The preparation of 2-O-[1′-14C]arachidonoyl-1-O-stearoyl- sn-glycerol

Article abstract of DOI:10.1002/jlcr.1609

2-O-Arachidonoyl-1-O-stearoyl-sn-glycerol is the most abundant molecular species of the 1,2-diacyl-sn-glycerol signaling lipids in neural tissue. The facile preparation of 2-O-[1′-¹⁴C]arachidonoyl-1-O-stearoyl-sn- glycerol from 2-O-[1′-^14<

Full text of DOI:10.1002/jlcr.1609

Research Article
Received 6 February 2009,
Revised 14 April 2009,
Accepted 15 April 2009
Published online 1 June 2009 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1609
The preparation of 2-O-[1’-14C]arachidonoyl-1-
O-stearoyl-sn-glycerol
Ã
Richard I. Duclos Jr,a S. John Gatley,b Shachi R. Bhattb and
Meghan Johnstona
2-O-Arachidonoyl-1-O-stearoyl-sn-glycerol is the most abundant molecular species of the 1,2-diacyl-sn-glycerol signaling
lipids in neural tissue. The facile preparation of 2-O-[1’-14C]arachidonoyl-1-O-stearoyl-sn-glycerol from 2-O-[1’-14C]arachi-
donoyl-1-O-stearoyl-sn-glycero-3-phosphocholine at a hexane and phosphate buffer interface with phospholipase C was
demonstrated on a 20 mCi scale in 83% radiochemical yield. The specific activity of the product 2-O-[1’-14C]arachidonoyl-1-
O-stearoyl-sn-glycerol was 57.0 mCi/mmol and the radiochemical purity was determined to be 499% by TLC. The hydrolysis
of this lipid biosynthetic intermediate with lipoprotein lipase was shown to produce 2-O-[1’-14C]arachidonoylglycerol
(2-AG). The 14C-radiolabeled monoacylglycerol 2-AG is an endogenous cannabinoid receptor-signaling molecule
(endocannabinoid).
Keywords: 2-O-arachidonoyl-1-O-stearoyl-sn-glycerol; 14C-labeled; diacylglycerol; 2-arachidonoylglycerol; 2-AG
hydrolysis by phospholipase C.26 The hydrolysis of the
phosphatidylcholine 1 with phospholipase C from Bacillus
Introduction
1,2-Diacyl-sn-glycerols have important physico-chemical and
juxtacrine signaling functions.1–4 Although diacylglycerols are
found in extremely low concentration in cell membranes,
2-O-arachidonoyl-1-O-stearoyl-sn-glycerol is the most abundant
molecular species in brain5 and peripheral nerve.6 The hydrolysis
of this lipid by diacylglycerol lipase (DAGL) produces the
endocannabinoid 2-O-arachidonoylglycerol (2-AG).7,8 The endo-
cannabinoid system is the focus of our research and includes the
two most abundant endocannabinoid lipids, 2-AG and
N-arachidonoylethanolamine (AEA), each having distinct signal-
ing functions.9,10 Radiolabeled 2-O-arachidonoyl-1-O-stearoyl-
sn-glycerol (2) (see Scheme 1) was required for our studies of
DAGL enzyme activity and the biosynthesis of 14C-labeled
endocannabinoid 2-AG (3). 2-O-[1’-14C]Arachidonoyl-1-O-stear-
oyl-sn-glycerol (2) has recently been used to assay the activity
of hDAGL-a preparations,11,12 including in the presence of
inhibitors, but is no longer commercially available.
cereus was performed at neutral pH. The mild reaction
conditions are critical for 1,2-diacyl-sn-glycerols, where variation
of pH from 7 or increased temperature results in rearrangement
of 1,2-diacyl-sn-glycerol to 1,3-diacyl-sn-glycerol.27–29 1,2-Diacyl-
glycerols are reportedly more stable in hydrocarbon solution.30
The 1,2-diacylglycerol and 1,3-diacylglycerol isomers are readily
separable by analytical TLC28,31,32 and HPLC.24,25 However, our
1,2-diacyl-sn-glycerol isolation following the enzymatic protect-
ing group cleavage required only flash chromatographic
filtration to remove trace amounts of unreacted starting material
as well as the phosphocholine salt byproduct origin materials.
We had previously used this methodology with unlabeled
2-O-arachidonoyl-1-O-stearoyl-sn-glycero-3-phosphocholine on
scales of up to 10 mg to give unlabeled 2-O-arachidonoyl-1-O-
stearoyl-sn-glycerol that was homogeneous by TLC (30:70
acetone/hexane, developing with phosphomolybdic acid
1
reagent) and characterized by H NMR.
This report describes the convenient preparation of radi-
olabeled 2-O-[1’-14C]arachidonoyl-1-O-stearoyl-sn-glycerol (2) via
a biomimetic route that uses the phosphocholine phosphodie-
ster group for protection. Syntheses of labeled 1,2-diacyl-
sn-glycerols have previously used the 3-phosphocholine
group,13–18 3-phosphate group,19 or 3-phosphoinositol group20
as protecting groups in chemo-enzymatic syntheses. Other
classical protecting groups have also been used in purely
chemical syntheses of unlabeled21–23 and labeled11 1,2-diacyl-
sn-glycerols and labeled rac-1,2-diacylglycerols,24,25 although
such routes involve the technical challenge of avoiding acyl
group migration during a microscale radiosynthesis.
Results and discussion
The substrate 2-O-[1’-14C]arachidonoyl-1-O-stearoyl-sn-glycero-
3-phosphocholine (1) (GE Healthcare CFA655) contained a
aCenter for Drug Discovery, Northeastern University, 360 Huntington Avenue,
116 Mugar Life Sciences Building, Boston, MA 02115, USA
bDepartment of Pharmaceutical Sciences, Northeastern University, 360
Huntington Avenue, 211 Mugar Life Sciences Building, Boston, MA 02115, USA
*Correspondence to: Richard I. Duclos Jr, Center for Drug Discovery, North-
eastern University, 360 Huntington Avenue, 116 Mugar Life Sciences Building,
Boston, MA 02115, USA.
Our conversion of the commercially available labeled
2-O-[1’-14C]arachidonoyl-1-O-stearoyl-sn-glycero-3-
substrate
phosphocholine (1) utilized the regiospecificity of the enzymatic
E-mail: duclos@neu.edu
J. Label Compd. Radiopharm 2009, 52 324–326
Copyright r 2009 John Wiley & Sons, Ltd.

