Synthesis of monophosphoryl lipid A using 2-naphtylmethyl ethers as permanent protecting groups

Article abstract of DOI:10.1016/j.carres.2020.108152

Lipid A, which is a conserved component of lipopolysaccharides of gram-negative bacteria, has attracted considerable interest for the development of immuno-adjuvants. Most approaches for lipid A synthesis rely on the use of benzyl ethers as permanent protecting groups. Due to the amphiphilic character of lipid A, these compounds aggregate during the hydrogenation step to remove benzyl ethers, resulting in a sluggish reaction and by-product formation. To address this problem, we have developed a synthetic approach based on the use of 2-naphtylmethyl ether (Nap) ethers as permanent protecting group for hydroxyls. At the end of a synthetic sequence, multiple of these protecting groups can readily be removed by oxidation with 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ). Di-allyl N,N-diisopropylphosphoramidite was employed to install the phosphate ester and the resulting allyl esters were cleaved using palladium tetrakistriphenylphosphine. The synthetic strategy allows late stage introduction of different fatty acids at the amines of the target compound, which is facilitated by Troc and Fmoc as orthogonal amino-protecting groups.

Full text of DOI:10.1016/j.carres.2020.108152

Journal Pre-proof
Synthesis of monophosphoryl lipid A using 2-naphtylmethyl ethers as permanent
protecting groups
Enrico C.J.M. Verpalen, Arwin J. Brouwer, Geert-Jan Boons
PII:
S0008-6215(20)30523-1
DOI:
https://doi.org/10.1016/j.carres.2020.108152
CAR 108152
Reference:
To appear in: Carbohydrate Research
Received Date: 7 August 2020
Revised Date: 7 September 2020
Accepted Date: 9 September 2020
Please cite this article as: E.C.J.M. Verpalen, A.J. Brouwer, G.-J. Boons, Synthesis of monophosphoryl
lipid A using 2-naphtylmethyl ethers as permanent protecting groups, Carbohydrate Research, https://
doi.org/10.1016/j.carres.2020.108152.
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Synthesis of monophosphoryl lipid A using 2-naphtylmethyl ethers as
permanent protecting groups
Enrico C.J.M. Verpalena, Arwin J. Brouwera, Geert-Jan Boonsa,b,*
a Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical
Sciences, and Bijvoet Center for Biomolecular Research, Utrecht University,
Universiteitsweg 99, 3584 CG Utrecht, The Netherlands.
b
Complex Carbohydrate Research Center and Department of Chemistry, University of
Georgia, Athens, GA 30602, USA.
Keywords: lipid A, adjuvant, protecting groups, glycosylation
ABSTRACT
Lipid A, which is a conserved component of lipopolysaccharides of gram-negative bacteria,
has attracted considerable interest for the development of immuno-adjuvants. Most
approaches for lipid A synthesis rely on the use of benzyl ethers as permanent protecting
group. Due to the amphiphilic character of lipid A, these compounds aggregate during the
hydrogenation step to remove benzyl ethers, resulting in a sluggish reaction and by-product
formation. To address this problem, we have developed a synthetic approach based on the use
of 2-naphtylmethyl ether (Nap) ethers as permanent protecting group for hydroxyls. At the
end of a synthetic sequence, multiple of these protecting groups can readily be removed by
oxidation with 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ). Di-allyl N,N-
diisopropylphosphoramidite was employed to install the phosphate ester and the resulting
allyl esters could van cleaved using palladium tetrakistriphenylphosphine. The synthetic
strategy allows late stage of the introduction of different fatty acids at the amines of the target
compound, which is facilitated by Troc and Fmoc as orthogonal amino-protecting groups.
1. Introduction
Adjuvants, which are molecules that can augment adaptive immune responses, can improve
vaccine performance [1-3]. The use of an adjuvant is particularly important for subunit
vaccines, such as proteins and polysaccharides, which often have reduced immunogenicity
because of a lack of lack of intrinsic immunostimulatory activity [4].
1

Advances in the understanding of innate immune responses has providing opportunities
to design better adjuvants. The innate immune system senses microbes through pattern-
recognition receptors, which include the Toll-like receptors (TLRs) and C-type lectin-like
receptors (CTRs) that are expressed by immune cells such as dendritic, neutrophiles and B-
cells. Activation of these receptors leads to the production of cytokines that provide early
defence during infection. Cytokines also regulate adaptive immunity by controlling the
quantity and quality of B- and T-cell activation, which in turn results in protective immune
responses [5]. Ligands for TLRs and CTRs are attractive compounds for the development of
adjuvants. The challenge, however, is to discover compounds that can enhance
immunogenicity without causing adverse effects.
Lipid A, which is a conserved component of lipopolysaccharides (LPS) of gram-negative
bacteria, has attracted considerable interest for the development of adjuvants [4]. It is
recognized by TLR4/MD2 complex of dendritic cells resulting in the activation of two
intracellular signalling cascades, namely the MyD88 and TRIF pathways that result in the
production of (pro)inflammatory mediators [5,6]. The lipid A moiety of E. coli consists of a
hexaacylated bis-1,4'-phosphorylated glucosamine disaccharide that has (R)-3-
hydroxymyristyl residues at C-2, C-2’, C-3, and C-3’. The (3)-hydroxyacyl chains of the
distal glucosamine moiety are further modified by lauric and myristic acids (Fig. 1). Lipid A
of S. minnesota has a different acylation pattern, it is hepta-acylated featuring an additional
bi-antennary palmitic acid on the (3)-hydroxyacyl of the C-2 amine. The endotoxic activity of
LPS and lipid A can result to overactivation of the immune system, which has precluded its
use as an adjuvant.
Systematic studies by Ribi and co-workers uncovered that the immunostimulatory
properties of lipid A can be separated from the endotoxic effects by hydrolytic treatment
using acid and base, resulting in a major species having six fatty acids and only one
phosphate moiety at the C-4’ position (Fig. 1) [7,8]. This preparation, which was named
mono-phosphoryl lipid A (MPLA), is employed by GSK Biologicals for several vaccine
formulations [9]. The favourable properties of MPLA probably arise from differences in gene
expression compared to native LPS and lipid A, and it induces mainly cytokines associated
with the TRIF-dependent pathway [10]. It has also been found that MPLA induces higher
levels of IL-10, which has anti-inflammatory properties and may contribute to its low toxicity
[11]. MPLA is structurally heterogeneous which may compromise consistent biological
performance. To address this shortcoming, chemical approaches have been developed for the
preparation of mono-phosphoryl lipid A derivatives [12-18]. In particular, a derivative from
2

the lipid A of E. coli (Fig. 1, MPL) has received considerable attention, and enhances
immunological response against co-administered antigen epitopes [19-23]. Furthermore,
higher Th1 responses are seen which are linked to a longer lasting protection in vaccination
[19,20,24]. Monophosphoryl lipid A has also been used for the development of self-
adjuvating vaccine in which it is covalently attached to an antigen [15,16,25].
O
P
O
P
OH
O
OH
O
HO
HO
HO
HO
O
O
O
O
O
O
O
O
NH
O
NH
O
O
O
O
O
HO
O
O
HO
O
P
O
OH
O
C13H27
C13H27
HN
OH
OH
NH
O
O
C11H23
O
C11H23
O
O
O
O
HO
O
C11H23
HO
O
C11H23
C11H23
HO
C11H23
C11H23
C11H23
HO
C11H23
C11H23
E. coli type
E. coli MPLA
lipid A
R515 (1)
O
HO
O
OH
O
OH
O
HO
P
P
HO
O
HO
O
O
O
O
O
O
O
NH
O
O
NH
O
HO
HO
O
O
O
O
O
HO
OH
O
O
NH
C13H27
C13H27
HN
OH
OH
O
C11H23
O
P
C11H23
O
O
O
O
O
O
C11H23
HO
O
C11H23
C11H23
C11H23
C11H23
O
C11H23
C15H31
O
C11H23
O
S. minnesota type
lipid A
S. minnesota MPLA
R595
C15H31
Fig. 1. Structures of E. coli and S. minnesota lipid A’s and their relevant
monophosphorylated derivatives.
The chemical synthesis of lipid A is challenging especially when an approach is needed
that can readily provide various derivatives [26]. Most approaches for lipid A synthesis rely
on the use of benzyl ethers as a permanent protecting group. Due to the amphiphilic character
of lipid A, these compounds aggregate during the hydrogenation step to remove benzyl
ethers, resulting in a sluggish reaction and by-product formation. In particular, we have
observed that the benzyl ether at C-4 of the reducing GlcN moiety is particularly resistant to
hydrogenation. To address these difficulties, we have employed allyloxycarbonates (Alloc) as
permanent protecting groups for the C-3 and C-4 hydroxy groups of the acceptor for MPL
synthesis [14]. This protecting group can easily be removed by treatment with Pd(PPh3)4
3

