Synthesis and biological investigation of pim mimics carrying biotin or a fluorescent label for cellular imaging

Article abstract of DOI:10.1021/bc3004974

Phosphatidyl inositol mannosides (PIMs) are constituents of the mycobacterial cell wall; these glycolipids are known to exhibit potent inhibitory activity toward the LPS-induced production of cytokines by macrophages, and therefore have potential as anti-

Full text of DOI:10.1021/bc3004974

Article
pubs.acs.org/bc
Synthesis and Biological Investigation of PIM Mimics Carrying Biotin
or a Fluorescent Label for Cellular Imaging
Sophie Front,† Marie-Laure Bourigault,‡ Stephanie Rose,‡ Segueni Noria,‡ Valerie F. J. Quesniaux,‡
́
́
́
and Olivier R. Martin*,†
†Institut de Chimie Organique et Analytique, Universite
‡Immunologie et Neurogen
etique Experimentales et Molec
45071 Orleans, France
* Supporting Information
́
d’Orlea
́
ns, CNRS UMR 7311, Rue de Chartres, 45067 Orlea
́
ns, France
́
́
́
́
ulaires, Universite
́
d’Orleans, CNRS UMR 7355, 3B Rue de la Ferollerie,
́
́
S
ABSTRACT: Phosphatidyl inositol mannosides (PIMs) are
constituents of the mycobacterial cell wall; these glycolipids
are known to exhibit potent inhibitory activity toward the LPS-
induced production of cytokines by macrophages, and
therefore have potential as anti-inflammatory agents. Recently,
heterocyclic analogues of PIMs in which the inositol is
replaced by a piperidine (aza-PIM mimics) or a tetrahydropyr-
an moiety (oxa-PIM mimics) have been prepared by short
synthetic sequences and shown to retain the biological activity
of the parent PIM structures. In this investigation, the aza-PIM
analogue was used as a convenient scaffold to link biotin or a
fluorescent label (tetramethyl-rhodamine) by way of an
aminocaproyl spacer, with the goal of using these conjugates
for intracellular localization and for the study of the mechanism of their antiinflammatory action. The synthesis of these
compounds is reported, as well as the evaluation of their activities as inhibitors of LPS-induced cytokine production by
macrophages (TNFα, IL12p40); preliminary investigations by FACS and confocal microscopy indicated that PIM−biotin
conjugate binds to macrophage membranes with rapid kinetics.
to be complex, not clearly understood, and may involve both
receptor recognition and direct interaction with the cell
membranes. There is a clear need to perform experiments
with labeled derivatives, for in-cell imaging and localization
studies.
INTRODUCTION
■
Mycobacteria express a number of molecules modulating the
host immune system. Among them, mannose-capped lip-
oarabinomannan (ManLAM), lipomannans (LM), and PIM are
recognized by various pattern recognition receptors present on
macrophages and dendritic cells such as Toll-like receptors
(TLRs)1−5 or C-type lectins like mannose receptor (CD206)
and DC-SIGN (CD209).6−9 Several molecules of mycobacte-
rial origin down-modulate the immune system, including the
ESAT-6 and ManLAM, but also diacylated forms of LM and
their glycosyl-phosphatidylinositol (GPI) anchor structure
phosphatidyl-myo-inositol mannosides (PIM).10−12 Dimanno-
side and hexamannoside PIM (PIM2 and PIM6), the two most
abundant classes of PIM found in M. tuberculosis H37Rv and M.
bovis BCG, inhibit endotoxin-induced proinflammatory re-
sponses, and we could reproduce this activity with synthetic
PIM1 and a mimetic of PIM2.12 We showed that the inhibition
of TNFα and IL-12p40 expression in response to TLR2 or
TLR4 agonists by synthetic PIM1 and PIM2 mimetic could be
partially explained by an inhibition of LPS binding to CD14 in
murine macrophages.13 However, some inhibitory activities of
PIM1 and PIM2 mimetic were independent of CD14, as seen
for the inhibition of IL-12p40 release in response to rough-LPS
stimulation of TLR4 pathway independent of CD14.13 The
mechanisms involved in PIM biological actions are thus likely
The synthesis of PIM1 or PIM2 derivatives carrying
appropriate substituents for labeling would require the
differentiation of the OH groups of the inositol moiety.
Although efficient methodologies are available for selective
protection of the OH groups in myo-inositol,14−16 the synthesis
of such compounds would require long sequences of reactions.
This difficulty could be avoided by taking advantage of our PIM
mimics based on an aza-cyclitol core. We have recently shown17
that the inositol moiety in PIM2 could be advantageously
replaced by a saturated heterocyclic unit, such as a piperidine
(as in IA, Figure 1) or a tetrahydropyran (as in IB): these
compounds retain the biological activity of the parent PIM
structures, provided the nitrogen group in IA is acylated. The
aza-cyclitol moiety present in IA and related compounds
provides thus a point of attachment readily available for
tethering groups which can be used to conjugate PIM mimics
Received: September 4, 2012
Revised: November 6, 2012
© XXXX American Chemical Society
A
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Figure 1. PIM2 mimics built on an heterocyclic core.13
with various markers or carriers. We report in this article the
synthesis of aza-PIM mimics carrying biotin or a fluorescent
label, as well as initial biological investigations of the
immunomodulating activity of the new compounds and of
their utility as imaging probes.
(80 mg) was added and the reaction mixture was stirred at rt
under H2 atmosphere for 18 h. The catalyst was removed by
filtration through a Millipore membrane and washed with
EtOH (2 × 10 mL). The organic phases were combined and
the solvent was evaporated under reduced pressure to give
essentially pure compound 6 (413 mg, 100%) as a white solid.
1H NMR (400 MHz, CD3OD): δ 2.90 (dd, 2H, H1A/5A, J =
12.8, 6.9 Hz), 3.24 (dd, 2H, H5B/1B, J = 12.8, 3.5 Hz), 3.47 (t,
1H, H3, J3,4 = J3,2 = 6.2 Hz), 3.87 (ddd, 2H, H2/4), 4.78 (s, 2H,
CH2Ph), 7.22−7.46 (m, 5H, Ph). 13C NMR (101 MHz,
CD3OD): δ 48.68 (C1/5), 68.38 (C2/4), 74.65 (CH2Ph),
81.04 (C3), 128.70, 128.98, 129.30 (CHAr), 139.70 (CqAr).
MS: calcd for C12H16NO3: 223.12; (+)IS-MS: m/z 224.5 [M
+H]+, 246.5 [M+Na]+.
EXPERIMENTAL PROCEDURES
■
SynthesisGeneral Procedures. Unless otherwise sta-
ted, all reactions requiring anhydrous conditions were carried
out under dry Ar. Dichloromethane (CH2Cl2) was distilled
from calcium hydride (CaH2). Diethyl ether (Et2O) was
distilled from CaH2 and stored at 4 °C, over 4 Å molecular
sieves, under Ar. All reagent-grade chemicals were obtained
from commercial suppliers (Sigma-Aldrich, Acros) and were
1
used as received. H, 13C, 19F, and 31P NMR spectra were
recorded on a Bruker DPX250 or a Bruker Avance 400
spectrometer at 25 °C. Proton chemical shifts (δ) are reported
in parts per million (ppm) relative to Me4Si (δ 0.000 ppm) as
internal standard. Carbon chemical shifts are referenced to the
central line of the CDCl3 triplet (δ 77.16 ppm), unless
otherwise stated. 31P and 19F chemical shifts are reported in
ppm downfield from the external standard, phosphoric acid and
boron trifluoride diethyl etherate, respectively. Coupling
constants (J) are reported in hertz (Hz). Spectral splitting
patterns are designated as follows: s, singlet; d, doublet; t,
triplet; q, quadruplet; m, multiplet; br, broad. Carbon
N-(6-Trifluoroacetamidohexanoyl)-3-O-benzyl-1,5-di-
deoxy-1,5-iminoxylitol (7). To a solution of compound 6
(413 mg, 1.85 mmol) in dry CH2Cl2 (10 mL), 6-
trifluoroacetamidohexanoic acid (462 mg, 2.03 mmol, 1.1
equiv) and 1-hydroxybenzotriazole (HOBt) (300 mg, 2.22
mmol, 1.2 equiv) were added. The mixture was cooled down to
0 °C and dicyclohexylcarbodiimide (DCC) (458 mg, 2.22
mmol, 1.2 equiv) was added. After having been stirred at rt for
18 h, the reaction mixture was diluted with CH2Cl2 (50 mL).
