The endotoxic portion of lipopolysaccharide (LPS), a glycophospholipid Lipid A, initiates the activation of the Toll-like Receptor 4 (TLR4)-myeloid differentiation factor 2 (MD-2) complex, which results in pro-inflammatory immune signaling. To unveil the structural requirements for TLR4·MD-2-specific ligands, we have developed conformationally restricted Lipid A mimetics wherein the flexible βGlcN(1→6)GlcN backbone of Lipid A is exchanged for a rigid trehalose-like αGlcN(1↔1)αMan scaffold resembling the molecular shape of TLR4·MD-2-bound E. coli Lipid A disclosed in the X-ray structure. A convergent synthetic route toward orthogonally protected αGlcN(1↔1)αMan disaccharide has been elaborated. The α,α-(1↔1) linkage was attained by the glycosylation of 2-N-carbamate-protected α-GlcN-lactol with N-phenyl-trifluoroacetimidate of 2-O-methylated mannose. Regioselective acylation with (R)-3-acyloxyacyl fatty acids and successive phosphorylation followed by global deprotection afforded bis- and monophosphorylated hexaacylated Lipid A mimetics. αGlcN(1↔1)αMan-based Lipid A mimetics (α,α-GM-LAM) induced potent activation of NF-κB signaling in hTLR4/hMD-2/CD14-transfected HEK293 cells and robust LPS-like cytokines expression in macrophages and dendritic cells. Thus, restricting the conformational flexibility of Lipid A by fixing the molecular shape of its carbohydrate backbone in the "agonistic" conformation attained by a rigid αGlcN(1↔1)αMan scaffold represents an efficient approach toward powerful and adjustable TLR4 activation.
The endotoxic portion of lipopolysaccharide (LPS), a glycophospholipid Lipid A, initiates the activation of the Toll-like Receptor 4 (TLR4)-myeloid differentiation factor 2 (MD-2) complex, which results in pro-inflammatory immune signaling. To unveil the structural requirements for TLR4·MD-2-specific ligands, we have developed conformationally restricted Lipid A mimetics wherein the flexible βGlcN(1→6)GlcN backbone of Lipid A is exchanged for a rigid trehalose-like αGlcN(1↔1)αMan scaffold resembling the molecular shape of TLR4·MD-2-bound E. coli Lipid A disclosed in the X-ray structure. A convergent synthetic route toward orthogonally protected αGlcN(1↔1)αMan disaccharide has been elaborated. The α,α-(1↔1) linkage was attained by the glycosylation of 2-N-carbamate-protected α-GlcN-lactol with N-phenyl-trifluoroacetimidate of 2-O-methylated mannose. Regioselective acylation with (R)-3-acyloxyacyl fatty acids and successive phosphorylation followed by global deprotection afforded bis- and monophosphorylated hexaacylated Lipid A mimetics. αGlcN(1↔1)αMan-based Lipid A mimetics (α,α-GM-LAM) induced potent activation of NF-κB signaling in hTLR4/hMD-2/CD14-transfected HEK293 cells and robust LPS-like cytokines expression in macrophages and dendritic cells. Thus, restricting the conformational flexibility of Lipid A by fixing the molecular shape of its carbohydrate backbone in the "agonistic" conformation attained by a rigid αGlcN(1↔1)αMan scaffold represents an efficient approach toward powerful and adjustable TLR4 activation.
Toll-like Receptor
4 (TLR4) is a mammalian transmembrane receptor
protein which, in complex with a myeloid differentiation factor 2
(MD-2), detects picomolar concentrations of Gram-negative bacterial
endotoxin (or lipopolysaccharide, LPS) (Figure 1A) and initiates an inflammatory signaling cascade aimed at the eradication
of bacterial infection.[1] Activation of
the innate immune response through TLR4·MD-2-LPS complex was
shown to contribute to the pathogenesis of numerous inflammatory,
autoimmune, and chronic diseases, such as sepsis syndrome, asthma,
arthritis, and cancer, which highlights the significance of TLR4-MD-2
complex as a therapeutic target.[2−5] Therapeutic modulation of the innate immune response
by intervention with TLR4·MD-2 signaling has grown to a “hot”
topic in the past decade.[4,6] Moreover, activation
of TLR4 has been proposed to bridge the innate and adaptive immunity,[7] emphasizing stimulation of the TLR4·MD-2
complex by nontoxic ligands as a straightforward way to efficient
vaccine adjuvants.[8−10]
Figure 1
(A) Structure of LPS, with Re-LPS and Lipid A.
(B) Structures of TLR4 agonist E. coli Lipid A and
MPLA. (C) Co-crystal structure of E. coliRa-LPS-hMD-2·TLR4 (PDB code: 3FXI; only Lipid A portion
of LPS is shown for clarity), top and side views. Phe126 (orange)
together with 2-N-acyl chain (yellow) creates a hydrophobic
patch at the dimerization interface with the second TLR4*·MD-2*
complex (brown). Positively charged Arg and Lys (blue) at the rim
of the binding pocket of MD-2 are involved in the ionic interactions
with the Lipid A phosphates. Images were generated with PyMol.
Lipid A, an amphiphilic membrane-bound portion
of LPS, represents
the major pathogen-associated molecular pattern which drives the activation
of TLR4 by binding to the co-receptor protein MD-2 and triggering
the dimerization of two TLR4·MD-2·LPS complexes.[11] Generally, the binding of hexaacylated bisphosphorylated
Lipid A (such as from Escherichia coli, Figure 1B) by human TLR4·MD-2 complex results in the
efficient activation of the innate immune response, whereas underacylated
Lipid A variants are either inactive or antagonistic (such as tetraacylated
lipid IVa, or the synthetic drug candidate Eritoran).[12,13] The co-crystal structures of the E. coliRe- and Ra-LPS with mouse (m) or human
(h) MD-2·TLR4 complex, respectively, unravel that only five long-chain
acyl residues of the hexaacylated Lipid A are incorporated into the
hydrophobic binding pocket of MD-2 whereas the sixth 2-N-acyl chain is exposed on the surface of MD-2 and is involved in
the dimerization interface with the second TLR4*·MD-2*-LPS complex
(Figure 1C).[14,15] The Phe126
residue of MD-2 is proposed to stabilize the presentation of an acyl
tail on the surface of the protein and to serve as hydrophobic switch
allowing dimerization to occur.[16,17] LPS-driven homodimerization
of TLR4·MD-2-LPS complexes initiates recruitment of adaptor proteins
to the intracellular TIR (Toll/interleukin-1 receptor) domains of
TLR4 which ultimately results in the induction of the intracellular
inflammatory signaling cascade.[18] In contrast,
submerging of all lipid chains of the ligand into the hydrophobic
binding groove of MD-2 results in an efficient binding without initiation
of signaling, which is a characteristic feature of TLR4·MD-2
antagonists.[12,13](A) Structure of LPS, with Re-LPS and Lipid A.