without effecting acyloxyacyl- or phosphate esters. MPLA has been synthesized by
employing the N-2,2,2-trichloroethoxycarbonyl (Troc) protecting group for various hydroxyls
and amino groups [12]. In both approaches, several alcohols were still protected as benzyl
ether necessitating a hydrogenation step. Furthermore, Troc and Alloc carbonates are very
base sensitive making compound handling difficult and restrict the range of chemical
manipulations that can be employed. To address these difficulties, we report here a synthesis
approach for monophosphoryl lipid A that is based on the use of 2-naphtylmethyl ether (Nap)
ethers [27-31] as a permanent protecting group that can readily be cleaved by oxidation with
2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) greatly simplifying the final deprotection
step. The approach allows late stage modification of the amines thereby offering the
possibility to easily prepare analogs having different fatty acid substitutions.
2. Results and discussion
We envisaged that E. coli monophosphoryl lipid A (1) can be synthesized from
monosaccharide building blocks 2 and 3 and (R)-3-hydroxymyristic acids 4, 5 and 6 (Fig 2).
The hydroxyls of the monosaccharides and fatty acid that require permanent protection are
modified as Nap ethers. A number of our previous studies have shown that multiple of these
protecting groups can be removed at a late stage of synthesis giving confidence in the
approach [32,33]. The C-3 hydroxyl of 2 and 3 were already modified by a (R)-3-
dodecanoyltetradecanoic and (R)-3-(2-naphthylmethoxy)tetradecanoic ester, respectively
because previous studies had shown that late stage installation of these lipids is challenging
and can lead to by-product formation [34]. The C-2 amine of 3 is protected as a Troc group
because the carbamate can perform neighbouring group participation during glycosylations
thereby providing only 1,2-trans-glycosides. It can easily be removed by Zn in acetic acid to
give a free amine that can then be acylated with 5. The amine of acceptor 3 is protected as
fluorenylmethyloxycarbamate (Fmoc), which can be cleaved under mild basic conditions and
is fully orthogonal with the Troc protecting group allowing selective acylation of the amines.
Finally, the phosphate is protected by allyl esters that can be cleaved by palladium
tetrakistriphenylphosphine (Pd(PPh3)4) , which was expected to be fully compatible with
other functionalities of the target compound.
4

Ph
ONap
O
O
P
N
HO
O
P
OH
O
AllylO
AllylO
HO
HO
O
O
NapO
O
CF3
OTDS
O
O
O
O
O
O
NHTroc
NHFmoc
O
O
O
NH
O
O
O
O
HO
OH
NapO
O
C13H27
C11H23
NH
O
C11H23
2
3
O
C11H23
O
C13H27
O
HO
O
C11H23
O
C11H23
C11H23
HO
R
C11H23
O
O
NapO
C11H23
O
C11H23
1
OH
OH
4. R = C13H27
5. R = C11H23
6
Fig. 2. Monophosphoryl lipid A (1) and building blocks (2-5) for chemical synthesis.
The syntheses of lipids 4-6 are described in the supporting information (Schemes S1 and
S2). Glycosyl donor 2 and acceptor 3 were prepared from common intermediate 7 [35,36].
The preparation of donor 2 started with a Steglich acylation [14] of the C-3 hydroxyl of 7
using N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDC) in the presence of a catalytic
amount of 4-(dimethylamino)pyridine (DMAP) to give, after silica gel column
chromatography, 8 in a yield of 78% (Scheme 1). The naphthylidene acetal of 8 was
regioselectively opened using trifluoromethanesulfonic acid (TfOH) in the presence of
triethylsilane (TES) [37] as a hydride donor to provide 9, having a Nap ether at C-6 and a
hydroxyl at C-4. The hydroxyl of the latter compound was phosphorylated using di-allyl N,N-
diisopropylphosphoramidite in the presence of 1H-tetrazole as the activator, followed by in-
situ of oxidation of the intermediate phosphite by m-chloroperbenzoic acid (mCPBA) to
obtain phosphotriester 10. The anomeric dimethylthexylsilyl (TDS) ether of 10 was removed
in near quantitative yield by treatment with HF in pyridine to obtain hemiacetal 11 which was
converted into 2,2,2-trifluoro-N-phenylacetimidate 2 by reaction with the corresponding
chloride in presence of cesium carbonate [26].
5

Et3SiH
TfOH
CH2Cl2
-78°C
4, EDC
DMAP
CH2Cl2
ONap
O
2-Naph
O
2-Naph
O
O
O
HO
O
O
OTDS
NHTroc
OTDS
NHTroc
OTDS
O
O
HO
(78%)
NHTroc
O
O
O
(72%)
O
7
8
9
C11H23
C11H23
O
O
C13H27
C13H27
2
4
2
4
1) i-Pr2NP(OAllyl)2
1H-tetrazole
CH2Cl2
CF3C(NPh)Cl
Cs2CO3
ONap
O
ONap
O
O
HF/pyr
pyridine
2) m-CPBA
CH2Cl2
AllylO
AllylO
AllylO
AllylO
P
P
O
O
O
CH2Cl2
OTDS
NHTroc
OH
O
O
2
O
O
O
(70%)
(98%)
NHTroc
(99%)
O
10
11
C11H23
C11H23
O
O
C13H27
C11H23
Scheme 1. Preparation of glycosyl donor 2.
The preparation of glycosyl acceptor 3 started by a Steglich acylation [14] of common
intermediate 7 using 2-naphthylmethylether protected lipid 6 to provide compound 12
(Scheme 2). The Troc protecting group of 12 was replaced by an Fmoc group involving a
two-step procedure entailing reductive removal of the Troc group, followed by coupling of 9-
fluorenylmethyloxycarbonyl chloride to the resulting amine to give 14 in a yield of 79%.
Glycosyl acceptor 3 was obtained after selective opening of the naphthylidene acetal of 14
using dichlorophenylborane in the presence of TES [38,39].
6
EDC
DMAP
CH2Cl2
Et3SiH
BPhCl2
CH2Cl2
-78°C
2-Naph
O
2-Naph
O
O
O
O
O
3
OTDS
NHTroc
OTDS
O
HO
(77%)
NHR
(63%)
O
7
NapO
C11H23
12. R = Troc
13. R = H
14. R = Fmoc
Zn, THF/AcOH
Fmoc-Cl, DIPEA, CH2Cl2
(79% over 2 steps)
Scheme 2. Preparation of glycosyl acceptor 3.
Glycosylation of donor 2 with acceptor 3 using TfOH as the promoter, afforded β-linked
disaccharide 15 in 73% yield (Scheme 3). The Troc-protecting group was removed using
standard conditions. Next, several attempts were made to introduce acylated lipid 5, which
6

turned out to be challenging, and low yields were obtained by for example using asymmetric
anhydride and HATU/DIPEA mediated coupling reaction conditions (≤25%). Under these
reaction conditions, lipid migration and residual acetyl coupling was observed. Gratifyingly,
16 was be obtained in a yield of 61% by pre-activation of lipid 5 with
dicyclohexylcarbodiimide (DCC) and K-OxymaPure [40], followed by addition of the
primary amine. Under these conditions, no migratory by-products were observed. Next, the
Fmoc protecting group of 16 was removed by treatment with DBU and the resulting amine
was efficiently acylated with 6 that was pre-activated with DCC and K-OxymaPure, to give
fully protected MPL 17 in 70% yield. Deprotection of 17 was performed by three-step
procedure entailing cleavage of the anomeric TDS group using HF-pyridine complex in THF
followed by removal of the allyl groups using Pd(PPh3)4. Finally, the Nap ethers were
cleaved by oxidation with DDQ in chloroform. DDQ mediated oxidation is usually
performed in a mixture of CH2Cl2 and aqueous buffer, however chloroform gave a cleaner
reaction profile and resulted in a facile purification. MPL (1) was obtained in 75% yield over
the three deprotection steps after purification by using size exclusion chromatography over
1
Sephadex LH20. The interpretation H and gHSQC spectra shows a clear indication that
target compound 1 was obtained, this was confirmed by high resolution MS. 31P NMR
confirmed the presence of only one phosphate group.
O
P
1) Zn, THF/AcOH
2) 5, K-OxymaPure
DCC, CH2Cl2
ONap
O
ONap
O
TfOH
CH2Cl2
-30°C
AllylO
AllylO
O
AllylO
AllylO
P
O
O
O
O
O
O
O
2
+ 3
O
NH
O
NHTroc
NapO
(61%)
OTDS
O
(73%)
O
O
NapO
O
OTDS
NHFmoc
O
O
2
8
C13H27
O
O
NHFmoc
O
C11H23
O
O
C11H23
C13H27
O
NapO
C11H23
NapO
C11H23
C11H23
C11H23
15
16
1) HF, pyr, THF
2) Pd(PPh3)4, PPh3
Et3N, HCOOH
O
P
ONap
O
1) DBU, CH2Cl2
2) 6, K-OxymaPure
DCC, CH2Cl2
AllylO
AllylO
O
O
3) DDQ, CHCl3
O
1
O
NH
O
O
(70%)
(75% over 3 steps)
O
NapO
O
OTDS
O
C13H27
NH
O
C11H23
O
O
NapO
C11H23
C11H23
O
C11H23
NapO
C11H23
17
Scheme 3. Assembly and deprotecting of MPLA (1).
7

