 170.55, 170.31,
tion, a strategy that gives access to future molecular probes con- 170.10, 169.98, 169.94, 169.67, 169.63, 117.63, 93.69, 93.57,
taining pure β-ᴅ-mannose, which was previously only acces- 93.36, 72.72, 72.66, 70.98, 69.78, 69.69, 65.99, 65.90, 62.67,
sible through a chemoenzymatic approach.
62.43, 59.20, 58.96, 58.78, 58.53, 44.07, 43.90, 43.77, 43.59,
24.61, 24.52, 24.43, 24.34, 24.04, 23.94, 20.80, 20.77, 20.68,
20.56, 20.34, 20.25, 20.14, 20.06; 31P NMR (121 MHz, CDCl3,
δ) 153.31, 150.00.
Experimental
Synthesis
Detailed descriptions of the synthesis and the analytical data
(NMR and MS spectra) are provided in Supporting Information MPC-1
File 1.
ETT (72 mg, 0.55 mmol, 1.5 equiv) was dissolved in an-
hydrous DCM (2 mL), β-4Ac-Man-CEP (223 mg, 0.41 mmol,
Cit-BZP-yne
1.1 equiv) was added, and the resultant solution was added to a
THP-Cit-BZP-yne (279 mg, 0.57 mmol, 1 equiv) was dis- solution of Cit-BZP-yne (150 mg, 0.37 mmol, 1 equiv) dis-
solved in anhydrous ethanol (8 mL), and pyridinium p-toluene- solved in anhydrous DCM (2 mL). The reaction mixture was
sulfonate (PPTS, 286 mg, 1.14 mmol, 2 equiv) was added. The stirred for 30 min at rt under argon. Afterwards, a t-BuOOH
solution was stirred at 60 °C for 2.5 h. The reaction mixture (at solution (201 μL, 1.11 mmol, 3 equiv, ≈5.5 M in decane) was
rt) was poured into a separating funnel containing diethyl ether added, and the mixture was stirred for an additional 15 min. The
(40 mL) and brine (16 mL). The organic layer was dried over reaction mixture was diluted with toluene (60 mL) and then
MgSO4, and the volatile components were removed under washed with a saturated sodium hydrogen carbonate solution
reduced pressure. The yellowish residue (crude product) was (30 mL) and brine (30 mL). The organic layer was dried over
purified by flash chromatography over silica gel, with a mix- MgSO4, and the volatile components were removed under
ture of n-hexane/ethyl acetate 3:2, v/v as eluent. The fractions reduced pressure. The resulting residue was dissolved in metha-
containing the product (Rf = 0.22 in n-hexane/ethyl acetate 3:2, nol and purified by PLC (using ethyl acetate/n-hexane 2:1, v/v
v/v) were combined, and the solvents were removed under as a mobile phase). The broad band, located at the very bottom
reduced pressure on a rotary evaporator to give Cit-BZP-yne of the plate (Rf ≈ 0.10 using ethyl acetate/n-hexane 1:1, v/v)
(154 mg, 0.38 mmol, 67%) as white solid. 1H NMR (300 MHz, was removed, and the intermediate β-4Ac-Man-P-Cit-BZP-
CDCl3, δ):7.85–7.72 (m, 4H), 7.09–6.99 (m, 2H), 7.02–6.91 (m, yne was extracted with methanol (150 mL). The solvent was re-
2H), 5.61–5.49 (m, 1H), 4.77 (d, J = 2.4 Hz, 2H), 4.47 (s, 2H), moved under reduced pressure on a rotary evaporator. Then, an
3.69 (m, 2H), 2.56 (t, J = 2.4 Hz, 1H), 2.21–1.98 (m, 2H), 1.74 ammonia solution (3 mL, 2.0 M in methanol) was added, and
(s, 3H), 1.70–1.50 (m, 1H), 1.50–1.15 (m, 4H), 0.92 (d, J = the reaction mixture was stirred overnight at rt. The resulting
6.4 Hz, 3H); 13C NMR (75 MHz, CDCl3, δ) 194.48, 162.39, solution was first diluted with methanol (2 mL), and the vola-
160.64, 132.22, 132.14, 131.64, 130.51, 130.29, 129.82, 114.37, tile components were removed under reduced pressure. Then,
1736

Beilstein J. Org. Chem. 2020, 16, 1732–1739.