The organic phase was washed with 1 N aqueous HCl (20 mL),
then with brine (20 mL), dried over MgSO4, and evaporated
under vacuum. The residue was purified by silica gel column
chromatography (CH2Cl2/acetone 3/1, Rf = 0.65) to give
1
multiplicities were assigned by DEPT experiments. H and
13C NMR signals were assigned with the help of H−H and H−
C correlations (COSY and HSQC spectra). Flash chromatog-
raphy was performed according to the established protocol on
silica gel 60 (40−63 μm). The purification of final compounds
was performed on Sephadex LH-20 from Sigma. Reactions
were monitored by thin layer chromatography (TLC) (silica gel
60F254 precoated aluminum plates) and the compounds were
detected under UV light and by staining with phosphomolybdic
acid solution. Electron spray-ionization mass spectrometry
(ESI-MS) was performed with a Perkin-Elmer SCIEX API 300
spectrometer. High-resolution mass spectra were recorded on a
1
compound 7 (480 mg, 60%). H NMR (400 MHz, CDCl3;
slow amide bond rotation promotes differentiation of 1/5 and
2/4 positions in the iminoxylitol ring): δ 1.25−1.39 (m, 2H,
2Hc), 1.50−1.68 (m, 4H, 2Hb, 2Hd), 2.30−2.48 (2 td, JAB = 15
Hz, 2H, 2Ha), 3.31 (app q, 2H, 2He, J = 6.6 Hz), 3.36 (dd, 1H,
H1A, J1A,2 = 1.5, J1A,1B = 13.6 Hz), 3.51−3.67 (m, 3H, H5A,
H5B, H3), 3.78 (br, 1H, H4), 3.89 (br, 1H, H2), 4.15 (dd, 1H,
H1B, J1B,2 = 4 Hz), 3.34 (br d, 1H, 4-OH, J = 7.2 Hz), 4.58 (br
s, 1H, 2-OH), 4.66 (AB, 2H, CH2Ph, J = 11.6 Hz), 7.26−7.37
(m, 5H, CHar), 7.53 (t, 1H, NH, J = 4.8 Hz). 13C NMR (101
MHz, CDCl3): δ 24.32 (Cb), 26.15 (Cc), 28.32 (Cd), 32.69
(Ca), 39.56 (Ce), 44.25 (C1), 48.47 (C5), 68.24 (C2, C4),
72.78 (CH2Ph), 76.98 (C3), 116.04 (q, CF3, J = 289 Hz),
127.66, 128.01, 128.57 (CHAr), 137.96 (CqAr), 157.51 (q,
COCF3, J = 37 Hz), 173.90 (CO). 19F NMR (376 MHz,
CDCl3): δ - 75.74. HRMS (ESI): [M+H]+ calcd for
C20H28F3N2O5: m/z = 433.19448; found m/z = 433.19491.
[M+Na]+ calcd for C20H27F3N2NaO5: m/z = 455.17643; found
m/z = 455.17693.
́
Bruker maXis UHR-Q-TOF spectrometer in Orleans.
N-Benzyl-3-O-benzyl-1,5-dideoxy-1,5-iminoxylitol (5).
This was obtained from diacetone glucose as described
recently;18 purification of 5 by silica gel column chromatog-
raphy using petroleum ether/ethyl acetate 2/1 as the eluent was
required for efficient selective N-debenzylation in the next step.
(Yield ∼31% over 4 steps, from 3-O-benzyl-1,2;5,6-diisopro-
pylidene-α-D-glucofuranose).
3-O-Benzyl-1,5-dideoxy-1,5-iminoxylitol (6). The pro-
cedure was improved from the one previously published.17
Compound 5 previously purified (582 mg, 1.85 mmol) was
dissolved in EtOH (15 mL). Palladium hydroxide (Pd(OH)2)
N-(6-Trifluoroacetamidohexanoyl)-3-O-benzyl-2,4-
bis-O-(2,3,4,6-tetra-O-methoxyacetyl-α-D-manno-pyra-
nosyl)-1,5-dideoxy-1,5-iminoxylitol (9). Compound 7 (481
B
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mg, 1.11 mmol) was previously dried overnight under high
vacuum and then dissolved under Ar in CH2Cl2 (10 mL). 4 Å
Molecular sieves were added and the mixture was stirred for 30
min; a solution of 2,3,4,6-tetra-O-methoxyacetyl-α-D-manno-
pyranosyl trichloroacetimidate (8)17 (2.081 g, 3.38 mmol, 3
equiv) in CH2Cl2 (5 mL) was added under argon flux. After
having stirred the mixture for 15 min, trimethylsilyl triflate
(TMSOTf) (266 μL, 1.35 mmol, 40% mol equiv vs the donor)
was added and the mixture was stirred for 1 h at rt under Ar.
The reaction was stopped by the addition of triethylamine (376
μL, 2 equiv vs TMSOTf), and the mixture was filtered through
a sintered glass filter containing Celite. The solvent was
removed under vacuum and the residue was purified by silica
gel column chromatography (toluene/acetone 2.5/1 to 1/1,
difficult separation) to give a pure fraction of compound 9 (553
2H2′, 2H3′, 2H4′), 7.12−7.22 (m, 0.5H, 0.5 NH), 7.23−7.34
1
(m, 0.5H, 0.5NH). H NMR (400 MHz, DMSO-d6, 90 °C):
δ1.30 (quint, 2H, Hc), 1.54 (2 overlapping quint, 4H, 2Hb,
2Hd), 2.36 (t, 2H, 2Ha), 2.6−3 (very broad, 1 × H1, 1 × H5),
3.18 (q, 1H, JHe,Hd ∼6.7 Hz, JHe,NH ∼6.7 Hz, 2He), 3.30, 3.31,
3.32, 3.325, 3.35, 3.36, 3.40, 3.41 (8s, 8 × 3H, 8 × OMe),
3.30−3.50, (br signals, 1 × H1, 1 × H5, H2, H4), 3.58 (dt, 1H,
J3,OH = 5.2 Hz, J2,3 = J3,4 = 8.8 Hz, H3), 3.86−4.13 (several s
and AB systems, 16H, 8 CH2OMe), 4.17 (m, 1H, H5′), 4.2−
4.26 (m, 3H, 2H6′A, H6′B), 4.29 (dd, 1H, J5′,6′A = 5.2 Hz,
J6′A,6′B = 12 Hz, H6′B), 4.53 (dt, 1H, J4′5′ =10 Hz, J5′,6′A= J5′,6′B
=
3.6 Hz, H5′), 5.15 (br s, 1H, H1′), 5.18−5.27 (overlapping
signals, 4H: 2H4′, H2′, H1′), 5.45 (dd, 1H, J2′,3′ = 3.2 Hz, J3′,4′
= 10 Hz, H3′), 5.41 (dd, 1H, J2′,3′ = 3.2 Hz, J3′,4′ = 10 Hz, H3′),
5.43 (1 narrow dd, H2′), 5.95 (d, 1H, J3,OH = 5.2 Hz, HO-3),
9.02 (br signal, 1H, NH). 13C NMR (62.9 MHz, CDCl3): δ
24.05 (Cb), 26.04 and 26.12 (Cc), 28.38 (Cd), 32.50 and 32.57
(Ca), 39.42 and 39.48 (Ce), 42.66, 44.08, 46.38, 47.91 (C1, C5,
4 signals), 59.39, 59.45 (CH3 MAc), 62.30, 62.38, 62.60 (2H6′,
3 signals), 66.00, 66.10, 66.18, 66.60, 68.53, 68.70, 68.38, 69.00,
69.13, 69.61, 69.63, 69.92 (2C2′, 2C3′, 2C4′, 2C5′), 69.27,
69.31, 69.41, 69.52 (8 CH2 MAc), 74.99, 75.72, 76.03, 76.07,
67.98, 78.38 (C2, C3, and C4, 6 signals), 95.93, 95.95, 98.80,
98.98 (2C1′, 4 signals), 118.50 (q, CF3, J = 288 Hz), 157.29 (q,
COCF3, J = 37 Hz), 169.25, 169.26, 169.27, 169.30, 169.38,
169.41, 169.43, 169.50, 169.53, 169.59, 169.77, 170.08, 170.19,
170.22 (8 COOR), 171.50, 171.70 (CONH). 19F NMR (376
MHz, CDCl3): δ −75.82, −75.81. HRMS (ESI): [M+H]+ calcd
for C49H74F3N2O31: m/z = 1243.42221; found m/z =
1243.42145.
