An expeditious procedure for the synthesis of isotopically labelled fatty acids: Preparation of 2,2-d2-nonadecanoic acid

Article abstract of DOI:10.1002/jlcr.1094

We describe a new synthetic path for the preparation of isotopically labelled saturated odd-numbered fatty acids by a homologation procedure for the direct conversion of carboxylic acids into their 2,2-dideuterated homologues. This process can be used to obtain both odd- and even-numbered 2,2-dideuterated fatty acids starting from carboxylic acids. Furthermore, the reiteration of this process can also be used for the synthesis of polydeuterated fatty acids in which the heavy atoms are placed on adjacent carbon atoms or differently arranged in the molecule. Copyright

Full text of DOI:10.1002/jlcr.1094

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 675–682.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1094
Research Article
An expeditious procedure for the synthesis of
isotopically labelled fatty acids: preparation
of 2,2-d2-nonadecanoic acid
Annalisa Guaragna1,*, Mauro De Nisco1, Silvana Pedatella1, Vittoria Pinto1,2
and Giovanni Palumbo1
1
`
Dipartimento di Chimica Organica e Biochimica, Universita di Napoli Federico II,
Via Cynthia 4, I-80126 Napoli, Italy
2 Chiron Vaccines, Via Fiorentina 1 Research Center, Bldg 35 I-53100 Siena, Italy
Summary
We describe a new synthetic path for the preparation of isotopically labelled saturated
odd-numbered fatty acids by a homologation procedure for the direct conversion
of carboxylic acids into their 2,2-dideuterated homologues. This process can be used
to obtain both odd- and even-numbered 2,2-dideuterated fatty acids starting from
carboxylic acids. Furthermore, the reiteration of this process can also be used for
the synthesis of polydeuterated fatty acids in which the heavy atoms are placed on
adjacent carbon atoms or differently arranged in the molecule. Copyright # 2006
John Wiley & Sons, Ltd.
Received 9 May 2006; Revised 13 May 2006; Accepted 13 May 2006
Key Words: deuterium; isotopically labelled; deuterated fatty acids
Introduction
There is a constantly increasing demand for the synthesis of commercially
unavailable labelled starting materials, for use in studies investigating several
major topics, such as the metabolic pathways of drugs and bioactive molecules
in biological media,1 or the order and dynamics of liquid crystals in their
different mesophases.1,2 Isotopically labelled fatty acids are of interest as
2
biological tracers in various different types of investigation.3 Indeed, H and
13C enriched fatty acids have been incorporated into phospholipids and
*Correspondence to: Annalisa Guaragna, Dipartimento di Chimica Organica e Biochimica, Universita di
Napoli Federico II, Via Cynthia 4, I- 80126 Napoli, Italy. E-mail: guaragna@unina.it
Copyright # 2006 John Wiley & Sons, Ltd.

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A. GUARAGNA ET AL.
glycolipids with extensive applications in NMR, Raman, and neutron
diffraction studies.4,5 However, preparation of deuterated lipids has involved
much more extensive chemical investigations than 13C labelled lipids, due
to the more numerous ways to introduce deuterium. Several syntheses
of deuterated lipids have been made, for use in studies of fragmentation in
mass spectrometry (MS).6–8 Basically, only two methods for the introduction
of deuterium into organic molecules are known: exchange and reduction.9–11
More methods are available for the synthesis of deuterium labelled lipids,
besides those just mentioned, including the use of chemical reactions to
connect labelled segments to the non-labelled part of the molecule,11,12 and
biosynthesis.13
Over recent years particular attention has been devoted to the study of
odd-numbered fatty acids, due to their presence in human tissues, in a
concentration generally not exceeding 5% of total fatty acids.14 Since the
normal synthetic pathway for fatty acids leads to only even numbers of carbon
atoms in the products, it may seem curious to find any odd-numbered chains
present in human tissues. Fatty acids with odd numbers of carbon atoms
are produced primarily by initiating the synthetic route with the three
carbon compound, propionic acid. Vitamin B12 (cobalamin) is required for the
conversion of propionate into succinate for oxidation in the central energy
pathways.15 As a consequence, deficiency of vitamin B12 leads to accumulation
of propionate and subsequent build up of the odd-numbered fatty acids. The
association between vitamin B12 and abnormal fatty acid synthesis provides a
rationale for the neuropathy of cobalamin deficiency.16,17 Vitamin B12 plays
an important role in DNA synthesis and neurologic functions. Cobalamin
deficiency can lead to a wide spectrum of haematologic and neuropsychiatric
disorders that can often be reversed by early diagnosis and prompt treat-
ment. Diagnosis of vitamin B12 deficiency is typically based on measurement
of serum vitamin B12 levels; however, about 50% of patients with subclinical
disease have normal B12 levels.18,19 The detection of low concentrations
of odd-numbered fatty acids could be a useful instrument to study pathologies
associated with cobalamin deficiency. Usually, stable isotope tracers asso-
ciated with mass spectrometry techniques are used to follow and quantify the
biosynthesis and metabolism of fatty acids.