(B) Structures of TLR4 agonist E. coli Lipid A and
MPLA. (C) Co-crystal structure of E. coliRa-LPS-hMD-2·TLR4 (PDB code: 3FXI; only Lipid A portion
of LPS is shown for clarity), top and side views. Phe126 (orange)
together with 2-N-acyl chain (yellow) creates a hydrophobic
patch at the dimerization interface with the second TLR4*·MD-2*
complex (brown). Positively charged Arg and Lys (blue) at the rim
of the binding pocket of MD-2 are involved in the ionic interactions
with the Lipid A phosphates. Images were generated with PyMol.The presence of both 1- and 4′-phosphate
groups of Lipid
A was shown to be crucial for the efficiency of the dimerization and
the potency of the initiated signaling.[19] The absence of 1-phosphate leads to less efficient dimerization[20,21] and dampened cytokine production while maintaining sufficient TLR4-mediated
immune activation and full adjuvant activity, which guided the development
of monophosphoryl Lipid A (MPLA), a licensed vaccine adjuvant (Figure 1B).[8,10]Despite tremendous intensive
research on the interaction of TLR4·MD-2
complex with isolated,[22,23] genetically engineered,[24] and synthetic Lipid A’s and analogues,[25−29] the structure–activity relationships of the LPS-triggered
TLR4 activation are not unambiguously established. Minor variations
in the length and distribution pattern of fatty acyl chains in Lipid
A typically result in dramatic amendment of TLR4-mediated immune signaling[25−27] which cannot be rationally predicted.We have addressed the
challenges associated with the exploration
of structural basis of LPS-induced TLR4 activation by development
of a novel type of Lipid A mimetics wherein the flexible βGlcN(1→6)GlcN
backbone of Lipid A is replaced by the conformationally restricted
(1↔1)-connected disaccharide scaffolds. Notably, all Lipid
A analogues synthesized so far were based either on the native β(1→6)-diglucosamine
or on the more flexible backbones wherein one or both GlcN residues
were replaced by a linear aglycon.[9,10,27,30] Previously we reported
on the synthesis and potent anti-endotoxic activity of tetraacylated
Lipid A mimetics derived from the β,α(1↔1)-linked
diglucosamine representing an “antagonistically” shaped
scaffold.[31]Taking advantage of a
striking similarity between the conformation
of the nonreducing sugar trehalose [αGlc(1↔1)αGlc] and the molecular shape of the β(1→6) diglucosamine
backbone of TLR4·MD-2-bound agonist E.
coli Lipid A disclosed in the X-ray structure, we have developed
novel agonistic conformationally confined Lipid A mimetics based on
the two-bond-linked rigid trehalose-type αGlcN(1↔1)αMan scaffold
(Figure 2).
Figure 2
Structure of Lipid A mimetics (α,α-GM-LAMs) 1–3 based on the conformationally confined
αGlcN(1↔1)αMan
scaffold.
Structure of Lipid A mimetics (α,α-GM-LAMs) 1–3 based on the conformationally confined
αGlcN(1↔1)αMan
scaffold.
Results and Discussion
X-ray Structure-Guided
Design of αGlcN(1↔1)αMan-Based
Lipid A Mimetics
The β(1→6) diglucosamine backbone
represents the most conserved part of Lipid A, whereas its acylation
and phosphorylation pattern varies within bacterial species.[22,32] The overall three-dimensional conformation of the intrinsically
flexible three-bond-linked βGlcN(1→6)GlcN backbone
of Lipid A is determined by the values of the dihedral
angles ω, ϕ, and ψ about (1→6) glycosidic and oxymethyl
linkages (Figure 3A). Thus, the relative orientation
of GlcN rings can be easily adapted
by rotation about glycosidic and oxymethyl linkages via altering the
corresponding torsion angles. This permits spontaneous adjustment
of the shape of Lipid A to the geometry of the binding pocket of MD-2,
which complicates the estimation of the “active” conformation
of the ligand in the [Lipid A-MD-2·TLR4]2 complex.
As seen in the co-crystal structures, the proximal (reducing) GlcN
ring of MD-2-bound hexaacylated Lipid A adopts an inclined (or “twisted”)
orientation which, as we assume, is essential for the exposure of
the long-chain 2-N-acyl residue on the surface of
MD-2 followed by dimerization with the second MD-2·TLR4 complex
(Figure 3A,B).[31]
Figure 3
X-ray
structure-based design of α,α-GM-LAMs. (A) Adjustment
of the torsion angles about the (1→6) glycosidic linkage in
the diglucosamine backbone of Lipid A upon binding by MD-2 results
in a “twisted” orientation of the proximal GlcN ring
for an agonist and in a coplanar orientation of the two GlcN rings
for antagonist. (B) The proximal GlcN moiety of MD-2-bound E. coli Lipid A (PDB code 3FXI) adopts inclined orientation which allows
the exposure of the 2-N-acyl chain.[31] Image was generated with PyMol. (C) The molecular shape
of α,α-trehalose (crystal structure)[35,37] resembles the three-dimensional arrangement of βGlcN(1→6)GlcN
backbone of the MD-2-bound E. coli Lipid A. (D) Structure
of αGlcN(1↔1)αMan-based Lipid A mimetic (α,α-GM-LAM) 1 and proposed interaction of 1 with MD-2.
X-ray
structure-based design of α,α-GM-LAMs. (A) Adjustment
of the torsion angles about the (1→6) glycosidic linkage in
the diglucosamine backbone of Lipid A upon binding by MD-2 results
in a “twisted” orientation of the proximal GlcN ring
for an agonist and in a coplanar orientation of the two GlcN rings
for antagonist. (B) The proximal GlcN moiety of MD-2-bound E. coli Lipid A (PDB code 3FXI) adopts inclined orientation which allows
the exposure of the 2-N-acyl chain.[31] Image was generated with PyMol. (C) The molecular shape
of α,α-trehalose (crystal structure)[35,37] resembles the three-dimensional arrangement of βGlcN(1→6)GlcN
backbone of the MD-2-bound E. coli Lipid A. (D) Structure
of αGlcN(1↔1)αMan-based Lipid A mimetic (α,α-GM-LAM) 1 and proposed interaction of 1 with MD-2.To explore the structural prerequisites
needed for an effective
receptor complex homodimerization, we have manipulated the flexibility
of the carbohydrate backbone of Lipid A by fixing its molecular shape
in an “agonistic” conformation. Since the relative spatial
arrangement of the two GlcN rings of MD-2-bound agonist E.
coli Lipid A disclosed in the co-crystal structures[14,15] resembles the arrangement of α,α-(1↔1)-connected
glucoses in the nonreducing disaccharide trehalose (Figure 3B,C), we have developed predictably agonistic Lipid
A mimetics based on the conformationally confined α,α-trehalose-like
αGlcN(1↔1)αMan scaffold (Figure 3D).The values for the torsion angles Φα
and Φ′α,
representing rings orientation about the α,α-(1↔1)
glycosidic linkage (Figure 3D), are governed
mostly by the anomeric and exo-anomeric effects and are only marginally
dependent on the nature of functional groups in variably substituted
α,α-trehaloses.[33] The existence
of a single conformational minimum with respect to the dihedrals about
glycosidic linkage in α,α-trehaloses was confirmed by
molecular dynamics simulations,[33,34] whereas the preferred gauche–gauche conformation of the substituted α,α-trehalose[35] and its αGlc(1↔1)αMan analogue[36] was corroborated by X-ray and conformational
analysis, respectively. Thus, conformationally restrained α,α-(1↔1) glycosidic
linkage in
αGlcN(1↔1)αMan-based Lipid A mimetics would impose
a specific relative orientation of sugar rings resembling the molecular
shape of the diglucosamine backbone of the agonistic MD-2-bound Lipid
A.αGlcN(1↔1)αMan-based Lipid A mimetics
(α,α-GM-LAMs) 1–3 were
designed such that the acylation
and phosphorylation pattern of the nonreducing (distal) GlcN residue
of E. coli Lipid A remains unaffected, whereas the β(1→6) glycosidic linkage
is substituted
by an α,α(1↔1) glycosidic
bond and the reducing (proximal) GlcN moiety of natural Lipid A is
exchanged for a nonreducing sugar (mannose) having a specific acylation
and phosphorylation pattern (Figure 3D). The
location of the phosphate functionality at C-6 of the Man residue
was selected to closely resemble the positioning of a 1-phosphate
group of E. coli lipid A at the secondary dimerization
interface of the TLR4-MD-2-Lipid A complex (PDB code 3FXI). The site of acylation
at the mannose moiety was chosen such that the attachment of the long-chain
(R)-3-acyloxyacyl residue at Man C-4 would provide
a sufficient hydrophobic patch to support the homodimerization and
the interaction with the second TLR4*·MD-2*-ligand complex.Upon interaction with the receptor complex, the tetraacylated
GlcN unit of α,α-GM-LAMs 1–3 was supposed to be fully accommodated within the hydrophobic pocket
of MD-2, whereas the “twisted” mannose ring should be
excluded from the binding site on MD-2 such that the two lipid chains
at Man C-4 are presented onto the surface of the protein and involved
in the secondary dimerization interface (Figure 3D). The axial configuration at C-2 of mannose should provide, according
to the crystal structures, a better fitting to the geometry of the
binding pocket of MD-2 and, simultaneously, ensure easier stereocontrol
in the 1,2-trans glycosylation step to α,α(1↔1)-linked
disaccharide.
