Synthesis of monophosphorylated lipid A precursors using 2-naphthylmethyl ether as a protecting group

Article abstract of DOI:10.3762/BJOC.16.162

Lipid A, the hydrophobic domain of lipopolysaccharide (LPS), is a strong immunostimulator and therefore a valuable target for the development of novel immunomodulators. Various lipid A derivatives have been chemically synthesized in order to reduce toxicity while retaining the immunostimulatory activity. In this work, we describe a novel approach to the frequently problematic synthesis of monophosphorylated mono- and disaccharide lipid X using a combination of established chemistry and a novel 2-naphthyl-methyl ether (Nap) protecting group for “permanent” protection of hydroxy groups. Of particular note is the fact that the key Nap protecting group is able to remain in the molecule until the final global deprotection step. Our synthetic strategy is not only efficient in regards to the yield of the various chemical transformations, but also robust in regards to the potential application of this route to the production of other lipid A analogs.

Full text of DOI:10.3762/BJOC.16.162

Synthesis of monophosphorylated lipid A precursors using
2-naphthylmethyl ether as a protecting group
Jundi Xue1, Ziyi Han2, Gen Li2, Khalisha A. Emmanuel3, Cynthia L. McManus3,
Qiang Sui*1,2, Dongmian Ge*4, Qi Gao2 and Li Cai*3
Letter
Open Access
Address:
Beilstein J. Org. Chem. 2020, 16, 1955–1962.
1Shanghai University of Engineering Science, 333 Long Teng Road,
Shanghai 201620, China, 2China State Institute of Pharmaceutical
Industry, 285 Gebaini Rd, Shanghai 201203, China, 3Department of
Chemistry, University of South Carolina Lancaster, 476 Hubbard
Drive, Lancaster, South Carolina 29720, USA, and 4Suzhou Jingye
Medicine & Chemical Co., Ltd, 88 Sanlian Street, Suzhou, Jiangsu
Province, 215129, China
doi:10.3762/bjoc.16.162
Received: 02 June 2020
Accepted: 28 July 2020
Published: 10 August 2020
Associate Editor: S. Bräse
Email:
© 2020 Xue et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Qiang Sui* - Chem_sq@163.com; Dongmian Ge* -
gedm001@sina.com; Li Cai* - caili@mailbox.sc.edu
* Corresponding author
Keywords:
lipid A; lipid X; lipopolysaccharide; 2-naphthylmethyl ether; synthesis
Abstract
Lipid A, the hydrophobic domain of lipopolysaccharide (LPS), is a strong immunostimulator and therefore a valuable target for the
development of novel immunomodulators. Various lipid A derivatives have been chemically synthesized in order to reduce toxicity
while retaining the immunostimulatory activity. In this work, we describe a novel approach to the frequently problematic synthesis
of monophosphorylated mono- and disaccharide lipid X using a combination of established chemistry and a novel 2-naphthyl-
methyl ether (Nap) protecting group for “permanent” protection of hydroxy groups. Of particular note is the fact that the key Nap
protecting group is able to remain in the molecule until the final global deprotection step. Our synthetic strategy is not only effi-
cient in regards to the yield of the various chemical transformations, but also robust in regards to the potential application of this
route to the production of other lipid A analogs.
Introduction
Bacterial cell surfaces are decorated with various types of Gram-negative bacterial cell wall and is crucial in bacterial
glycoconjugates (in the form of glycoproteins and glycolipids) pathogenicity [2]. LPS is a complex molecule that is composed
that are known to participate in many biological processes, of three structural regions: lipid A (endotoxin), a non-repeating
especially in the interactions between bacteria and the environ- core oligosaccharide, and O-antigen [2]. While O-antigen and
ment [1]. For example, lipopolysaccharide (LPS) comprises the the core oligosaccharide are exposed to the external environ-
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Beilstein J. Org. Chem. 2020, 16, 1955–1962.