Results and discussion
We report a new methodology for the synthesis of 2,2-dideuterated saturated
odd-numbered fatty acids, which can also be applied to even-numbered fatty
acids. The early literature20 of deuterium chemistry features a large number of
papers on the exchange behaviour H/D of carboxylic acids. Kinetic studies
were most common, followed by combustion analysis, and neither enabled
exchange to be definitively located in such compounds. Recently, there has
Copyright # 2006 John Wiley & Sons, Ltd.
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DOI: 10.1002/jlcr

SYNTHESIS OF ISOTOPICALLY LABELLED FATTY ACIDS
677
been renewed interest in the liability of the a-protons of carboxylic acids and
early exchange21 reactions with D2O. The latter make possible a one-step
preparation of a wide variety of a-deutero carboxylic acids. Additional steps
intended to introduce the second deuterium atom into a-position have proved
to be more difficult and low yielding. These syntheses are in fact quite complex
and not generally available on the large scale.
We have devised a new process for the direct conversion of carboxylic acids
into their 2,2-dideuterated homologues under smooth, clean, quick conditions
and on the large scale. This process can be used to obtain both odd- and even-
numbered 2,2-dideuterated fatty acids starting from carboxylic acids. The
reiteration of this process can also be used for the synthesis of polydeuterated
fatty acids in which the heavy atoms are placed on adjacent carbon atoms or
differently arranged in the molecule. Some of the isotopically labelled fatty
acids which can be obtained are shown in Scheme 1.
In this paper, we describe the preparation of the 2,2-dideuterated
nonadecanoic acid starting from stearic acid. The current path is based on
the homologation of carboxylic acids by reduction and then substitution of the
hydroxyl group of the corresponding primary hydroxyl function with a cyano
group (Scheme 2). We began our synthesis of isotopically labelled compounds
by treating stearic acid with methyl chloroformate: the corresponding in situ
formed carbomethoxy anhydride was next reduced and labelled by means
of NaBD4 (98% atom D) in D2O, affording a high yield (91%) of
Scheme 1.
Copyright # 2006 John Wiley & Sons, Ltd.
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678
A. GUARAGNA ET AL.
Scheme 2.
1,1-d2-octadecanol (1) with a 98% deuterium incorporation (determined by 1H
NMR spectroscopy). On the basis of previously acquired knowledge,22 the
dideuterated alcohol 1 was then converted into its corresponding alkyl iodide 2
using a suspension of triphenyl phosphine polymer-bound/iodine complex
(polystyryl diphenyl iodophosphonium iodide) in anhydrous acetone. The
phosphine oxide, which under our conditions is the only by-product of the
reaction, is linked to the polymeric matrix and can thus be easily separated by
filtration. The triphenylphosphine polymer-bound/halogen complex is a Lewis
acid and a dehydrating agent widely used in miscellaneous reactions23 with low
environmental impact. In fact it avoids contamination from by-products and
use of non environmentally friendly solvents in the purification processes.
Triphenylphosphine polymer-bound/iodine complex is an easy to prepare,24
convenient, semicrystalline solid; when dried and kept properly, it can be
stored for weeks at room temperature, under nitrogen atmosphere. The
somewhat high cost of the starting triphenylphosphine polymer-bound does
not actually represent a limitation of this procedure, if one considers that the
polymer-linked phosphine oxide generally obtained from the reaction can be
readily filtered off and reduced to the original phosphine form with
trichlorosilane.25 Compound 2 so obtained was directly employed in the next
step, and the iodine atom was easily replaced by a cyano group in the presence
of Et3N+CNꢀ. Finally, hydrolysis of nitrile 3 using conc. aq HCl in dioxane
directly afforded, following the common purification procedures, the 2,2-d2-
nonadecanoic acid 4. Furthermore, acid catalysed alcoholysis of 3 led to the
corresponding 2,2-d2-methylnonadecanoate 5. It is noteworthy that the
deuterium content percentage was maintained unchanged during the whole
synthetic route, at a rate shown by MS spectrometry and 1H NMR
spectroscopy to be not less than 98%.