Synthetic Strategy
The assembly
of αGlcN(1↔1)αMan,
a 1,1-glycosidically connected (nonreducing) disaccharide, represents
a formidable synthetic challenge with regard to simultaneous stereocontrol
at two anomeric centers. Typically, approaches involving conventional
glycosylation procedures for the synthesis of trehalose provide moderate
stereoselectivity and low yields.[38] Since
we aimed to establish the α,α-(1↔1) glycosidic
linkage between an amino sugar and a manno-configured
monosaccharide, we could hardly rely on the intramolecular aglycon
delivery approach[39] or on the versatile
synthetic desymmetrization of the natural trehalose.[40]For the synthesis of bis- and monophosphorylated Lipid
A mimetics 1–3 (Figure 2)
having non-symmetrically distributed acyloxyacyl functional groups,
a convergent approach involving first the preparation of the orthogonally
protected αGlcN(1↔1)αMan disaccharide
scaffold followed by regioselective phosphorylation
and acylation with (R)-3-acyloxyacyl fatty acids
of variable chain lengths was envisaged (Scheme 1). For the assembly of the αGlcN(1↔1)αMan backbone,
a 2-N-carbamate-protected glucosamine-based
lactol was chosen as acceptor, and a 2-O-levulinoyl
(Lev, 4-oxopentanoyl)-protected mannose was selected to serve as glycosyl
donor. The participating protecting group at C-2 (Lev) should allow
for a preferable 1,2-trans mannosylation. Since the
2-O-Lev protection had to be exchanged for a 2-O-Me group later in the synthesis, an alternative glycosylation
approach using non-participating methyl protection at C-2 of the mannose-based
donor was planned to be explored as well.
The 2-N-carbamates of variably protected GlcN-based
lactols revealed the highest α/β ratio (up to 9:1) of
the anomeric 1-OH group, which highlighted these intermediates as
the most “stereoselective” glycosyl acceptors. Apparently,
the presence of a carbamate N–H capable of hydrogen bonding
with the axial oxygen at C-1 is responsible for the substantial enrichment
with the α-anomer.
Synthesis of αGlcN(1↔1)αMan
Scaffold
To minimize the number of required orthogonal protecting
groups,
we intended the use of a GlcN acceptor with the pre-installed phosphate
group at C-4, whereas C-6 was permanently and C-3 was temporarily
protected. To this end, the 2-N-Troc-protected 4,6-di-O-benzylidene acetal 4(41) was first acetylated to provide 3-O-acetate 5, which was subjected to regioselective reductive opening
of benzylidene acetal with Et3SiH/TfOH in CH2Cl2 to furnish 6-O-benzylated compound 6 (Scheme 2). Phosphitylation of 4-OH
with bisbenzyl(diisopropylamino)phosphoramidite
with 1H-tetrazole as catalyst followed by in situ oxidation with meta-chloroperbenzoic
acid (mCPBA) afforded phosphotriester 7. Compoud 7 was either desilylated at C-1 by
treatment with pyridinium hydrofluoride (HF·Py) in THF
to provide a 2-N-Troc-protected axially configured
lactol 8 (α/β = 9:1) or processed to the
2-N-(9-fluorenylmethyl)carbamate (Fmoc)-protected
counterpart 9, which was similarly deprotected at C-1
to furnish anomeric lactol 10, again with a high preponderance
of the α-anomer (α/β = 9:1).
Scheme 2
Synthesis of Glucosamine
Lactol Acceptors 8 and 10
Reagents and conditions: (a)
Ac2O, DMAP, pyridine, 97%; (b) Et3SiH, TfOH,
4 Å MS, −78 °C, CH2Cl2, 68%;
(c) 1. (BnO)2PN(iPr)2, 1H-tetrazole, CH2Cl2, 2. mCPBA, −78 °C, 90%;
(d) HF·Py, THF, 91% for 8, 94% for 10; (e) 1. Zn, AcOH, CH2Cl2, 2. FmocCl, EtN(iPr)2, CH2Cl2, 89%.
Synthesis of Glucosamine
Lactol Acceptors 8 and 10
Reagents and conditions: (a)
Ac2O, DMAP, pyridine, 97%; (b) Et3SiH, TfOH,
4 Å MS, −78 °C, CH2Cl2, 68%;
(c) 1. (BnO)2PN(iPr)2, 1H-tetrazole, CH2Cl2, 2. mCPBA, −78 °C, 90%;
(d) HF·Py, THF, 91% for 8, 94% for 10; (e) 1. Zn, AcOH, CH2Cl2, 2. FmocCl, EtN(iPr)2, CH2Cl2, 89%.The synthesis of the required mannose-based donors
commenced with
Zemplén deacetylation of the peracetylated thioethyl glycoside 11,[42] followed by introduction
of a 4,6-di-O-tert-butylsilylene
(DTBS) group in 12 (Scheme 3).
Regioselective benzylation at C-3 via stannylene acetal intermediate
and the agency of benzyl bromide in the presence of (nBu)4NI furnished alcohol 13 in 93% yield.
The 2-OH group was either levulinoyl-protected by reaction with 4-oxopentanoic
acid, N,N′-diisopropylcarbodiimide
(DIC), and a catalytic amount of 4-N,N-(dimethylamino)pyridine (DMAP) to give 2-O-levulinate
ester 14 (90%) or methylated by reaction with MeI/NaH
in DMF to provide 2-O-methyl ether 15 in 84% yield. Anomeric deprotection with N-bromosuccinimide
(NBS) in aqueous acetone afforded lactols 16 and 17, respectively, which were converted to trichloroacetimidates
(TCA) 18 and 20 or to N-phenyl-trifluoroacetimidate (NPTFA) donors[43]19 and 21.
Scheme 3
Synthesis of Mannose-Based
Donors
Reagents and conditions: (a)
1. NaOMe, MeOH, 2. (tBu)2Si(OTf)2, pyridine, DMF, −35 °C, 89%; (b) 1. (nBu)2SnO, toluene, 2. BnBr, (nBu)4NI, DMF, toluene, reflux, 93%; (c) →14: LevOH, DIC, DMAP, CH2Cl2, 90%; (d) →15: MeI, NaH, DMF, 84%; (e) NBS, acetone–H2O, 24:1, 0 °C, 77% for 16 and 89% for 17; (f) →18 and 20: CCl3CN, DBU, CH2Cl2, 0 °C, 90% for 18 and 94% for 20; (g) →19 and 21: CF3(NPh)CCl, K2CO3, acetone,
99% for 19 and 93% for 21.