ment, lipid A, the hydrophobic domain of LPS, is embedded in monosaccharide precursor of lipid A biosynthesis (structure 1,
the cell wall. The lipid A substructure is relatively conserved Figure 1). It was found that lipid X retained some immunomod-
that consists of a β-1,6-linked diglucosamine with 1,4′-di-O- ulatory activity while having drastically reduced toxicity
phosphorylation and 2,2′-N- and 3,3′-O-acylation (Figure 1). [10,11]. Lipid X was also found to give partial protection
The associated fatty acid acyl chains may be conserved within a against a 100% lethal dose of endotoxin in mice [11]. However,
species but can vary significantly in terms of the chain number there were also studies with conflicting results that showed that
and length for lipid A of different bacterial origins [3,4]. Lipid synthetic lipid X could be contaminated with small amounts of
A represents a particularly important subject to research given disaccharide-1-phosphate containing four (R)-3-hydroxytetrade-
the continued rise of problematic bacterial infections. Notably, canoic acids at the 2,2’ and 3,3’ positions (structure 2,
the LPS pathogenicity is almost entirely due to lipid A because Figure 1). This disaccharide precursor 2 was identified as the
it leads to immunostimulatory effects when LPS dissociates main immunostimulatory side product [12,13]. While the
from bacterial membranes within a host [5]. While these research suggested chemically pure lipid X had no immunos-
immunostimulatory effects can be beneficial in the setting of timulatory properties of lipid A, it did behave as a competitive
localized infections, the occurrence of severe sepsis causes inhibitor of LPS [13].
systemic release of inflammation mediators and stimulatory
molecules, thus leading to various pathophysiological effects In this paper we describe a synthesis of lipid X (1) and the
[6]. Accordingly, structure–activity relationship studies of lipid disaccharide lipid A precursor 2 (2,2′-N;3,3′-O-tetra[(R)-3-
A which examine or facilitate the examination of how one hydroxytetradecanoyl]-β(1→6)-ᴅ-glucosamine disaccharide
might harness these immunostimulatory effects are particularly 1-phosphate) (Figure 1). The synthesis of such precursors is
valuable as they can provide basis for the development of particularly important as it will facilitate the aforementioned
vaccines and adjuvants. For example, recent studies have goal of harnessing the immunostimulatory effects of lipid A
disclosed that both the fatty acid structure and the phosphoryla- through development of a clear understanding of the
tion degree can affect the activity and endotoxic effects [7-9].
structure–activity relationship. More importantly, we employed
the 2-naphthylmethyl ether (Nap) group for protection of
Various lipid A derivatives have since been synthesized to dis- various hydroxy groups on the carbohydrate and acyl moieties,
sociate endotoxic effects from beneficial immunomodulatory aiming to provide an advantage over previous methods that
activities. Lipid X, 2-N;3-O-di[(R)-3-hydroxytetradecanoyl]-ᴅ- mainly used the benzyl group [4,14-16] in synthesizing lipid A
glucosamine-1-phosphate, is the naturally occurring early derivatives. We also aim at developing a robust strategy in
Figure 1: Chemical structures of hexa-acylated Escherichia coli lipid A, monophosphorylated lipid X (the reducing monosaccharide lipid A precursor),
and disaccharide lipid A precursor (“disaccharide lipid X”).
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Beilstein J. Org. Chem. 2020, 16, 1955–1962.
regards to the potential application of our route to the produc- rated NaHCO3 during reaction work-up seemed necessary to
tion of other lipid A analogs.