1
mg, 37%) as a colorless syrup. H NMR (400 MHz, CDCl3;
presence of amide rotamers and of nonequivalent mannosyl
residues): δ 1.32−1.45 (m, 2H, 2Hc), 1.52−1.75 (m, 4H, 2Hd,
2Hb), 2.25−2.45 (m, 2H, 2Ha), 2.98 (dd, 0.5 H, J = 9.4, 13.0
Hz), 3.07 (dd, 0.5 H, J = 8.8, 12.8 Hz), 3.13 (dd, 0.5 H, J = 9.2,
13.2 Hz), 3.27 (dd, 0.5H, J = 9.0, 13.8 Hz) (4 dd for H1A and
H5A), 3.31−3.53 (m, 26H, 8 × CH3, 2He), 3.55−3.76 (m, 3H,
H2, H3, H4), 3.77−4.21 (21H, 8 × CH2, 2H5′, 2H6′, 2 × 0.5
H1B/5B), 4.21−4.29 (m, 1H, H6′), 4.30−4.35 (m, 0.5H, 0.5
H1B/5B), 4.36−4.46 (m, 1H, H6′), 4.47−4.54 (m, 0.5H, 0.5
H1B/5B), 4.73−4.88 (m, 2H, CH2Ph), 5.01 (s, 0.5H, H1′),
5.06 (s, 0.5H, H1′), 5.11 (s, 0.5H, H1′), 5.15 (s, 0.5H, H1′),
5.21−5.52 (m, 6H, 2H2′, 2H3′, 2H4′), 7.11−7.21 (m, 0.7H,
NH), 7.23−7.44 (m, 5H, 5CHar). 13C NMR (62.9 MHz,
CDCl3): δ 23.91 and 23.98 (Cb), 26.03 and 26.16 (Cc), 28.33
(Cd), 32.39 and 32.49 (Ca), 39.44 (Ce), 41.23, 44.19, 45.63,
47.97 (C1 and C5, 4 signals), 59.34 (CH3 MAc), 59.42 (CH3
MAc), 59.46 (CH3 MAc), 61.78 and 61.88 (C6′), 62.38 and
62.55 (C6′), 65.50, 65.69, 66.05, 66.71, 68.35, 68.60, 68.74,
68.92, 69.07 (2C2′, 2C3′, 2C4′), 69.18−69.80 (6 signals for
MeOCH2), 71.38 and 72.96 (C2 or 4), 75.08 and 75.21
(CH2Ph), 76.57 (C3), 80.35 and 81.91 (C2 or 4), 94.38 and
94.82 (C1′), 98.18 and 98.66 (C1′), 116.05 (q, CF3, J = 289
Hz), 127.7, 128.04, and 128.62 (CHAr), 137.31 (CqAr),
157.27 (q, COCF3, J = 36 Hz), 169.20, 169.34, 169.45, 169.78,
169.90, 170.01, 170.04, 170.19, 171.59, and 171.84 (CO). 19F
NMR (376 MHz, CDCl3): δ −75.81. HRMS (ESI): [M+H]+
calcd for C56H80F3N2O31: m/z = 1333.46916; found m/z =
1333.46832.
N-(6-Trifluoroacetamidohexanoyl)-2,4-bis-O-(2,3,4,6-
tetra-O-methoxyacetyl-α-D-mannopyranosyl)-1,5-di-
deoxy-1,5-iminoxylitol (10). Compound 9 (542 mg, 0.407
mmol) was dissolved in a mixture of CH2Cl2 (15 mL) and
EtOH (20 mL). 10% Palladium on carbon (100 mg) was added
and the reaction mixture was vigorously stirred at rt under an
atmosphere of H2 for 18 h. The reaction mixture was then
filtered through a Millipore membrane to remove the catalyst;
the black solid was washed with a 1:1 mixture of CH2Cl2 and
EtOH (2 × 20 mL). The solvents were then evaporated to give
compound 10 (496 mg, 98%). Rf = 0.59 (CH2Cl2/acetone 2:1).
1H NMR (250 MHz, CDCl3): δ 1.30−1.48 (m, 2H, 2Hc),
1.53−1.76 (m, 4H, 2Hb, 2Hd), 2.29−2.48 (m, 2H, 2Ha),
2.49−2.71 (m, 1H, 0.5 H1A, 0.5 H5A), 2.90−3.21 (m, 1H, 0.5
H1A, 0.5 H5A), 3.30−3.60 (m, 27H, 8 × CH3 MAc + 2He +
1H), 3.60−3.75 (m, 1H), 3.81−4.40 (m, 22H, 8 × CH2 MAc,
4H6′, H5′, H1B/H5B), 4.40−4.53 (m, 1H, H5′), 4.60−4.78
(m, 1H, H1B/H5B), 5.02 (s, 0.5H, H1′), 5.03 (s, 0.5H, H1′),
5.20 (s, 0.5H, H1′), 5.23 (s, 0.5H, H1′), 5.25−5.52 (m, 6H,
N-(6-Trifluoroacetamidohexanoyl)-2,4-bis-O-(2,3,4,6-
tetra-O-methoxyacetyl-α-D-mannopyranosyl)-3-O-
[(2R)(2-hexadecanoyloxy-3-octadecanoyloxypropyl)-
(benzyl)phosphoryl]-1,5-dideoxy-1,5-iminoxylitol (12).
Phosphoramidite 1117 (197 mg, 0.236 mmol, 1.4 equiv) was
added to a solution of compound 10 (210 mg, 0.169 mmol) in
dry CH2Cl2 (11 mL). The mixture was cooled down to 0 °C
and a commercial solution of 1H-tetrazole in acetonitrile
(∼0.40 M), previously dried over 3 Å molecular sieves, was
added (1.27 mL, 0.507 mmol, 3 equiv). The reaction mixture
was stirred for 2 h at rt and then was cooled down to −17 °C. A
solution of mCPBA (purity ∼50%) (175 mg, 0.507 mmol, 3
equiv) in CH2Cl2 (4 mL) dried over some MgSO4 was then
added dropwise. After keeping the mixture for 10 min at −10 to
−15 °C and 1 h at rt, the reaction was quenched by the
addition of saturated aqueous Na2S2O3 (20 mL) and the entire
mixture was extracted with Et2O (2 × 80 mL). The organic
phases were combined and washed successively with saturated
aqueous Na2S2O3 (3 × 15 mL), aqueous NaHCO3 (15 mL),
and brine (15 mL), and then dried over MgSO4. The solvent
was removed in vacuum and the residue was purified by silica
gel chromatography (toluene/acetone 3:1) to give compound
12 (160 mg, 47%). Rf Phosphite = 0.41 and Rf Phosphate = 0.31
(toluene/acetone 2:1). Note: the NMR data of 12 are complex
because of amide rotamers, P*-stereoisomers (≠1:1), and the
1
nonequivalence of the mannosyl residues. H NMR (400 MHz,
CDCl3): δ 0.88 (t, 6H, 2CH3, J = 6.6 Hz), 1.03−1.50 (m, 54H,
26 CH2, 2Hc), 1.51−1.78 (m, 8H, 2Hd, 2Hb, 2CH2 acyl chain),
2.14−2.51 (m, 6H, 2Hb, 2CH2 acyl chain), 3.19−3.55 (m, 27H
including 8 OCH3), 3.55−4.49 (32H, including 8 CH2OMe),
4.95−5.59 (m, 11H), 7.27−7.54 (m, 6H, 5CHar, NH). 13C
NMR (62.9 MHz, CDCl3): δ 14.12 (CH3), 22.69 (CH2), 23.77
and 23.87, 25.96 and 26.13, and 28.23 (Cb, Cc, Cd), 24.85
C
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(CH2), 29.12, 29.17, 29.33, 29.36, 29.55, and 29.71 (CH2),
31.92 (CH2), 32.26 (Ca), 33.97 and 34.12 (CH2), 39.39 (Ce),
39.87, 43.09, 44.45, and 47.14 (C1 and C5), 59.12, 59.17,
59.23, 59.31, 59.33, 59.37, 59.42, 59.47 (8 signals OCH3), 61.71
(CH2), 61.89 (CH2), 62.16−62.58 (broad signal, CH2), 65.35
(CH), 65.97 (broad, CH2), 66.88 (CH), 68.45 (CH), 68.79
(CH), 68.92 (CH), 69.15 (CH), 69.25 (CH2), 69.33 (CH2),
69.43 (CH2), 69.67 (CH), 69.70 (CH), 70.02 (CH), 70.26−
70.59 (broad signal, CH2), 71.41 (broad, CH), 73.69−73.85
(broad, CH), 74.07−74.29 (broad, CH), 74.29−74.58 (broad,
CH), 75.25−75.50 (broad, CH), 75.63−75.96 (broad, CH),
76.72 (CH), 76.94 (CH), 94.38 and 94.40, 94.99 and 95.03,
97.32 and 97.44, 98.34 and 98.40 (C1′, 4 × 2 signals), 116.05
(q, CF3, J = 288 Hz), 128.30, 128.35, 128.44, 128.77, 128.81,
128.87, 128.88, 128.97 (CHAr), 135.49, 135.57 (CqAr), 157.21
(q, COCF3, J = 36 Hz), 169.15, 169.23, 169.31, 169.37, 169.43,
169.52, 169.78, 169.82, 169.95, 170.04, 170.13 (CO), 171.86
and 171.89, 172.08 and 172.14, 172.86 and 172.89, 173.24 and
173.26 (CONH, COOR). 31P NMR (162 MHz, CDCl3): δ
−1.30, −1.08, −1.03, −1.01. 19F NMR (376 MHz, CDCl3): δ
−75.83, −75.80. HRMS (ESI): [M+H]+ calcd for
C93H151F3N2O38P: m/z = 1991.96291; found m/z =
1991.96052.