Synthesis of Mannose-Based
Donors
Reagents and conditions: (a)
1. NaOMe, MeOH, 2. (tBu)2Si(OTf)2, pyridine, DMF, −35 °C, 89%; (b) 1. (nBu)2SnO, toluene, 2. BnBr, (nBu)4NI, DMF, toluene, reflux, 93%; (c) →14: LevOH, DIC, DMAP, CH2Cl2, 90%; (d) →15: MeI, NaH, DMF, 84%; (e) NBS, acetone–H2O, 24:1, 0 °C, 77% for 16 and 89% for 17; (f) →18 and 20: CCl3CN, DBU, CH2Cl2, 0 °C, 90% for 18 and 94% for 20; (g) →19 and 21: CF3(NPh)CCl, K2CO3, acetone,
99% for 19 and 93% for 21.The key point in our initial approach was to obtain a
good double
α-stereoselectivity in the glycosylation reaction between reducing
acceptor 8 (α/β = 9:1) and the 2-O-levulinoyl-protected imidate donors 18 or 19. The participating levulinoyl group at C-2 of the manno-configured donors 18 and 19 should allow for a selective 1,2-trans glycosylation.
A survey of the literature revealed that a complete α-manno selectivity upon application of 2-O-Lev-protected mannosyl donors could be obtained with a variety of
acceptors.[44,45]In an initial glycosylation
attempt comprising coupling the TCA
donor 18 and acceptor 8 using trimethylsilyl
trifluoromethanesulfonate (TMSOTf) as promoter, an orthoester 23 was obtained as the major product (23%) along with a minor
proportion of the target α,α-disaccharide 22 (8%) and a concurrently formed donor self-coupling product 24 (Scheme 4A, Supplementary SI-Table 1). With the less reactive NPTFA donor 19 or with the thioethyl donor 14 the formation
of the orthoester was not observed; however, the desired product 22 was isolated only in trace amounts.
Scheme 4
Initial Attempts
toward the Synthesis of αGlcN(1↔1)αMan
Scaffold
Initial Attempts
toward the Synthesis of αGlcN(1↔1)αMan
Scaffold
Reagents and conditions: (a)
1. HF·Py, THF, 2. Ac2O, DMAP, pyridine, 85%; (b) NBS,
acetone–H2O, 24:1, 0 °C, 89%; (c) CCl3CN, DBU, CH2Cl2, 67%; (d) TMSOTf (0.05 equiv),
4 Å MS, CH2Cl2, 0 °C; (e) (CF3CO)2O, CF3COOH, Et3SiH, CH2Cl2, 0 °C, 91%; (f) 1. (BnO)2PN(iPr)2, 1H-tetrazole, CH2Cl2, 2. mCPBA, −78 °C,
91%; (g) 1. Zn, AcOH, CH2Cl2, 0 °C, 2.
FmocCl, EtN(iPr)2, CH2Cl2, 88%; (h) 1. [Ir(COD)(Ph2MeP)2]PF6, H2, THF, 2. I2, aq. THF, 86%.We have hypothesized that the diminished reactivity
of the torsionally
disarmed 4,6-di-O-cyclic-protected mannose donor
was responsible for the glycosylation failure.[46] Accordingly, the 4,6-di-O-DTBS group in 14 was cleaved and substituted for two acetates to provide 25, wherein the C5–C6 bond was unlocked from the disarming trans–gauche conformation.[46,47] To provide consistency with the previously performed imidate-mediated
glycosylations, the thioglycoside at C-1 was exchanged for a TCA group
to furnish 27 (Scheme 4B). The
coupling of 8 and the torsionally unlocked 4,6-di-O-acetyl donor 27 resulted in the isolation
of the α,α-configured disaccharide 28, albeit
in a similarly low yield (8%).Thereafter, our attention was
turned to the apparently low reactivity
of the lactol acceptor 8 affected by the H-bonding between
the α-1-OH and the carbamate NH groups. To increase the nucleophilicity
of the lactol acceptor and to reduce steric constraints, the disarming
acetate at C-3 was exchanged for a TBDMS group, and the sterically
demanding 2-N-Troc group was replaced by Fmoc protection,
which provided α-lactol acceptor 33. To this end,
compound 30, made by a highly selective reductive opening
of benzylidene acetal in the allyl glycoside 29,[31] was phosphorylated at C-4 to give 31 (Scheme 4C). Reductive cleavage of 2-N-Troc protection with Zn in acetic acid followed by reaction
with Fmoc chloride in the presence of a EtN(iPr)2 furnished 32, which was anomerically deprotected
using [Ir(COD)(Ph2MeP)2]PF6-catalyzed
isomerization of the allyl group followed by oxidative cleavage of
the 1-propenyl group with I2 in THF–H2O to afford the “armed” acceptor 33. Reaction
of the 2-O-levulinoyl TCA donor 18 with 33, however, reproducibly resulted in the formation of the
orthoester 34 as the major product (Scheme 4C).In retrospect, we assume that the failure of the
2-O-levulinoyl-protected mannose-based donors to
provide the desired
αGlcN(1↔1)αMan compound in a glycosylation reaction
with GlcN-based lactol acceptors was rather related to a particular
conformation of the arising trehalose-type α,α-disaccharide.
A successful glycosylation would lead to a sterically hindered coupling
product 22, having overlapping bulky 2-N-Troc/Fmoc groups at the GlcN moiety and a linear 4-oxopentanoate
(levulinate) ester group at C-2 of mannose, while reciprocal repulsion
of the N-carbamate and electron-rich levulinoyl groups
could contribute as well.Given the failure of 2-O-levulinoyl-protected
mannosyl donors to provide the desired coupling products, the participating
protecting group at C-2 of mannose was exchanged for the 2-O-Me group as in the donors 20 and 21 (Scheme 3). Successful application of non-participating
groups in the α-selective mannosylation has been extensively
reported.[48,49] Gratifyingly, the coupling of the 2-O-Me-Man imidate donor 21 with the GlcN-lactol
acceptor 8 allowed for a much higher isolated yield (51%)
of the α,α-disaccharide 35 (Scheme 5, Table 1, entry 2). Owing
to the low reactivity of acceptor 8, the NPTFA donor 21 was found to be superior to the TCA donor 20 due to the propensity of the latter to form substantial amounts
of the rearranged glycosylamide 45 (52%), which is characteristic
for glycosylations involving acceptors of diminished reactivity
(Table 1, entries 1 and 2). The isolation of
the α,α-disaccharide 35 was complicated by
the concomitant formation of the co-migrating αGlcN−βMan 38 and βGlcN−αMan 41 byproducts.
Glycosylation of the 2-N-Fmoc-protected acceptor 10 by the NPTFA donor 21 afforded a similar isolated
yield (52%) of the target α,α-configured disaccharide 36 (Scheme 5, Table 1, entry 3).