3. Conclusion
In conclusion, we have developed an efficient synthetic approach for the preparation of
monophosphoryl lipid A derived from E .coli. A key feature of the approach is the use of Nap
ethers as permanent protecting group for hydroxyls that could readily be cleaved at the final
step of deprotection by oxidation with DDQ. The use of di-allyl N,N-
diisopropylphosphoramidite for installation of the phosphate ester ensured that no
hydrogenation step was required for deprotection. The synthetic strategy is convergent and
allows at a late stage of synthesis the introduction of different fatty acids at the amines of the
target compound. The latter is facilitated by the selection of Troc and Fmoc as orthogonal
amino-protecting groups.
4. Experimental section
General synthetic methods. Unless stated otherwise, all reagents were purchased from
Sigma-Aldrich and Fischer Scientific. Carbohydrates were purchased from Carbosynth
Limited (UK). Petroleum ether (boiling range 40-60 °C) was purchased from Biosolve BV
(The Netherlands). Organic solvents for reactions were dried for at least 2 days over
molecular sieves (3 or 4 Å). 1H and 13C NMR spectra were recorded on either an Agilent
400 instrument (400 and 101 MHz) or a Bruker Avance Neo 600 spectrometer (600 and 125
MHz). Chemical shifts are reported in parts per million (ppm) relative to TMS (0.00 ppm for
1H NMR), MeOD (3.31 ppm for 1H NMR, 49.2 ppm for 13C NMR) or CDCl3 (77.0 ppm for
13C NMR) as the internal standard. NMR data are presented as follows: Chemical shift,
multiplicity (s = singlet, d = doublet, 2d = 2 doublets, t = triplet, dd = doublet of doublet, sept
= septet, m = multiplet and/or multiple resonances); coupling constants are reported in Hertz
(Hz). All NMR signals were assigned on the basis of 1H NMR, COSY and HSQC
experiments. Signals marked with L1 and L2 are of the bi-antennary lipids of the C-3’ and C-
2’ respectively, the signals marked as L1’ and L2’ are of their corresponding lipid side chain.
Signals marked with L3 and L4 are of the mono-antennary lipids attached to the C-3 and C-2
respectively. High resolution mass spectra were recorded on an Agilent technologies 6560 Ion
mobility Q-TOF spectrometer. Sephadex LH20 (Sigma Aldrich) column chromatography was
performed using a mixture of CH2Cl2 and MeOH(1:1, v/v) as the eluent. Silica column
chromatography was performed using silica gel SiliaFlash P60 (SiliCycle, Canada, 40-63 µm,
239-400 mesh). TLC analysis was conducted on SiliaPlate TLC Aluminium Backed TLC
F254 (SiliCycle) with examination under UV light (254 nm) where applicable, and with 5%
sulfuric acid in ethanol or an aqueous solution of Ce(NH4)2(NO3)6 and
8

(NH4)6Mo7O24.4H2O (20 g/L and 48 g/L, respectively) with 5% sulfuric acid, followed by
heating. All reactions were carried out under nitrogen gas atmosphere unless when water was
present in the reaction. All reactions were carried out at room temperature (RT) in glassware
with magnetic stirring, unless when stated otherwise.
Dimethylthexylsilyl
2-deoxy-4,6-naphthylidene-3-O-(R)-
(tetradecanoyloxy)tetradecanoate-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-
glucopyranoside (8). To a solution of 7 (3.29 g, 5.06 mmol) and 4 (2.96 g, 4.60 mmol) in
CH2Cl2 (50 mL) were added EDC (1.42 g, 7.40 mmol) and catalytic DMAP (20 mg, 0.2
mmol) and the reaction was stirred for 16 h. The reaction mixture was concentrated under
reduced pressure and the residue was purified by silica gel column chromatography (eluent:
PE/Et2O, 7.5/2.5) to give 8 as a clear colorless oil (3.85 g, 3.59 mmol, 78%) Rf = 0.8
1
(PE/EtOAc, 4/1). H NMR (400 MHz, CDCl3): δ 7.83, 7.49 (2m, 7H, aromatic (Nap)), 5.65
(s, 1H, CH (Nap)), 5.38 (t, 1H, J = 10.0 Hz, H-3), 5.23 (d, 1H, J = 9.0 Hz, N-H), 5.18 (m, 1H,
CHCH2COOC (L1)) 4.92 (d, 1H, J = 7.8 Hz, H-1), 4.77 (d, 1H, J = 12.0 Hz, CH2a-CCl3),
4.60 (d, 1H, J = 12.0 Hz, CH2b-CCl3), 4.35 (dd, 1H, J6a,6b = 10.5 Hz, J6a,5 = 5.0 Hz, H-6a),
3.85 (t, 1H, J6b,6a = 10.5 Hz, J6b,5 = 10.1 Hz, H-6b), 3.76 (t, 1H, J = 9.4, H-4), 3.57 (m, 2H, H-
2, H-5), 2.60 (dd, 1H, Jgem = 15.2 Hz, J = 7.2 Hz, CH2aCOOC (L1)), 2.52 (dd, 1H, Jgem
=
15.2 Hz, J = 5.5 Hz, CH2bCOOC (L1)), 2.12 (t, 2H, J = 7.5 Hz, CH2COOC (L1’)) 1.64 (m,
1H, CH (TDS)) 1.49 (m, 4H, CH2CHCH2COO (L1), CH2CH2COOC(L1’)), 1.20 (m, 38H,
13
19x CH2), 0.86 (m, 18H, 2x CH3, 4x CH3 (TDS)), 0.17, 0.14 (2s, 6H, 2x CH3 (TDS)). C
NMR (101 MHz, CDCl3): δ 173.4, 170.1 (2x C=O), 154.1 (COCH2CCl3), 134.3, 134.3,
133.7, 133.6, 132.8, 132.8, 128.3, 128.1, 128.0, 127.6, 126.4, 126.1, 125.8, 125.6, 123.7,
123.6 (aromatic (Nap)), 101.7 (CH (Nap)), 96.6 (C-1), 95.3 (CCl3), 79.1 (C-4), 74.7 (CH-
2CCl3), 71.2 (C-3), 70.0 (CHCH2COOC (L1)), 68.7 (C-6), 66.4 (C-5), 59.1 (C-2), 39.2
(CH2COOC (L1)), 34.3 (CH2COOC (L1’)), 33.9 (CH2CHCH2COO (L1), 33.9 (CH (TDS)),
31.9, 29.7, 29.7. 29.6, 29.6, 29.4, 29.3, 29.3, 29.3, 29.2, 29.0, 29.0, 25.0, 25.0, 24.9, 24.8,
24.7, 22.7 (20x CH2), 19.9, 19.9, 18.5, 14.1 (4x CH3 (TDS) + CH2CH3), -2.0, -3.4 (2x CH3
(TDS)). HR MS (m/z) calcd for C56H90Cl3NNaO10Si [M + Na]+, 1092.5297; found,
1092.5382.
Dimethylthexylsilyl
2-deoxy-6-O-naphthalen-2-ylmethoxy-3-O-(R)-
(tetradecanoyloxy)tetradecanoate-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-
9