(164 μL) was added. The mixture was stirred at 0 °C for 10
min then warmed up to rt and stirred for an additional 2 h;
solvents were evaporated without heating and the residue was
purified by flash chromatography (CHCl3/MeOH/H2O 70/
40/1) to give compound 1 (73 mg, 78%) as an amorphous
solid. Note: Under these conditions, the COCF3 group is not
1
cleaved. H NMR (400 MHz, CDCl3/CD3OD 0.35/0.2 mL;
lock on CD3OD; presence of amide rotamers in a ratio of about
2:1): δ 0.86 (t, 6H, CH3, J = 6.8 Hz), 1.17−1.35 (m, 52H, 26
CH2), 1.35−1.43 (m, 2H, 2Hc), 1.52−1.69 (m, 8H, 2Hd, 2Hb,
2CH2 acyl chain), 2.31 (2 t, 4H, 2CH2 acyl chain, J = 7.6 Hz),
2.44 (t, 2H, 2Ha, J = 7.2 Hz), 3.28 (app t, 2H, He, J = 7.1 Hz),
3.39−3.95 (m, 16H), 3.95−4.03 (m, 2H, 2H1a), 4.08−4.28 (m,
2H incl. H3aA), 4.28−4.41 (m, 2H), 4.44 (dd, 1H, H3aB, J =
2.6, 12.1 Hz), 4.95 (s, 0.3H, H1′), 4.98 (s, 0.7H, H1′), 5.02 (s,
0.3H, H1′), 5.09 (s, 0.7H, H1′), 5.18−5.29 (m, 1H, H2a). 13C
NMR (62.9 MHz, CDCl3/CD3OD 0.35/0.2 mL; lock on
CD3OD): δ 14.39 (CH3), 25.45 and 25.50, 27.09, 29.13, 33.16,
and 33.30 (Ca-Cd), 23.33, 25.59, 25.64, 29.8−30.4, 32.63,
34.73, 34.88 (acyl CH2), 40.27 and 40.32 (Ce), ∼48−49
(broad, C1/C5), 61.89 (broad, C6′), 62.27 (broad, C6′), 63.44
(C3a), 64.36 (broad, C1a), 67.61, 68.26, 71.05, 71.19, 71.30,
71.69, 71.92,72.27, 74.06, 74.29, 74.73 (all CH), 97.57 (C1′),
N-(6-Trifluoroacetamidohexanoyl)-2,4-bis-O-(2,3,4,6-
tetra-O-methoxyacetyl-α-D-mannopyranosyl)-3-O-
[(2R)(2-hexadecanoyloxy-3-octadecanoyloxypropyl)-
phosphoryl]-1,5-dideoxy-1,5-iminoxylitol (13). Com-
pound 12 (158 mg, 0.079 mmol) was dissolved in a mixture
of CH2Cl2 (6 mL) and EtOH (8 mL). 10% Palladium on
charcoal (50 mg) was added and the reaction mixture was
stirred at rt under H2 atmosphere for 4 h. The catalyst was
removed by filtration through a Millipore membrane. The solid
was washed with a 1:1 mixture of CH2Cl2 and EtOH (2 × 10
mL). The organic phases were combined and the solvents were
evaporated to give compound 13 as a white solid (142 mg,
95%). 1H NMR (250 MHz, CDCl3): δ 0.88 (t, 6H, CH3, J = 6.8
Hz), 1.17−1.36 (m, 52H, 26CH2), 1.36−1.52 (m, 2H, 2Hc),
1.52−1.78 (m, 8H, 2Hd, 2Hb, 2CH2 acyl chain), 2.23−2.54 (m,
6H, 2Ha, 2CH2 acyl chain), 3.21−3.51 (m, 26H, 8xOCH3,
2He), 3.51−4.48 (m, 33H), 5.04 (s, 0.5H), 5.10 (s, 0.5H), 5.11
(s, 0.5H), 5.17 (s, 0.5H) (2 × H1′), 5.21−5.54 (m, 7H, 2H2′,
2H3′, 2H4′, H2a), 7.34 (br, 0.5 NH), 7.49 (br, 0.5 NH). 13C
NMR (62.9 MHz, CDCl3): δ 14.15 (CH3), 23.83 and 23.95,
26.03 and 26.20, 28.23 (br) and 32.27 (br) (4CH2, Ca-Cd),
22.72, 24.91, 24.98, 29.2−29.8, 31.96, 32.27, 34.06, and 34.20
(CH2 acyl chains), 39.43 and 39.45 (Ce), 39.70−39.89, 42.97−
43.14, 44.28−44.67, 47.86−47.31 (4 broad signals for C1,C5),
59.11, 59.15, 59.27, 59.30, 59.39, 59.44 (6 signals for OCH3),
62.10−62.56, 64.98−65.34, 65.74−66.05, 66.74, 68.42−70.42
(several signals), 71.55−71.74, 73.71−73.98, 74.60−74.97,
94.36 and 95.06 (C1′), 97.06 and 98.31 (C1′), 116.10 (q,
CF3, JC−F = 288 Hz), 157.25 (q, COCF3, JC−F = 37 Hz),
169.35−169.78, 170.18, 170.25 (CO), 172.12, 172.42, 173.13,
173.43 (CONH, COOR). 31P NMR ({1H}, 162 MHz, CDCl3):
δ −1.57 −1.44. 19F NMR (376 MHz, CDCl3): δ −75.78,
−75.75. HRMS (ESI): [M+H]+ calcd for C86H145F3N2O38P:
m/z = 1901.91596; found m/z = 1901.91178.
99.25 (C1′), 99.81 (C1′), 101.39 (C1′), 116.94 (q, CF3, JC−F =
287 Hz), 158.61 (s, COCF3, JC−F = 37 Hz), 174.12, 174.41,
174.60, 174.76 (CO′s). 31P NMR (162 MHz, CDCl3/CD3OD
0.35/0.2 mL): δ −0.49. 19F NMR (376 MHz, CDCl3): δ
−76.66, −76.78. HRMS (ESI): [M+H]+ calcd for
C62H113F3N2O22P: m/z = 1325.74692; found m/z =
1325.74709.
N-(6-Trifluoroacetamidohexanoyl)-2,4-bis-O-(2,3,4,6-
tetra-O-methoxyacetyl-α-D-mannopyranosyl)-3-O-
[(2R)(2,3-bis(hexadecyloxy)propyl)(benzyl)phosphoryl]-
1,5-dideoxy-1,5-iminoxylitol (15). Compound 1419 (318
mg, 0.409 mmol, 1.4 equiv) and compound 10 (358 mg, 0.288
mmol) were separately dried overnight under vacuum over
P2O5. CH2Cl2 (6 mL) was added to compound 10 and CH2Cl2
(3 mL) was added to compound 14 under Ar. The solution of
compound 14 was added to the solution of compound 10 and
the flask rinsed with 1 mL of CH2Cl2. Molecular sieves (3 Å)
were added, and after 15 min, the solution was cooled down to
0 °C. A commercial solution of 1H-tetrazole in acetonitrile
(∼0.45 M) (1.92 mL, 0.864 mmol, 3 equiv), previously dried
over 3 Å molecular sieves, was then added. The reaction
mixture was stirred for 2 h at rt and was then cooled to −17 °C.
A solution of mCPBA (purity ∼50%) (298 mg, 0.864 mmol, 3
equiv) in CH2Cl2 (4 mL) dried over some MgSO4 was then
added dropwise. After having been stirred for 10 min at −15 to
−10 °C and 1 h at rt, the reaction was quenched by the
addition of saturated aqueous Na2S2O3 (20 mL). The whole
mixture was extracted with Et2O (2 × 90 mL). The organic
phases were combined and washed successively with saturated
aqueous Na2S2O3 (3 × 20 mL), aqueous NaHCO3 (20 mL),
and brine (20 mL), and then dried (MgSO4). The solvent was
removed in vacuum and the residue was purified by silica gel
column chromatography (CH2Cl2/acetone 3/1) to give
compound 15 (238 mg, 43%) as a syrup. Rf phosphite = 0.42
and Rf phosphate = 0.27 (CH2Cl2/acetone 4/1). NMR data are
complex because of amide rotamers, P*-isomers, and non-
equivalence of the mannosyl residues. 13C NMR (62.9 MHz,
CDCl3): δ 14.12 (CH3), 23.72 and 23.82, 25.94 and 26.1, 28.12
and 28.25, and 32.16 (Ca-Cd), 22.68, 26.05, 26.09, 29.4−30.0,
31.92 (CH2 acyl chains), 39.38 (Ce), 38.92−39.20 (broad),
N-(6-Trifluoroacetamidohexanoyl)-2,4-bis-O-(α-D-
mannopyranosyl)-3-O-[(2R)(2-hexadecanoyloxy-3-
octadecanoyloxypropyl)phosphoryl]-1,5-dideoxy-1,5-
iminoxylitol (1). Compound 13 (135 mg, 0.071 mmol) was
dissolved in a 1:4 mixture of CHCl3 and MeOH (2 mL). The
reaction mixture was cooled down to 0 °C and t-butylamine
D
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Bioconjugate Chemistry
Article
42.39−42.91 (broad), 43.83−44.21 (broad), 46.61−47.07
(broad) (C1 and C5), 59.00−59.45 (6 signals for OCH3),
61.56−61.97 (broad, CH2, C6′), 62.30 and 62.53 (CH2, C6′),
65.29 (CH), 65.89 (CH), 67.04 (CH), 67.73−68.07 (broad,
CH2), 68.16−68.36 (broad, CH), 68.85 (CH), 69.08 (CH),
69.21 (CH2), 69.32 (CH2), 69.42 (CH2), 69.90 (CH), 70.06
(CH2), 70.55 (CH2), 70.63 (CH2), 71.19 (CH), 71.82 (CH2O
alkyl chain), 72.0−75.0 (several broad signals, CH), 76.99
(CH), 77.36 (CH), 94.36 (C1′), 95.14 (C1′), 96.28 and 96.47
(C1′), 97.80 and 98.01 (C1′), 116.08 (q, CF3, J = 288 Hz),
128.1−128.8 (CHAr), 135.56 and 135.60 (CqAr), 135.65 and
135.68 (CqAr), 157.19 (q, COCF3, J = 36 Hz), 157.22 (q,
COCF3, J = 36 Hz), 169.07−170.08 (12 lines for MAc-CO),
C67H119F3N2O20P: m/z = 1359.80404; found m/z =
1359.80231.