Scheme 5
Synthesis of GlcN(1↔1)Man Disaccharides
Table 1
Outcome of (1↔1)
Glycosylation
Utilizing 2-O-Me-Protected Imidate Donors 20 and 21 (Scheme 5)
Enhancement of the acceptor
reactivity by the use of 3-O-TBDMS-protected “armed”
lactol 33 did not improve the yields in the coupling
reactions with either
donor 20 or 21 (Table 1, entries 4 and 5), furnishing αGlcN(1↔1)αMan
disaccharide 37 in 25% and 50% yield, respectively. The
yield of the αGlcN(1↔1)αMan disaccharide was related
to the ease of its chromatographic purification, which, in turn, was
strongly dependent on the protection group pattern. Since isolation
of the 2-N-Fmoc-3-O-Ac-protected 36 from the mixture of anomeric products was the most straightforward,
this disaccharide was chosen for further transformation to the target
α,α-GM-LAMs 1–3.The configurations at the anomeric centers of the (1↔1)-linked
disaccharides were assigned on the basis of 1H and 13C NMR shifts at the anomeric positions and the 1JC1,H1 coupling constants[50] (SI-Table 2). To
circumvent the severe peak broadening in the 1H NMR spectrum,
the Fmoc group in 42 was replaced by a 2-N-acetate to give acetamide 44 appropriate for the unambiguous
signal assignment. The α,α-linkage in 35–37 was confirmed by the large 1JC1,H1 coupling constant values of the anomeric carbons
(1JC1,H1 = 170–174 Hz
for α-manno- and 1JC1,H1 = 173–178 Hz for α-gluco-anomers) and by the downfield shifts and the corresponding vicinal
proton coupling constants 3JH1,H2 of the anomeric H-1 signals (δ 5.02–5.12, 3J1,2 = 1.5 Hz for α-manno- and 5.08–5.23, 3J1,2 ≈ 3.7 Hz for α-gluco-anomers).[51] The β-manno linkage in 38–40 was corroborated by the typically
smaller 1JC,H coupling constants
for the anomeric carbons (1JC1,H1 = 155–157 Hz) and by the upfield shifts of the anomeric H-1
signals (4.6 ppm) and H-5 signals (3.28 ppm), characteristic for β-mannosides.[52]
Synthesis of Lipid A Mimetics 1–3 Based on αGlcN(1↔1)αMan
Scaffold
Having
orthogonally protected αGlcN(1↔1)αMan disaccharide
scaffold 36 in hand, we next approached the stepwise
deprotection and acylation at C-2 and C-3 of the GlcN fragment. The
feasibility of regioselective deacetylation at C-3 in the presence
of the adjacent 2-N-Fmoc carbamate and the base-labile
phosphotriester group at C-4 in 36 was first examined
(SI-Scheme 1). Among a variety of tested
conditions, application of aqueous hydroxylamine provided the best
reproducible results for the exclusive removal of 3-O-acetate (SI-Table 3). Cleavage of the
3-O-Ac group to furnish 46 was accompanied
by the migration of the phosphate from C-4 to the liberated hydroxyl
group at C-3 to give 47 (Scheme 6). Besides, a partial hydrolytic loss of one benzyl protecting group
in the phosphotriester 46 leading to formation of the
phosphodiester 48 was also observed. Further improvements
could be achieved through optimization of reaction conditions. Indeed,
when the reaction was terminated prior to completion (48 h), the formation
of the undesired byproducts 47 and 48 could
be largely avoided (2% and 9%, respectively), providing 3-O-deacylated compound 46 in 53% yield (SI-Table 3). Repeated chromatographic purifications
of the disaccharide 46 partly account for the relatively
low yield.
Scheme 6
Synthesis of αGlcN(1↔1)αMan-Based
Lipid A Mimetics
(α,α-GM-LAMs) 1–3
Synthesis of αGlcN(1↔1)αMan-Based
Lipid A Mimetics
(α,α-GM-LAMs) 1–3
Reagents and conditions: (a)
aq. 50% NH2OH, THF, 0 °C: 46 (53%), 47 (2%), 48 (9%), recovered 36 (15%);
(b) 49, DIC, DMAP, CH2Cl2, 0 °C,
83%; (c) DBU, CH2Cl2; (d) 50, EDC,
CHCl3; (e) HF·Py, THF; (f) →57 + 59, DIC, DMAP, 50, CH2Cl2, 0 °C;
(g) →58 + 60, DIC, DMAP, 51, CH2Cl2, 0°C; (h) 1. (BnO)2PN(iPr)2, 1H-tetrazole,
CH2Cl2, 2) mCPBA, −78
°C, 91% for 61, 89% for 62; (i) Pd
black, toluene–MeOH, 1:1, 72% for 1, 87% for 2, 81% for 3.The first
(R)-3-(tetradecanoyloxy)tetradecanoyl
residue at C-3 of the GlcN moiety was introduced by reaction of 46 with β-acyloxyacyl acid 49 under the
agency of DIC and a catalytic amount of DMAP to provide 52 in 83% yield (Scheme 6). Notably, strictly
equimolar amounts of DIC and fatty acid 49, and a catalytic
quantity of DMAP at 0 °C, had to be applied to suppress the concomitant
formation of the co-migrating 3-O-tetradecanoyl (53) and 3-O-alkenoyl (54) byproducts
(11% and 3%, respectively). Application of higher amounts of DIC and/or
fatty acid aimed to accelerate the transformation resulted in augmented
formation of 53 and 54, which could be rationalized
by a probable β-elimination or rearrangement[53] of the in situ-formed O-acyl-oxyacylisourea intermediate. Formation of a 3-O-alkanoyl side product similar to 53 in the DIC/DMAP-mediated
condensation had been previously reported, though no elimination byproduct
such as 54 was detected.[30] Since esterification with β-acyloxyacyl fatty acids under
Steglich conditions is routinely applied in the synthesis of Lipid
A’s and analogues which often display high bioactivity at picomolar
doses, this finding is of importance for the preparation of Lipid
A-based compounds of the uppermost purity.Subsequent Fmoc cleavage
in 52 with DBU followed by
EDC-mediated N-acylation with (R)-3-(dodecanoyloxy)tetradecanoic acid 50 provided
tetraacylated disaccharide 55. Conversion of 4,6-di-O-DTBS derivative 55 into diol 56 was performed under standard conditions with HF·Py in THF.
To introduce acyl and phosphate functional groups at mannose C-4 and
C-6, respectively, without additional protecting group manipulation,
the regioselectivity of the acylation of the diol 56 with
the acyloxyacyl acids 50 or 51 was first
examined. Since hydroxyl groups in the substituted trehaloses are
known to differ in reactivity due to both steric and electronic effects,[38,40] we expected that, in a heavily substituted αGlcN(1↔1)αMan
disaccharide 56, positions C-4 and C-6 at the Man moiety
could be discriminated in a subsequent acylation procedure.Indeed, the major outcome of DIC/DMAP-mediated acylation of 56 with the acids 50 or 51 was not
the intrinsically expected primary 6-OH-derived acylation products,
but the 4-O-acyloxyacyl derivatives 57 and 58 in 51% and 65% yield, respectively, having a
6-OH group at the mannose unit accessible for the ensuing phosphorylation.
Minor amounts of the 4,6-bis-O-acylated derivatives 59 and 60 (24% and 13%, respectively) were isolated
as well. Although the 6-OH group of the mannose residue in αGlcN(1↔1)αMan
disaccharide 56 is somewhat remote from the 2-NH of the
GlcN moiety, the crystal structures of α,α-trehalose-based
compounds indicate spatial proximity of the two groups. Thus, it could
be assumed that the intramolecular hydrogen bonding (2-NHGlcN–6-OHMan) exerts an adverse effect on the reactivity
of the primary hydroxyl group at Man C-6. Furthermore, the bulkiness
of the in situ-formed acyloxyacyl-activated ester
resulting from the reaction of the fatty acids 50 or 51 with DIC/DMAP could also explain the limited access to
the sterically hindered 6-OH group in the α,α-trehalose-like
disaccharide 56.Next, the free 6-OH group in the
hexaacylated disaccharides 57 and 58 was
phosphorylated by reaction with
dibenzyl-(N,N′-diisopropylamino)phosphoramidite
in the presence of a mild acid catalyst, 1H-tetrazole,
and subsequent oxidation with mCPBA at −78
°C to furnish the bisphosphorylated hexaacylated products 61 and 62 in 91% and 89% yield, respectively.
