Synthesis of deuterated γ-linolenic acid and application for biological studies: Metabolic tuning and Raman imaging

Article abstract of DOI:10.1039/d0cc07824g

γ-Linolenic acid (GLA) is reported to show tumor-selective cytotoxicity through unidentified mechanisms. Here, to assess the involvement of oxidized metabolites of GLA, we synthesized several deuterated GLAs and evaluated their metabolism and cytotoxicity towards normal human fibroblast WI-38 cells and VA-13 tumor cells generated from WI-38 by transformation with SV40 virus. Deuteration of GLA suppressed both metabolism and cytotoxicity towards WI-38 cells and increased the selectivity for VA-13 cells. Fully deuterated GLA was visualized by Raman imaging, which indicated that GLA is accumulated in intracellular lipid droplets of VA-13 cells. Our results suggest the tumor-selective cytotoxicity is due to GLA itself, not its oxidized metabolites. This journal is

Full text of DOI:10.1039/d0cc07824g

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Synthesis of deuterated c-linolenic acid and
application for biological studies: metabolic
tuning and Raman imaging†
Cite this: Chem. Commun., 2021,
57, 2180
Received 1st December 2020,
Accepted 26th January 2021
abcd
ab
ab
Kosuke Dodo,
*
Ayato Sato,
Yuki Tamura,
Syusuke Egoshi,ac
Koichi Fujiwara,ac Kana Oonuma,d Shuhei Nakao,
Naoki Terayama
and
ac
acd
abcd
DOI: 10.1039/d0cc07824g
Mikiko Sodeoka
*
rsc.li/chemcomm
c-Linolenic acid (GLA) is reported to show tumor-selective cytotoxicity
Among the PUFAs, g-linolenic acid (GLA) was reported to
through unidentified mechanisms. Here, to assess the involvement of show anticancer activity via an unidentified mechanism.4 In
oxidized metabolites of GLA, we synthesized several deuterated GLAs contrast to its cytotoxic effects towards various tumor cell lines,
and evaluated their metabolism and cytotoxicity towards normal GLA was much less toxic to normal cells, and free radicals
human fibroblast WI-38 cells and VA-13 tumor cells generated from generated from GLA were proposed to mediate the cytotoxicity
WI-38 by transformation with SV40 virus. Deuteration of GLA sup- in a cell-line-specific manner.5–7 However, the details remain
pressed both metabolism and cytotoxicity towards WI-38 cells and unclear.
increased the selectivity for VA-13 cells. Fully deuterated GLA was
Recent studies indicate that deuteration of the bis-allylic
visualized by Raman imaging, which indicated that GLA is accumulated methylene structure suppresses both enzymatic and non-
in intracellular lipid droplets of VA-13 cells. Our results suggest the enzymatic metabolism of PUFAs.8–11 In metabolic reactions of
tumor-selective cytotoxicity is due to GLA itself, not its oxidized PUFAs, the abstraction of a hydrogen atom from the bis-allylic
metabolites.
methylene is a critical step, producing a delocalized radical.
The kinetic isotope effect (KIE) of deuterium inhibits this
Polyunsaturated fatty acids (PUFAs) are transformed into meta- abstraction step, and thus inhibits the subsequent metabolism
bolites that regulate various biological phenomena. Among of PUFAs. In this work, we planned to examine the effect of
them, arachidonic acid (AA) is a precursor of various eicosa- metabolic inhibition of GLA on the cytotoxicity by introducing
noids, including prostaglandins and leukotrienes, which are deuterium into the bis-allylic positions (Fig. 1). Evaluation of
both physiologically and pathologically important.1 Initial trans- deuterated sites that affect the activity of GLA could help to
formation of AA is mainly mediated by oxidizing enzymes, such identify the key reaction sites for generating putative cytotoxic
as cyclooxygenase (COX) and lipoxygenase (LOX). Non-enzymatic metabolites.