glucopyranoside (9). To a cooled (-78 °C) solution of compound 8 (2.82 g, 2.63 mmol) in
CH2Cl2 (10 mL) was added triethylsilane (2.5 mL, 15.8 mmol). Next, TfOH (1.2 mL, 13.2
mmol) was added dropwise and the resulting reaction mixture was stirred at -78 °C for 0.5 h.
The reaction was quenched with Et3N and MeOH. The reaction mixture was concentrated in
vacuo followed by partitioning in EtOAc and saturated aqueous sodium bicarbonate. The
organic layer was dried with MgSO4, filtered and concentrated. The residue was purified by
column chromatography (PE/Et2O, 7.5/2.5) to give compound 9 (2.05 g, 1.91 mmol, 72%) as
1
a clear oil. Rf = 0.5 (PE/EtOAc, 4/1) H NMR (400 MHz, CDCl3): δ 7.82, 7.48 (2m, 7H,
aromatic (Nap)), 5.14 (m, 2H, CHCH2COOC (L1), NH), 5.01 (t, 1H J = 9.8 Hz, H-3), 4.74
(m, 4H, H-1, CH2a-CCl3 CH2 (Nap)), 4.58 (d, 1H, J = 11.8 Hz, CH2b-CCl3), 3.81 (m, 2H,
CH2-6), 3.70 (m, 1H, H-4), 3.57 (m, 2H, H-2, H-5), 3.35 (d, 1H, J = 2.5, OH), 2.54 (m, 2H,
CH2COOC (L1)), 2.28 (t, 2H, J = 7.5 Hz, CH2COOC (L1’)), 1.60 (m, 5H, CH (TDS),
CH2CHCH2COO (L1), CH2CH2COOC (L1’)), 1.27 (m, 38H, 19x CH2), 0.86 (m, 18H,
2xCH3, 4x CH3 (TDS)), 0.19, 0.14 (2s, 6H, 2x CH3 (TDS)). 13C NMR (101 MHz, CDCl3): δ
174.3, 171.7 (2x C=O) 154.2 (COCH2CCl3), 135.5, 135.2, 133.2, 133.2, 133.0, 133.0, 128.2,
128.1, 127.8, 127.7, 127.7 126.4, 126.2, 126.1, 126.1, 125.9, 125.8, 125.5 (aromatic (Nap)),
96.3 (C-1), 95.4 (CCl3), 76.0 (C-3), 74.9, 74.7, 74.6, 74.6, 74.2 (CH2CCl3 + C-5), 73.8, 73.7
(CH2 (Nap)), 70.9 (CHCH2COOC (L1)), 70.4 (C-4), 70.1, 70.0 (C-6), 57.8 (C-2), 40.0
(CH2COOC (L1)), 34.5 (CH2COOC (L1’)), 33.9 (CH (TDS)), 34.3, 31.9, 31.9, 29.7, 29.7.
29.6, 29.6, 29.6, 29.4, 29.3, 29.3, 29.3, 29.2, 29.1, 29.0, 25.1, 24.9, 24.8, 22.7 (20x CH2),
20.0, 18.5, 14.1 (4x CH3 (TDS) + CH2CH3), -1.8, -3.4 (2x CH3 (TDS)). HR MS (m/z) calcd
for C56H92Cl3NNaO10Si [M + Na]+, 1094.5454; found, 1094.5486.
Dimethylthexylsilyl 2-deoxy-4-O-di-O-allylphosphate-6-O-(naphthalen-2-ylmethyl)-3-O-
(R)-(tetradecanoyloxy)tetradecanoate-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-
glucopyranoside (10). 1H-tetrazole (669 mg, 9.55 mmol) was added to a solution of 9 (2.05
g, 1.91 mmol) under an atmosphere argon. Di-allyl diisopropylphosphoramidite (1.08 mL,
4.08 mmol) was added dropwise and the resulting reaction mixture was stirred at RT for 16 h.
The reaction mixture was cooled (-40 °C) and m-CPBA (396 mg, 2.29 mmol) was added.
After the addition, the reaction mixture was allowed to warm up to RT and stirring was
continued for 1 h. The reaction mixture was concentrated and the crude was purified by silica
gel column chromatography (PE/Et2O, 7/3) to give 10 as a clear colorless oil (1.65 g, 1.34
1
mmol, 70%) Rf = 0.6 (PE/EtOAc, 4/1). H NMR (400 MHz, CDCl3): δ 7.81, 7.47 (2m, 7H,
10

aromatic (Nap)), 5.82 (m, 2H, 2x CH2=CH), 5.38 (t, 1H, J = 9.8 Hz, H-3), 5.24 (m, 6H,
CHCH2COOC (L1), NH, 2x CH2=CH), 4.97 (d, 1H, J = 7.9 Hz, H-1), 4.74 (m, 3H, CH2a-
CCl3, CH2 (Nap)), 4.62 (d, 1H, J = 12.0 Hz, CH2b-CCl3), 4.41 (m, 5H, 2x CH2-CH=CH2, H-
4), 3.84 (dd, 1H, J6a,6b = 10.9 Hz, J = 1.6 Hz, H-6a), 3.71 (m, 2H, H-6b, H-5), 3.44 (m, 1H, H-
2), 2.61 (m, 2H, CH2COOC (L1)), 2.28 (t, 2H, J = 7.5 Hz, CH2COOC (L1’)), 1.62 (m, 5H,
CH (TDS), CH2CHCH2COO (L1), CH2CH2COOC (L1’)), 1.27 (m, 38H, 19x CH2), 0.86 (m,
18H, 2x CH3, 4x CH3 (TDS)), 0.20, 0.14 (2s, 6H, 2x CH3 (TDS)). 13C NMR (101 MHz,
CDCl3): δ 173.5, 170.3 (2x C=O) 153.9 (COCH2CCl3), 135.7, 133.2, 133.0, 132.3, 132.2,
132.0, 128.1, 127.8, 127.7, 126.2, 126.0, 125.8, 125.6 (aromatic (Nap) + CH=CH2), 118.5,
118.4 (2x CH2=CH), 95.5 (C-1), 95.3 (CCl3), 74.6 (CH2CCl3), 74.1 (C-4), 74.1 (C-5), 73.6
(CH2 (Nap)), 72.5 (C-3), 70.0 (CHCH2COOC (L1)), 68.7, 68.6 (C-6), 68.4, 68.4 (CH2-
CH=CH2), 58.6 (C-2), 39.6 (CH2COOC (L1)), 34.5 (CH2COOC (L1’)), 34.2
(CH2CHCH2COO (L1) 34.0 (CH (TDS)), 31.9, 29.6, 29.6. 29.5, 29.3, 29.2, 25.2, 25.0, 24.8,
22.7 (20x CH2), 20.0, 20.0, 18.5, 14.1 (4x CH3 (TDS) + CH2CH3), -1.9, -3.4 (2x CH3 (TDS)).
31P NMR (162 MHz, CDCl3): δ -2.0. HR MS (m/z) calcd for C62H105Cl3N2O13PSi [M +
NH4]+, 1249.6189; found, 1249.6182.
2-deoxy-4-O-di-O-allylphosphate-6-O-(naphthalen-2-ylmethyl)-3-O-(R)
(tetradecanoyloxy)tetradecanoate-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-
glucopyranose (11). To a solution of compound 10 (800 mg, 648 µmol) in pyridine (16 mL)
was added HF-pyridine (1.15 mL, 45.6 mmol). The reaction mixture was stirred for 16 h,
after it was poured in water (200 mL) and stirring was continued for 2 h. The resulting
precipitate was filtered off and dried in vacuo to give 11 as a white amorphous solid (697 mg,
1
639 µmol, 99%) Rf = 0.5 (PE/EtOAc, 7/3). H NMR (400 MHz, CDCl3): δ 7.78, 7.46 (2m,
7H, aromatic (Nap)), 5.80 (m, 2H, 2x CH2=CH), 5.65 (d, 1H, J = 9.1 Hz, NH), 5.24 (m, 7H,
H-3, CHCH2COOC (L1), H-1, 2x CH2=CH), 4.80 (d, 1H, J = 12.2 Hz, CH2a (Nap)), 4.67 (m,
3H, CH2-CCl3, CH2b (Nap)), 4.37 (m, 5H, 2x CH2-CH=CH2, H-4), 4.22 (m, 1H, H-5), 4.04
(bs, 1H, OH), 3.97 (m, 1H, H-2), 3.76 (m, 2H, CH2-6), 2.64 (dd, 1H, Jgem = 16.1 Hz,
CH2aCOOC (L1)), 2.55 (dd, 1H, Jgem = 16.1 Hz, CH2bCOOC (L1)), 2.24 (t, 2H, J = 7.6 Hz,
CH2COOC (L1’)), 1.56 (m, 4H, CH2CHCH2COO (L1), CH2CH2COOC(L1’)), 1.27 (m, 38H,
19x CH2), 0.88 (t, 6H, J = 6.8 Hz, 2x CH3). 13C NMR (101 MHz, CDCl3): δ 173.4, 170.7 (2x
C=O) 154.3 (COCH2CCl3), 135.1, 133.2, 133.0, 132.2, 132.2, 132.0, 132.0, 129.4, 128.2,
127.9, 127.7, 126.7 126.3, 126.1, 125.9, 125.8, 120.5 (aromatic (Nap) + CH=CH2), 118.7,
11

