An improved procedure for the synthesis of labelled fatty acids utilizing diethyl malonate

Article abstract of DOI:10.1002/jlcr.1034

An improved procedure for the preparation and purification of labeled fatty acids by malonic ester synthesis has been developed. This method uses the different hydrolysis rates of the monoalkylmalonic ester intermediate and its dialkylmalonic ester side p

Full text of DOI:10.1002/jlcr.1034

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 171–176.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1034
Note
An improved procedure for the synthesis of labelled
fatty acids utilizing diethyl malonate
Takhar Kasumov* and Henri Brunengraber
Department of Nutrition, Case Western Reserve University, Cleveland, OH 44106, USA
Summary
An improved procedure for the preparation and purification of labeled fatty acids by
malonic ester synthesis has been developed. This method uses the different hydrolysis
rates of the monoalkylmalonic ester intermediate and its dialkylmalonic ester side
product. A convenient GC–MS monitoring technique allows performing this 3-step
procedure without isolation of intermediates and gives a good yield. Copyright #
2006 John Wiley & Sons, Ltd.
Key Words: carbon-13; hydrogen-2; malonic ester synthesis; fatty acids
Introduction
Diethyl malonate is a valuable synthetic equivalent of the synthons
ꢀCH2CO2H or 2ꢀCHCO2H, and is widely used for the preparation of a
2
variety of carboxylic acids labeled with H and 13C isotopes in different
positions of the aliphatic chain.1 When treated with one equivalent of base,
such as NaH or C2H5ONa, diethyl malonate is converted to a mono metal
enolate. This mesomeric anion undergoes an SN2 reaction with an alkyl halide
or alkylsulfonate to give a monoalkylmalonic ester. Alkaline hydrolysis of this
malonic ester, followed by acidic decarboxylation, produces a carboxylic acid
with the extension of the aliphatic chain by two carbon units. When 2
equivalents of diethyl malonate enolate react with 1 mol of terminal
dihaloalkane, dicarboxylic acids with 4 more carbons than dihaloalkane can
be prepared.
A reaction starting with an accurate stoichiometry of substrates and the
correct combination of base and solvent usually produces a good yield
*Correspondence to: Takhar Kasumov, Department of Nutrition, Case Western Reserve University,
Cleveland, OH 44106-7139, USA. E-mail: tmk2@case.edu
Contract/grant sponsor: National Institutes of Health; contract/grant number: R33DK070291
Contract/grant sponsor: American Heart Association; contract/grant number: 0465221B
Copyright # 2006 John Wiley & Sons, Ltd.
Received 10 October 2005
Revised 2 November 2005
Accepted 4 November 2005