distilled water (2 mL) was added, and the material was and the intermediate β-4Ac-Man-P-Cit-Dod-NBD was
lyophilized overnight to afford MPC-1 (110 mg, 0.17 mmol, extracted with acetonitrile (180 mL). The solvent was removed
46%) as yellowish solid. 1H NMR (400 MHz, DMSO-d6, δ) under reduced pressure on a rotary evaporator. The material was
7.75–7.66 (m, 4H), 7.17–7.04 (m, 4H), 5.58 (t, J = 6.7 Hz, 1H), then treated overnight with an ammonia solution (2 mL, 2.0 M
4.92 (d, J = 2.4 Hz, 2H), 4.87 (d, J = 8.5 Hz, 1H), 4.51 (s, 2H), in methanol). Afterwards, the reaction mixture was diluted with
3.77–3.61 (m, 5H), 3.62–3.32 (m, 2H), 3.33–3.25 (m, 2H), 2.05 methanol (2 mL), the volatile components were removed under
(m, 2H), 1.69 (s, 3H), 1.62–1.48 (m, 2H), 1.42–1.26 (m, 2H), reduced pressure, and the orange-brownish residue (redissolved
1.26–1.13 (m, 1H), 0.87 (d, J = 6.2 Hz, 3H); 13C NMR in 1.5 mL methanol) was purified by PLC (using chloroform/
(101 MHz, DMSO-d6, δ) 193.64, 162.46, 160.86, 132.29, methanol/water 60:25:4, v/v/v as the mobile phase). The broad
132.13, 131.24, 130.63, 130.30, 129.66, 115.07, 115.02, 96.07, band, centered at around an Rf of about 0.22, was removed, and
96.04, 79.25, 79.20, 74.05, 73.98, 71.53, 71.48, 67.39, 63.02, the product was extracted with methanol (170 mL). The solvent
62.97, 61.82, 56.20, 37.85, 37.78, 36.76, 36.36, 29.33, 25.14, was removed under reduced pressure on a rotary evaporator, the
19.69, 14.17; 31P NMR (121 MHz, DMSO-d6, δ) 25.54, 4.74, residue dissolved in distilled water (1.3 mL), and the material
−2.89.
was lyophilized overnight to give MPC-2 (65 mg, 0.09 mmol,
45%). 1H NMR (300 MHz, D2O, δ) 8.27 (s, 1H), 6.17 (s, 1H),
5.35 (s, 1H), 5.13 (d, J = 8.4 Hz, 1H), 4.14–3.53 (m, 10H),
Cit-Dod-NBD
DMT-Cit-Dod-NBD (787 mg, 0.96 mmol) was dissolved in a 3.50–3.19 (m, 4H), 2.05–1.99 (m, 2H), 1.78–1.42 (m, 9H),
trichloroacetic acid solution (3% in DCM/MeOH 1:1, v/v) at rt. 1.33–1.08 (m, 21H), 0.91 (d, J = 6.0 Hz, 3H); 13C NMR
The reaction mixture was stirred for 2 h. The mixture was (101 MHz, D2O, δ) 131.98, 131.89, 131.83, 128.40, 128.33,
diluted with DCM, washed with brine, and the organic phase 95.42, 95.38, 76.90, 76.65, 72.73, 71.17, 71.12, 69.33, 69.32,
was dried with Na2SO4. The purification by column chromatog- 66.19, 64.79, 61.20, 60.82, 37.11, 29.76, 29.61, 29.52, 29.39,
raphy on silica gel (hexane/EtOAc 6:4, v/v) gave Cit-Dod-NBD 29.23, 26.26, 25.09, 18.99, 13.57; 31P NMR (121 MHz, D2O, δ)
(455 mg, 92%) as red oil (Rf = 0.5 in hexane/EtOAc 1:1, v/v). 7.22, −1.64.