R. I. Duclos et al.
O
O
O
O
*
O
*
O
H
(CH2)16-CH3
+
O
H
(CH2)16-CH3
phospholipase C
O
O
N-(CH3)3
O-P-O
OH
OH
-
OH
2
1
2- - [1'-14C]arachidonoyl-1- -stearoyl-sn-glycerol
2-O- [1'-14C]arachidonoyl-1-O-stearoyl-sn-glycero-
O
O
3-phosphocholine
83% radiochemical yield
>99% radiochemical purity
0.29 mg (19.95 µCi)
O
*
O
OH
OH
diacylglycerol lipase
(CH2)16-CH3
O
+
HO
3
4
2- - [1'-14C]arachidonoylglycerol
stearic acid
O
Scheme 1. Enzymatic preparations of 2-O-[1’-14C]arachidonoyl-1-O-stearoyl-sn-glycerol (2) and 2-O-[1’-14C]arachidonoylglycerol (3, 2-AG).
14C-label in the carbonyl of the arachidonoyl group, and the
[1’-14C]arachidonoyl group was assayed to be 97.5% in the sn-2
position. This substrate 1 had a specific activity of 57.0 mCi/
mmol and was found to be nearly homogeneous (498%
radiochemical purity) by TLC (Rf 0.00, 30:70 acetone/hexane; Rf
0.33, 60:30:5 chloroform/methanol/water). The phospholipase C
(Sigma P9439 from B. cereus) was reconstituted with water to
give a phosphate buffered solution that could be frozen, stored
at À201C, and reused. Generally, PLC hydrolyses have been run
at an interface of diethyl ether and aqueous buffer.13,16,17,33,34
We found that the reaction proceeds when hexane is used,
although the enzyme activity does fall off and the conversion
must be driven to completion by adding fresh enzyme solution
at 20 min intervals over 2 h. The phase separation of hexane
from aqueous buffer was easier than when using diethyl ether.
There is still some solubility of the oily phosphocholine substrate
1 in hexane even though it carries the zwitterionic phosphocho-
line head group. The complete removal of any trace starting
material and salts was performed by adding acetone to reach a
30:70 acetone/hexane ratio followed by flash filtration through a
Figure 1. Left: TLC (60:30:5 chloroform/methanol/water) of 2-O-[1’-14C]arachido-
noyl-1-O-stearoyl-sn-glycerol (2, Rf 0.95) (filtration elution Lane 1 and column wash
Lane 2) from the phospholipase hydrolysis of the phosphodiester 2-O-
short plug of silica gel. The column was then washed with an
equal volume of fresh 30:70 acetone/hexane. Both elution
fractions were found to contain product with no starting
material (see Figure 1) and were ultimately combined to give an
overall radiochemical yield of 83%. The specific activity of the
product 2-O-[1’-14C]arachidonoyl-1-O-stearoyl-sn-glycerol (2)
was 57.0 mCi/mmol and the radiochemical purity was deter-
mined to be 499% by TLC (Rf 0.28, 4:96 acetone/chloroform; Rf
0.49, 30:70 acetone/hexane; Rf 0.95, 60:30:5 chloroform/metha-
nol/water). The tailing of the 1,2-diacyl-sn-glycerol 2 spot was
