Synthesis of a deuterated probe for the confocal Raman microscopy imaging of squalenoyl nanomedicines

Article abstract of DOI:10.3762/bjoc.12.109

The synthesis of ω-di-(trideuteromethyl)-trisnorsqualenic acid has been achieved from natural squalene. The synthesis features the use of a Shapiro reaction of acetone-d₆ trisylhydrazone as a key step to implement the terminal isopropylidene-d<

Full text of DOI:10.3762/bjoc.12.109

Synthesis of a deuterated probe for the confocal Raman
microscopy imaging of squalenoyl nanomedicines
Eric Buchy1, Branko Vukosavljevic2,3, Maike Windbergs2,3, Dunja Sobot1,
Camille Dejean4, Simona Mura1, Patrick Couvreur1 and Didier Desmaële*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2016, 12, 1127–1135.
1Institut Galien (UMR CNRS 8612) Faculté de Pharmacie, Université
Paris-Sud, 5, rue Jean-Baptiste Clément, 92296 Châtenay-Malabry,
France, 2Department of Drug Delivery, Helmholtz Centre for Infection
Research and Helmholtz Institute for Pharmaceutical Research
Saarland, Campus E8.1, 66123 Saarbruecken, Germany,
3Biopharmaceutics and Pharmaceutical Technology, Saarland
University, Campus A 4.1, 66123 Saarbruecken, Germany and
4BIOCIS (UMR CNRS 8076) Faculté de Pharmacie, Université
Paris-Sud, 5, rue Jean-Baptiste Clément, 92296 Châtenay-Malabry,
France
doi:10.3762/bjoc.12.109
Received: 25 January 2016
Accepted: 19 May 2016
Published: 06 June 2016
Associate Editor: S. C. Zimmerman
© 2016 Buchy et al; licensee Beilstein-Institut.
License and terms: see end of document.
Email:
Didier Desmaële* - didier.desmaele@u-psud.fr
* Corresponding author
Keywords:
deuterium labelling; nanomedicine; Raman spectroscopy; Shapiro
reaction; squalene
Abstract
The synthesis of ω-di-(trideuteromethyl)-trisnorsqualenic acid has been achieved from natural squalene. The synthesis features the
use of a Shapiro reaction of acetone-d6 trisylhydrazone as a key step to implement the terminal isopropylidene-d6 moiety. The ob-
tained squalenic acid-d6 has been coupled to gemcitabine to provide the deuterated analogue of squalenoyl gemcitabine, a powerful
anticancer agent endowed with self-assembling properties. The Raman spectra of both deuterated and non-deuterated squalenoyl
gemcitabine nanoparticles displayed significant Raman scattering signals. They revealed no differences except from the deuterium
peak patterns in the silent spectral region of cells. This paves the way for label-free intracellular trafficking studies of squalenoyl
nanomedicines.
Introduction
Application of nanotechnology to medicine holds promises to cokinetics and the biodistribution of many active compounds,
profoundly impact healthcare especially to treat severe diseases thus increasing specificity, therapeutic efficacy and reducing
such as cancer, intracellular infections, neurodegenerative systemic exposure and toxicity [1,2]. In recent years, many drug
diseases, etc. Indeed, the nanometric size confers to drug delivery systems have been developed covering all aspects of
delivery systems unique properties which improve the pharma- medicine. Among them, lipid drug conjugates (LDC) were
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especially developed for the delivery of hydrophilic drugs by lular tracking of the loaded drug after carrier disassembling.
covalent coupling with lipid components [3,4]. In this context Thus, specific tools such as fluorescence resonance energy
we recently found that the chemical conjugation of squalene, a transfer (FRET) and fluorescence quenching, have been de-
natural and biocompatible triterpene, to a drug led to the forma- veloped to study the stability of nanoparticles [23].