1H NMR (DMSO-d6, 300 MHz, δ) 9.55 (s, 1H), 8.50 (d, J =
8.8 Hz, 1H), 6.49–6.31 (m, 1H), 5.33 (t, J = 7.2 Hz, 1H), 4.28 MPC-3
(t, J = 5.1 Hz, 1H), 3.73 (s, 2H), 3.59–3.36 (m, 4H), 3.26 (t, J = Compound α-4Ac-Man-CEP was prepared according to
6.4 Hz, 2H), 1.74–1.61 (m, 2H), 1.55 (s, 3H), 1.52–1.40 (m, published procedures [22] (for more information see Support-
4H), 1.24 (s, 19H), 0.84 (d, J = 6.5 Hz, 3H); 13C NMR (DMSO- ing Information File 1).
d6, 75 MHz, δ) 132.41, 127.51, 99.47, 79.67, 76.21, 69.19,
63.78, 59.24, 37.01, 33.97, 30.68, 29.63, 29.47, 29.42, 29.18, α-4Ac-Man-CEP (252 mg, 0.46 mmol, 1.1 equiv) and ETT
28.81, 28.06, 26.85, 26.19, 25.00, 24.92, 19.86, 19.09, 14.06, (82 mg, 0.63 mmol, 1.5 equiv) were dissolved in anhydrous
13.99.
DCM (1 mL) and Cit-BZP-yne (170 mg, 0.42 mmol, 1 equiv,
dissolved in 1.5 mL anhydrous DCM) was added. The reaction
mixture was stirred for 30 min at rt under argon. Afterwards, a
MPC-2
ETT (38 mg, 0.29 mmol, 1.5 equiv) was dissolved in an- t-BuOOH solution (182 μL, 1.31 mmol, 3 equiv, 70 wt % in
hydrous DCM (2 mL), β-4Ac-Man-CEP (116 mg, 0.21 mmol, H2O) was added, and the solution was stirred for an additional
1.1 equiv) was added, and the resultant solution was added to 15 min. The reaction mixture was diluted with toluene (15 mL)
Cit-Dod-NBD (100 mg, 0.19 mmol, 1 equiv) dissolved in an- and poured into a separating funnel containing a saturated sodi-
hydrous DCM (1 mL). The reaction mixture was stirred for um hydrogen carbonate solution (10 mL). The organic phase
30 min at rt under argon. Afterwards, a t-BuOOH solution was washed with brine, dried with MgSO4, and the volatile
(110 μL, 0.61 mmol, 3 equiv, ≈5.5 M in decane) was added, and components were removed under reduced pressure. The result-
the mixture was stirred for an additional 15 min. The reaction ing material was then dissolved in DCM and purified by flash
mixture was poured into a separating funnel containing DCM chromatography on silica gel using ethyl acetate/n-hexane 7:3,
(15 mL) and a saturated sodium hydrogen carbonate solution v/v as eluent. The fractions containing the intermediate α-4Ac-
(15 mL). The organic layer was further washed with brine Man-P-Cit-BZP-yne (Rf = 0.33 using the same solvent system
(15 mL) and then dried with MgSO4. The volatile components as for the column) were combined, and the solvents were re-
were removed under reduced pressure, the brownish residue moved under reduced pressure on a rotary evaporator. To the
was redissolved in acetonitrile (1.5 mL) and purified by PLC white residue, an ammonia solution (4 mL, 2.0 M in methanol)
(using a mixture of ethyl acetate/n-hexane 1:1, v/v as the mobile was added, and the reaction mixture was stirred overnight at rt.
phase). The broad band located at the very bottom of the plate Afterwards, the volatile components were removed under
(Rf ≈ 0.10 using ethyl acetate/n-hexane 1:1, v/v) was removed, reduced pressure, the residue was dissolved in methanol (2 mL),
1737

Beilstein J. Org. Chem. 2020, 16, 1732–1739.