Copyright # 2006 John Wiley & Sons, Ltd.
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SYNTHESIS OF ISOTOPICALLY LABELLED FATTY ACIDS
679
Experimental
General
All moisture-sensitive reactions were conducted under dry nitrogen using
oven-dried glassware. THF and diethyl ether were distilled from sodium/
benzophenone immediately prior to use.
1H and 13C NMR spectra: Varian Gemini 300 MHz and Varian Inova
500 MHz spectrometers. MS (ESI) analyses were performed on Waters Micromass
ZQ spectrometer. Reactions were monitored by TLC (precoated silica gel plate
F254, Merck). Column chromatography: Merck Kieselgel 60 (70–230 mesh).
Melting points are uncorrected and were determined with a capillary apparatus.
Triphenyl phosphine polymer-bound was purchased from Fluka Chemical
Co. Deuterium oxide minimum isotopic purity 99.9 at.% D and NaBD4
98 at.% D were used.
1
The position of deuteration was established by H and 13C NMR experi-
ments of compounds 2, while the complete deuterium incorporation was
determined by mass spectrometry.
1,1-d2-octadecanol (1)
To a magnetically stirred solution of 4-methyl morpholine (NMM) (3.1 ml,
28.1 mmol) and stearic acid (4.0 g, 14.0 mmol) in anhydrous THF (80 ml), methyl
chloroformate (MeOCOCl) (28.1 mmol, 2.17 ml) was added dropwise at 08C.
After 40 min the solution was filtrated through a glass sinter funnel and the
N-methylmorpholinium salt so obtained was washed with anhydrous THF
(3 ꢁ 70 ml).
A suspension of NaBD4 (1.2 g, 28.0 mmol) in D2O (10 mL) was then added
to the filtrate, under magnetic stirring and at 08C. The reaction mixture was
stirred for 10 min at room temperature and then evaporated under reduced
pressure. The crude residue was diluted with ethyl acetate (200 ml) and washed
with water (100 ml) until neutral. The organic layer dried (Na2SO4), and
evaporated under reduced pressure afforded a solid residue whose chromato-
graphy on silica gel (1:15, CHCl3/CH3OH=95:5) gave pure 1 (3.5 g, 91%
1
yield); m.p. 57.0–59.08C (from CH3CN). H NMR (300 MHz, CDCl3) d: 0.89
(t, JCH ;CH ¼ 6:6; 3 H, CH3), 1.2–1.36 (m, 30 H, 15 ꢁ CH2), 1.44–1.63 (m, 2 H,
3
2
CH2CD2), 1.62 (bs, 1 H, OH), 3.61–3.67 (m, 0.04 H, CH2OH); 13C NMR
(75 MHz, CDCl3) d: 13.9 (CH3), 22.5 (CH2CH3), 25.6 (CH2CH2CD2OH), 29.4
(12 ꢁ CH2), 31.8 (CH2CH2CH3), 32.5 (CH2CD2OH), 62.2 (CD2). MS (ESI+)
m/z 271.42 (M-1).+
2,2-d2-nonadecanonitrile (3)
To a magnetically stirred suspension of dry polystyryl diphenyl phosphine
(4.5 g, ꢂ 13.4 phosphine units) in anhydrous CH2Cl2 (50 ml) at room
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temperature, a solution of I2 (3.4 g, 13.2 mmol) in the same solvent (50 ml) was
added dropwise in the dark and under dry nitrogen atmosphere. After 15 min,
solid octadecanol-1,1-d2 (1) (3.0 g, 11.2 mmol) was added in one portion to the
suspension. TLC monitoring (CHCl3/CH3OH, 9:1) showed that the starting
material was completely consumed within 30 min. The reaction mixture was
then filtered through a glass sinter funnel and washed with CH2Cl2. The
solvent was removed under reduced pressure to afford a crude residue which
was used without purification in the next synthetic steps.