N-(6-Trifluoroacetamidohexanoyl)-2,4-bis-O-(α-D-
mannopyranosyl)-3-O-[(2R)-(2,3-bis(hexadecyloxy)-
propyl)phosphoryl]-1,5-dideoxy-1,5-iminoxylitol (2).
Compound 16 (31 mg, 0.023 mmol) was dissolved in a 2:1
mixture of CH2Cl2/EtOH (6 mL). 10% Palladium on charcoal
(50 mg) was added and the reaction was stirred at rt under H2
atmosphere for 3.5 h. The solid catalyst was removed by
filtration through a Millipore membrane. The catalyst was
washed with a 1:1 mixture of CH2Cl2 and EtOH (2 × 20 mL).
The solvents were evaporated to give compound 2 (26 mg,
90%) as a solid. Rf (16) = 0.65, Rf (2) = 0.20 (CH2Cl2/MeOH
4:1). The NMR data are complex because of amide rotamers
(∼70/30) and nonequivalence of the mannosyl residues. 1H NMR
(400 MHz, CDCl3/CD3OD 0.35/0.2 mL + 1 drop D2O − lock
on CD3OD): δ 0.85 (t, 6H, 2CH3, J = 6.8 Hz), 1.14−1.41 (m,
69H), 1.48−1.67 (m, 9H), 2.33−2.44 (m, 2H, 2Ha), 3.26 (app
t, 2H, 2He, J = 6.6 Hz), 3.39−3.93 (m, 28H), 3.93−4.08 (m,
2H), 4.31−4.46 (m, 1H), 4.98 (several br s, 2H, 2H1′). 13C
NMR (101 MHz, J-Mod, CDCl3/CD3OD 0.35/0.2 mL + 1
drop D2O − lock on CD3OD): δ 14.33 (CH3), 25.12 and
25.17, 26.79, 28.84 and 28.84, 32.93, and 33.06 (Ca-Cd), 23.14,
26.52, 26.57, 29.84, 30.02, 30.14, 30.41, 30.89, 32.41 (CH2 acyl,
alkyl chains), 40.04 and 40.10 (Ce), 40.59, 43.85, 45.69, and
47.87 (4 signals, C1 and C5), 61.94 and 62.14 (2C6′), 66.87
(CH2), 67.48, 67.56 (CH), 67.84 (small broad CH), 70.50
(CH), 70.55 (CH2), 70.64 (CH), 70.98 and 71.02 (CH), 71.32
(OCH2), 71.40 (CH), 71.65 (CH), 72.38 (OCH2), 72.90
(CH), 73.91 (CH), 74.14 (CH), 74.57 (CH), 78.11 (C3),
97.42, 99.14, 100.06, and 101.77 (4 signals, C1′), 116.67 (q,
CF3, J = 288 Hz), 158.46 (q, COCF3, J = 37 Hz), 173.79 and
174.28 (CONH). 31P NMR (162 MHz, CDCl3): δ −1.45 ppm.
19F NMR (376 MHz, CDCl3): δ −76.45 (small pic) and
−76.44. HRMS (ESI): [M-H]− calcd for C60H111F3N2O20P: m/
z = 1267.74254; found m/z = 1267.74310.
172.19 and 172 25 (CONH), 172.40 and 172.49 (CONH). 31
P
NMR (162 MHz, CDCl3): δ −1.04. 19F NMR (376 MHz,
CDCl3): δ −75.79, −75.75, and −75.74. HRMS (ESI): [M
+Na]+ calcd for C91H150F3N2NaO36P: m/z = 1957.95502;
found m/z = 1957.95012.
N-(6-Trifluoroacetamidohexanoyl)-2,4-bis-O-(α-D-
mannopyranosyl)-3-O-[(2R)(2,3-bis(hexadecyloxy)-
propyl)(benzyl)phosphoryl]-1,5-dideoxy-1,5-iminoxyli-
tol (16). Compound 15 (56 mg, 0.029 mmol) was dissolved in
a 1:4 mixture of CHCl3 and MeOH (1 mL). The reaction
mixture was cooled down to 0 °C and t-butylamine (164 μL)
was added. After having been stirred at 0 °C for 45 min, the
mixture was evaporated without heating and the residue was
purified by flash chromatography (CHCl3/MeOH 8/1 to 4/1)
to give compound 16 (32 mg, 82%) as an amorphous solid. Rf
(15) = 0.81 and Rf (16)= 0.38 (CH2Cl2/MeOH 6:1). NMR
data are complex because of amide rotamers, P*-isomers, and
1
nonequivalence of the mannosyl residues. H NMR (400 MHz,
CDCl3/CD3OD 0.35/0.2 mL, lock on CD3OD, calibration
TMS ; amide rotamers: ∼2:1 ratio): δ 0.89 (t, 6H, 2CH3, J =
6.6 Hz), 1.18−1.48 (m, 58H), 1.48−1.70 (m, 8H), 2.34−2.48
(m, 2H, 2Ha), 3.30 (t, 2H, 2He, J = 6.8 Hz), 3.38−3.96 (m,
25H) 3.97−4.09 (m, 1.7H), 4.09−4.18 (m, 1H), 4.23 (m,
0.36H), 4.37−4.48 (m, 1H), 4.85−5.01 (6 lines, 2H, 2H1′),
5.11−5.20 (m, 2H, CH2Ph), 7.33−7.48 (m, 5H, 5CHAr). 13C
NMR (101 MHz, CDCl3/CD3OD 0.35/0.2 mL; sm = small
signals, probably due to minor rotamer form): δ 14.29 (CH3),
25.01 and 25.07, 26.70, 28.79 and 28.84, 32.87, and 33.00 (Ca-
Cd), 23.09, 26.45, 26.48, 26.50, 29.79, 30.00, 30.13, 30.36,
32.37 (CH2 acyl chain), 39.96 and 40.02 (Cf), 40.76, 43.8
(broad), 45.66, 47.85 (4 signals, C1/5), 62.05, 62.12, 62.35, and
62.05 (4 signals, 2 × C6′), 67.46 (sm, CH), 67.56 (CH), 67.67
(CH), 68.07 (CH2), 68.14 (CH2), 68.20 (CH2), 68.74 (CH2),
69.90 (CH2), 69.93 (CH2), 70.12 (sm, CH), 70.17 (sm, CH),
70.22 (sm, CH), 70.27 (sm, CH), 70.52 (CH), 70.69−70.75
(broad, CH2Ph), 70.93 (CH), 71.19 (CH2O alkyl chain), 71.28
(sm, CH), 71.39 (CH), 71.60 (CH), 72.04 (sm, CH), 72.09
(sm, CH), 72.14 (sm, CH), 72.34 (CH2O alkyl chain), 73.36
(broad, CH), 73.97 (CH), 74.05 (CH), 74.15 (CH), 74.26
(sm, CH), 74.62 (CH), 74.67 (CH), 74.75 (sm, CH), 74.81
(sm, CH), 74.85 (sm, CH), 76.42 (sm, CH), 76.48 (sm, CH),
76.58 (sm, CH), 76.66 (sm, CH), 77.26 (sm, CH), 77.50
(CH), 77.57 (CH), 97.42 and 97.47, 99.17, 100.70 and 100.72,
102.27 and 102.33 (7 signals, C1′), 116.62 (q, CF3, J = 288
Hz), 128.73, 128.78, 128.27, 129.19, 129.25, 129.38, 129.42
(CHAr) 135.88−135.96 (CqAr), 158.40 (q, COCF3, J = 37
Hz), 173.57 and 174.03 (CONH). 31P NMR (162 MHz,
CDCl3): δ −2.17 ppm. 19F NMR (376 MHz, CDCl3): δ
−76.43 and −76.44 (∼2:1). HRMS (ESI): [M+H]+ calcd for
N-(6-Aminohexanoyl)-2,4-bis-O-(α-D-mannopyrano-
syl)-3-O-[(2R)(2,3-bis(hexadecyloxy)propyl)phosphoryl]-
1,5-dideoxy-1,5-iminoxylitol (17). Compound 2 (23 mg,
0.018 mmol) was dissolved under Ar in MeOH (2 mL). A
solution of MeONa was prepared (∼8 mg Na in MeOH (2
mL)) under Ar. This solution was transferred to the solution of
compound 2. The reaction mixture was then stirred for 40 h at
25 °C under vacuum (∼0.4 bar) to trap methyl trifluoroacetate
(bp = 43−43.5 °C) until complete deprotection. The base was
neutralized with AcOH (2 drops) and the solvent was evapored
to give compound 17 containing sodium acetate. Rf (2) = 0.78,
Rf (17) ∼0.1 (CH2Cl2/MeOH/D2O 70/40/6) (pink in
1
ninhydrin spot test). H NMR (400 MHz, CDCl3/CD3OD
0.4/0.3 mL − lock on CD3OD): δ 0.85 (CH3), 1.15−1.34 (m,
52H), 1.34−1.47 (m, 2H, 2Hc), 1.47−1.59 (m, 4H, 2CH2 alkyl
chain), 1.59−1.74 (m, 4H, 2Hb, 2Hd), 2.25−2.39 (m, 1H,
HaA), 2.42−2.63 (m, 1H, HaB), 2.80−3.00 (m, 2H, 2He),
3.01−3.16 (m, 1H), 3.39−3.50 (m, 4H, OCH2 alkyl chain
+2H), 3.50−3.99 (several m, 21H), 4.26−4.63 (m, 2H), 4.93−
5.03 (2 br s, 1.4H, H1′), 5.08−5.16 (m, 0.6H, H1′). 31P NMR
(162 MHz, CDCl3): δ 0.197 ppm. 19F NMR (376 MHz,
CDCl3/CD3OD 0.4/0.3): δ −76.39 (trace of fluorinated
impurity, probably CF3COONa). HRMS (ESI): [M+H]+
calcd for C58H114N2O19P: m/z = 1173.77479; found m/z =
1173.77413.
