Synthesis of 2-(5Z,8Z,11Z,14Z)-Icosa-5,8,11,14-tetraenamidoethyl-d 4 dihydrogen phosphate, tetra-deuterated pAEA

Article abstract of DOI:10.1002/jlcr.1540

A labile intermediate phospho-anandamide (2-(5Z,8Z,11Z,14Z)-icosa-5,8,11, 14-tetraenamidoethyl dihydrogen phosphate, pAEA) has been identified in mouse brain and macrophages, but its precise quantitation was difficult because of its low concentration and chemical instability. We report the synthesis of tetra-deuterated pAEA from 2-aminoethyl dihydrogen phosphate-1,1,2,2-d ⁴ and (5Z,8Z,11Z,14Z)-2,5-dioxopyrrolidin-1-yl icosa-5,8,11,14- tetraenoate. The compound will be used to quantitate the pAEA necessary for a novel biosynthetic pathway.

Full text of DOI:10.1002/jlcr.1540

Research Articles
Received 12 July 2008,
Revised 23 July 2008,
Accepted 12 August 2008
Published online 9 October 2008 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1540
Synthesis of 2-(5Z,8Z,11Z,14Z)-Icosa-
5,8,11,14-tetraenamidoethyl-d4 dihydrogen
phosphate, tetra-deuterated pAEAy
Kejun Cheng,a Bijali Saha,b A. Mahadevan,b Raj K. Razdan,b
Ã
George Kunos,c Arthur E. Jacobson,a and Kenner C. Ricea
A labile intermediate phospho-anandamide (2-(5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenamidoethyl dihydrogen phosphate,
pAEA) has been identified in mouse brain and macrophages, but its precise quantitation was difficult because of its low
concentration and chemical instability. We report the synthesis of tetra-deuterated pAEA from 2-aminoethyl dihydrogen
phosphate-1,1,2,2-d4 and (5Z,8Z,11Z,14Z)-2,5-dioxopyrrolidin-1-yl icosa-5,8,11,14-tetraenoate. The compound will be used
to quantitate the pAEA necessary for a novel biosynthetic pathway.
Keywords: deuterium labeling; deuterated phospho-anandamide; pAEA; endocannabinoid
gen phosphate-1,1,2,2-d4 (2). Compound 2 was obtained as a
crystalline solid from a water–ethanol mixture. (5Z,8Z,11Z,14Z)-
Introduction
Endocannabinoids are endogenous lipid ligands that interact
with the same receptors that recognize the D9-tetrahydrocan-
nabinol in marijuana to produce similar biological effects. The
first endocannabinoid was isolated from porcine brain and
identified as arachidonoyl ethanolamide (anandamide, AEA) in
1992.1 Shortly thereafter a two step biosynthetic process was
proposed to account for the in vivo generation of AEA, whereby
a transacylase transfers the arachidonic acid from the sn-1
position of phosphatidylcholine to the nitrogen in phosphati-
dylethanolamine (PE) to generate N-arachidonoyl PE (NAPE),
which is then hydrolyzed by a NAPE-specific phospholipase D
(NAPE-PLD) to yield AEA.2 Unexpectedly, tissue AEA levels were
found unaffected by genetic deletion3 or knockdown of NAPE-
PLD,4 suggesting the existence of alternative biosynthetic
pathways involved in the conversion of NAPE to anandamide.
One such recently identified pathway involves the hydrolysis of
NAPE by a phospholipase C (PLC) to yield the labile intermediate
2,5-dioxopyrrolidin-1-yl icosa-5,8,11,14-tetraenoate (3) was
added to an alkaline solution of 2 to give, after workup, 2-
(5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenamidoethyl-d4 dihydro-
gen phosphate (tetra-deuterated pAEA, 4) as a waxy solid. The
availability of the tetra-deuterated compound 4 in reasonable
overall yield via a simple two-step reaction will enable the
detailed examination of interesting biosynthetic pathways.
Experimental
General
The starting material 2-aminoethanol-1,1,2,2-d4 was obtained
from CDN Isotopes (88 Leacock St., Pointe-Claire; Quebec,
Canada H9R 1H1; www.CDNISOTOPES.com). Chemical ionization
phospho-anandamide (pAEA), which is then dephosphorylated aDrug Design and Synthesis Section, Chemical Biology Research Branch,
by a phosphatase.4,5 pAEA was definitively identified in mouse
National Institute on Drug Abuse and the National Institute on Alcohol Abuse
brain and macrophages using HPLC/ESI-MS/MS, but its precise
quantitation was difficult because of its low concentration and
chemical instability.4,5 In order to facilitate the quantitation of
pAEA that is necessary for the further analysis of this novel
biosynthetic pathway, we have introduced a method for the
synthesis of tetra-deuterated pAEA.
Department of Health and Human Services, 5625 Fishers Lane, Room 4N03,
and Alcoholism, National Institutes of Health, MSC 9415, Bethesda, MD 20852,
USA
bOrganix Inc., 240 Salem Street, Woburn, MA 01801, USA
cNeuroendocrinology Section, Laboratory of Physiologic Studies, Department of
Health and Human Services, 5625 Fishers Lane, Room 2S24, National Institute
on Alcohol Abuse and Alcoholism, National Institutes of Health, MSC 9413,
Rockville, MD 20852, USA
*Correspondence to: Kenner C. Rice, Drug Design and Synthesis Section,
Chemical Biology Research Branch, Department of Health and Human Services,
5625 Fishers Lane, Room 4N03, National Institute on Drug Abuse and the
Results and discussion
Based on the procedure for the preparation of the analogous
National Institute on Alcohol Abuse and Alcoholism, National Institutes of
C14-labelled compound,6 2-aminoethanol-1,1,2,2-d4 (1, Scheme
1) was added to a solution of dilute phosphoric acid and water
Health, MSC 9415, Bethesda, MD 20852, USA.
E-mail: kr21f@nih.gov.
yThis article is a US Government work and is in the public domain in the USA.
was removed to obtain the intermediate 2-aminoethyl dihydro-
J. Label Compd. Radiopharm 2008, 51 389–390
Published in 2008 by John Wiley & Sons, Ltd.