Final debenzylation by hydrogenation of 57, 61, and 62 on Pd-black followed by purification with gel
permeation chromatography on Sephadex SX1 in toluene–methanol
(2:1) afforded target bisphosphorylated Lipid A mimetics 1 and 2 and a monophosphorylated counterpart 3. In contrast to native Lipid A, compounds 1 and 2 do not possess a labile anomeric phosphate functionality;
consequently, they were isolated and biologically assessed as free
acids at the phosphates and could be stored in aqueous solution at
4 °C for several months without any noticeable sign of degradation,
which was confirmed by MALDI-TOF analysis.
Activation of TLR4·MD-2
Complex by α,α-GM-LAMs 1–3
The propensity of the α,α-GM-LAMs 1–3 to stimulate TLR4-mediated immune
signaling was first assessed in the hTLR4/hMD-2/CD14 transfected human
embryonic kidney (HEK) 293 cells (HEK-Blue). Since we were particularly
interested in the molecular recognition mechanisms implicated in the
binding of LPS by the MD-2·TLR4 complex wherein the Lipid A/Re-LPS portion of LPS is exclusively involved, we evaluated
the activities of α,α-GM-LAMs 1–3 compared to E. coliRe-LPS (Figure 1A). It has been previously shown
that the minimum structural requirement for the expression of the
highest cytokine-inducing potency resides in Re-LPS,
which entails two 3-deoxy-d-manno-oct-2-ulosonic
acid (Kdo) residues in addition to Lipid A (Supporting
Information, SI-Figure 1A).[54,55] Moreover,
the Re-LPS has a defined molecular weight (MW) similar
to the MW range for Lipid A mimetics (1.8–2.2 kDa), in contrast
to wild-type LPS having variable MW (10–15 kDa), so that the
direct comparison of a dose-dependent response between compounds 1–3 and Re-LPS is more
appropriate. The TLR4-stimulating activity of Lipid A mimetics 1–3 was examined over a wide concentration
range by monitoring of the activation of the NF-κB regulated
signal transduction pathway via measuring the induction of secreted
embryonic alkaline phosphatase (SEAP) and compared to the responses
elicited by E. coliRe-LPS and SM-MPLA.Remarkably, the glycosylation at C-6′ of Lipid A with Kdo
residues was reported to be responsible for the 10–20-fold
enhancement of the activity of the Kdo/Kdo2-Lipid A (Re-LPS) compared to Lipid A alone in the nanomolar concentration range.[28,54] Notably, conformationally confined α,α-GM-LAM 2 based on just a disaccharide scaffold displayed NF-κB
activation (EC50 = 0.08 nM) similar to those of Re-LPS (EC50 = 0.04 nM) and E. coli LPS (EC50 = 0.08 nM, SI-Figure 1A), and the TLR4 saturation plateau was reached at a concentration
of 1 ng/mL for both ligands (Figure 4). Compound 1, having a 2×CH2-longer acyl side chain at
Man C-4 compared with α,α-GM-LAM 2, was a
less efficient activator of NF-κB (EC50 = 0.4 nM),
so that its TLR4 saturation plateau was reached at a concentration
of 5 ng/mL. Thus, shortening of a lipid side chain at C-4 of the mannose
moiety by 2×CH2 resulted in a 5-fold increase of TLR-4
stimulating activity, which underlines the significance of hydrophobic
interactions at the dimerization interface. Along this line, synthetic
manipulation of the length of the acyl side chain at Man C-4 could
be used for fine-tuning of the hTLR4-mediated activity in α,α-GM-LAMs.
The monophosphate 3 was, as expected, significantly less
active (EC50 = 31 nM) than its bisphosphorylated counterpart 1, but it showed a more potent activation profile than SM-MPLA
at concentrations above 10 ng/mL.
Figure 4
Dose-dependent activation of TLR4 signaling
in hTLR4/hMD-2/hCD14-transfected
HEK293 cells (HEK-Blue) by Lipid A mimetics 1–3 compared to E. coliRe-LPS and S. minnesota MPLA.
Dose-dependent activation of TLR4 signaling
in hTLR4/hMD-2/hCD14-transfected
HEK293 cells (HEK-Blue) by Lipid A mimetics 1–3 compared to E. coliRe-LPS and S. minnesota MPLA.
Modulation of the Expression of Cytokines by α,α-GM-LAMs 1–3 in Human and Mouse Macrophages
Lipid A mimetics 1–3 were examined
for the ability to initiate the expression of tumor necrosis factor-α
(TNF-α), interleukin-8 (IL-8), and monocyte chemotactic protein-1
(MCP-1) in the human monocytic macrophage-like cell line THP-1, which
expresses MD-2, CD14, and a variety of cell surface receptors, including
TLR4.The dose-dependent stimulating activity of synthetic Lipid
A mimetics was cytokine-specific, revealing higher potency in the
induction of the expression of TNF-α and IL-8 by α,α-GM-LAMs 1 and 2 than by Re-LPS/LPS (Figure 5A,B, SI-Figures 2 and 3). The release of MCP-1 induced by α,α-GM-LAM 2 was clearly more effective than the production of this chemokine
by Re-LPS and compound 1 (Figure 5C). Expression of MyD88-dependent chemokine MCP-1
is associated with the activation of the intracellular TLR4·MD-2
complex.[56] The dampened induction of the
expression of cytokines by α,α-GM-LAM 3 correlates
to its chemical structure missing a phosphate group at Man C-6. Our
results indicate that both α,α-GM-LAMs 1 and 2 are more potent activators of the MyD88 signaling pathway
than Re-LPS/LPS (Figure 5, SI-Figure 4).
Figure 5
Dose-dependent expression of cytokines
induced by α,α-GM-LAMs 1–3 in human macrophage cell line THP-1
compared to E. coliRe-LPS. (A)
Production of TNF-α. (B) Induction of the expression of IL-8.
(C) Induction of release of MCP-1.
Dose-dependent expression of cytokines
induced by α,α-GM-LAMs 1–3 in human macrophage cell line THP-1
compared to E. coliRe-LPS. (A)
Production of TNF-α. (B) Induction of the expression of IL-8.
(C) Induction of release of MCP-1.The ability of Lipid A mimetics 1–3 to induce the production of TNF-α and IL-6 from bone
marrow-derived
macrophages (BMDM) in mice was subsequently examined and compared
to that of synthetic E. coli Lipid A and E. coli MPLA, which are reliable positive controls due to
their chemical purity and homogeneity (Figure 6). The maximum level of TNF-α (4000 pg/mL) was detected in
BMDM cultivated in the presence of 1 nM α,α-GM-LAM 1 or 2, whereas the same quantity of the “parent”
Lipid A resulted in release of half the amount of TNF-α (2000
pg/mL). On the other hand, monophosphoryl α,α-GM-LAM 3 exhibited a dampened ability to express TNF-α (500
pg/mL) at a concentration of 1 nM. Likewise, 1 nM (1.8 ng/mL) of 1 or 2 triggered the release of a 3-fold higher
amount of IL-6 (1500 pg/mL) compared to Lipid A (500 pg/mL). Monophosphate 3 revealed a dose-dependent cytokine induction profile showing
only marginal expression level of IL-6 at a concentration of 1 nM
but higher IL-6 release than MPLA at concentrations above 1 nM.
Figure 6
Induction of
expression of TNF-α and IL-6 by Lipid A mimetics 1–3 in mBMDM compared to synthetic E.
coli Lipid A and E. coli MPLA. E.
coli O111:B4 LPS was used as positive control (1 nM).
Induction of
expression of TNF-α and IL-6 by Lipid A mimetics 1–3 in mBMDM compared to synthetic E.
coli Lipid A and E. coli MPLA. E.
coli O111:B4 LPS was used as positive control (1 nM).