tion of a prodrug that spontaneously self-assembled as nanopar-
ticles in water. The advantage of this approach is a very high In this context, Raman spectroscopy is an interesting technique
drug loading into the nanoparticles and the absence of burst which is based on the detection of scattered laser light upon
release [5]. The proof of concept of this method has been done irradiating the sample. Nevertheless, because of the low intensi-
using gemcitabine (2), an anticancer chemotherapeutic drug ty of the Raman scattering, efficient in vivo confocal Raman
used to treat various solid tumors [6]. Remarkably, the squa- microspectroscopy of cells had to wait until laser technology
lene conjugate of gemcitabine (GemSQ) self-assembled in and mathematical image processing have made enough progress
aqueous media as nanoassemblies of around 100 nm mean par- [24,25]. In contrast to fluorescence spectroscopy, Raman spec-
ticle size with a low polydispersity index. The nanosuspension troscopy is label-free, as its scattering effect is unique for a spe-
exhibited impressively greater anticancer activity than free cific molecular structure. Raman spectra of cells usually consist
gemcitabine against different experimental tumor models [7-11] of spectral contributions from proteins, lipids and polysaccha-
overcoming the main drawbacks of the parent drug such as its rides. For the simultaneous detection of both the drug and the
short biological half-life and its low intracellular diffusion cell components with the aim to investigate how they interact, a
[12,13]. Following these initial results, the squalenoylation significant spectral contrast is required. Unfortunately, it can be
method was extended to other nucleoside analogues such as hardly achieved when dealing with low drug concentrations or
antiretroviral agents, ddC, ddI and AZT [14] and to siRNA biological-like structures such as peptide drugs or nucleoside
oligonucleotides [15]. More notably, squalenoylation of adeno- analogues. To overcome this issue, deuterium can be intro-
sine and the subsequent formation of NAs, allowed prolonged duced to a sample molecule, as it exhibits a significant Raman
circulation of this nucleoside, providing neuroprotection in mice signal at around 2200 cm−1, which is in a so called “silent
with induced focal cerebral ischemia and in rats undergoing region” (1800–2800 cm−1) of most biological molecules. For
spinal cord injury [16]. Interestingly, the “squalenisation plat- example as early as 1976, specifically deuterated stearic acids
form” initially developed with highly hydrophilic therapeutics have been used by Sunder et al. in Raman studies and Stiebing
has been further extended to hydrophobic drugs such as beta- et al. studied the uptake of arachidonic acid in human macro-
lactam antibiotics [17], paclitaxel [18], indolinone kinase inhib- phages [26,27]. Furthermore, deuterium does not change the
itors [19] or doxorubicin [20].
physicochemical properties and thus does not perturb the struc-
ture of the described NAs. Consequently, deuterated squalenic
For the elucidation of the mechanisms involved in the efficacy acid is expected to be excellent as bioorthogonal Raman tag for
of these promising nanomedicines, the precise knowledge squalene-based NAs. Since the Raman signal intensity is ex-
regarding the cellular uptake, the intracellular localization and pected to increase with the number of deuterium atoms in the
the determination of the subcellular interactions and trafficking same chemical environment, we have undertaken an effort
is crucial. To fulfill this task, radioactive labeling or fluorescent directed towards the synthesis of ω-di-(trideuteromethyl)tris-
probes have been thoroughly used. For example, the subcellu- norsqualenic acid (SQCO2H-d6, 1) bearing six deuterium atoms
lar localization of the 3H-radiolabeled GemSQ conjugate has on a non-labile position via isotopic exchange. We disclose
been evaluated by micro-autoradiography coupled to confocal herein the synthesis of this deuterated Raman probe from
imaging of fluorescently labeled cellular structures [21]. A dual natural squalene, its coupling with gemcitabine and the Raman
radioactive labeling 3H,14C has been taken into profit to study spectra of the deuterated GemSQ nanoassemblies, opening the
the pharmacokinetics, the biodistribution and the metabolism of way to perform intracellular imaging of squalenoyl nanomedi-
squalenoyl adenosine nanoparticles [22]. Nevertheless, the syn- cine (Figure 1).