N-(6-Biotinylamidohexanoyl)-2,4-bis-O-(α-D-manno-
pyranosyl)-3-O-[(2R)(2,3-bis(hexadecyloxypropyl)-
E
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Bioconjugate Chemistry
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phosphoryl]-1,5-dideoxy-1,5-iminoxylitol (3). Compound
17 (20 mg, 0.0167 mmol) was dissolved in dry DMF (0.5 mL)
under Ar; 0.5 mL of CH2Cl2 was then added to increase
solubility. Et3N (116 μL, 0.084 mmol, 5 equiv) was added, then
(+)-biotin N-hydroxysuccinimide ester (6.9 mg, 0.02 mmol, 1.2
equiv). The mixture was stirred for 5 h under Ar. The solvents
were evaporated under vacuum. The residue was purified by gel
permeation chromatography on Sephadex LH-20 (diam = 25
mm, H = 400) with MeOH/CH2Cl2 2/1 as the eluent (V0 = 15
mL, fractions = 5 mL, collecting compound in tubes 14 to 19)
to give the expected biotinylated derivative 3 (17 mg, 74%) as a
white solid. Rf (3) = 0.35 (CH2Cl2/MeOH/D2O 70/40/6). 1H
NMR (400 MHz, CDCl3/CD3OD 0.35/0.2 mL + 1 drop D2O
− lock on CD3OD; amide rotamers in ratio ∼2:1): δ 0.85
(CH3), 1.15−1.45 (m, 58H), 1.45−1.75 (m, 10H), 2.19 (t, 2H,
2HI), 2.26−2.49 (m, 2H, 2Hb), 2.71 (d, 1H, HDA, J = 12.4 Hz),
2.9 (dd, 1H, HDB, J = 4.8, 12.4 Hz), 3.06−3.22 (m, 4H, 2Hf,
2HE), 3.39−4.0 (several m, 28H), 4.26−4.34 (m, 1H, HB),
∼4.5 (hidden by HOD, HC), 4.92 (s, 0.31H, H1′), 4.95 (s,
0.69H, H1′), 5.01 (s, 0.3 H, H1′), 5.09 (s, 0.7H, H1′). 13C
NMR (62.9 MHz, CDCl3/CD3OD 0.35/0.2 mL + 1 drop of
D2O − lock on CD3OD): δ 14.37 (CH3), 23.14, 25.23, 26.53,
26.53, 26.60, 27.06, 28.60, 28.86, 29.35, 29.84, 30.06, 30.20,
30.88, 32.41, 32.92, 36.15, 39.74 and 39.81, 40.72 (all CH2),
56.24 (CH), 60.81 (CH), 61.87 (broad, CH2), 62.51, 65.74
(broad), 67.35 (CH), 68.11 (broad, CH), 68.83 (CH2), 70.17−
70.45 (broad, CH2), 70.85 (broad, CH2), 71.13 (CH), 71.22
(OCH2 alkyl), 71.38 (CH), 71.53 (CH), 71.71 (CH), 72.32
(OCH2 alkyl), 74.00 (CH), 74.48 (CH), 78.16 (CH), 97.36
(C1′), 98.28 (C1′), 100.57 (C1′), 103.50 (C1′), 165.07 and
165.10 (CO biotin), 174.61 (CONH), 175.34 (CONH). 31P
NMR (162 MHz, CDCl3/CD3OD 0.35/0.2 mL + 1 drop of
D2O): δ − 1.856 ppm. 19F NMR (376 MHz): no signal. HRMS
(ESI): [M+H]+ calcd for C68H128N4O21PS: m/z = 1399.85239;
found m/z = 1399. 85097.
70.38 (OCH2), 70.50, 70.69, 70.91, 71.47 (OCH2), 72.93,
73.13, 73.55, 77.35, 77.65, 77.71, 96.23 (CHAr-Rh), 96.33
(broad, C1′), 97.54 (broad, C1′), 112.72 (CHAr-Rh), 121.79
(broad, CAr-Rh), 122.58 (broad, CAr-Rh), 129.77 (CHAr-Rh),
131.34 (CHAr-Rh), 141.88 (broad, CAr-Rh), 156.05 (minor
rotamer), 156.14 (major), 156.61 (minor), 156.69 (major)
(CAr-Rh), 171.09 (COOH-Rh), 173.11 (CONH minor
rotamer), 173.57 (CONH major), 180.24 (CS). 31P NMR
(162 MHz, CDCl3/CD3OD 0.35/0.2 mL − lock CDCl3): δ
4.05 (minor rotamer), 4.26 (major rotamer). 19F NMR (376
MHz): no signal. HRMS (ESI): [M+H]+ calcd for
C83H135N5O22PS: m/z = 1616.905155; found m/z =
1616.903886.
BIOLOGICAL PROCEDURES
■
Bioassays. Murine bone marrow cells harvested from
femurs were cultivated (106/mL) for 7 days in Dulbecco’s
minimal essential medium (DMEM) supplemented with 2 mM
L-glutamine, 20% horse serum, and 30% L929 cell-conditioned
medium as a source of M-CSF. After three further days in fresh
medium, the bone marrow-derived macrophages (BMDM; 105
cells/well) in DMEM supplemented with 2 mM L-glutamine, 2
× 10−5 M β-mercapto-ethanol, and 0.1% fetal calf serum (FCS)
were stimulated with 100 ng/mL of LPS (Escherichia coli,
serotype O111:B4, Sigma, St. Louis, MO) in the presence of
IFN-γ (500 U/mL). Lyophilized PIM preparations were
solubilized in DMSO and used at a noncytotoxic, maximum
1% DMSO final concentration. PIM or DMSO vehicle controls
were added 30 min prior to LPS stimulation (100 ng/mL).
Absence of cytotoxicity was controlled using MTT incorpo-
ration. Cell culture supernatants were harvested after 24 h at 37
°C and TNFα and IL-12p40 cytokine content were measured
using commercially available ELISA Kits (Duoset R&D
Systems, Abingdon, U.K.). Nitrite was determined using the
Griess reaction (3% phosphoric acid, 1% p-aminobenzenesul-
fonamide, 1% N-(1-naphthyl)ethylenediamine). Results are
expressed as percent of inhibition as compared with full release
in the presence of LPS plus DMSO vehicle.
Tetramethylrhodamine-Labeled N-(6-aminohexano-
yl)-2,4-bis-O-(α-D-mannopyranosyl)-3-O-[(2R)(2,3-bis-
(hexadecyloxy)propyl)phosphoryl]-1,5-dideoxy-1,5-imi-
noxylitol (4). Compound 17 (21 mg, 0.0176 mmol) was
dissolved in dry DMF (1 mL) under Ar. Et3N (100 μL, 0.724
mmol, 41 equiv) was added followed by tetramethylrhodamine
isothiocyanate (TRITC, mixture of 5- and 6-isomers; 10 mg,
0.02 mmol, 1.1 equiv). The mixture was stirred for 15 h under
Ar. The solvent was then removed under vacuum. The residue
was first submitted to gel permeation chromatography on
Sephadex LH-20 (diam = 25 mm, H = 400 mm) with MeOH/
CH2Cl2 2/1 as the eluent. A second purification by flash
chromatography (CH3Cl3/MeOH/D2O 70/40/5) was neces-
sary to give pure compound 4 (15 mg, 52%) as an amorphous
Immunoassay. Biotinylated PIM2 derivative 3 at the
indicated concentrations in PBS (100 μL/well) was coated
on a microtiter plate and incubated overnight at room
temperature. After saturation with 1% BSA in PBS (200 μL/
well) for 1 h at room temperature and three washings with
0.05% Tween 20-PBS, the bound PIM−biotin was revealed by
incubation with horseradish peroxidase−streptavidin D con-
jugate (1/2000, Vector Laboratories) diluted in 1% BSA in
PBS, for 20 min at room temperature. After addition of the
substrate (ABTS, 2,2′-azinobis[3-ethylbenzothiazoline-6-sul-
fonic acid] diammonium salt, at 0.3 g/L in 0.1 M anhydrous
citric acid containing 0.3% H2O2) for 20 min at room
temperature, absorbance at 405 nm was measured with a
microplate reader (Bio-Tek Instrument, Inc.).