5. Orlean, P.; Menon, A. K. J. Lipid Res. 2007, 48, 993–1011.
and the material was purified by PLC (using chloroform/metha-
nol/water 60:25:4, v/v/v as the mobile phase). The broad band,
centered at around Rf ≈ 0.19, was removed, and the product was
extracted with methanol (100 mL). The solvent was removed
under reduced pressure on a rotary evaporator to give MPC-3
(65 mg, 0.10 mmol, 24%) as white solid. 1H NMR (400 MHz,
DMSO-d6, δ) 7.75–7.64 (m, 4H), 7.16–7.04 (m, 4H), 5.58 (t,
J = 7.2 Hz, 1H), 5.17 (dd, J = 7.8, 1.9 Hz, 1H), 4.91 (d, J =
2.4 Hz, 2H), 4.88–4.87 (m, 3H), 4.76–4.71 (m, 2H), 4.50 (s,
2H), 3.77–3.63 (m, 2H), 3.63–3.51 (m, 4H), 3.42–3.23 (m, 2H),
doi:10.1194/jlr.r700002-jlr200
6. Anand, M.; Rush, J. S.; Ray, S.; Doucey, M.-A.; Weik, J.; Ware, F. E.;
Hofsteenge, J.; Waechter, C. J.; Lehrman, M. A. Mol. Biol. Cell 2001,
12, 487–501. doi:10.1091/mbc.12.2.487
7. Neubert, P.; Strahl, S. Curr. Opin. Cell Biol. 2016, 41, 100–108.
doi:10.1016/j.ceb.2016.04.010
8. Hofsteenge, J.; Müller, D. R.; de Beer, T.; Löffler, A.; Richter, W. J.;
Vliegenthart, J. F. G. Biochemistry 1994, 33, 13524–13530.
doi:10.1021/bi00250a003
9. Rush, J. S.; Waechter, C. J. Biochemistry 2004, 43, 7643–7652.
doi:10.1021/bi036083o
2.04 (m, 2H), 1.69 (s, 3H), 1.60–1.45 (m, 2H), 1.41–1.24 (m, 10.Rush, J. S.; Subramanian, T.; Subramanian, K. L.; Onono, F. O.;
Waechter, C. J.; Spielmann, P. H. Curr. Chem. Biol. 2015, 9, 123–141.
2H), 1.24–1.10 (m, 1H), 0.85 (d, J = 6.4 Hz, 3H); 13C NMR
doi:10.2174/2212796810666160216221610
(101 MHz, DMSO-d6, δ) 193.74, 162.47, 160.84, 132.31,
11.Murale, D. P.; Hong, S. C.; Haque, M. M.; Lee, J.-S. Proteome Sci.
132.15, 131.22, 130.61, 130.26, 129.71, 115.07, 115.03, 96.05,
2016, 15, 14. doi:10.1186/s12953-017-0123-3
95.99, 79.24, 79.14, 74.51, 74.06, 71.46, 71.38, 70.92, 67.64,
12.Smith, E.; Collins, I. Future Med. Chem. 2015, 7, 159–183.
62.81, 62.76, 61.80, 56.17, 37.90, 37.83, 36.75, 29.30, 25.11,
doi:10.4155/fmc.14.152
13.Rush, J. S.; Waechter, C. J. Partial Purification of
Mannosylphosphorylundecaprenol Synthase From Micrococcus
Luteus. In Glycobiology Protocols; Brockhausen, I., Ed.; Methods in
Molecular Biology, Vol. 347; Humana Press: Clifton, NJ, U.S.A., 2006;
pp 13–30. doi:10.1385/1-59745-167-3:13
19.68, 14.16; 31P NMR (121 MHz, DMSO-d6, δ) −2.86.
