Effect of carbon chain length in acyl coenzyme A on the efficiency of enzymatic transformation of okadaic acid to 7-O-acyl okadaic acid

Article abstract of DOI:10.1016/j.bmcl.2016.05.027

Okadaic acid (OA), a product of dinoflagellate Prorocentrum spp., is transformed into 7-O-acyl OA in various bivalve species. The structural transformation proceeds enzymatically in vitro in the presence of the microsomal fraction from the digestive gland of bivalves. We have been using LC-MS/MS to identify OA-transforming enzymes by detecting 7-O-acyl OA, also known as dinophysistoxin 3 (DTX3). However, an alternative assay for DTX3 is required because the OA-transforming enzyme is a membrane protein, and surfactants for solubilizing membrane proteins decrease the sensitivity of LC-MS/MS. The present study examined saturated fatty acyl CoAs with a carbon chain length of 10 (decanoyl), 12 (dodecanoyl), 14 (tetradecanoyl), 16 (hexadecanoyl) and 18 (octadecanoyl) as the substrate for the in vitro acylation reaction. Saturated fatty acyl CoAs with a carbon chain length of 14, 16 and 18 exhibited higher yields than those with a carbon chain length of 10 or 12. Acyl CoAs with carbon chain lengths from 14 to 18 and containing either a diene unit, an alkyne unit, or an azide unit in the carbon chain were synthesized and shown to provide the corresponding DTX3 with a yield comparable to that of hexadecanoyl CoA. The three functional units can be conjugated with fluorescent reagents and are applicable to the development of a novel assay for DTX3.

Full text of DOI:10.1016/j.bmcl.2016.05.027

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Effect of carbon chain length in acyl coenzyme A on the efficiency of
enzymatic transformation of okadaic acid to 7-O-acyl okadaic acid
Sachie Furumochi a, Tatsuya Onoda a, Yuko Cho a, Haruhiko Fuwa b, Makoto Sasaki b,
Mari Yotsu-Yamashita a, Keiichi Konoki a,
⇑
a Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan
b Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Okadaic acid (OA), a product of dinoflagellate Prorocentrum spp., is transformed into 7-O-acyl OA in var-
ious bivalve species. The structural transformation proceeds enzymatically in vitro in the presence of the
microsomal fraction from the digestive gland of bivalves. We have been using LC–MS/MS to identify OA-
transforming enzymes by detecting 7-O-acyl OA, also known as dinophysistoxin 3 (DTX3). However, an
alternative assay for DTX3 is required because the OA-transforming enzyme is a membrane protein, and
surfactants for solubilizing membrane proteins decrease the sensitivity of LC–MS/MS. The present study
examined saturated fatty acyl CoAs with a carbon chain length of 10 (decanoyl), 12 (dodecanoyl), 14
(tetradecanoyl), 16 (hexadecanoyl) and 18 (octadecanoyl) as the substrate for the in vitro acylation reac-
tion. Saturated fatty acyl CoAs with a carbon chain length of 14, 16 and 18 exhibited higher yields than
those with a carbon chain length of 10 or 12. Acyl CoAs with carbon chain lengths from 14 to 18 and con-
taining either a diene unit, an alkyne unit, or an azide unit in the carbon chain were synthesized and
shown to provide the corresponding DTX3 with a yield comparable to that of hexadecanoyl CoA. The
three functional units can be conjugated with fluorescent reagents and are applicable to the development
of a novel assay for DTX3.
Received 10 March 2016
Revised 25 April 2016
Accepted 10 May 2016
Available online xxxx
Keywords:
Okadaic acid
Dinophysistoxin 3
Detoxification
Bivalves
Ó 2016 Elsevier Ltd. All rights reserved.
Okadaic acid (OA, 1) and dinophysistoxin 1 (DTX1, 2) are pro-
duced by the dinoflagellates Prorocentrum spp.1 and Dinophysis
spp.,2 respectively, and cause diarrheic shellfish poisoning (DSP)
(Fig. 1).3,4 Poisoning occurs by the specific inhibition of serine/thre-
onine protein phosphatases 1 (PP1) and 2A (PP2A), although the
relationship between inhibitory activity against PP1/PP2A and
DSP has not been clarified.5 The bivalves accumulate the toxins in
the digestive gland during dinoflagellate breakouts. The safety level
of DSP for humans was defined by the European Union in 2004.6 A
toxicity level above 160 ng OA equiv weight/g edible shellfish
results in a voluntary suspension of shipping of shellfish in Japan,
thus potentially economically impacting the aquaculture industry.