thesis of labeled compounds is chemically challenging, expen-
Results and Discussion
sive and submitted to drastic regulation rules. In addition, the
Chemical synthesis of squalenic acid-d6 and
GemSQ-d6 conjugate
In a first approach we decided to explore the selective Wittig
mono-olefination of dialdehyde 5 readily accessible from the
known 2,3;22,23-epoxysqualene (7) [28]. As depicted in
Scheme 1, the synthetic sequence began with the treatment of
squalene with two equivalents of NBS in a water/THF mixture.
use of fluorescent probes requires the covalent binding of large
dye molecules (bodipy, cyanine, rhodamine etc, ...) to the drug
conjugate, thus potentially modifying its physicochemical
profile as well as the in vivo fate and the pharmacological activ-
ity. A simple encapsulation of an amphiphilic fluorochrome in
LDC nanoparticles can be used as far as the colloidal stability
of the nanocarrier is preserved, but cannot address the intracel-
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Figure 1: Structure of squalenic acid-d6, gemcitabine and GemSQ-d6 conjugate.
Scheme 1: Retrosynthetic route to SQCO2H-d6 (1) and synthetic routes for the preparation of dialdehyde 5 and squalene-d6 11.
After separation of the bis-bromohydrin from the monoproduct, THF, −78 °C) did not afford any amount of the desired deuter-
potassium carbonate treatment gave the expected diepoxide 7. ated olefin but only polar material that could not be character-
Oxidative cleavage with periodic acid provided the correspond- ized. In an attempt to find more efficient reaction conditions, we
ing dialdehyde 5 in 17% overall yield from squalene. The investigated this reaction using the simple aldehyde 10 as a
perdeuterated phosphonium salt 9 was obtained by simple con- model compound, easily accessible from squalene according to
densation of commercially available 2-bromopropane-d7 (8) the van Tamelen procedure [30]. The condensation of ylide 4
with triphenylphosphine [29]. To our surprise, condensation of with 10 seemed to be an easy task, but many well-established
dialdehyde 5 with one equivalent of the ylide 4 (9, n-BuLi, procedures using various bases (n-BuLi, LiHMDS, NaH/
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DMSO, PhLi) [29,31,32] gave only intractable materials. We group, we turned our attention to the application of this method
finally found that the treatment of 10 with the “instant ylide to a two-end functionalized squalene derivative. However,
mixture” of Schlosser made by grinding a solid mixture of 9 when applied to dialdehyde 5 the Shapiro reaction led to a mix-
and NaNH2 [33] delivered the desired squalene-d6 (11) al- ture of starting material (30%), allylic alcohol 18 (15%) and
though in a low 18% yield. However, when applied to the diol 17 (20%). This result could not by improved using reverse
dialdehyde 5 this procedure failed to give the expected Wittig addition conditions (Scheme 2). Therefore, selective monopro-
adduct.
tection of the dialdehyde 5 was next attempted. Unfortunately,
treatment of the latter either with ethylene glycol or 2,2-
Whatever the origin of this problem, we decided to explore a dimethylpropandiol gave a mixture of di- and monoacetal what-
new strategy based on a more nucleophilic deuterated synthon. ever the conditions, as a new example of the lack of chemose-
In this regard we targeted prop-1-en-2-yllithium-d5 (15) which lectivity of this long polyisoprenyl chain derivatives.
is easily accessible through the Shapiro reaction of sulfonylhy-
drazone of acetone-d6 [34,35]. Thus condensation of trisylhy- The differentiation of both ends of squalene was thus per-
drazine with acetone-d6 (99.8% D) gave the expected hydra- formed starting from trisnorsqualenaldehyde 10. Protection of
zone 14 in 55% yield. To our delight, upon treatment with two 10 as 2,2-dimethyl-1,3-dioxane derivative gave 19 in 96% yield
equivalents of n-BuLi and warming to 0 °C, the trisylhydra- which was further elaborated into aldehyde 20 in 16% overall
zone 14 afforded the vinyllithium reagent 15 (along with N2 and yield according to the three-step van Tamelen sequence
the trisyl anion) which upon condensation with squalenalde- (i. NBS, THF, H2O; ii. K2CO3, MeOH; iii. H3IO6, Et2O) [30].