avoid cleavage of the TBS ether in compound 13. Then,
(2-naphthyl)methylene acetal [21] was used to protect the
C-4,6-hydroxy groups using 2-naphthaldehyde dimethyl acetal
Results and Discussion
The acyl chain (R)-3-(2-naphthylmethoxy)tetradecanoic acid 7 and 0.2 equiv of camphorsulfonic acid (CSA). These protecting
was prepared via an enantioselective route as previously re- group manipulations resulted in the exposure of the C-3
ported (Scheme 1) [16]. Lauroyl chloride (3) was treated with hydroxy group in compound 14 for further acylation [4]. They
Meldrum’s acid (2,2-dimethyl-1,3-dioxane-4,6-dione) followed are also essential for orthogonal protection of glucosamine,
by decarboxylation in methanol to give methyl 3-oxotetrade- allowing the specific deprotection in subsequent steps (for
canoate (4) in 77% yield. The enantioselective hydrogenation of example, the arylidene acetals at O4 and O6 could be regiose-
the β-carbonyl group using (R)-Ru(OAc)2(BINAP) at 65 °C and lectively opened and transformed into Nap ethers) [19]. The C-3
under 1.5 MPa H2 afforded methyl (R)-3-hydroxytetrade- hydroxy group in compound 14 was then acylated with
canoate (5) in 98% yield. The same hydrogenation reaction was (R)-3-(2-naphthylmethoxy)tetradecanoic acid (7) using 1-ethyl-
carried out using the (S)-Ru(OAc)2(BINAP) catalyst. Then both 3-(3-dimethylaminopropyl)carbodiimide (EDC) and
the R and S products were compared using chiral HPLC to 4-dimethylaminopyridine (DMAP) as the activation reagents
confirm the absolute configuration and enantiomeric purity [14] to give the key/common building block 15 in good yield
(Figure S1, Supporting Information File 1). The 3-hydroxy (Scheme 2).
group in 5 was then protected as a Nap ether through a
TMSOTf-catalyzed one-pot reductive naphthylmethylation Glycosyl acceptor 18 and donor 20 were thus conveniently pre-
process [17,18], by which free hydroxy groups were first pared from the common building block 15 through multiple
trimethylsilylated in situ with hexamethyldisiloxane ((TMS)2O) protecting group manipulations (Scheme 2). The N-Troc group
before being naphthylmethylated by treatment with 2-naphth- in 15 was removed by treatment with zinc in a mixture of acetic
aldehyde, trimethylsilyl trifluoromethanesulfonate (TMSOTf), acid and CH2Cl2. The resulting amine 16 was protected imme-
and triethylsilane (Et3SiH) [17,19]. On a 10 g scale, the pro- diately as fluorenylmethylenoxy (Fmoc) carbamate by reaction
tected methyl ester 6 could be purified by recrystallization fol- with FmocCl in the presence of diisopropylethylamine (DIPEA)
lowed by filtration to remove the major byproduct 2-methyl- to give the fully protected compound 17. The regioselective
naphthalene. Subsequent saponification of the methyl ester 6 opening of the arylidene acetal at O6 with Et3SiH and PhBCl2
with LiOH gave (R)-3-(2-naphthylmethoxy)tetradecanoic acid in the presence of molecular sieves at −78 °C [22] gave com-
(7) in 78% yield over two steps.
pound 18 in good yield (80%) having a free C-6 hydroxy group.
Compound 18 is the glycosyl acceptor for the synthesis of the
The glucosamine building block 14 was synthesized using the disaccharide lipid A precursor (Scheme 4). For the synthesis of
procedures described in previous literature [20] (Scheme 2). donor 20, first removal of the anomeric TBS in building block
The protection of the free amine of glucosamine with a 2,2,2- 15 was achieved by treatment with HF-pyridine followed by
trichloroethoxycarbonyl (Troc) group under basic conditions conversion of the resulting lactol into the desired N-phenyltri-
followed by peracetylation afforded compound 10 on a ≈150 g fluoroacetimidate glycosyl donor 20 by reaction with 2,2,2-
scale. The regioselective anomeric deacetylation with hydrazine trifluoro-N-phenylacetimidoyl chloride in the presence of base
and reprotection of the anomeric hydroxy group as tert-butyl- DBU [14].