Many research studies have revealed that OA (1) or DTX1 (2) is
transformed into the 7-O-acyl derivative (dinophysistoxin 3, DTX3,
3)7 when accumulated in bivalves (Fig. 1). The ratio of the acylated
form (DTX3, 3) to the non-acylated form (OA (1) or DTX1 (2)) var-
ies depending on the species of bivalve.8 DTX3 exhibits low bind-
ing ability to PP1 and PP2A9 but retains acute toxicity and
potency to cause diarrhea.10 Identification of the OA-transforming
enzyme is therefore important for elucidating the physiological
significance of this acylation.
The enzymatic reaction undergoes regioselectively at 7-OH,
although four hydroxy groups are present in OA. Moreover, the
lipid composition of DTX3 does not appear to reflect that of the
digestive gland of scallop and mussel.8 For example, 7-O-tetrade-
canoyl OA, 7-O-hexadecanoyl OA and 7-O-hexadecenoyl OA are
the primary DTX3s found in bivalves, whereas bivalves are rich
in hexadecanoic acid, hexadecenoic acid and polyunsaturated fatty
acid (20:5, 22:6). These results suggest that the enzyme transfers
acyl CoA(s) of a certain carbon chain length(s) to 7-OH of OA (1)
or DTX1 (2) in a regioselective manner. If the enzyme was identi-
fied and the reaction mechanism was revealed, the enzymatic reac-
tion would provide useful feedback to organic chemistry.
We attempted to purify the OA-transforming enzyme by detect-
ing DTX3 with liquid chromatography/mass spectrometry/mass
spectrometry (LC–MS/MS) analysis and successfully demonstrated
the in vitro acylation reaction of OA (1) in the presence of acyl CoA,
ATP, and the microsomal fraction of the digestive gland of a bivalve
(Fig. 1). We also revealed that a membrane protein(s) in the micro-
somal fraction catalyzes this structural transformation.11 However,
LC–MS/MS analysis decreases sensitivity when surfactants are
⇑
Corresponding author. Tel.: +81 22 717 8819.
E-mail address: konoki@m.tohoku.ac.jp (K. Konoki).
http://dx.doi.org/10.1016/j.bmcl.2016.05.027
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
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S. Furumochi et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 1
In vitro acylation of OA (1) using various saturated fatty acyl CoA (4–8) and functional
fatty acyl CoA (9–11) as the substrate. The relative yield was calculated from the ratio
of the raw yield of DTX3 (any one of 12–16, 22, 29, 30) from OA (1) and the designated
fatty acyl CoA (4–8, 9–11) to the raw yield of DTX3 (15) from OA (1) and
hexadecanoyl CoA 7
Fatty acyl CoA
Fatty acyl OA (DTX3)
Relative
yields
Figure 1. Diarrhetic shellfish poisoning toxins and in vitro acylation reaction.
Okadaic acid (1, OA) and dinophysistoxin 1 (2, DTX1) were converted to dinoph-
ysistoxin 3 (3, DTX3) in the presence of acyl CoA, ATP and microsomal fraction of
the digestive gland of bivalves.
0.11
0.14
1.06
1.00
0.61
used to solubilize and purify the membrane protein(s). Several MS-
compatible surfactants are commercially available for research
studies in proteomics and were developed to improve the analysis
of membrane-embedded regions, but protein solubilization using
these surfactants results in loss of biological activity.
Purpose of the present study is to characterize the OA-trans-
forming enzyme for developing an assay for DTX3 (3) alternative
to the aforementioned assay using LC/MS. We investigated the
optimal carbon chain length in saturated fatty acyl CoA (4–8) as
the substrate for OA-transforming enzyme. We then synthesized
and evaluated three functional fatty acyl CoAs (9–11) that can be
labeled with fluorescent reagents after an in vitro acylation reac-
tion. We demonstrate that this approach is applicable to the devel-
opment of a novel assay for DTX3.
0.94
1.39
0.32
As previously described,11 in vitro acylation of OA (1) proceeded
in the presence of hexadecanoyl CoA (7), ATP, and the microsomal
fraction of the digestive gland from a scallop.11 The reaction mix-
ture was directly partitioned with hexane to recover 7-O-hexade-
canoyl OA (15) as DTX3 (3). The reaction yield was determined
by LC/MS in negative ion mode and ranged from 0.1% to 15.4%,
indicating the inconsistency of the in vitro acylation reaction. We
assessed the efficiency of the transformation of OA (1) to DTX3
(3) for decanoyl CoA (4), dodecanoyl CoA (5), tetradecanoyl CoA
(6), hexadecanoyl CoA (7), and octadecanoyl CoA (8) (Table 1).