auto-oxidation reactions of AA also produce bioactive metabo-
In addition, deuterium serves as a tag for Raman imaging.12
lites via the generation of lipid radicals.2 Other PUFAs such as Tumor-selective effects of GLA might be due to differences in
eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) the cellular uptake or distribution of GLA. Therefore, we aimed
also afford bioactive metabolites.3 Therefore, it is possible that to examine this possibility by employing Raman imaging. The
unidentified metabolites derived from PUFAs are biologically
active.
a Synthetic Organic Chemistry Laboratory, RIKEN Cluster for Pioneering Research,
2-1 Hirosawa, Wako, Saitama 351-0198, Japan. E-mail: sodeoka@riken.jp,
dodo@riken.jp
b Sodeoka Live Cell Chemistry Project, ERATO, Japan Sciences and Technology
Agency, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
c AMED-CREST, Japan Agency for Medical Research and Development,
2-1 Hirosawa, Wako, Saitama 351-0198, Japan
d RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa,
Wako, Saitama 351-0198, Japan
Fig. 1 Applications of deuterated g-linolenic acid (GLA) derivatives for
† Electronic supplementary information (ESI) available: Experimental proce-
mechanistic studies.
dures, supplemental data, and spectral data. See DOI: 10.1039/d0cc07824g
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C–D bond has a specific Raman peak in the silent region where
cellular endogenous molecules have no Raman signal.
Although the Raman signal of C–D is very weak compared with
that of an alkyne moiety, which was previously developed as a
Raman tag for live-cell imaging,13–16 highly deuterated fatty
acids have been used as probes for live-cell Raman
imaging.17,18 Therefore, we planned to apply deuterated GLA
for live-cell Raman imaging (Fig. 1).
We firstly introduced deuterium at the bis-allylic methylenes,
C-8 and/or C-11, in order to examine the effects on metabolism.
As shown in Scheme 1, deuterated methylene unit 2 was synthe-
sized from TMS-acetylene 1. Lithium acetylide derived from 1
was reacted with deuterated paraformaldehyde, and then protec-
tion of the alcohol with THP and deprotection of a TMS
group afforded 2. In the same manner, normal or deuterated
paraformaldehyde was reacted with lithium acetylide generated
from 1-heptyne 3, and the alcohol group was tosylated19 to afford
4a or 4b having the C-11 methylene group of GLA. The C-8
methylene group was then introduced by nucleophilic substitu-
tion of the OTs group with copper acetylide.20,21 Compound 4a
was reacted with 2, and then deprotection of the THP group
followed by tosylation afforded 5a. Compound 4b was reacted
with propargyl alcohol, and tosylation afforded 5b. Compound
5c was obtained from 4b and 2 according to the same procedure
used for 5a. Compounds 5a–5c were reacted with cuprate derived
from methyl 6-heptynoate and successive hydrogenation
with Ni(OAc)2–NaBH4 afforded compounds 6a–6c.22 Finally,
hydrolysis of the methyl ester gave the deuterated GLA deriva-
tives 7a–7c.
Fig. 2 Cytotoxic activities of GLA and its partially deuterated derivatives
7a–7c towards VA-13 and WI-38 cells.
VA-13 cells than WI-38 cells, confirming the reported tumor-
selective cytotoxicity, although the difference between the
effective doses for the two cell lines was not large. Next, the
effects of metabolism on the cytotoxicity were examined by
using deuterated GLA derivatives 7a–7c. Interestingly, none of
these partially deuterated GLA was cytotoxic to WI-38 cells even
at 200 mM, indicating that the cytotoxicity of GLA to normal
cells depends on metabolic and/or chemical reaction at the
bis-allylic positions. To support this speculation, we also com-
pared the reactivities between GLA and deuterated GLA to the
enzymatic oxidation, which confirmed the metabolic inhibition
of GLA by the deuteration of bis-allylic methylenes (Fig. S1,
ESI†). In contrast, deuteration at the C8 and/or C11 positions
only slightly decreased the cytotoxicity of GLA to VA-13 cells.