hyde 10 furnished the desired allylic alcohol 16 in 59% yield. Interestingly enough, the 1,3-dioxane group survived the
Reduction of the hydroxy group of 16 was straightforwardly strongly acidic conditions of the oxidative cleavage. We next
achieved in 47% yield by treatment with a large excess of turned to the elaboration of the isopropylidene-d6 moiety. In the
thionyl chloride followed by LiAlD4 reduction [36]. Having event, the Shapiro reaction using trisylhydrazide 14 delivered
secured an efficient method to reinstall the isopropylidene end- the expected allylic alcohol 21 in 70% yield. The latter afforded
Scheme 2: Implementation of the deuterated isopropylidene end-group by the Shapiro reaction of trisylhydrazone of acetone-d6.
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the deuterated ketal 22 in 52% yield, upon sequential treatment of SQCO2H-d6 1. Mass spectral analysis confirmed the pres-
with thionyl chloride and LiAlD4 as described above. With the ence of the six deuterium atoms (m/z = 405.3631 for
success of the implementation of the terminal deuterated iso- [C27H37D6O2−]) along a small amount (~5%) of SQCO2H-d5.
propylidene group we turned our attention to the deprotection of GemSQ-d6 3 was next synthetized using activation with ethyl
the ketal and the oxidation of the aldehyde group. This seem- chloroformate as previously reported [7]. However, to optimize
ingly trivial task, turned out to be unexpectedly challenging. All the process a large excess of gemcitabine was used in the reac-
conditions tried (HCl 3 N, THF, 20 °C; HCl 3 N, THF, reflux; tion to increase the yield to 72% in respect of the more valu-
HCl 6 N, dioxane, 20 °C; HCO2H, reflux; FeCl3·6H2O/SiO2 able SQCO2H-d6 (Scheme 4).
[37], Jones reagent) either let the starting material unchanged or
Nanoparticle formulation of the GemSQ-d6
induced a complete decomposition. Even treatment with 1,2-
ethanedithiol in the presence of BF3·OEt2 failed to give the cor- conjugate and Raman spectroscopy
responding thioketal [38]. These results clearly showed that The GemSQ and GemSQ-d6 nanoassembly suspensions
another protecting group must be used, avoiding the use of an (2 mg·mL−1) were prepared in a single step by nanoprecipita-
acidic catalyst that triggered the cyclization cascade of the poly- tion of an ethanolic solution (2–4 mg·mL−1) in milli-Q water
isoprenyl chain (Scheme 3).
[7]. After spontaneous formation of the NAs the organic sol-
vent was evaporated under vacuum (Figure 2A). Single Raman
Despite this setback, the synthetic route seemed suitable to spectra of the raw substances as well as of the particles were re-
produce the desired material. Thus, the synthetic pathway de- corded (Figure 2B). As depicted in Figure 2C and Figure 2D,
scribed above was reimplemented starting from tert- the Raman spectra of the deuterated and non-deuterated com-
butyldiphenylsiloxysqualene 23 readily obtained in 85% yield pounds revealed no differences except the deuterium peaks in
from squalenaldehyde 10 by NaBH4 reduction followed by the silent region.
protection with tert-butyldiphenylsilyl chloride. Functionalisa-
tion of the opposite extremity of the polyisoprenoid chain using Conclusion
the van Tamelen sequence (i. 1 equiv NBS, THF, H2O; ii. A synthesis of squalenic acid-d6 was developed through the
K2CO3, MeOH; iii. H3IO6, Et2O) afforded the aldehyde 26 in Shapiro reaction of the sulfonylhydrazone of acetone-d6 with an
16% overall yield. Uneventfully, the Shapiro reaction with ω-silyloxysqualene aldehyde derivative followed by a regiose-
trisylhydrazone 14 produced the allylic alcohol 27. Thionyl lective reduction of the obtained allylic alcohol. This material
chloride treatment followed by LiAlD4 reduction delivered was obtained from natural squalene in 0.6% yield over 12 steps.