The relative yield was calculated from the ratio of the raw yield
of DTX3 (any one of 12–16) from OA (1) and the designated fatty
acyl CoA (4–8) to the raw yield of DTX3 (15) from OA (1) and hex-
adecanoyl CoA 7; these relative yields were 0.11, 0.14, 1.06, 1.00
and 0.61, respectively, suggesting that fatty acyl CoAs with a car-
bon chain length of 14 (6), 16 (7) and 18 (8) provided higher yields
than those with a carbon chain length of 10 (4) and 12 (5). We pre-
viously reported the in vitro acylation reaction of OA (1) using only
acyl CoA 7. The present results clearly revealed that various fatty
acyl CoAs can be substrates for the in vitro acylation reaction,
and that the OA-acylating enzyme has selectivity to acyl CoAs of
specific carbon chain length.
the alcohol 18, which was subjected to TEMPO oxidation14 to
afford an aldehyde. Takai olefination15 converted the aldehyde to
the vinyl iodide 19, which was subjected to Suzuki–Miyaura cou-
pling16 with vinylboronic acid pinacol ester to provide (E)-pen-
tadeca-12,14-dienoic acid ethyl ester (20). The ethyl ester 20 was
hydrolyzed under basic conditions, and the resulting carboxylic
acid 21 was conjugated with CoA in the presence of PyBOP and
K2CO3 to deliver the acyl CoA 9.
Based on these results, we embarked on developing an alterna-
tive to LC–MS/MS analysis to assay for DTX3 (3). Three functional
fatty acyl CoAs were synthesized possessing either a diene unit 9,
an alkyne unit 10, or an azide unit 11 in the acyl chain to facilitate
fluorescent labeling after the in vitro acylation reaction (Table 1).
The acyl CoAs 9, 10 and 11 have carbon chain lengths of 15, 16
and 15, respectively, and would provide high yields in the
in vitro acylation reaction.
When 2.0 lg of OA (1) was subjected to in vitro acylation with
the acyl CoAs 9 and 7, the corresponding DTX3s 22 and 15 were
produced in 0.29% and 0.31% yield, respectively (Table 1). The rel-
ative yield of 22 was 0.94, comparable to that of 1.06 for 14 and
1.00 for 15. This comparable yield would be explained by the car-
bon chain length of 15 (C15) in the acyl part of the acyl CoA 9,
which is longer by one carbon than that of the acyl CoA 6 (C14
)
and shorter by one carbon than that of the acyl CoA 7 (C16). Follow-
ing Diels–Alder reaction of DTX3 (22) with DMEQ-TAD (Fig. 3A),
the product mixture was analyzed by LC/MS. The reaction did
not appear to deliver the desired product 23. When the in vitro
acylation of OA (1) was subsequently scaled up 10-fold, the com-
pound 23 was detected at 22.3 min on LC/MS at m/z 1368.7427
We first synthesized the (E)-pentadeca-12,14-dienoyl CoA (9)
(Fig. 2A). We chose a diene functionality because a diene under-
goes Diels–Alder reaction with the fluorescent dienophile, DMEQ-
TAD (Fig. 3A), used to quantify vitamin D3 and yessotoxin.13
12
Monoethyl dodecandioate 17 was reacted with BH3ÁTHF to yield
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S. Furumochi et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
A
B
O
O
O
O
a
b, c
d
OH
OH
O
EtO
EtO
EtO
I
5
5
5
5
O
17
20
18
21
19
9
O
f
e
EtO
OH
SCoA
5
5
O
O
O
j
g
k
h, i
MeO
HO
OH
MeO
OH
6
6
6
26
25
27
O
O
OH
SCoA
6
6
28
10
Figure 2. Syntheses of (A) (E)-pentadeca-12,14-dienoyl CoA (9) and (B) hexadec-15-ynoyl CoA (10). (a) BH3ÁTHF, rt, 5 h; (b) TEMPO, NaOCl/CH2Cl2, rt, 1.5 h, 67%; (c) CHI3,
CrCl2/1,4-dioxane, rt, 1.5 h, 30% in 2 steps; (d) vinylboronic acid pinacol ester, Pd(dppf)Cl2, Ag2O/10:1 THF–H2O, rt, 2.5 h, 82%; (e) 1.0 M aq NaOH, rt, 5 h; 1.0 M aq HCl, 81%; (f)
CoA, PyBOP, K2CO3/7:3 THF–H2O, rt, 4 h, 59%; (g) 0.6 M trimethylsillyldiazomethane/THF, rt, 30 min; (h) TEMPO, NaOCl/CH2Cl2, rt, 30 min; (i) dimethyl (1-diazo-2-oxopropyl)
phosphonate/MeOH, rt, 8 h, 23% in 3 steps; (j) 1.0 M aq NaOH, 4 h; 1.0 M aq HCl, 46%; (k) CoA, PyBOP, K2CO3/7:3 THF–H2O, 83%.