118.4 (2x CH2=CH), 95.3 (CCl3), 91.5 (C-1), 74.6 (CH2CCl3), 73.8 (C-4), 73.6 (CH2 (Nap)),
70.8 (C-3), 69.9 (CHCH2COOC (L1)), 69.5 (C-5), 68.7, 68.6, 68.5, 68.4, 68.3 (C-6, CH2-
CH=CH2), 54.3 (C-2), 39.1 (CH2COOC (L1)), 34.4 (CH2COOC (L1’)), 34.1
(CH2CHCH2COO (L1) 31.9, 29.7, 29.6, 29.6, 29.6, 29.5, 29.5, 29.4, 29.3 29.3, 29.2, 25.2,
31
25.0, 22.7 (20x CH2), 14.1 (CH2CH3). P NMR (162 MHz, CDCl3): δ -1.9. HR MS (m/z)
calcd for C54H87Cl3N2O13P [M + NH4]+, 1107.5011; found, 1107.5003.
2,2,2-trifluoro-N-phenylacetimidoyl 2-deoxy-4-O-di-O-allylphosphate-6-O-(naphthalen-
2-ylmethyl)-3-O-(R)-(tetradecanoyloxy)tetradecanoate-2-(2,2,2-
trichloroethoxycarbonylamino)-β-D-glucopyranoside (2). To a solution of 11 (670 mg,
614 µmol) in CH2Cl2 (10 mL) were added 2,2,2-trifluoro-N-phenyl acetimidoyl chloride (0.6
mL, 4.40 mmol) and cesium carbonate (400 mg, 1.23 mmol). After stirring at RT for 1 h, the
reaction mixture was directly loaded on a silica gel column (PE/Et2O, 9/1 – 4/1) to give 2 as a
clear colorless oil (756 mg, 599 µmol, 98%). 1H NMR (400 MHz, CDCl3): δ 7.81, 7.48 (2m,
7H, aromatic (Nap)), 7.15, 6.74 (m, d, 5H, aromatic (NPh)), 5.83 (m, 2H, 2x CH2=CH), 5.67
(d, 1H, JNH,2 = 8.2 Hz, NH), 5.26 (m, 6H, H-3, CHCH2COOC (L1), 2x CH2=CH), 4.70 (m,
5H, CH2-CCl3, CH2 (Nap), H-4), 4.43 (m, 4H, 2x CH2-CH=CH2), 4.19 (m, 1H, H-2), 4.02
(m, 1H, H-5), 3.76 (m, 2H, CH2-6), 2.62 (m, 2H, CH2COOC (L1)), 2.27 (m, 2H, J = 7.6 Hz,
CH2COOC (L1’)), 1.61 (m, 4H, CH2CHCH2COO (L1), CH2CH2COOC(L1’)), 1.25 (m, 38H,
19x CH2), 0.88 (t, 6H, J = 6.2 Hz, 2x CH3). 13C NMR (101 MHz, CDCl3): δ 173.3, 170.0 (2x
C=O) 154.3 (COCH2CCl3), 135.3, 133.2, 133.0, 128.7, 128.1, 127.9, 127.6, 126.5, 126.4,
126.1, 125.9, 125.7, (aromatic (Nap)), 132.1 (CH=CH2), 118.6 (CH=CH2), 74.7 (CH2CCl3),
73.6 (CH2 (Nap)), 72.8 (C-4), 72.3 (C-5), 70.3 (C-3), 69.9 (CHCH2COOC (L1)), 68.8, 68.7,
68.6, 68.5 (CH2-CH=CH2) 67.7 (C-6), 53.7 (C-2), 39.2 (CH2COOC (L1)), 34.4 (CH2COOC
(L1’)) 34.2 (CH2CHCH2COO (L1), 31.9, 29.6, 29.6, 29.3, 29.2, 25.2, 25.0, 25.0, 22.7 (20x
CH2), 14.1 (CH2CH3). 19F NMR (376 MHz, CDCl3): δ 65.5. The anomeric carbon and proton
could not be assigned due to signal overlap.
Dimethylthexylsilyl
2-deoxy-4,6-naphthylidene-3-O-(R)-((naphthalen-2-
ylmethyl))tetradecanoate-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranoside
(12). To a solution of 7 (5.19 g, 8.00 mmol) and 6 (2.80 g, 7.28 mmol) in CH2Cl2 (50 mL)
were added EDC (2.51 g, 13.1 mmol) and catalytic DMAP (20 mg, 0.2 mmol) and the
reaction was stirred for 16 h. The reaction mixture was concentrated under reduced pressure
12

and the residue was purified by silica gel column chromatography (eluent: PE/Et2O, 7.5/2.5)
1
to give 13 as a clear colorless oil (5.60 g, 5.59 mmol, 77%) Rf = 0.8 (PE/EtOAc, 4/1). H
NMR (400 MHz, CDCl3): δ 7.81, 7.69, 7.39 (s, m, m, 14H, aromatic (2x Nap)), 5.52 (s, 1H,
CH (Nap)), 5.40 (t, 1H J = 9.9 Hz, H-3), 5.07 (d, 1H, J= 9.3 Hz, N-H), 4.88 (d, 1H, J = 7.9
Hz, H-1), 4.62 (m, 4H, CH2-CCl3, CH2 (Nap)), 4.33 (dd, 1H, J6a,6b = 10.4 Hz, J6a,5 = 5.0 Hz,
H-6a), 3.82 (m, 2H, H-6b, H-4), 3.72 (t, 1H, J = 9.2 Hz, CHCH2COOC (L3)), 3.64 (m, 1H, H-
2), 3.55 (m, 1H, H-5), 2.69 (dd, 1H, Jgem = 14.9 Hz, J = 6.2 Hz, CH2aCOOC (L3)), 2.51 (dd,
1H, Jgem = 14.9 Hz, J = 5.8 Hz, CH2bCOOC (L3)), 1.61 (m, 1H, CH (TDS)) 1.53 (m, 2H,
CH2CHCH2COO (L3)), 1.21 (m, 18H, 9x CH2), 0.86 (m, 15H, CH3, 4x CH3 (TDS)), 0.17,
0.14 (2s, 6H, 2x CH3 (TDS)). 13C NMR (101 MHz, CDCl3): δ 171.5 (C=O), 154.0
(COCH2CCl3), 135.9, 134.2, 133.6, 133.2, 132.9, 132.8, 128.3, 128.0, 128.0, 127.9, 127.6,
126.3, 126.3, 126.1, 126.0, 125.8, 125.7, 125.7, 123.6 (aromatic (Nap)), 101.7 (CH (Nap)),
96.8 (C-1), 95.3 (CCl3), 79.0 (CHCH2COOC (L3)), 75.5 (C-4), 74.7 (CH2CCl3), 71.2 (CH2
(Nap)), 71.1 (C-3), 68.6 (C-6), 66.6 (C-5), 59.1 (C-2), 39.5 (CH2COOC (L3)) 34.5
(CH2CHCH2COO (L3)) 33.9 (CH (TDS)), 31.9, 29.6, 29.6, 29.3, 25.2, 24.8, 22.7 (9x CH2),
19.9, 18.5, 14.1 (4x CH3 (TDS) + CH2CH3), -1.9, -3.4 (2x CH3 (TDS)). HR MS (m/z) calcd
for C53H72Cl3NNaO9Si [M + Na]+, 1022.3940; found, 1022.3967.
Dimethylthexylsilyl 2-deoxy-2-(9-fluorenylmethoxycarbonylamino)-4,6-naphthylidene-
3-O-(R)-(naphthalen-2-ylmethoxy)tetradecanoate-β-D-glucopyranoside (14). To
a
solution of 13 (5.60 g, 5.59 mmol) in THF/AcOH 4/1 (50 mL) was added Zn (5.46 g, 84.0
mmol) and was stirred for 2 h. After sonication the Zn was filtered off over a celite pad and
afforded after evaporation the crude free amine 14. To a solution of crude 14 in CH2Cl2 (50
mL) were added 9-fluorenylmethyloxycarbonyl chloride (1.74 g, 6.73 mmol) and N,N-
diisopropylethylamine (2.3 mL, 13.4 mmol). The reaction mixture was stirred for 16 h, and
the resulting product was washed with 1 M HCl (aq. 2x 50 mL). The organic layer was dried
with MgSO4, filtered and concentrated in vacuo. Column chromatography (PE/EtOAc, 95/5 –
9/1) resulted in formation of compound 15 over two steps as a pale yellow oil (2.25 g, 2.15
1
mmol, 38%) Rf = 0.7 (PE/EtOAc, 4/1). H NMR (400 MHz, CDCl3): δ 7.50 (m, 22H,
aromatic (2x Nap, Fmoc)), 5.53 (s, 1H, CH (Nap)), 5.43 (t, 1H J = 9.7 Hz, H-3), 4.92 (d, 2H,
J = 5.3 Hz, H-1, NH), 4.63 (d, 1H, J = 11.9 Hz, CH2a (Nap)), 4.50 (d, 1H, J = 11.9 Hz, CH2b
(Nap)), 4.30 (m, 3H, H-6a, CH2 (Fmoc)), 4.18 (m, 1H, CH (Fmoc)), 3.82 (m, 2H, H-6b, H-4),
3.74 (t, 1H, J = 10.0 Hz, CHCH2COOC (L3)), 3.64 (m, 1H, H-2), 3.58 (m, 1H, H-5), 2.69
13