These results indicate that different cell-death-inducing
mechanisms are operative in normal and tumor cells. In the
case of VA-13 tumor cells, reaction at the bis-allylic position is
not critical. GLA itself or its metabolites generated by reaction
at some other position(s) might be involved in the cytotoxicity
towards tumor cells. To clarify this point, we next synthesized
fully deuterated GLA (all-D-GLA). If abstraction of any of the
hydrogen atoms in GLA is critical for the production of the key
metabolite(s), the cytotoxicity of all-D-GLA should be further
decreased.
To evaluate the tumor-selective cytotoxicity of GLA, we used
two cell lines, WI-38 and VA-13. WI-38 cells are normal human
lung fibroblast cells, while VA-13 cells are tumor cells generated
from WI-38 by transformation with SV40 virus. Therefore, the
genetic background of the two cell lines is the same. First, WI-
38 and VA-13 cells were treated with GLA for 48 h, and cell
viability was determined by means of alamarBlue assay.
As shown in Fig. 2, GLA showed stronger cytotoxicity towards
As shown in Scheme 2, deuterated methyl 6-heptynoate 9
was prepared from deuterated cyclohexene 8.23,24 The fully
Scheme 1 Synthesis of partially deuterated GLA derivatives. Reagents and
conditions: (a) (i) nBuLi, (CD2O)n, THF, 0 1C, (ii) DHP, p-TsOHÁH2O, DCM,
Scheme 2 Synthesis of fully deuterated GLA. Reagents and conditions: (a)
(i) O3, NaHCO3, DCM, CD3OD, À78 1C, (ii) Ac2O, NEt3, DCM, (iii) Ohira–
Bestmann reagent, K2CO3, CD3OD, 0 1C; (b) (i) nBuLi, C5D11Br, HMPA, THF,
(ii) p-TsOHÁH2O, MeOH, (iii) TsCl, K2CO3, 1-hexadecylimidazole, H2O; (c) (i)
2, CuI, K2CO3, NaI, DMF, (ii) p-TsOHÁH2O, MeOH, (iii) TsCl, K2CO3,
1-hexadecylimidazole, H2O; (d) (i) 9, CuI, K2CO3, NaI, DMF, (ii) D2, NaBD4,
Ni(OAc)2, ethylenediamine, CD3OD; (e) LiOHÁH2O, THF, H2O.
(iii) TBAF, THF,
0 1C; (b) (i) nBuLi, (CX2O)n, THF, (ii) TsCl, K2CO3,
1-hexadecylimidazole, H2O; (c) (i) propargyl alcohol, CuI, K2CO3, NaI,
DMF, (ii) TsCl, K2CO3, 1-hexadecylimidazole, H2O; (d) (i) 2, CuI, K2CO3,
NaI, DMF, (ii) p-TsOHÁH2O, MeOH, (iii) TsCl, K2CO3, 1-hexadecylimidazole,
H2O; (e) (i) methyl 6-heptynoate, CuI, K2CO3, NaI, DMF, (ii) H2, NaBH4,
Ni(OAc)2, ethylenediamine, MeOH; (f) LiOHÁH2O,THF, H2O.
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deuterated C8–C18 unit 11 was synthesized from 10, which was
prepared from deuterated pentyl bromide and 2. Then 11 was
converted to 12 by coupling reaction with 9 and partial
reduction using D2. Finally, all-D-GLA was obtained by hydro-
lysis of the ester moiety.
All-D-GLA was tested for cytotoxicity towards WI-38 and
VA-13 cells (Fig. 3). It proved to be cytotoxic to VA-13 cells,
but not WI-38 cells, like the partially deuterated derivatives
7a–7c. This result suggests that GLA itself, but not its metabo-
lites, is cytotoxic to tumor cells.