Conversion of compound 2 into cyanide 3. To a magnetically stirred solution of
the crude reaction product 2 in anhydrous CH2Cl2 (30 ml), at room
temperature and under nitrogen atmosphere, solid tetraethylammonium
cyanide (2.5 g, 14.6 mmol) was added in one portion. The mixture was
refluxed for 4 h, until complete consumption of the starting product (TLC
monitoring, petroleum ether/EtOAc=7:3), then cooled and, after addition of
silica gel (10 g), evaporated under reduced pressure. The solid residue was
transferred onto a short silica gel (20 g) column and eluted with petroleum
ether/EtOAc (5 ! 20%) affording 3 as a crystalline white solid (2.8 g, yield
1
88%); m.p. 41.6–44.38C (from CH3CN). H NMR (500 MHz, CDCl3) d: 0.88
(t, 3 H, JCH ;CH ¼ 6:8; CH3), 1.20–1.36 (m, 28 H, 14xCH2), 1.40–1.48 (m, 2 H,
3
2
CH2CD2), 1.64 (t, 2 H, JCH ;CH ¼ 7:8; CH2CH3); 13C NMR (125 MHz,
2
3
CDCl3) d: 14.3 (CH3), 22.9 (CD2CN), 25.4 (CH2CH3), 28.8, 29.0, 29.5, 29.7,
29.9 (14xCH2), 32.2 (CH2CH2CH3), 119.9 (CN).
2,2-d2-nonadecanoic acid (4)
A magnetically stirred solution of a pure 3 (1.9 g, 6.7 mmol) in dioxane (10 ml)
and conc. aq hydrogen chloride (10 ml) was heated under reflux for 8 h. After
cooling, the solution was diluted with water (25 ml) and extracted with
Et2O (3 ꢁ 30 ml). The organic layer was then washed with water until neutral,
dried (Na2SO4), and evaporated under reduced pressure. The crude reaction
product was recrystallized (CH3CN) to afford 4 as a crystalline white solid
1
(1.9 g, yield 93%); m.p. 69.0–71.08C (from CH3CN). H NMR (500 MHz,
CDCl3) d: 0.89 (t, 3 H, JCH ;CH ¼ 6:5; CH3), 1.18–1.42 (m, 30 H, 15 ꢁ CH2),
3
2
1.52–1.70 (m, 2 H, CH2CD2); 13C NMR (125 MHz, CDCl3) d: 14.0 (CH3),
22.6 (CH2CH3), 24.4 (CH2CH2CD2), 28.9, 29.1, 29.2, 29.3, 29.6, 31.8 (16 ꢁ CH2
and CD2), 31.8 (CH2CH2CH3), 179.4 (C=O). MS (ESI+) m/z 301.24
(M+1)+.
Methyl-2,2-d2-nonadecanoate (5)
A magnetically stirred solution of a pure 3 (0.8 g, 2.84 mmol) in anhydrous
methanol (10 ml), at ꢀ158C, is saturated with HCl (g) dry and then kept at
room temperature for 36 h. To the solution are added few drops of water
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SYNTHESIS OF ISOTOPICALLY LABELLED FATTY ACIDS
681
(76 mg, 4.26 mmol) and the solvent is evaporated with Et2O (3 ꢁ 10 ml). The
desired compound (5) is obtained as a crystalline white solid (0.8 g, yield 94%);
m.p. 39.0–41.08C (from CH3CN); 1H NMR (500 MHz, CDCl3) d: 0.95 (t, 3 H,
JCH ;CH ¼ 6:8; CH3), 1.18–1.40 (m, 30 H, 15 ꢁ CH2), 1.45–1.70 (m, 2 H,
3
2
CH2CD2), 3.70 (s, 3 H, CH3O). 13C NMR (125 MHz, CDCl3) d: 13.9 (CH3),
22.6 (CH2CH3), 24.7(CH2CH2CD2), 28.9, 29.1, 29.2, 29.3, 29.6, 31.8 (16 ꢁ CH2
and CD2), 31.8 (CH2CH2CH3), 51.3 (CH3O), 174.3 (C=O).
Conclusion
We have described the preparation of the 2,2-d2-nonadecanoic acid, as a
generic example, to be used as standard material for mass spectrometric
analysis. The efficiency of our method lies in the wide flexibility of synthetic
strategy: it is possible to choose how many heavy atoms to incorporate and in
which positions, replacing NaBD4 with NaBH4 at the reduction step during
the homologation process, thereby obtaining dideuterated or polydeuterated
fatty acids. To date we have used the method exclusively for synthesis of
saturated odd-numbered fatty acids, but it should also be applicable, as
previously shown in Scheme 1, to even-numbered fatty acids and to
unsaturated compounds.
Work is also in progress to prepare, by the same strategy, 2,2-dideuterated
b-amino acids to be used as linker moieties in new isotope-coded affinity tags
(ICAT) reagents for quantitative proteome analysis.
Acknowledgements
1H NMR spectra were performed at Centro Interdipartimentale di Metodo-
logie Chimico-Fisiche, Universita di Napoli Federico II.
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