(dd, 1H, Jgem = 14.9 Hz, J = 6.4 Hz, CH2aCOOC (L3)), 2.49 (dd, 1H, Jgem = 14.9 Hz, J = 5.7
Hz, CH2bCOOC (L3)), 1.59 (m, 1H, CH (TDS)), 1.46 (m, 2H, CH2CHCH2COO (L3)), 1.20
(m, 18H, 9x CH2), 0.87 (t, 3H, J = 7.0 Hz, CH3), 0.82 (m, 12H, 4x CH3 (TDS)), 0.15, 0.11
(2s, 6H, 2x CH3 (TDS)). 13C NMR (101 MHz, CDCl3): δ 171.7 (C=O) 154.0 (C=O (Fmoc)),
143.8, 141.2, 135.9, 134.2, 134.2, 133.6, 133.1, 133.1, 132.8, 132.7, 132.7, 129.0, 128.3,
128.3, 128.2, 128.0, 127.9, 127.8, 127.6, 127.0, 126.3, 126.2, 126.0, 125.9, 125.8, 125.6,
125.1, 123.6, 119.9 (aromatic (Nap, Fmoc)), 101.6 (CH (Nap)), 97.0 (C-1), 79.0
(CHCH2COOC (L3)), 75.6 (C-4), 71.3 (CH2 (Nap)), 71.2 (C-3), 68.7 (C-6), 67.2 (CH2
(Fmoc)), 66.5 (C-5), 58.9 (C-2), 47.0 (CH (Fmoc)) 39.7 (CH2COOC (L3)) 34.5
(CH2CHCH2COO (L3)) 33.9 (CH (TDS)), 31.9, 29.6, 29.6, 29.5, 29.3, 25.1, 24.7, 22.7 (9x
CH2), 19.9, 19.8, 18.5, 14.1 (4x CH3 (TDS) + CH2CH3), -1.9, -3.4 (2x CH3 (TDS)). HR MS
(m/z) calcd for C65H81NNaO9Si [M + Na]+, 1070.5578; found, 1070.5570.
Dimethylthexylsilyl 2-deoxy-2-(9-fluorenylmethoxycarbonylamino)-4-O-(naphthalen-2-
ylmethyl)-3-O-(R)-(naphthalen-2-ylmethoxy)tetradecanoate-β-D-glucopyranoside (3).
To a cooled (-78 °C) solution of compound 14 (2.25 g, 2.15 mmol) in dry CH2Cl2 (20 mL)
containing 4Å molecular sieves were added triethylsilane (1.0 mL, 6.26 mmol) and
dichlorophenylborane (1.1 mL, 8.12 mmol) The reaction mixture was stirred at -78 °C for 0.5
h. The reaction was quenched by addition of MeOH and Et3N, and pure glycosyl acceptor 3
was obtained as a pale yellow oil after column chromatography (PE/Et2O, 7/3) (780 mg, 743
1
µmol, 33%) Rf = 0.4 (PE/EtOAc, 4/1). H NMR (400 MHz, CDCl3): δ 7.52 (m, 22H,
aromatic (2x Nap, Fmoc)), 5.32 (t, 1H J = 9.8 Hz, H-3), 4.89 (d, 1H, JNH,2 = 9.1 Hz, NH),
4.80 (d, 1H, J = 7.5 Hz, H-1), 4.72 (q, 2H, Jgem = 11.7 Hz, CH2 (Nap)), 4.57 (q, 2H, Jgem
=
11.8 Hz, CH2 (Nap)), 4.25 (m, 2H, CH2 (Fmoc)), 4.15 (m, 1H, CH (Fmoc)), 3.84 (m, 2H, H-
6a, CHCH2COOC (L3)), 3.70 (m, 2H, H-6b, H-4), 3.60 (m, 1H, H-2), 3.50 (m, 1H, H-5), 2.54
(dd, 1H, Jgem = 15.5 Hz, J = 7.1 Hz, CH2aCOOC (L3)), 2.39 (dd, 1H, Jgem = 15.5 Hz, J = 4.9
Hz, CH2bCOOC (L3)), 1.85 (t, 1H, J = 6.2 Hz, OH), 1.54 (m, 1H, CH (TDS)) 1.44 (m, 2H,
CH2CHCH2COO (L3)), 1.21 (m, 18H, 9x CH2), 0.88 (t, 3H, J = 7.0 Hz, CH3), 0.80 (m, 12H,
13
4x CH3 (TDS)), 0.14, 0.09 (2s, 6H, 2x CH3 (TDS)). C NMR (101 MHz, CDCl3): δ 171.1
(C=O) 155.7 (C=O (Fmoc)), 143.8, 141.2, 135.9, 135.0, 133.2, 133.1, 132.9, 132.9, 128.2,
128.0, 127.9, 127.8, 127.6, 127.6, 127.0, 126.6, 126.2, 126.1, 125.9, 125.9 125.8, 125.7,
125.2, 119.9 (aromatic (Nap, Fmoc)), 96.4 (C-1), 75.7 (CHCH2COOC (L3)), 75.7 (C-4), 75.2
(C-5), 74.7 (C-3), 74.5 (CH2 (Nap)), 71.4 (CH2 (Nap)), 67.2 (CH2 (Fmoc)), 62.0 (C-6), 58.4
14

(C-2), 47.0 (CH (Fmoc)) 39.7 (CH2COOC (L3)) 34.1 (CH2CHCH2COO (L3)), 33.9 (CH
(TDS)), 31.9, 29.6, 29.6, 29.6, 29.5, 29.3, 25.1, 24.8, 22.7, 22.6, 22.3 (9x CH2), 19.9, 18.4
(4x CH3 (TDS)), 14.1 (CH2CH3), -1.8, -3.4 (2x CH3 (TDS)). HR MS (m/z) calcd for
C65H83NNaO9Si [M + Na]+, 1072.5735; found, 1072.5729.
Dimethylthexylsilyl [2-deoxy-4-O-di-O-allylphosphate-6-O-(naphthalen-2-ylmethyl)-3-
O-(R)-(tetradecanoyloxy)tetradecanoate-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-
glucopyranosyl]-(1‰6)-2-deoxy-2-(9-fluorenylmethoxycarbonylamino)-4-O-
(naphthalen-2-ylmethyl)-3-O-(R)-(naphthalen-2-ylmethoxy)tetradecanoate-β-D-
glucopyranoside (15). To a cooled (-30 °C) solution of N-phenyl-trifluoroacetimidate 2 (1.88
g; 1.49 mmol) and acceptor 3 (1.09 g; 1.03 mmol) in dry CH2Cl2 (25 mL) was added acid
washed molecular sieves 4Å (1.09 g). Next, a 100-fold diluted solution of TfOH in dry
CH2Cl2 (1.4 mL, 155 µmol) was added dropwise and the resulting reaction mixture was
stirred at -30 °C for 0.5 h. The reaction mixture was quenched with Et3N. After column
chromatography (PE/EtOAc, 1/0 – 8/2) disaccharide 15 was obtained as a clear yellow oil
(1.59 g; 749 µmol; 73%) Rf = 0.3 (PE/EtOAc, 4/1). 1H NMR (600 MHz, CDCl3): δ 7.53 (m,
29H, aromatic (3x Nap, Fmoc)), 5.78 (m, 2H, 2x CH2=CH), 5.45 (d, 1H, J = 7.0 Hz,
NHTroc), 5.38 (t, 1H, J = 9.8 Hz, H-3’), 5.22 (m, 6H, CHCH2COOC (L1), H3, 2x CH2=CH),
4.81 (m, 2H, NHFmoc, H-1’), 4.69 (m, 7H, CH2-CCl3, 2x CH2 (Nap), H1), 4.57 (m, 3H, CH2
(Nap), H-4’), 4.57 (m, 5H, 2x CH2-CH=CH2, H-5’), 4.24 (m, 2H, CH2 (Fmoc)), 4.14 (d, 1H,
J = 7.3 Hz, CH (Fmoc)), 4.06 (d, 1H, J = 10.4 Hz, H6a), 3.76 (m, 5H, H-6b, CH2-6’, H-4,
CHCH2COOC (L3)), 3.60 (m, 2H, H-2, H-5’), 3.40 (dd, 1H, J2’,NH = 8.8 Hz, H-2’), 2.60 (m,
2H, CH2COOC (L1)), 2.49 (dd, 1H, Jgem = 15.7 Hz, J = 7.1 Hz, CH2aCOOC (L3)), 2.34 (dd,
1H, Jgem = 15.7 Hz, J = 5.2 Hz, CH2bCOOC (L3)), 2.28 (t, 2H, J = 7.5 Hz, CH2COOC (L1’)),
1.57 (m, 7H, CH (TDS), CH2CHCH2COO (L1), CH2CH2COOC(L1’), CH2CHCH2COO
(L3)), 1.27 (m, 56H, 28x CH2), 0.87 (t, 9H, J = 6.9 Hz, 3x CH3), 0.79 (m, 12H, 4x CH3
13
(TDS)), 0.17, 0.12 (2s, 6H, 2x CH3 (TDS)). C NMR (151 MHz, CDCl3): δ 173.7, 171.8,
170.3 (3x C=O) 155.7, 154.0 (2x NCO), 143.8, 141.2, 135.9, 135.6, 135.3, 135.0, 133.2,
133.2, 133.1, 132.9, 132.7, 132.2, 132.1, 132.1, 128.1, 128.0, 127.9, 127.9, 127.9, 127.8,
127.6, 127.6, 127.6, 127.0, 126.2, 126.0, 125.9, 125.9, 125.9, 125.8, 125.7, 125.6, 125.2,
119.9 (aromatic (Nap, Fmoc) + CH=CH2), 118.5, 118.3 (2x CH2=CH), 100.1 (C-1’), 96.4 (C-
1), 95.4 (CCl3), 76.1 (CHCH2COOC (L3)), 75.7 (C-4), 75.2, 74.8, 74.6, 74.4, 74.3, 74.1,
74.1, 74.0, 73.9, 73.6, 73.6 (CH2CCl3, 2x CH2 (Nap), C-3, C-4, C-5, C-4’), 72.4 (C-3’), 71.4
15