TLR4 Stimulating Activities
of α,α-GM-LAMs 1 and 3 in Human
Dendritic Cells
To
test the impact of selected α,α-GM-LAMs 1 and 3 on maturation of human dendritic cells (hDCs),
immature monocyte-derived hDCs were stimulated with 1 and 3 in a wide concentration range or with E. coli LPS as positive control. DCs are able to persistently
sense pathogen-associated molecular patterns and present antigens
to T lymphocytes, thereby initiating an adaptive immune response.[7] DCs treated with LPS acquired a distinctive morphologic
phenotype and, when analyzed by flow cytometry, displayed characteristic
markers of mature DCs.Stimulation of DCs with 1 (1 mg/mL) was as potent as that with LPS in inducing DCs maturation
and up-regulation of the co-stimulatory molecules CD86, as well as
the antigen-presenting structures MHC class I and MHC class II, which
are necessary for the induction of an adaptive immune response (SI-Figure 5). None of the α,α-GM-LAMs
exerted cytotoxic effects on DCs, as determined by propidium iodide
staining (data not shown).Activated DCs were examined for the
production of pro-inflammatory
cytokines TNF-α, IL-6, and IL-12, which contribute to the modulation
of the T cell response and innate effector functions.[57] The release of TNF-α and IL-6 reached nearly maximum
levels (attained with 10 ng/mL LPS) at the α,α-GM-LAM 1 concentration of 1 ng/mL (Figure 7A). Also the expression of IL-12, which promotes the development
of adaptive immune cells and is involved in coordinating innate and
adaptive immunity,[58] was efficiently induced
by α,α-GM-LAM 1, indicating its potential
adjuvant capacity. The release of TNF-α, IL-6, and IL-10, a
unique cytokine with a wide spectrum of anti-inflammatory effects,
in DCs induced by monophosphate 3 was, in agreement with
the experiments in the recombinant hTLR4/hMD-2 signaling system, less
efficient compared to that induced by 1 (Figure 7B).
Figure 7
Expression of cytokines induced by α,α-GM-LAMs 1 and 3 in human dendritic cells. (A) Induction
of cytokine production by 1 in DCs. (B) Expression of
TNF-α, IL-6, and IL-10 induced by monophoshorylated α,α-GM-LAM 3 compared to its bisphosphorylated counterpart 1. E. coli LPS (10 ng/mL) was used as a positive
control.
Expression of cytokines induced by α,α-GM-LAMs 1 and 3 in human dendritic cells. (A) Induction
of cytokine production by 1 in DCs. (B) Expression of
TNF-α, IL-6, and IL-10 induced by monophoshorylated α,α-GM-LAM 3 compared to its bisphosphorylated counterpart 1. E. coli LPS (10 ng/mL) was used as a positive
control.
Conclusions and Perspectives
We have rationally designed, synthesized, and biologically evaluated
an entirely novel type of TLR4 agonists based on the conformationally
restrained disaccharide scaffold which mimics the spatial arrangement
of (1→6) diglucosamine backbone
of MD-2-bound E. coli Lipid A, revealed in the co-crystal
structures of the TLR4·MD-2-LPS complexes. A convergent synthetic
approach toward the αGlcN(1↔1)αMan scaffold
and hexaacylated Lipid A mimetics based thereon
has been developed. Orthogonally protected nonreducing αGlcN(1↔1)αMan disaccharide
was assembled via imidate-mediated glycosylation by taking advantage
of the axial orientation of the anomeric OH group in the 2-N-carbamate-protected GlcN-lactol acceptors. The protecting
group pattern was fine-tuned to ensure stereospecific α,α-(1↔1) glycosylation
and to
afford efficient isolation of the α,α-disaccharide from
the anomeric mixtures. Replacement of the labile anomeric 1-phosphate
functionality (as in natural Lipid A) for a primary phosphate group
in α,α-GM-LAMs 1 and 2 provides
unambiguous advantages with respect to hydrolytic stability.In spite of the lack of Kdo moieties which were demonstrated to
be responsible for a 10–20-fold enhancement of the activity
of Re-LPS (Kdo-Kdo-Lipid A) compared to Lipid A alone,
the NF-κB activation efficacy and cytokine inducing capacity
of bisphosphorylated Lipid A mimetics 1 and 2 in DCs and human macrophages were comparable to or higher than those
of Re-LPS/LPS. The inherent rigidity of the α,α-(1↔1)
glycosidic linkage in 1–3 would not
allow for extensive conformational adjustment of the αGlcN(1↔1)αMan
backbone to the shape of the binding pocket of MD-2, such that the
three-dimensional arrangement of an α,α-GM-LAM molecule
should remain preserved also in the protein-bound state. Accordingly,
our results indicate that restricting the flexibility of the carbohydrate
backbone of Lipid A in an “agonistic” conformation,
as in α,α-GM-LAMs, allows the prearrangement of the phosphate
and acyl groups in the “flipped” Man moiety in a defined
conformation, which results in a very potent TLR4 activation (SI-Figure 6). The shortening of a secondary acyl
chain at Man C-4 and the presence of a phosphate group at Man C-6
significantly enhanced the TLR-4 stimulating activity, which indicates
the involvement of these functionalities at the dimerization interface
with the second TLR4*·MD-2*-ligand complex and opens opportunities
for fine-tuning the activities of α,α-GM-LAMs by chemical
modifications.Since the molecular shape of αGlcN(1↔1)αMan-based
Lipid A mimetics is believed to resemble the conformation of the MD-2-bound E. coli Lipid A in the active [TLR4·MD-2-LPS]2 complex, further immuno-biological studies of the interaction of
α,α-GM-LAMs with TLR4·MD-2 would provide deeper insight
into the molecular basis of TLR4 activation by LPS.Along these
lines, application of a conformationally restricted
“agonistically” shaped disaccharide scaffold in place
of the native βGlcN(1→6)GlcN Lipid A backbone appears
to provide a useful tool for modulation of TLR4·MD-2-mediated
immune signaling. Thus, synthetic αGlcN(1↔1)αMan-based
Lipid A mimetics represent the key structures for the advanced development
of pharmaceutically applicable immuno-therapeutics or vaccine adjuvant
candidates.
Experimental Section
General Synthetic Methods
Reagents and solvents were
purchased from commercial suppliers and used without further purification
unless otherwise stated. Dichloromethane was distilled from CaH2 and stored over activated 4 Å molecular sieves (MS).
THF was distilled over Na/benzophenone directly prior to use. Other
solvents were dried by storage over activated MS for at least 48 h
prior to use [toluene (4 Å), acetonitrile (3 Å), and DMF
(3 Å)]. Residual moisture was determined by colorimetric titration
on a Mitsubishi CA-21 Karl Fischer apparatus and did not exceed 20
ppm for dry solvents. Reactions were monitored by TLC performed on
silica gel 60 F254 HPTLC precoated glass plates with a 25 mm concentration
zone (Merck). Spots were visualized by UV light followed by dipping
into a H2SO4–p-anisaldehyde
solution or a ninhydrin–EtOH solution and subsequent charring
at 250 °C. Solvents were removed under reduced pressure at ≤30
°C. Preparative HPLC was performed with linear solvent gradients
on a YMC Pack SIL-06 250 × 20 mm, S-5 μm, 6 nm column (Column
A, loadings 50–150 mg), or on a YMC Pack SIL-06 250 ×
10 mm, S-5 μm, 6 nm column (Column B, loadings 5–50 mg).