1
magenta solid. Rf ∼0.63 (CH3Cl3/MeOH/D2O 70/40/6). H
NMR (250 MHz, CDCl3/CD3OD 0.35/0.2 mL; lock CDCl3;
calibration TMS): δ 0.88 (t, 6H, 2CH3, J = 6.5 Hz), 1.11−1.46
(m, 54H), 1.47−1.74 (m, 8H), 2.27−2.55 (m, 2H, 2Ha), 3.00−
3.31 (m, 12H, 4CH3−Rh), 3.39−4.00 (30H), 4.96 (br s, 0.3H,
H1′), 4.99 (br s, 0.7H, H1′), 5.08 (br s, 0.3H, H1′), 5.18 (br s,
0.7H, H1′), 6.67 (s, 2H, 2CH-Rh), 6.80−6.87 (m, 2H, 2CH-
Rh), 7.20−7.45 (m, 3H, 3CH-Rh), 7.87−8.18 (2H, 2CH-Rh).
13C NMR (126 MHz, CDCl3/CD3OD 0.35/0.2 mL− lock
CDCl3 − Calibration CDCl3): δ 13.58 (CH3), 22.34, 24.59,
24.78, 25.78, 25.82, 26.43, 28.12, 29.0−29.7, 31.62, 32.14 (Cb,
major rotamer), 32.39 (Ca, minor rotamer), 38.93, 40.09
(CH3−Rh), 43.84, 46.70, 60.23, 61.11, 61.33, 64.98, 66.22,
66.41, 67.29, 68.05−68.47 (broad), 69.74, 69.91, 70.14, 70.33,
Flow Cytometry. Macrophages (1 × 106 cells in 50 μL
DMEM containing 0.2% FCS) were incubated with biotiny-
lated PIM2 derivative 3 at the indicated concentrations in PBS
(100 μL/well) for 1−30 min at 37 °C under gentle agitation.
Cells were washed with ice-cold PBS and stained with
streptavidin−Alexa A532 conjugate (diluted 100× in cold
PBS) for 15 min. After further washings and fixation with 3%
paraformaldehyde (PFA), fluorescent cells were analyzed on a
FACS Canto II equipment (BD Bioscience, San Jose, Ca, USA)
using a Tree Star software entitled FlowJo (Trustees of Leland
F
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Stanford Jr. University, Stanford, CA, USA). Cytotoxicity was
controlled by 7-amino-actinomycin (7-AAD) staining.
Scheme 2. Synthesis of Diester Precursor 1
Confocal Microscopy. Macrophages adsorbed on glass
microslides (1 × 105 cells in 200 μL DMEM containing 5%
FCS) overnight at 37 °C and rinsed once with PBS were
incubated with biotinylated-PIM2 conjugate 3 (5 μg/mL in
DMEM containing 1% FCS for 10 min at 37 °C). After rapid
fixation (1 min at RT in 4% PFA) and rinsing in PBS, cells were
incubated with Streptavidin−Alexa 532 fluorescent conjugate
(10 μg/mL for 15 min at RT) and fixed (10 min at RT in 4%
PFA). The microslides were then washed twice with PBS,
mounted in Vectashield medium (Vector Laboratories, Inc.,
Burlingame, CA, USA), and analyzed by confocal microscopy.
Conditions: (a) 11, CH2Cl2, 1H-tetrazole, then mCPBA, 47%; (b) H2,
10% Pd/C, CH2Cl2/EtOH, 95%; (c) MeOH/CH2Cl2, tBuNH2, 45
min, −17 °C, 78%.
RESULTS AND DISCUSSION
Synthesis of a N-ω-Aminocaproyl Derivative of aza-
PIM2 in Ester Series (1). The first objective of the project was
■
Figure 3. Ether series: N-TFAC precursor 2.
Scheme 3. Synthesis of Diether Precursor 2
Figure 2. Ester series: N-TFAC (N-ω-TriFluoroAcetamidoCaproyl)
precursor 1.
Scheme 1. Synthesis of Precursor 10 (Diester Series)
Conditions: (a) 14, CH2Cl2, 1H-tetrazole, then mCPBA, 43%; (b)
MeOH/CHCl3, tBuNH2, −17 °C, 82%; (c) H2, CH2Cl2/EtOH, 10%
Pd/C, 90%.
N-Acylation followed by double glycosylation using the
trichloroacetimidate of tetra-O-methoxyacetyl-α-D-mannopyra-
nose (8) and O-debenzylation afforded the protected precursor
10 in good yield (Scheme 1).
Compound 10 was then phosphorylated by reaction with
phosphoramidite 1117 and oxidation of the resulting phosphite
a
to give phosphotriester 12 in 47% yield (Scheme 2). The
Conditions: (a) EtOH, H2, 20% Pd(OH)2/C, quant.; (b) CF3CONH-
(CH2)5COOH, CH2Cl2, HOBT, DCC, 60%; (c) 8 (2 equiv),
TMSOTf, CH2Cl2, 37%; (d) CH2Cl2/EtOH, H2, 10% Pd/C, 98%.
benzyl phosphate group was then cleaved by hydrogenolysis
and the mannosyl residues deprotected from methoxyacetate
groups by reaction with t-butylamine in methanol, to provide
the diester precursor 1 in 78% yield.
the synthesis of an aza-PIM2 mimic carrying an N-aminocaproyl
residue protected with a COCF3 group (N-TFAC precursor 1,
Figure 2) which could be used to tether various reporter groups
after deprotection. This compound was prepared from
iminoxylitol 6 by the sequence of reactions described in
Schemes 1 and 2.
The synthesis of the previously described iminoxylitol
derivative 617,18 was significantly improved: selective cleavage
of the N-benzyl group20 could be achieved quantitatively by
catalytic hydrogenolysis of purified 5 using Pearlman’s catalyst.
We showed earlier that a mimetic of PIM2 in which the
cyclitol is replaced by a 3-C glycerol scaffold could recapitulate
the inhibitory activity of natural PIM2 purified from M. bovis
BCG on LPS-induced macrophage release of IL-12p40 and IL-
10.12 PIM2 mimetic also reproduced the inhibitory activity of
natural PIM2 on TNF and NO release, while being non-
cytotoxic (data not shown). Aza-PIM2-NTfa (IA) and Oxa-
PIM2 (IB) retained the activity of PIM2 mimetic and were at
least as active as PIM2 mimetic for inhibition of TNF, IL-12
p40, and NO release by LPS-induced macrophages, with IB
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Figure 4. Both ester and ether forms of PIM2 heterocyclic analogues inhibit TNFα, IL-12p40, and NO release by LPS-stimulated macrophages. Bone
marrow-derived macrophages were incubated for 24 h with LPS in the presence of increasing concentrations of oxa-PIM2 analogue (IB), aza-PIM2
analogue (IA), compound 1 (TFAC-AzaPIM2-diester), and compound 2 (TFAC-AzaPIM2-diether). TNFα (A), IL-12p40 (B), and NO (C)
concentrations were measured in the supernatants. Results are expressed as percent of inhibition of the control stimulation performed with LPS plus
vehicle and represent the mean SEM of n = 4 values from two independent experiments.
(Oxa-PIM2) being slightly more active than IA (AzaPIM2-
NTfa) (Supporting Information Figure 1). The ability of
AzaPIM2-diester derivative 1 to inhibit LPS-induced pro-
inflammatory response was tested on murine bone marrow-
derived macrophages (see Figure 4A−C). Compound 1
inhibited the release of TNFα, IL-12p40, and NO essentially
as strongly as precursor IA. The aza-derivatives were slightly
less potent than the oxa PIM2 analogue IB, which is shown as a
reference. Thus, the addition of the aminocaproyl spacer did
not alter the immuno-modulating activity of the parent aza-
PIM2.
Further elaboration of 1 revealed, however, that the chemical
synthesis of conjugated derivatives from 1 would not be
successful: indeed under the basic conditions required to
remove the N-COCF3 group,21 the cleavage of the fatty acid
esters occurred more rapidly. We therefore decided to replace
H
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Scheme 4. Synthesis of 3 and 4 from 2
Conditions: (a) MeOH, NaOMe, 40 h, 25 °C, 0.4 bar; (b) DMF, Et3N, CH2Cl2, (+)-biotin N-succinimide ester, 5 h, 74% ; (c) DMF, Et3N, TRITC,
15 h, 52%.
the glycerol esters with ethers and to proceed with PIM mimics
carrying a di-O-hexadecyl glycerol unit as in compound 2
(Figure 3). Previous work has already shown that esters can be
replaced by ether linkages in PIMs without loss of biological
activity;16,19 subtle differences, however, have been recently
observed between PIM2 analogues carrying one alkyl chain at
sn-2 or at sn-1 glycerol position or two alkyl chains in their
ability to induce cytokine (IL-6) production by macrophages
(agonist effect), but all three types of compounds retained the
ability of the diester to inhibit LPS-induced production of this
cytokine by the same immune system cells.16
reduced pressure to eliminate methyl trifluoroacetate. This
provided the key aza-PIM2 derivative 17 functionalized for
conjugation or coupling with various markers (Scheme 4). The
fully deprotected PIM2 mimic 17 could be coupled in one step
to (+)-biotin using biotin N-succinimidyl ester derivative to
give conjugate 3 in 74% yield. Compound 17 was also engaged
in a coupling reaction with TRITC (tetramethylrhodamine
isothiocyanate) and gave the corresponding labeled derivative 4
(as a mixture of two isomers at the phenyl group). Both
compounds were fully characterized before they were engaged
in biological studies.