dimethylsilyl ether (TBS) led to compound 12. Compound 12
was then treated with sodium methoxide in guanidine hydro- The monoacylated derivative 15 is also the key building block
chloride buffer solution (pH ≈ 9) to remove the O-3,4,6-acetyl for the synthesis of lipid X monosaccharide 1 (Scheme 3). After
groups [14]. Because the deacetylation reaction was later neu- the N-Troc protecting group was removed as described above,
tralized with cation exchange resin, extra washing with satu- the free amine was immediately acylated with (R)-3-(2-naph-
Scheme 1: Enantioselective synthesis of Nap-protected (R)-3-hydroxytetradecanoic acid (7). Conditions: (a) Meldrum's acid, pyridine, CH2Cl2, 0 °C;
(b) CH3OH, reflux, 77% over two steps; (c) (R)-Ru(OAc)2(BINAP), H2, CH3OH, 65 °C, 98%; (d) NapCHO, TMSOTf, (TMS)2O, Et3SiH, THF, 0 °C;
(e) LiOH, THF, H2O, 65 °C, 78% (over two steps).
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Beilstein J. Org. Chem. 2020, 16, 1955–1962.
Scheme 2: Synthesis of monoacylated glucosamine building blocks. Conditions: (a) NaHCO3, TrocCl, H2O, 0 °C, 94% ; (b) Ac2O, pyridine, rt, 96%;
(c) N2H4, AcOH, DMF, rt, 89%; (d) TBSCl, imidazole, DMF, rt, 93%; (e) guanidine hydrochloride buffer, rt; (f) NapC(OMe)2, camphorsulfonic acid,
CH3CN, rt, 68% (2 steps); (g) acid 7, EDC·HCl, DMAP, CH2Cl2, rt, 85%; (h) Zn/AcOH, CH2Cl2, rt; (i) DIPEA, FmocCl, CH2Cl2, rt, 80% (2 steps);
(j) PhBCl2, Et3SiH, CH2Cl2, MS 4 Å, −78 °C, 80%; (k) HF/pyridine, THF,−40 °C to rt, 93%; (l) DBU, ClCN(Ph)CF3, CH2Cl2, 95%.
thylmethoxy)tetradecanoic acid (7) using EDC and DMAP as amine was immediately acylated with (R)-3-(2-naphthyl-
the activation reagents to give the diacylated compound 21 in methoxy)tetradecanoic acid (7) using EDC and DMAP as the
good yield. The anomeric TBS ether of 21 was then cleaved coupling reagents to afford triacylated disaccharide 26. Then,
with HF, and the resulting anomeric hydroxy group was phos- the N-Fmoc group (reducing end) in 26 was removed by treat-
phorylated using tetrabenzyl diphosphate in the presence of lith- ment with triethylamine, and the resulting amine again was
ium bis(trimethyl)silylamide (LHMDS) in THF at −78 °C [23] immediately acylated with (R)-3-(2-naphthylmethoxy)tetrade-
to afford the anomeric phosphate 23 exclusively as the canoic acid (7) to afford disaccharide 28 with four fatty acid
α-anomer. Finally, global deprotection of 23 (benzyl phosphate, chains. After cleavage of the anomeric TBS moiety employing
Nap ethers, and naphthylidene acetal) were accomplished by HF in pyridine, the resulting anomeric hydroxy group of 29 was
catalytic hydrogenolysis over Pd/C under 15 kg/cm2 of H2 to phosphorylated using tetrabenzyl diphosphate in the presence of
give the target lipid X monosaccharide 1 (as triethylammonium LHMDS in THF at −78 °C. Then finally global deprotection
salt) in good yield.
(hydrogenation over Pd-black) was carried out to remove the
naphthylidene acetal, Nap ethers, and the benzyl phosphate
Having the glycosyl donor 20 and acceptor 18 at hand groups in compound 30. By this route the target disaccharide
(Scheme 2), in order to prepare the disaccharide precursor, the lipid A precursor 2 (as triethylammonium salt) was obtained in
glycosylation reaction was performed first, followed by depro- 88% yield.
tection, acylation, and phosphorylation reactions (Scheme 4).