Preparative MPLC was performed on silica gel 60 (230–400 mesh,
Merck). Size exclusion chromatography was performed on Sephadex LH20
or Bio-Beads SX1 (BioRad) supports. NMR spectra were recorded at 298
K on a Bruker Avance III 600 spectrometer (1H at 600.22
MHz; 13C at 150.92 MHz; 31P at 242.97 MHz) or
on a Bruker DPX 400 spectrometer (1H at 400.13 MHz; 13C at 100.61 MHz; 31P at 161.68 MHz) using standard
Bruker NMR software. Chemical shifts are reported in ppm, where 1H NMR spectra in CDCl3 are referenced to internal
TMS and 13C-spectra are referenced to the corresponding
solvent signal (77.0 ppm for CDCl3). NMR spectra in other
solvents are referenced to residual solvent signals (for acetone-d6, 2.05 and 29.84 ppm; for MeOD, 3.31 and 49.00
ppm, 1H and 13C NMR, respectively). 31P NMR spectra in CDCl3 are referenced according to IUPAC
recommendations from 2001 from a referenced 1H NMR spectrum.
In the disaccharides, the mannose NMR signals are indicated by primes.
The purity (>95%) was determined by LC-MS and HRMS. HPLC-MS was
performed
by injections of 0.01–0.1% CH3CN solutions into
a Shimadzu LC-10AD VP system equipped with two gradient pumps, a degasser,
a Shimadzu LCMS 2020 detector, and an AllTech 3300 ELSD detector.
Analytes were eluted over a Phenomenex Jupiter 5μ C4 300A column
using mobile phase A = H2O (0.1% HCOOH) and mobile phase
B = CH3CN (0.1% HCOOH) in linear gradients from 5% B to
100% B and a flow rate of 0.5 mL/min. High-resolution mass spectrometry
(HRMS) was carried out on 1–10 mg/L acetonitrile solutions
via LC-TOF MS (Agilent 1200SL HPLC and Agilent 6210 ESI-TOF, Agilent
Technologies). The mass spectrometer was tuned with Agilent tune mix
to provide a mass accuracy below 2 ppm. The data were analyzed using
Agilent Mass Hunter Software. MALDI-TOF MS was performed in the negative-ion
mode using a Bruker Autoflex Speed instrument with 6-aza-2-thiothymine
(ATT) as matrix. Optical rotation was measured on a PerkinElmer 243B
polarimeter equipped with a Haake water circulation bath and a Haake
D1 immersion circulator for temperature control. All [α]D20 values are reported in units of deg·dm–1·cm3·g–1.
HEK293
stably expressing human TLR4, MD-2,
CD14, and a secreted NF-κB-dependent reporter (HEKBlue hTLR4), E. coli O111:B4 LPS, E. coli serotype R515 Re-LPS, S. minnesota R595 MPLA (SM-MPLA),
and synthetic E. coli MPLA were purchased from InvivoGen.
Synthetic E. coli lipid A was purchased from Peptide
Institute. The THP-1 human monocyte-like cell line was obtained from
Dr. Rene Devos (Roche Research Ghent) and originally purchased from
ATCC. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate
(TPA) was purchased from Sigma. Lipid A mimetics 1–3 were reconstituted in DMSO to provide 1 mg/mL stock solutions.
Further dilutions were made with cell medium (RPMI or DMEM) supplemented
with 10% FCS so that the final amount of DMSO in the cell culture
did not exceed 0.01%.
Growth conditions and activation assay were set as
recommended by InvivoGen. The cells were stimulated with the solutions
of compounds 1–3 or LPS/Re-LPS in
DMEM supplemented by 10% FCS at the indicated concentrations. The
final amount of DMSO in the cell culture did not exceed 0.005% for 1 and 2 or 0.01% for 3. The compounds
were added in a total volume of 20 μL to 25000 HEK-Blue hTLR4
cells in 180 μL plates and were incubated for 20–24 h
at 37 °C and 5% CO2. SEAP levels were determined by
incubation of 20 μL of challenged cell supernatants with 180
μL of detection reagent (QUANTI-Blue), and the color development
was measured at 650 nm using a spectrophotometer (SpectraMAX 190).
Data were combined from n = 3 independent experiments;
error bars indicate standard error of the mean.
Differentiation
and Stimulation of THP-1 Cells
THP-1
cells were grown in RPMI-1640 cell-culture medium (Life Technologies)
that was supplemented with 2 mM l-glutamine, 100 U/mL penicillin,
100 μg/mL streptomycin, and 10% FCS. Cells were seeded in a
96-well plate at 105 cells/well in 150 μL of complete
medium and stimulated by 200 nM TPA for 24 h to induce the differentiation
into macrophage-like cells.[59] On the next
day the cells were washed twice with complete culture medium to discard
the cells that did not adhere, refreshed with 200 μL of complete
medium, and left for 1 h to recover. Cells were stimulated with α,α-GM-LAMs 1–3 at the indicated concentration and
with E. coliRe-LPS (or E. coli O111:B4 LPS), which were added as solutions in 10
μL of complete medium. The total volume of the well after stimulation
reached 220 μL. The cells were incubated for 18 h, and the supernatants
were analyzed for TNF-α, IL-8, and MCP-1 by ELISA (BD Biosciences).
Activation of Mouse Bone Marrow-Derived Macrophages by α,α-GM-LAMs 1–3
Bone-marrow-derived microphages
(BMDMs) were isolated and differentiated from the bone marrow of C57BL/6J
mice. The bone marrow was flushed from femur and fibia with RPMI media.
The erythrocytes were lysed with 0.88% ammonium chloride, 15 min at
37 °C. A single-cell suspension of the bone marrow cells was
then seeded in cell culture flasks at a concentration of 1 ×
106 cells/mL in RPMI supplemented with 20% FBS and 40 ng/mL
recombinant M-CSF. The cell culture medium was changed on day 3. On
day 6, the differentiated cells were trypsinized, counted, and seeded
in 24-well plates at a concentration of 1 × 106 cells/mL
in RPMI supplemented with 10% FBS. After 24 h, the cells were stimulated
with Lipid A mimetics 1–3 and with
synthetic E. coli Lipid A/MPLA (E. coli O111:B4 LPS was used as positive control) for 16 h. The nanomolar
concentrations of 1–3 were calculated
according to the MW of the synthetic compounds. The supernatants were
then tested for cytokines using Ready-Set-Go ELISA kits (eBioscience).
Induction of Cytokine Production by α,α-GM-LAMs 1 and 3 in hDCs
Human peripheral blood
monocytes were cultured for 6 days in GM-CSF and IL-4 to receive immature
monocyte-derived DCs and were then stimulated with the indicated concentrations
of 1 and 3 using 10 ng/mL LPS as positive
control (solutions in PRMI containing 10% FCS and 0.005–0.01%
DMSO). The amounts of IL-6, IL-12, IL-10, and TNF-α in the supernatants
of the cells were analyzed after 24 h by Luminex. Results are representative
of three independent experiments for 1 and of two experiments
for 3. Mean values of duplicate examinations ± SD
are presented.
Authors: Ralph Hollaus; Simon Ittig; Andreas Hofinger; Mira Haegman; Rudi Beyaert; Paul Kosma; Alla Zamyatina Journal: Chemistry Date: 2015-01-28 Impact factor: 5.236
Authors: Holger Heine; Florian Adanitsch; Tina Tinkara Peternelj; Mira Haegman; Christoph Kasper; Simon Ittig; Rudi Beyaert; Roman Jerala; Alla Zamyatina Journal: Front Immunol Date: 2021-03-18 Impact factor: 7.561
Authors: Jesús Arenas; Elder Pupo; Coen Phielix; Dionne David; Afshin Zariri; Alla Zamyatina; Jan Tommassen; Peter van der Ley Journal: Vaccines (Basel) Date: 2020-10-09
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