Synthesis of a N-ω-Aminocaproyl Derivative of aza-
PIM2 in Ether Series (2). The synthesis of the di-O-hexadecyl
analogue of 1, compound 2, was achieved from 10 by coupling
with phosphoramidite 14,19 oxidation, and deprotection of the
resulting benzyl phosphate to afford compound 16 (Scheme 3).
The methoxyacetyl groups of 16 could be cleaved selectively
without affecting the trifluoroacetamido group in high yield
(82%) using t-butylamine in MeOH, thus providing diether
precursor 2. The biological activity of AzaPIM2-diether 2 was
tested on LPS-stimulated macrophages, and a clear, dose-
dependent inhibition of TNFα, IL-12p40, and NO release
documented (Figure 4A−C). Therefore, diether 2 retained the
immuno-modulating activity of the corresponding diester 1 and
of the parent aza-PIM2 (IA).
The biological activity of biotinylated-PIM2 conjugate 3 was
first tested in terms of inhibition of pro-inflammatory cytokine
release by LPS-stimulated macrophages. The dose-dependent
inhibition of TNFα, IL-12p40, and NO release was at least as
good as the activity measured with the diether precursor 2
(Figure 5A−C). Dipalmitoyl phosphatidyl inositol PI, used as a
non-mannosylated, negative control, exhibited strongly reduced
inhibitory activity. None of the compounds induced cytotox-
icity at the concentrations investigated, as measured by MTT
assay (data not shown). Therefore, biotinylated PIM2 mimics 3
retain the immuno-modulating activity of the parent PIM2
diether 2.
In contrast, the derivative labeled with rhodamine (4)
appeared to be cytotoxic for macrophages in our culture
conditions, as observed by microscopy, while standard MTT or
NO release assays were hampered by the strong absorption of
the compound. Although IL-12p40 production seemed to be
Synthesis of 3 and 4 from 16. Cleavage of the
trifluoroacetyl group from 2 required the use of sodium
methoxide in MeOH. This reaction was performed under
I
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Figure 5. Biotinylated PIM2 derivative 3 retains inhibitory activity on TNFα, IL-12p40, and NO release by LPS-stimulated macrophages. Bone
marrow-derived macrophages were incubated for 24 h with LPS in the presence of increasing concentrations of biotinylated-PIM2 conjugate 3
(AzaPIM2-Biot), precursor 2 (TFAC-AzaPIM2-diether), and dipalmitoyl phosphatidyl inositol (PI) as a negative control. TNFα (A), IL-12p40 (B),
and NO (C) concentrations were measured in the supernatants. Results are expressed as percent of inhibition of the control stimulation performed
with LPS plus vehicle and represent the mean SEM of n = 6 values from 3 independent experiments representative of 4 independent experiments.
slightly decreased in the presence of labeled derivative 4, an
effect which may be linked to the cytoxicity of this compound,
it is worth noting that TNFα release was not inhibited at
concentrations up to 20 μg/mL (data not shown). Further
titration and kinetic studies will be needed to fully assess how
this compound might be used. PIM cellular labeling was thus
further investigated with biotinylated-PIM2 conjugate 3.
Functional Characterization of Biotinylated PIM
Derivative. For functional characterization, aza-PIM-biotin
conjugate 3 was first titrated in a solid phase assay (Figure 6A).
Biotinylated PIM was adsorbed onto a solid phase in a
microtiter plate overnight at room temperature and revealed by
incubation with a streptavidin−peroxidase conjugate, followed
by substrate addition and spectrophotometric detection of the
colored product. Dose-dependent coating of the biotinylated
PIM was detected at concentrations from 0.05 to 1 μg/mL.
There was no further increase in PIM-biotin adsorption at
concentrations of 1−20 μg/mL (not shown). Thus, the biotin
moiety grafted on the PIM molecule was stable in our
J
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Figure 6. Biotinylated-PIM2 derivative 3 is detected by fluorescent streptavidin conjugate and labels macrophages: (A) Aza-PIM2-biotin conjugate 3
at increasing concentrations (0.001−1 μg/mL) was adsorbed onto a microtiter plate overnight at room temperature. After saturation of the free
binding sites and washings, bound PIM-biotin was revealed with a streptavidin−peroxidase conjugate, followed by substrate addition and
spectrophotometric detection of the colored product at 406 nm. (B) Macrophages were incubated with biotinylated-PIM2 conjugate 3 (1, 3, and 10
μg/mL) for 30 min at 37 °C and cell-bound biotin detected by flow cytometry using a streptavidin−Alexa 532 fluorescent conjugate (10 μg/mL).
(C) Kinetic study of biotinylated-PIM2 binding to macrophages revealed a strong labeling already after 1 min incubation with conjugate 3 (1 μg/
mL), which was less pronounced after 10 and 30 min. Results from 2 independent macrophage populations from two different mice are shown in B
and C.
experimental conditions and could be detected by binding of a
labeled streptavidin conjugate.
To further evaluate whether biotinylated PIM functionally
bind immune cells, bone marrow-derived macrophages were
K
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ability to inhibit LPS-induced production of pro-
inflammatory cytokines such as TNFα and IL12p40.
(4) In contrast with 3, the aza-PIM2 mimic carrying a
fluorescent label, compound 4, exhibits significant
cytotoxicity which will limit its use in further biological
studies.
(5) In addition, our first experiments with biotin-aza-PIM2
conjugate 3 clearly showed that PIMs rapidly associate
with macrophage cell membrane. This compound will be
an invaluable tool to further analyze the mode of action
of the PIMs. Among the many questions that are
amenable to study with this conjugate or related
molecules are the colocalization with cell-surface
receptors, co-receptors, co-stimulatory molecules, or
with molecules associated with their downstream path-
ways, the kinetics of internalization, and cellular
compartmentalization.
Figure 7. Subcellular localization of biotinylated PIM conjugate 3 on
macrophages. Macrophages adsorbed on glass microslides overnight at
37 °C were incubated with biotinylated-PIM2 conjugate 3 (5 μg/mL
for 10 min at 37 °C). Cell-bound biotin was detected using a
streptavidin−Alexa 532 fluorescent conjugate (10 μg/mL). Red
labeling indicative of a membrane localization of the PIM−biotin
conjugate (left) and the corresponding cell morphology (right) are
shown. Negative controls with streptavidin conjugate alone showed no
background fluorescence (not shown).
ASSOCIATED CONTENT
■
S
* Supporting Information
Supplementary Figure 1. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: Olivier.Martin@univ-orleans.fr. Phone: +33 238 49 45
81. Fax: +33 238 41 72 81.
incubated in the presence of increasing concentrations of PIM-
biotin and cell-associated biotin detected by flow cytometry
using a streptavidin−Alexa 488 fluorescent conjugate (Figure
5B). A dose-dependent labeling of macrophages was evident at
PIM−biotin conjugate concentrations of 1 to 10 μg/mL after
30 min incubation at 37 °C. No cytotoxicity was associated
with incubation at these concentrations of biotinylated PIM
derivative 3, although higher concentration (20 μg/mL) yielded
some cell mortality, as detected by 7-AAD labeling (not
shown). The kinetics of the association of biotinylated PIM to
macrophages (Figure 5C) revealed a strong binding already at 1
min of incubation. PIM-biotin labeling was less pronounced at
10 and 30 min, which might be due to desorption or
internalization. Thus, the PIM−biotin conjugate binds macro-
phages with a rapid kinetics.
Confocal microscopy was then used to localize the
biotinylated PIM derivative at the subcellular level. Macro-
phages incubated with PIM−biotin conjugate 3 were revealed
with a streptavidin−Alexa 532 fluorescent conjugate (Figure 7).
The clear membrane labeling indicated that under these
conditions PIM conjugates associated preferentially with the
membrane on macrophages.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by CNRS, University of Orlea
European Union FEDER no. 1649-32264.
■
́
ns, and
ADDITIONAL NOTE
■
a
Because of the presence of slow-exchanging amide rotamers,
of P*-diastereoisomers, and of the nonequivalence of the sugar
moieties, the NMR spectrum of 12 is highly complex, exhibiting
in particular eight anomeric signals of nearly equal intensities.
The 1H NMR spectrum of precursor 10 in DMSO was
recorded at 90 °C; at this temperature, the spectrum showed
clearly a single species present with signals of two non-
equivalent mannosyl units.
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