comparable with that seen using a related solvent system.29
A small amount of 1-O-[1’-14C]arachidonoyl-2-O-stearoyl-sn-
glycerol may be present from the starting phosphocholine.
Rearrangement of 2-O-[1’-14C]arachidonoyl-1-O-stearoyl-sn-
glycerol (2) to 3-O-[1’-14C]arachidonoyl-1-O-stearoyl-sn-glycerol
would have given a slightly higher Rf spot for this byproduct in
the TLC analysis with 30:70 acetone/hexane.2,23 The presence of
the rearrangement impurity could affect the study of the DAGL
enzyme where the 1,3-diacylglycerol byproduct could act as a
substrate or inhibitor.
C
[1’-14C]arachidonoyl-1-O-stearoyl-sn-glycero-3-phosphocholine (1). Right: TLC
(86:14:0.75 chloroform/methanol/aqueous ammonium hydroxide) of the enzy-
matic conversion of 2-O-[1’-14C]arachidonoyl-1-O-stearoyl-sn-glycerol (2, Rf 0.92) to
2-O-[1’-14C]arachidonoylglycerol (3, Rf 0.59) after 15 min with 58 units of
lipoprotein lipase (from Pseudomonas sp.).
lipase activity with high selectivity for the sn-1 acyl group.35–37 The
lipoprotein lipase readily hydrolyzed 2-O-[1’-14C]arachidonoyl-1-O-
stearoyl-sn-glycerol (2) enzymatically at a hexane and phosphate
buffer interface. Analytical TLC (86:14:0.75 chloroform/methanol/
aqueous ammonium hydroxide) shows the enzymatic hydrolysis of
the radiolabeled 1,2-diacyl-sn-glycerol 2 (Rf 0.92) to give 53.8%
conversion to 2-O-[1’-14C]arachidonoylglycerol (3, Rf 0.59) with the
release of only 0.4% of [1-14C]arachidonic acid (Rf 0.15) after 15 min
at 371C (see Figure 1). The 2-acylglycerol product 3 was not further
characterized18,22,38 as this radiolabeled 2-AG 3 was prepared only
on an analytical scale.
Thus, the facile preparation of the 14C-radiolabeled 1,2-diacyl-
sn-glycerol signaling lipid molecule 2 free of any 1,3-diacyl-sn-
glycerol rearrangement byproduct was demonstrated on a
20 mCi scale. This 2-O-[1’-14C]arachidonoyl-1-O-stearoyl-sn-gly-
cerol (2) activates protein kinase C and is the biosynthetic
We used the 2-O-[1’-14C]arachidonoyl-1-O-stearoyl-sn-glycerol (2)
for our preliminary experiments with lipoprotein lipase (from
Pseudomonas sp., Sigma), which has excellent 1,2-diacyl-sn-glycerol
J. Label Compd. Radiopharm 2009, 52 324–326
Copyright r 2009 John Wiley & Sons, Ltd.
www.jlcr.org

R. I. Duclos et al.
precursor of the endocannabinoid signaling molecule 2-AG 3. Makriyannis, Shawntelle Dillon, and Jack Price for their support and
The regioselective hydrolysis of 14C-radiolabeled 1,2-diacyl-sn- assistance.
glycerol 2 by an enzyme with DAGL activity to give 2-AG 3 was
also demonstrated.
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www.jlcr.org
Copyright r 2009 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2009, 52 324–326