Supporting Information
14.Rush, J. S.; Shelling, J. G.; Zingg, N. S.; Ray, P. H.; Waechter, C. J.
J. Biol. Chem. 1993, 268, 13110–13117.
Supporting Information File 1
Complete descriptions of the syntheses, including the
precursors THP-Cit-BZP-yne and DMT-Cit-Dod-NBD,
additional information about α-4Ac-Man, and the
analytical data: NMR and HRMS spectra.
15.Dotson, S. B.; Rush, J. S.; Ricketts, A. D.; Waechter, C. J.
Arch. Biochem. Biophys. 1995, 316, 773–779.
doi:10.1006/abbi.1995.1103
16.Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001,
40, 2004–2021.
[https://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-16-145-S1.pdf]
doi:10.1002/1521-3773(20010601)40:11<2004::aid-anie2004>3.0.co;2-
5
17.Beaucage, S. L.; Caruthers, M. H. Tetrahedron Lett. 1981, 22,
1859–1862. doi:10.1016/s0040-4039(01)90461-7
18.Ogawa, T.; Seta, A. Carbohydr. Res. 1982, 110, c1–c4.
doi:10.1016/0008-6215(82)85040-4
Funding
Financial support by the Swiss National Science Foundation
(SNSF) is gratefully acknowledged: This research was funded
by the Sinergia Project: Molecular identification of lipid trans-
porters for protein glycosylation, Grant CRSII5_170923.
19.Majumdar, D.; Elsayed, G. A.; Buskas, T.; Boons, G.-J. J. Org. Chem.
2005, 70, 1691–1697. doi:10.1021/jo048443z
20.Rutschow, S.; Thiem, J.; Kranz, C.; Marquardt, T. Bioorg. Med. Chem.
2002, 10, 4043–4049. doi:10.1016/s0968-0896(02)00269-9
21.Hara, R. I.; Kobayashi, S.; Noro, M.; Sato, K.; Wada, T. Tetrahedron
2017, 73, 4560–4565. doi:10.1016/j.tet.2017.06.015
22.Elsayed, G. A.; Boons, G.-J. Synlett 2003, 1373–1375.
doi:10.1055/s-2003-40344
ORCID® iDs
Oleg Khorev - https://orcid.org/0000-0001-6128-3831
Peter Bütikofer - https://orcid.org/0000-0001-9360-1013
Anant K. Menon - https://orcid.org/0000-0001-6924-2698
Robert Häner - https://orcid.org/0000-0001-5014-4318
23.Delbianco, M.; Bharate, P.; Varela-Aramburu, S.; Seeberger, P. H.
Chem. Rev. 2016, 116, 1693–1752. doi:10.1021/acs.chemrev.5b00516
24.Nikolaev, A. V.; Ivanova, I. A.; Shibaev, V. N. Carbohydr. Res. 1993,
242, 91–107. doi:10.1016/0008-6215(93)80024-9
25.Hermans, J. P. G.; de Vroom, E.; Elie, C. J. J.; van der Marel, G. A.;
van Boom, J. H. Recl. Trav. Chim. Pays-Bas 1986, 105, 510–511.
doi:10.1002/recl.19861051108
References
1. Rush, J. S.; Waechter, C. J. J. Cell Biol. 1995, 130, 529–536.
26.Naundorf, A.; Natsch, S.; Klaffke, W. Tetrahedron Lett. 2000, 41,
189–192. doi:10.1016/s0040-4039(99)02020-1
doi:10.1083/jcb.130.3.529
2. Sanyal, S.; Menon, A. K. Proc. Natl. Acad. Sci. U. S. A. 2010, 107,
11289–11294. doi:10.1073/pnas.1002408107
27.Nikolaev, A. V.; Ivanova, I. A.; Shibaev, V. N.; Kochetkov, N. K.