K. Cheng et al.
O
D
D
1) 0 oC, 30 min
D
D
O
D
D
HO
OH
D
O
P OH
OH
P
2) 138 oC, 18 h
38%
+
H2N
OH
H2N
D
HO
1
2
O
O
O
D
D
O
O
P OH
OH
D
THF/NaHCO3/H2O
79%
N
N
O
H
D
2
+
O
3
4
Scheme 1.
mass spectra (CIMS) were obtained using a Finnigan 4600 mass (2 ꢁ 20 mL), dried (Na2SO4) and concentrated in vacuo to afford
spectrometer unless otherwise noted. 2H nuclear magnetic 4 as a waxy light yellow solid (184 mg, 79%). 1HNMR (Jeol
resonance (2H NMR, 500 MHz) was recorded on a Bruker Avance Eclipse, 300 MHz, CDCl3) d 5.26–5.36 (m, 8H), 2.76–2.80 (m, 6H),
500 instrument in deuterium depleted water (2–3 ppm deuter- 2.25 (brt, 2H), 2.0–2.06 (m, 4H), 1.66–1.68 (m, 2H), 1.24–1.33 (m,
ium, Cambridge Isotope Laboratories, Inc.) with the values given 6H), 0.87 (t, J = 6.7 Hz, 3H); MS (Agilent 1100 Series HPLC-MS, CI)
in ppm (D2O as internal standard at 4.80 ppm). 31P nuclear m/z 430 (Mꢀ1) and 432 (M11). Anal. calcd. for
magnetic resonance (31P NMR) spectra were recorded on a C22H34D4NO5Pꢂ0.7CHCl3: C, 52.93; H and D as H, 7.58; N, 2.72.
Varian Gemini-300 instrument in D2O with the values given in Found: C, 52.90; H and D as H, 7.71; N, 2.65. The presence of
ppm (H2O as internal standard at 4.80 ppm). The melting point chloroform was confirmed by the NMR of 4 in DMSO.
was determined on a Bu¨chi B-545 melting point apparatus and
is uncorrected. Combustion analyses were determined at Acknowledgement
Atlantic Microlabs, Atlanta, GA.
We would like to thank Dr Klaus Gawrisch and Dr Walter Teague
of the Laboratory of Membrane Biochemistry and Biophysics
2-Aminoethyl dihydrogen phosphate-1,1,2,2-d4 (2)
(LMBB) in NIAAA for 2H NMR spectral data. The authors also
To an ice-cold solution of 2-aminoethanol-1,1,2,2-d4 (1, 1.0 g,
express their appreciation to Noel Whittaker and Wesley White
15.4 mmol) in water (20 mL), was added dropwise, over 10 min, a
of the Laboratory of Analytical Chemistry for mass spectral data
solution of phosphoric acid (3.0 g, 30.8 mmol) in water (20 mL).
and 31P NMR spectral data. The work of the Drug Design and
After the mixture was stirred at 01C for 30 min, most of the water
Synthesis Section, CBRB, NIDA, & NIAAA, was supported by the
was removed in vacuo. A desiccant flask holding phosphorus
NIH Intramural Research Programs of the National Institute on
pentoxide was attached, and the flask assembly was evacuated
Drug Abuse (NIDA) and the National Institute of Alcohol Abuse
to a pressure of 3–5 mm. The reaction flask was heated to 1381C
and Alcoholism, and the work of the Neuroendocrinology
(oil bath) and kept at this temperature for 18 h. After cooling to
Section, Laboratory of Physiologic Studies, was supported by the
room temperature, the residue was dissolved in water (7 mL)
NIH Intramural Research Programs of the National Institute of
and ethanol (3 mL) was added. The white crystalline solid that
Alcohol Abuse and Alcoholism. The authors (B.S., A.M., R.K.R.)
formed was filtered, washed with 95% ethanol, and dried in
thank NIDA Grant DA-05488 for financial support.
vacuo to give 2 (445 mg). The filtrate was concentrated and
dissolved in a water–ethanol mixture to give a second crop of
crystals of 2 (400 mg, 38.0% overall). Mp 238–2391C; 2H NMR
(H2O, deuterium depleted): d 3.99 (s, 2D), 3.17 (s, 2D); 31P NMR
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16.56; H1D, 5.56; H, 9.65; Found: C, 16.60; H1D, 5.51; H, 9.62.
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icosa-5,8,11,14-tetraenoate7
(3,
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CHCl3 (3 mL). The organic layer was washed with water
www.jlcr.org
Published in 2008 by John Wiley & Sons, Ltd
J. Label Compd. Radiopharm 2008, 51 389–390