(CH2 (Nap)), 70.1 (CHCH2COOC (L1)), 68.6, 68.5, 68.4, 68.3 (C-6, C-6’, CH2-CH=CH2),
67.2 (CH2 (Fmoc)), 58.4 (C-2), 56.8 (C-2’), 47.0 (CH (Fmoc), 39.7 (CH2COOC (L1),
CH2COOC (L3)), 34.5 (CH2COOC (L1’)), 34.4 (CH2CHCH2COO (L1), 34.1
(CH2CHCH2COO (L3)), 33.9 (CH (TDS)), 31.9, 29.6, 29.6, 29.3, 29.2, 25.2, 25.1, 25.0, 24.8,
24.7, 22.7 (28x CH2), 20.0, 19.9, 18.5, 18.4 (4x CH3 (TDS)), 14.1 (CH2CH3), -1.6, -1.8, -3.4,
-3.5 (2x CH3 (TDS)). 31P NMR (162 MHz, CDCl3): δ -2.0, -2.1. HR MS (m/z) calcd for
C119H168Cl3N3O21PSi [M + NH4]+, 2139.0743; found, 2139.0697.
Dimethylthexylsilyl
[2-deoxy-4-O-di-O-allylphosphate-2-N-
(dodecanoyloxy)tetradecanoyl)-6-O-naphthalen-2-ylmethoxy-3-O-(R)-
(tetradecanoyloxy)tetradecanoate-β-D-glucopyranosyl]-(1‰6)-2-(9-
fluorenylmethoxycarbonylamino)-2-deoxy-4-O-(naphthalen-2-ylmethyl)-3-O-(R)-
(naphthalen-2-ylmethoxy)tetradecanoate-β-D-glucopyranoside (16). To a solution of 15
(1.59 g, 749 µmol) in THF/AcOH (7.5 mL, 4/1) was added Zn (3.00 g; 45.9 mmol). The
reaction mixture was stirred for 2 h. After sonication the Zn was filtered off over a celite pad.
The THF/AcOH was evaporated and the crude amine was subsequently partitioned in
saturated sodium bicarbonate (50 mL) and EtOAc (50 mL) to give the free amine. The
organic layer was further washed with brine and dried with MgSO4, filtered and concentrated
in vacuo. To a solution of the crude amine in dry CH2Cl2 (10 mL) were added 5 (766 mg,
1.88 mmol), DCC (370 mg, 1.88 mmol) and K-OxymaPure (324 mg; 1.88 mmol). This
mixture was stirred for 16 h. Compound 16 was obtained as a yellow oil after column
chromatography (PE/EtOAc, 1/0 – 8/2) (1.07 g, 454 µmol, 61% over two steps) Rf = 0.4
1
(PE/EtOAc, 4/1). H NMR (600 MHz, CDCl3): δ 7.49 (m, 29H, aromatic (3x Nap, Fmoc)),
6.17 (d, 1H, J = 7.6 Hz, NH’), 5.77 (m, 2H, 2x CH2=CH), 5.44 (t, 1H, J = 9.4 Hz, H-3’), 5.19
(m, 8H, CHCH2COOC (L1), CHCH2COOC (L2), H-1’, H3, 2x CH2=CH), 4.87 (d, 1H, J =
9.7 Hz, NHFmoc), 4.71 (m, 5H, 2x CH2 (Nap), H1), 4.50 (m, 3H, CH2 (Nap), H-4’), 4.37 (m,
4H, 2x CH2-CH=CH2), 4.22 (m, 2H, CH2 (Fmoc)), 4.12 (d, 1H, J = 7.5 Hz, CH (Fmoc)), 4.06
(d, 1H, J = 10.9 Hz, H6a), 3.83 (m, 2H, H6b, H6a’), 3.76 (t, 1H, J = 6.0 Hz, CHCH2COOC
(L3), 3.67 (m, 5H, H-6b’, H-4, H-5’, H-2, H-5), 3.52 (dd, 1H, J2’,NH = 8.5 Hz, H-2’), 2.59 (m,
2H, CH2COOC (L1)), 2.46 (m, 1H, CH2aCOOC (L3)), 2.29 (m, 7H, CH2COOC (L1’),
CH2COOC (L2’) CH2bCOOC (L3)), 1.55 (m, 11H, CH (TDS), CH2CHCH2COO (L1),
CH2CH2COOC(L1’), CH2CHCH2COO (L2), CH2CH2COOC(L2’), CH2CHCH2COO (L3)),
1.14 (m, 90H, 45x CH2), 0.87 (t, 15H, J = 6.8 Hz, 5x CH3), 0.79 (m, 12H, 4x CH3 (TDS)),
16

0.17, 0.11 (2s, 6H, 2x CH3 (TDS)). 13C NMR (151 MHz, CDCl3): δ 173.7, 173.5, 170.1,
170.0 (5x C=O) 155.7 (CO (Fmoc)), 143.9, 141.2, 136.0, 135.7, 135.2, 133.2, 133.1, 132.9,
132.3, 128.1, 128.0, 127.9, 127.8, 127.6, 127.6, 127.0, 126.3, 126.2, 126.0, 125.9, 125.8,
125.7, 125.6, 125.2, 119.9 (aromatic (Nap, Fmoc) + CH=CH2), 118.4, 118.2 (2x CH2=CH),
100.0 (C-1’), 96.4 (C-1), 76.3 (CHCH2COOC (L3)), 75.5 (C-4), 74.8, 74.4, 74.2, 74.1, 74.0,
74.0, 73.6 (2x CH2 (Nap), C-3, C-4. C-5, C-4’), 72.9 (C-3’), 71.3 (CH2 (Nap)), 70.5
(CHCH2COOC (L2)), 70.3 (CHCH2COOC (L1)), 68.6, 68.5, 68.5, 68.3, 68.2 (C-6, C-6’,
CH2-CH=CH2), 67.1 (CH2 (Fmoc)), 58.3 (C-2), 56.1 (C-2’), 47.0 (CH (Fmoc), 41.4, 39.8,
39.6 (CH2COOC (L1), CH2COOC (L2), CH2COOC (L3)), 34.5, 34.5 (CH2COOC (L1’),
CH2COOC (L2’)), 34.3, 34.2 (CH2CHCH2COO (L1), CH2CHCH2COO (L2), 34.1
(CH2CHCH2COO (L3)), 33.9 (CH (TDS)), 31.9, 30.9, 29.7, 29.6, 29.4, 29.4, 29.3, 25.2, 25.1,
25.0, 24.7, 22.7 (48x CH2), 20.0, 18.5 (4x CH3 (TDS)), 14.1 (CH2CH3), -1.5 -3.4 (2x CH3
31
(TDS)). P NMR (162 MHz, CDCl3): δ -2.1. HR MS (m/z) calcd for C142H211N2NaO22PSi
[M + Na]+, 2378.4858; found, 2378.4764. C-1’ was determined on basis of HSQC, because it
was not clearly visible in the 13C spectrum.
Dimethylthexylsilyl
6-O-[2-N-(dodecanoyloxy)tetradecanoyl)-2-deoxy-4-O-di-O-
allylphosphate-6-O-(naphthalen-2-ylmethyl)-3-O-(R)-(tetradecanoyloxy)tetradecanoate-
β-D-glucopyranosyl]-2-N-(3-O-(R)-(naphthalen-2-ylmethoxy)tetradecanoyl)-2-deoxy-4-
O-(naphthalen-2-ylmethyl)-3-O-(R)-(naphthalen-2-ylmethoxy)tetradecanoate-β-D-
glucopyranoside (17). To a solution of 16 (203 mg, 86.1 µmol) in CH2Cl2 (2 mL) was added
1,8-diazabicyclo[5.4.0]undec-7-ene (26 µL, 172 µmol). The reaction mixture was stirred at
RT for 1 h. To a solution of lipid 6 (50 mg, 129 µmol) in CH2Cl2 (2 mL) were added
dicyclohexylcarbodiimide (27 mg, 129 µmol) and K-OxymaPure (23 mg, 129 µmol). The
pre-activated lipid 6 was diluted with CH2Cl2 (2.0 mL) and added to Fmoc-cleaved 16 and
the resulting mixture was stirred overnight at RT. The reaction mixture was directly loaded on
a silica gel column and after eluting (PE/EtOAc, 95/5 — 8/2) 17 was obtained as a clear
1
colorless oil (151 mg, 60.4 µmol, 70%). H NMR (400 MHz, CDCl3): δ 7.51 (m, 28H,
aromatic (4x Nap)), 6.16 (d, 2H, J = 8.7 Hz, NH, NH’), 5.76 (m, 2H, 2x CH2=CH), 5.44 (t,
1H, J = 9.5 Hz, H-3’), 5.16 (m, 8H, CHCH2COOC (L1), CHCH2COOC (L2), H-1’, H3, 2x
CH2=CH), 4.66 (m, 6H, 3x CH2 (Nap)), 4.49 (m, 3H, CH2 (Nap), H-4’), 4.37 (m, 4H, 2x
CH2-CH=CH2), 4.30 (d, 1H, JH1-H2 = 7.6 Hz, H-1), 3.97 (d, 1H, J = 11.3 Hz, H6a), 3.91 (m,
1H, H-2), 3.74 (m, 7H, H-6b, CH2-6’, H-4, CHCH2COOC (L3), CHCH2COOC (L4), H-5’),
17