directly the alcohol 28 in 41% yield through concomitant reduc- The synthesized deuterated squalenic acid was coupled to
tion of the intermediate allylic chloride and cleavage of the silyl gemcitabine to provide the corresponding deuterated squalenoyl
protecting group. The reductive cleavage of tert-butyldiphenyl- conjugate. Raman spectra of the nanoassemblies made of this
silyl ethers by LiAlH4 has been previously noticed [39]. conjugate were recorded, showing significant Raman peaks in
Jones oxidation straightforwardly completed the synthesis the silent region of the cells thus making this material a poten-
Scheme 3: Attempted synthesis of 1 via the protection of the aldehyde 10 as a 5,5-dimethyl-1,3-dioxane.
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Scheme 4: Synthesis of squalenic acid-d6 1 and conjugation to gemcitabine.
tial Raman probe. The use of this material as Raman probe in crude product was purified by chromatography over silica gel
the study of the intracellular trafficking of GemSQ-based NAs eluting with petroleum ether/Et2O 80:20 to give tris-
is currently in progress and will be reported in due course.
norsqualenic acid-d6 (1) as a colorless oil (31.5 mg, 55%).
1H NMR (CDCl3, 300 MHz) δ 5.19–5.07 (m, 5H, =CH), 2.45
(t, J = 7.6 Hz, 2H, CH2CO2H), 2.30 (t, J = 7.6 Hz, 2H,
Experimental
(4E,8E,12E,16E)-21-(2H3)methyl-4,8,13,17-tetra- CH2CH2CO2H), 2.15–1.95 (m, 16H, =HCCH2CH2C(CH3)),
methyl(22,22,22-2H3)docosa-4,8,12,16,20-pentaenoic acid 1.62 (s, 3H, (CH3)C=), 1.60 (br s, 9H, (CH3)C=); 13C NMR
(1): An ice-cooled solution of alcohol 28 (55 mg, 0.14 mmol) in (CDCl3, 75 MHz) δ 179.2 (C, CO2H), 135.3 (C, CH2(CH3)C=),
acetone (2 mL) was treated with a few drops of Jones reagent 135.1 (C, CH2(CH3)C=), 135.0 (C, CH2(CH3)C=), 133.0 (C,
(CrO3/H2SO4 6 M) until the mixture took a persistent dark red CH2(CH3)C=), 131.2 (C, (CD3)2C=), 125.5 (CH, HC=), 124.6
color. After complete disappearance of the starting material a (2CH, HC=), 124.4 (2CH, HC=), 39.9 (2CH2), 39.7 (CH2),
few drops of isopropanol were added. The mixture was taken 34.4(CH2, CH2CH2CO2H), 33.0 (CH2, CH2CO2H), 28.4
into brine (10 mL) and extracted with Et2O (4 × 15 mL), dried (2CH2), 26.9 (CH2), 26.8 (2CH2), 16.1 (3CH3), 16.0 (CH3); IR
over MgSO4 and concentrated under reduced pressure. The (film, cm−1) ν: 3500–2600 (broad), 2962, 2916, 2856, 2222,
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Figure 2: A) Sketch depicting the procedure of preparing the NAs. B) Single Raman spectra of GemSQ-d6 NAs, GemSQ NAs, gemcitabine and
squalenic acid, respectively. C) Single Raman spectra of deuterated GemSQ-d6 NAs (red) and GemSQ NAs (black). D) Close-up showing the differ-
ence between deuterated and non-deuterated compounds.