(calculated for C74H106N5O19 as [MÀH]À: m/z 1368.7488, Figs. 3B
and S11) but was not detected by a fluorescence detector con-
nected to LC (LC-FLD). Two major peaks were clearly apparent in
the LC-FLD chromatogram and were attributed to DMEQ-TAD
(closed arrow, Fig. 3B) and the compound 24 (open arrow,
Fig. 3B) from Diels–Alder reaction of the carboxylic acid 21 and
DMEQ-TAD. This observation suggested that most of the acyl CoA
9 was hydrolyzed in situ to the carboxylic acid 21 and functioned
as the dienophile (Figs. 3B and S12). Many minor peaks were also
apparent in the LC-FLD chromatogram. DMEQ-TAD might have
undergone nucleophilic attack by biological compounds, thus pro-
viding many undesired products that hampered identification of
the Diels–Alder adduct.
As shown in the present study, fluorescent derivatives of bio-
logical compounds, including proteins, peptides, small molecules,
have been used to evaluate their mode of action. This trend
towards the use of fluorescent derivatives has been accompanied
by the emergence of chemical biology, in which a bioorthogonal
click reaction between an azide unit and an alkyne unit is a key
reaction for labeling molecules of interest. Like phosphorylation
and dephosphorylation, acylation is an important modification
of biological compounds and enables binding to proteins and
promotes signal transductions.17,18 For example, cholesterol
acylated at the 3b-OH binds to low/high density lipoproteins
(LDL/HDL),19 and all-trans-retinol (vitamin A) must be acylated
for conversion to 11-cis-retinol.20 Chemical biology has also
targeted lipid metabolism to provide various chemical probes.21
We therefore examined hexadec-15-ynoyl CoA (10)22 and
15-azidopentadecanoyl CoA (11)23 as alternatives to (E)-pentadeca-
12,14-dienoyl CoA (9) as the substrate in the in vitro acylation
reaction. 15-Azidopentadecanoyl CoA (11) was synthesized
according to the literature23 and hexadec-15-ynoyl CoA (10)
was synthesized via a new route as follows (Fig. 2B). Hexadec-
15-ynoic acid (25) was converted to methyl ester 26 with
trimethylsilyldiazomethane. The methyl ester 26 was oxidized
with TEMPO to the corresponding aldehyde, which was reacted
with Ohira–Bestmann reagent24 to afford hexadec-15-ynoic acid
methyl ester (27), then 27 was hydrolyzed under basic condi-
tions to give hexadec-15-ynoic acid (28). The carboxylic acid
28 was converted to acyl CoA 10 in the presence of CoA, PyBOP
and K2CO3.
Hexadec-15-ynoyl CoA (10) and 15-azidopentadecanoyl CoA
(11), together with hexadecanoyl CoA (7), were used as substrates
for the in vitro acylation reaction and converted OA (1) to the cor-
responding DTX3 29, 30 and 15 in 0.43%, 0.10% and 0.31% yield,
respectively. The relative yields of 29 and 30 were 1.39 and 0.32,
respectively (Table 1). The relative yield of 29 was comparable to
that of 15, presumably because the acyl CoAs 7 and 10 have the
same carbon chain length (C16), and unsaturation in the carbon
chain of the acyl CoA may not affect enzymatic activity. On the
other hand, the relative yield of 30 (C15) was about one third that
of 14 (C14) or 15 (C16). If an azide unit is counted as being three car-
bons in size, then acyl CoA 11 can be considered as a mimic of acyl
CoA 8 because the acyl chain of 11 is composed of 18 atoms (15 C
and 3 N). In fact, the yield of DTX3 (16) from OA (1) and acyl CoA 8
was comparable to the yield of DTX3 (30) from OA (1) and acyl CoA
11. As described earlier, DMEQ-TAD might have acted as an accep-
tor during Michael reaction for endogenous compounds and left
un-identified fingerprints in the LC-FLD chromatogram. In contrast,
the alkyne unit in 29 and the azide unit in 30 should react specif-
ically according to the intended click chemistry. Thus, the acyl
CoAs 10 and 11 would be favored over the acyl CoA 9.