3.46 (m, 1H, H-2’), 3.31 (m, 1H, H-5), 2.59 (m, 2H, CH2COOC (L1)), 2.31 (m, 10H,
CH2COOC (L1’), CH2COOC (L2), CH2COOC (L2’), CH2COOC (L3), CH2COOC (L4)),
1.50 (m, 13H, CH (TDS), CH2CHCH2COO (L1), CH2CH2COOC(L1’), CH2CHCH2COO
(L2), CH2CH2COOC(L2’), CH2CHCH2COO (L3), CH2CHCH2COO (L4)), 1.14 (m, 108H,
54x CH2), 0.88 (t, 18H, J = 6.8 Hz, 6x CH3), 0.79 (m, 12H, 4x CH3 (TDS)), 0.09, -0.01 (2s,
6H, 2x CH3 (TDS)). 13C NMR (101 MHz, CDCl3): δ 173.7, 173.5, 171.6, 170.7, 170.1, 170.0
(6x C=O), 136.1, 135.8, 135.3, 133.3, 133.2, 133.2, 133.1, 133.0 132.9, 132.9, 132.8, 132.3,
132.2, 132.2, 132.1, 128.3, 128.0, 127.9, 127.9 127.8, 127.7, 127.6, 127.6, 126.5, 126.4,
126.3, 126.2, 126.2, 126.0, 125.8, 125.8, 125.7, 125.6 (aromatic (Nap) + CH=CH2), 118.4,
118.2 (2x CH2=CH), 99.8 (C-1’), 96.3 (C-1), 76.2 (C-4), 75.7 (CHCH2COOC (L3)), 75.5
(CHCH2COOC (L4)), 74.9 (C-3), 74.4 (C-5), 74.2, 74.2, 74.0, 73.9 (2x CH2 (Nap), C-5’, C-
4’), 72.8 (C-3’), 71.3 (CH2 (Nap)), 70.6 (CH2 (Nap)), 70.4 (CHCH2COOC (L2)), 70.3
(CHCH2COOC (L1)), 68.6, 68.5, 68.5, 68.3, 68.2 (C-6, C-6’, CH2-CH=CH2), 56.3 (C-2’),
55.9 (C-2), 41.3, 41.2, 39.8, 39.6 (CH2COOC (L1), CH2COOC (L2), CH2COOC (L3)),
CH2COOC (L4)), 34.6, 34.6, 34.5, 34.3, 34.3, 33.9 (CH2COOC (L1’), CH2COOC (L2’)),
(CH2CHCH2COO (L1), CH2CHCH2COO (L2), (CH2CHCH2COO (L3)), (CH2CHCH2COO
(L4)), 33.7 (CH (TDS)), 31.9, 29.7, 29.7, 29.6, 29.6, 29.5, 29.4, 29.4, 29.3, 25.2, 25.1, 25.0,
24.6, 22.7 (58x CH2), 20.0, 18.6 (4x CH3 (TDS)), 14.1 (CH2CH3), -1.5 -3.4 (2x CH3 (TDS)).
31P NMR (162 MHz, CDCl3): δ -2.1.
[2-N-(dodecanoyloxy)tetradecanoyl)-2-deoxy-4-O-
phosphate-
3-O-(R)-
(tetradecanoyloxy)tetradecanoate-β-D-glucopyranosyl]-
(1-6)-2-N-(3-(R)-
hydroxytetradecanoyl)-2-deoxy-3-(R)-hydroxytetradecanoate-β-D-glucopyranose (1). To
a solution of 17 (49 mg, 19.6 µmol) in THF (10 mL) was added pyridine (3 mL, 37.2 mmol)
and this mixture was cooled to -40 °C. Next, HF·pyridine (1 mL, 39.9 mmol, 70%) was
added dropwise and the resulting mixture was stirred overnight at RT. The reaction mixture
was diluted with water (100 mL) and extracted with EtOAc (2x 100 mL), the organic layer
was dried with MgSO4, filtered and concentrated. The residue was purified using Sephadex
LH-20 column chromatography (CH2Cl2/MeOH, 1/1). To this pyranose (44 mg, 18.6 µmol)
in THF (1.5 mL) were added triphenylphosphine (27 mg, 103 µmol), Et3N (144 µL, 1.03
mmol) and formic acid (72 µL, 1.89 mmol). After stirring for 10 min at RT,
tetrakis(triphenylphosphine)palladium (24 mg, 20.8 µmol) was added and stirring was
continued for 4 h. Concentration in vacuo was followed by Sephadex LH-20 column
18

chromatography (CH2Cl2/MeOH, 1/1), affording the free phosphate. To a solution of this
phosphate (40 mg, 17.6 µmol) in CHCl3 (8 mL) was added 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone (40 mg, 176 µmol), and the mixture was stirred for 16 h at RT. The crude
reaction mixture was diluted with MeOH (2 mL), and transferred to a Sephadex LH-20
column (CH2Cl2/MeOH, 1/1). Title compound 1 (25 mg, 14.5 µmol, 76% over three steps)
was obtained as a colorless oil. 1H NMR (600 MHz, CDCl3/MeOD 1/1): δ 5.15 (m, 4H, H-3,
CHCH2COOC (L1), CHCH2COOC (L2), H-3’), 5.10 (d, 1H, J = 3.6 Hz, H-1), 4.62 (s, 1H,
H-1’), 4.22 (m, 1H, H-4’), 4.09 (m, 3H, H-2, H-5, H6a), 4.00 (m, 2H, H6a’, CHCH2COOC
(L3)), 3.89 (m, 1H, CHCH2COOC (L4)), 3.83 (t, 1H, J = 9.5 Hz, H-2’), 3.73 (m, 2H, H6b’,
H6b), 3.46 (t, 1H, J = 9.8 Hz, H-4), 3.36 (m, 1H, H-5’), 2.65 (m, 2H, CH2COOC (L1), 2.55 -
2.13 (m, 10H, CH2COOC (L1’), CH2COOC (L2), CH2COOC (L2’), CH2COOC (L3),
CH2COOC (L4)), 1.60 (m, 12H, CH2CHCH2COO (L1), CH2CH2COOC(L1’),
CH2CHCH2COO (L2), CH2CH2COOC(L2’), CH2CHCH2COO (L3), CH2CHCH2COO (L4)),
1.46, 1.31 (m, 108H, 54x CH2), 0.89 (m, 18H, 6x CH3). 31P NMR (162 MHz, CDCl3 : MeOD
1:1): δ 4.4. The proton chemical shifts of CH-1 and CH-5’ (4.62, 3.36) were determined
based on the gHSQC spectrum. HR MS (m/z) calcd for C94H176N2O22P [M - H]-, 1716.2458;
found, 1716.2357.
Acknowledgements
This research was supported by Utrecht University
Appendix A. Supplementary data
Supplemental data related to this article is available.
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Graphical abstract
O
P
ONap
OH
O
O
HO
HO
AllylO
AllylO
P
O
O
O
O
O
O
O
O
O
NHTroc
NapO
NH
O
O
O
O
HO
O
O
OTDS
NHFmoc
O
O
C13H27
OH
NH
O
O
C11H23
O
C11H23
C13H27
O
O
O
HO
C11H23
NapO
C11H23
C11H23
HO
C11H23
C11H23
monophosphoryl lipid A
Nap-protected precursor
23

Highlights
•
•
•
Synthetic strategy for the preparation of monophosphoryl lipid A
2-Naphtylmethyl ether as permanent protecting group
Opportunities to develop tailormade adjuvants