2188, 1709, 1666, 1450, 1411, 1302, 1299, 1259, 1211, 1096, over silica gel eluting with cyclohexane/AcOEt 4:1 followed by
1049, 955, 846, 736, 704; HRMS–ESI−: calcd for C27H37D6O2: neat AcOEt to provide GemSQ-d6 (3) as a colorless oil
405.3645; found: 405.3631.
(36.0 mg, 72%). 1H NMR (CDCl3, 400 MHz) δ 9.15 (br s, 1H,
NHCO), 8.10 (d, J = 7.5 Hz, 1H, H-6), 7.47 (d, J = 7.5 Hz, 1H,
(4E,8E,12E,16E)-N-{1-[(2R,5R)-3,3-difluoro-4-hydroxy-5- H-5), 6.18 (t, J = 7.4 Hz, 1H, H-1’), 5.20–5.06 (m, 5H, =CH),
(hydroxymethyl)oxolan-2-yl]-2-oxo-1,2-dihydropyrimidin-4- 4.55–4.41 (m, 1H, H-3’), 4.15–3.95 (m, 3H, H-4’, H-5’, OH),
yl}-21-(2H3)methyl-4,8,13,17-tetramethyl(22,22,22- 3.91 (d, 1H, J = 10.8 Hz, H-5’), 2.55 (2H, t, J = 7.6 Hz,
2H3)docosa-4,8,12,16,20-pentaenamide (3): To a solution CH2CON), 2.32 (2H, t, J = 7.4 Hz, CH2CH2CON), 2.10–1.91
cooled at −5 °C of trisnorsqualenic acid-d6 (1) (31.5 mg, (m, 16H, =CCH2CH2C(CH3)), 1.60 (3H, s, =C(CH3)), 1.59 (s,
0.077 mmol) in dry THF (0.6 mL) was sequentially added tri- 6H, =C(CH3)), 1.58 (3H, s, =C(CH3)); 13C NMR (CDCl3, 75
ethylamine (60 mg, 0.23 mmol) and ethyl chloroformate MHz) δ 173.6 (C, CONH), 163.1 (C, C-4), 155.8 (C, C-2),
(10 mg, 0.093 mmol). The reaction mixture was stirred for 145.6 (CH, C-6), 135.3 (C, (CH3)C= CH2(CH3)C=), 135.0 (2C,
30 min at this temperature and a solution of gemcitabine base CH2(CH3)C=), 132.8 (C, CH2(CH3)C=), 131.2 (C, (CD3)2C=),
(60.6 mg, 0.23 mmol) in DMF (2 mL) was added. The reaction 126.0 (CH, HC=), 124.5 (2CH, HC=), 124.4 (2 HC=), 122.5
mixture was stirred for 4 days at 20 °C and concentrated under (CF2, t, J = 258 Hz, C-2’), 97.8 (CH, C-5), 81.8 (CH, C-4’),
reduced pressure. The residue was directly chromatographed 69.3 (CH, m, C-3’), 60.0 (CH2, C-5’), 39.9 (2CH2), 39.7 (CH2),
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Beilstein J. Org. Chem. 2016, 12, 1127–1135.
36.7 (CH2, NHCOCH2CH2), 34.5 (CH2, NHCOCH2CH2), 29.8 Agreement no.249835. The financial supports of the Ministère
(CH2), 28.4 (2CH2), 27.0 (CH2), 26.9 (2CH2), 26.8 (2CH2), de la Recherche et de la Technologie (fellowship to E.B.) is
16.2 (2CH3), 16.1 (CH3), 16.0 (CH3); IR (film, cm−1) ν: gratefully acknowledged.
3500–3000 (broad), 2979, 2932, 2872, 2852, 2222, 2191, 1724,
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Supporting Information
Supporting Information File 1
Experimental procedures and 1H and 13C NMR spectral
data for compounds 1, 3, 11, 25, 27, 28.
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Acknowledgements
The research leading to these results has received funding from
the European Research Council under the European Commu-
nity's Seventh Framework Program FP7/2007-2013 Grant
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