The detection of 23 showed that the sensitivity of MS is superior
to that of FLD for this family of compounds. An alternative assay for
DTX3 (3) using FLD should provide increased efficiency of the
in vitro acylation reaction. In fact, Suzuki et al. reported that the
ratio of the acylated form (DTX3, 3) to the non-acylated form
(OA (1) or DTX1 (2)) was more than 10 in scallops.8 We believed
that the in vitro acylation reaction of OA (1) was not optimized;
indeed, the administered acyl CoA was hydrolyzed in situ and thus
unavailable as the substrate for the OA-acylating enzyme. We
therefore attempted to prevent hydrolysis of the acyl CoA to
improve the efficiency of the in vitro acylation reaction. However,
the inhibitors for acyl CoA hydrolase tested (clofibrate,25 gemfi-
brozil,26 b-NADP,27 and ADP28) did not increase the production of
DTX3 (3). It is possible that there is a physiological or ecological
factor(s) involved in the generation of OA-transforming enzyme
in the digestive gland of scallops. DTX3 (3) diminishes binding
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S. Furumochi et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Figure 3. Diels–Alder reaction of DMEQ-TAD with DTX3 (22) or carboxylic acid 21. (A) Diels–Alder reaction of DMEQ-TAD with DTX3 (22) or carboxylic acid 21, a hydrolyzed
product in situ from acyl CoA 9. (B) Detection of the desired product 23 with (upper) extracted ion chromatogram (EIC) at m/z 1368.7427 and with (lower) LC-FLD at Ex.
370 nm/Em. 440 nm. The closed and open arrows represented peaks for DMEQ-TAD and 24, respectively.
affinity of OA-acylating enzyme to PP1 and PP2A, and the conver-
sion of OA (1)/DTX1 (2) to DTX3 (3) in the presence of the OA-acy-
lating enzyme would be a detoxification mechanism in bivalves.
The scallops used in the present study were purchased at a fish
market and therefore did not contain toxins. Overexpression of
OA-transforming enzyme could be triggered by the uptake of OA
(1) or DTX3 (3) by scallops, and scallops purchased at a fish market
may not be inherently rich in OA-transforming enzymes.
Investigation of mode of action has recently been admitted as
one of the core subjects in the natural product chemistry. We did
not investigate mode of action for a therapeutic purpose as in
many other research studies but aimed to elucidate mode of action
in natural environments, which has been extensively studied for
plant hormones or insect pheromones. In the present study, we
focused the OA-transforming enzyme identified in the digestive
gland of shellfish. Digestive gland in shellfish would be considered
as liver in humans, which let us imagine that the structural conver-
sion of OA (1) is analogous to that of cholesterol in human liver.
Cholesterol is acylated at 3b-OH and conveyed outside of the organ
as a complex with low-density lipoprotein (LDL).19 It should be
noted that various shellfish toxins are also acylated in shellfish.29
Thus, the acylation of shellfish toxins could be inherent in shellfish,
although these intriguing phenomena will be thoroughly investi-
gated after isolation of the enzyme in the future.
In summary, we assessed various saturated fatty acyl CoAs as
substrates in the in vitro acylation reaction of OA (1) and verified
the optimal carbon chain length in acyl CoA for the production of
DTX3 (3). Furthermore, the three functional acyl CoAs 9–11 were
synthesized and confirmed to be substrates for the in vitro acyla-
tion reaction. These functional acyl CoAs can be conjugated with
fluorescent reagents and the products can be detected in the pres-
ence of surfactants. Acyl CoA 9, however, did not support the fluo-
rescent detection of DTX3 (23) in LC-FLD after conjugation with
DMEQ-TAD. Thus, the acyl CoAs 10 and 11 are favored because
DTX3 (3) possessing the fatty acyl part of 10 and 11 can be con-
verted into fluorescent derivatives by bioorthogonal click reaction.
As shown in the present study, LC/MS provides higher sensitivity
than LC-FLD. However, in the presence of surfactants, LC-FLD could
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S. Furumochi et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
5
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This research was supported by JSPS KAKENHI (Grant Numbers
21603003 and 24510291 to K.K.), the NOVARTIS Foundation
(Japan) for the Promotion of Science (to K.K.), and the JSPS Funding
Program for the Next Generation of World-Leading Researchers
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