Carbohydr. Res. 1990, 204, 65–78. doi:10.1016/0008-6215(90)84021-l
28.Sharipova, R. R.; Garifullin, B. F.; Sapunova, A. S.; Voloshina, A. D.;
Kravchenko, M. A.; Kataev, V. E. Russ. J. Bioorg. Chem. 2019, 45,
155–164. doi:10.1134/s1068162019020110
3. Schenk, B.; Fernandez, F.; Waechter, C. J. Glycobiology 2001, 11,
61R–70R. doi:10.1093/glycob/11.5.61r
4. Sanyal, S.; Menon, A. K. ACS Chem. Biol. 2009, 4, 895–909.
doi:10.1021/cb900163d
1738

Beilstein J. Org. Chem. 2020, 16, 1732–1739.
29.Garegg, P. J.; Hansson, J.; Helland, A.-C.; Oscarson, S.
Tetrahedron Lett. 1999, 40, 3049–3052.
doi:10.1016/s0040-4039(99)00362-7
30.Balcom, B. J.; Petersen, N. O. Biophys. J. 1993, 65, 630–637.
doi:10.1016/s0006-3495(93)81106-8
31.Chattopadhyay, A. Chem. Phys. Lipids 1990, 53, 1–15.
doi:10.1016/0009-3084(90)90128-e
32.Kern, N. R.; Lee, H. S.; Wu, E. L.; Park, S.; Vanommeslaeghe, K.;
MacKerell, A. D., Jr.; Klauda, J. B.; Jo, S.; Im, W. Biophys. J. 2014,
107, 1885–1895. doi:10.1016/j.bpj.2014.09.007
33.McIntyre, J. C.; Sleight, R. G. Biochemistry 1991, 30, 11819–11827.
doi:10.1021/bi00115a012
34.Chang, Q.-l.; Gummadi, S. N.; Menon, A. K. Biochemistry 2004, 43,
10710–10718. doi:10.1021/bi049063a
35.Bonner, W. A. J. Am. Chem. Soc. 1958, 80, 3372–3379.
doi:10.1021/ja01546a045
36.Warren, C. D.; Liu, I. Y.; Herscoves, A.; Jeanloz, R. W. J. Biol. Chem.
1975, 250, 8069–8078.
37.Wu, Y.-W.; Alexandrov, K.; Brunsveld, L. Nat. Protoc. 2007, 2,
2704–2711. doi:10.1038/nprot.2007.401
38.Lee, B.; Sun, W.; Lee, H.; Basavarajappa, H.; Sulaiman, R. S.;
Sishtla, K.; Fei, X.; Corson, T. W.; Seo, S.-Y. Bioorg. Med. Chem. Lett.
2016, 26, 4277–4281. doi:10.1016/j.bmcl.2016.07.043
39.Ren, T.; Zhang, G.; Liu, D. Tetrahedron Lett. 2001, 42, 1007–1010.
doi:10.1016/s0040-4039(00)02221-8
40.Yu, B.; van Ingen, H.; Vivekanandan, S.; Rademacher, C.;
Norris, S. E.; Freedberg, D. I. J. Magn. Reson. 2012, 215, 10–22.
doi:10.1016/j.jmr.2011.09.037
41.Kotowycz, G.; Lemieux, R. U. Chem. Rev. 1973, 73, 669–698.
doi:10.1021/cr60286a004
42.Bock, K.; Pedersen, C. J. Chem. Soc., Perkin Trans. 2 1974, 293–297.
doi:10.1039/p29740000293
License and Terms
This is an Open Access article under the terms of the
Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0). Please note
that the reuse, redistribution and reproduction in particular
requires that the authors and source are credited.
The license is subject to the Beilstein Journal of Organic
Chemistry terms and conditions:
(https://www.beilstein-journals.org/bjoc)
The definitive version of this article is the electronic one
which can be found at:
doi:10.3762/bjoc.16.145
1739