The non-specific cytotoxicity of GLA may be due to the
products of enzymatic oxidation or non-specific peroxidation
of GLA, and suppression of these reactions by deuteration may
account for the increased tumor-selectivity. Thus, all-D-GLA
may be preferable to GLA for analyzing the mechanism of the
tumor-selectivity.
Fig. 4 Raman spectra of GLA and all-D-GLA.
In addition to the improved activity, all-D-GLA is expected to
show a sufficiently strong Raman signal for live-cell imaging,
which would be useful to gain mechanistic insights into the
tumor-selective cytotoxicity of GLA. To confirm the Raman
signals resulting from deuteration, Raman spectra of GLA
and deuterated GLAs were obtained by means of Raman
microscopy (Fig. 4 and Fig. S2, ESI†). As shown in Fig. 4,
all-D-GLA showed strong Raman signals due to C–D bonds
(2000–2300 cmÀ1) in the cellular silent region. In contrast,
the strong Raman signals corresponding to C–H bonds
(2800–3100 cmÀ1) seen in the spectrum of GLA were completely
absent in the spectrum of all-D-GLA. These observations con-
firmed the successful complete deuteration of GLA. Moreover,
deuteration shifted the Raman signal of the carbon–carbon
double bond (CQC) from 1660 cmÀ1 (GLA) to 1630 cmÀ1
(deuterated GLA), in good agreement with
a previous
report.17,25 Therefore, we also examined whether the Raman
signal of the DCQCD bond could be used as a Raman tag for
live-cell imaging, in addition to the C–D bond signal.
Fig. 5 Raman images of VA-13 cells treated with 100 mM all-D-GLA.
VA-13 cells were treated with all-D-GLA (100 mM) and
observed by Raman microscopy at each indicated time (Fig. 5 obtained by using the signal of DCQCD at 1630 cmÀ1, indicating
and Fig. S3, ESI†). Raman images were constructed from the the potential of the DCQCD bond as a Raman tag for live-cell
Raman peaks at 1630, 2115, 2854, 3015 cmÀ1. The cellular imaging.
distribution of all-D-GLA was successfully visualized by
Compared with the cellular distributions of the H–C–H
using the Raman signal at 2115 cmÀ1, corresponding to the signal at 2854 cmÀ1 and the CQC–H signal at 3015 cmÀ1
,
D–C–D bond. Time-dependent accumulation of GLA in the cells all-D-GLA showed good colocalization with the CQC–H distri-
was observed. Interestingly, essentially identical images were bution, which was reported to be a marker of lipid droplets
(LDs) corresponding to triglyceride.26 Therefore, all-D-GLA is
thought to accumulate in LDs to exert its cytotoxicity. Although
the precise mechanism is still unclear, several reports indicate
that the formation of LDs is important for the proliferation of
cancer cells.27,28 LDs store fatty acids as triglyceride or choles-
teryl ester, and cancer cells use LD-derived fatty acids as
an energy source for proliferation through mitochondrial
b-oxidation. All-D-GLA remained in LDs, and was not incorpo-
rated into mitochondria (Fig. S3, ESI†), suggesting that it might
inhibit cancer cell proliferation, leading to induction of
cell death.
Next, Raman imaging of WI-38 cells was examined (Fig. 6
Fig. 3 Cytotoxic activities of GLA and all-D-GLA towards VA-13 and
WI-38 cells.
and Fig. S4, ESI†). WI-38 cells were treated with all-D-GLA
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This work was supported by AMED-CREST, AMED under
Grant Number JP18gm071004 (M. S.). This work was also
supported by JST CREST Grant Number JPMJCR1925 (M. S.),
MEXT KAKENHI Grant Number JP26110004 (M. S. and K. D.),
and JSPS KAKENHI Grant Number JP18K14360 and JP20K15418
(S. E.). S. E. was supported by the Special Postdoctoral Researchers’
Program in RIKEN.
Conflicts of interest
There are no conflicts to declare.
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