The triflic acid (TfOH)-mediated glycosylation of donor 20 and Conclusion
acceptor 18 in the presence of molecular sieves in CH2Cl2 at As described, we have developed an efficient approach for the
−20 °C gave disaccharide 24 [14] in excellent yield (β-anomer chemical synthesis of two monophosphorylated lipid A precur-
only). The N’-Troc protecting group (non-reducing end) was sors. Lipid X (1) could be prepared from the common building
first removed using Zn dust in acetic acid, and the resulting free block 15 via deprotection, acylation, phosphorylation, and
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Beilstein J. Org. Chem. 2020, 16, 1955–1962.
Scheme 3: Synthesis of lipid X monosaccharide 1. Conditions: (a) Zn, AcOH, CH2Cl2, rt; (b) acid 7, EDC·HCl, DMAP, CH2Cl2, rt, 67.5% (2 steps);
(c) HF/Py, THF, −40 °C to rt, 78%; (d) tetrabenzyl pyrophosphate, LHMDS, THF, −78 °C, 91%; (e) H2 (15 kg/cm2), Pd/C, THF/H2O, 38 °C, 86%.
global deprotection. The glycosyl acceptor and donor for the mainly used the benzyl group in synthesizing lipid A deriva-
synthesis of the disaccharide precursor could also be readily ob- tives. Of particular note is the fact that the key Nap protecting
tained starting from the same key building block. After glyco- group is able to remain in the molecule until the final global
sylation, the disaccharide lipid A precursor 2 was synthesized deprotection step. The presence of this protecting group until
following a similar reaction sequence of deprotection, acylation, such a late stage likely helps avoiding the problematic acyl
phosphorylation, and global deprotection.
migration often observed with similar molecules [28]. In addi-
tion, the 4,6-O-naphthylidene acetal (e.g., in compounds 23 and
The Nap protecting group has emerged as a particularly valu- 30) can be regioselectively opened at O6 or O4 under different
able addition to carbohydrate chemistry [24,25]. Not only does conditions [19,29]. This could potentially allow the incorpora-
it not significantly alter carbohydrate reactivity, it also can be tion of other functionalities in target molecules for the synthe-
readily cleaved under hydrogenolytic conditions as well as a sis of glycoconjugates. Based on the synthetic strategy de-
variety of oxidative [26] and acid-mediated conditions [25,27] scribed in this work (a common building block and Nap ether
that are orthogonal to benzyl ethers. Therefore, we employed protection), we have already designed a route to MPLA, a clini-
the Nap ether as a “permanent” protecting group for the carbo- cally safe [30] monophosphoryl lipid A derivative with one
hydrate and the 3-hydroxy group of the acyl chain, aiming to phosphate group linked to the 4′-OH group. This work is cur-
provide an advantage over literature reported methods that rently underway in our lab.
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Beilstein J. Org. Chem. 2020, 16, 1955–1962.
Scheme 4: Synthesis of the disaccharide lipid A precursor 2. Conditions: (a) TfOH, 4 Å MS, dry CH2Cl2, 94%; (b) Zn, AcOH, CH2Cl2; (c) acid 7,
EDC·HCl, DMAP, CH2Cl2, 88% (2 steps); (d) Et3N, DMF; (e) acid 7, EDC·HCl, CH2Cl2, 82% (2 steps); (f) HF/pyridine, pyridine, THF, −40 °C to rt,
92%; (g) tetrabenzyl pyrophosphate, LHMDS, dry THF, −78 °C, 82%; (h) H2, Pd-black, THF, 38 °C, 88%.
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Beilstein J. Org. Chem. 2020, 16, 1955–1962.
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Funding
This work was partially supported by the National Natural
Science Foundation of China (21602134) and the Shanghai
Science and Technology Innovation – International Collabora-
tion Fund of Shanghai Science and Technology Committee
(18430721800). LC would like to acknowledge the partial
financial support through an RISE (Research Initiative for
Summer Engagement) grant from the Office of the Vice Presi-
dent for Research at the University of South Carolina (2020).
doi:10.1016/s0040-4039(00)73357-0
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