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T. KASUMOV AND H. BRUNENGRABER
(80–90%) after distillation of the monoalkylmalonic ester.2 When applied to
the synthesis of carboxylic acids in large quantities, this is an excellent
technique. However, because of the high cost of labeled compounds,
particularly 13C-labeled diethylmalonate or alkylhalides, purification of the
alkylmalonic ester by distillation is not cost-effective. Therefore, column
chromatography is the most common separation method.3 Column chroma-
tography purification of the alkylmalonic ester is not only time consuming and
relatively costly, it is also associated with the uncertainties of thin-layer
chromatography (TLC) monitoring of diethylmalonate and alkyl substituted
(both mono- and di-) malonic esters.
Attempts to limit dialkylation and avoid the use of column chromatography
to purify alkylmalonic ester have been described.4,5 The use of excess
diethylmalonate minimizes the formation of dialkylated side products.2 The
unreacted substrate can be partially eliminated by a water extraction of free
malonic acid after the hydrolysis of the diethyl ester. However, the high cost of
carbon-labeled diethylmalonate prohibits such an approach when it is applied
to the synthesis of [1-13C]-, [2-13C]- and [1,2-13C2]-carboxylic acids. Even when
the cost of substrates is not prohibitive, the complete removal of malonic acid
by extraction is not possible.
2
Scheme 1. Synthesis of 13C and H labeled short-chain, medium-chain, long-
chain fatty acids and aromatic fatty acids
Scheme 2. Synthesis of [1,2,11,12-13C4]dodecanedioic acid
Copyright # 2006 John Wiley & Sons, Ltd.
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SYNTHESIS OF LABELLED FATTY ACIDS
173
As part of our study on the regulation of both peroxisomal and
mitochondrial fatty acid oxidation6,7 we developed an easy and convenient
2
method for the synthesis of 13C or H-labeled short, medium, and long chain
fatty acids. We also describe a reliable gas chromatography–mass spectro-
metry (GC–MS) technique for the monitoring of substrates, reaction
intermediates, and products that makes it possible to conduct a 3-step
reaction without isolation of intermediates and in good yield (Schemes 1
and 2).
Results and discussion
It is well known that the rate of saponification of malonate esters depends
upon the structure of these compounds.1 Particularly, the hydrolysis of diethyl
monoalkylmalonates is much easier than that of dialkylmalonates because of
the steric effects of the dialkyl groups. We took advantage of this phenomenon
when attempting to prepare a small scale amount of monoalkylmalonic acids,
such as hexylmalonic acid and found that overnight refluxing with excess of
2 M KOH solution in water resulted in their complete hydrolysis. Surprisingly,
the dialkyl side product of these reactions was not hydrolyzed at all under
these conditions. Our test run using unlabeled substrates revealed that the
saponification of diethyl 2, 2-dihexylmalonate, a potential dialkyl side
product, under these conditions required more than three days. We found
that the ethyl ether extraction of the reaction mixture resulted in the complete
transfer of the dialkylmalonate ester in the organic phase. After this step, the
water solution contained monoalkylmalonate disodium salt. A GC–MS assay
of trimethylsilyl (TMS) derivative of the acidified water solution demonstrated
the presence of trace amounts (less than 1%) of malonic acid that presumably
derived from the unreacted diethylmalonate.
The decarboxylation of the malonic acid, the next step in this synthesis, was
performed by refluxing the reaction mixture with acid for 36 h until no starting
material was detected by GC–MS of the TMS derivative. These conditions
decarboxylated unreacted malonic acid to acetic acid, which could be washed
out with water or vacuum-evaporated. Because of their high boiling points and
hydrophobic properties, long-chain and medium-chain fatty acids could be
purified easily by this technique. Using this procedure, [3-13C]octanoic acid
was isolated without distillation with 99% purity. The scope of this method
was tested through the synthesis of short and long chain fatty acids:
[3,3,3-2H3]propionic, [1-13C]docosanoic, 4-phenyl[1,2-13C2]butyric, and [1,2,
11,12-13C4]dodecanedioic acids. Based on the data of these preparations, we
concluded that increasing the length of aliphatic chain decreases the
probability of the formation of dialkyl substituted side products. On the
other hand, it is difficult to monitor the hydrolysis of the diethyl ester of long
chain eicosylmalonate and the complete decarboxylation of eicosylmalonate
Copyright # 2006 John Wiley & Sons, Ltd.
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T. KASUMOV AND H. BRUNENGRABER
by GC–MS because of the unstability of di-TMS derivative at 3208C, the GC
column temperature necessary for the elution of these compounds. It was
found that under these conditions, di-TMS of eicosylmalonate decarboxylates
to TMS of docosanate.
Experimental
Materials
Unlabeled reagents, including solvents were purchased from Aldrich. Diethyl-
[1,2-13C2]malonate, diethyl-[1,2,3-13C3]malonate [1-13C]hexylbromide, and
[2H3]methyliodide were supplied by ISOTEC. N,N-dimethylformamide
(DMF) was dried immediately before the reaction.
Analysis of products
Chemical purity of all compounds was analyzed by GC–MS both as the ethyl
ester and the TMS derivatives using a HP-5 column. The structures and
isotopic purities were verified by both GC–MS as well as by nuclear magnetic
resonance (NMR) spectroscopy.
GC–MS analyses were carried out on an Agilent 5973 mass spectrometer,
equipped with a Model 6890 gas chromatograph, auto sampler, and HP-5MS
5% phenyl methyl siloxane fused silica capillary column (60 m, 25 mm id,
0.25 mm film thickness). The injector temperature was set at 2708C and the
transfer line at 3058C. The GC temperature program was as follows: start at
808C, hold for 1 min, increase by 68C/min to 2008C, increase by 258C/min to
3108C. The ion source was set at 2008C and the quadruple at 1068C. Under
ammonia positive chemical ionization, m/z+1 and m/z+18 ions were
monitored.
All proton magnetic resonance (1H NMR) were recorded on a Varian
Gemini spectrometer operating at 300 MHz. Residual protons of CDCl3
(d ¼ 7:24) was used as an internal reference. Chemical shifts were reported in
parts per million (ppm). Carbon magnetic resonance (13C NMR) spectra were
recorded on the same instrument at 75 MHz. These spectra are reported in
ppm d scale relative to CDCl3. Each time NMR spectra of synthetic labeled
compound was compared with the spectra of unlabeled compound. This
2
comparison and analysis of 13C and H coupling was used for the elucidation
of the structure labeled carboxylic acid.
[3-13C]Octanoic acid. Sodium hydride 60% dispersion in mineral oil (0.5 g,
12.5 mmol) was suspended in 10 ml of DMF under nitrogen gas. Diethyl
malonate (2.00 g, 12.48 mmol) in 3 ml DMF was added slowly over 15 min.
The reaction mixture was heated at 558C until no more hydrogen gas was
released, and the reaction mixture became clear. After cooling the reaction
Copyright # 2006 John Wiley & Sons, Ltd.
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SYNTHESIS OF LABELLED FATTY ACIDS
175
mixture to 408C, freshly distilled [1-13C]hexylbromide (1.98 g, 12.4 mmol) in
3 ml DMF was added slowly over 20 min. The reaction mixture was stirred for
another 1 h at 608C. GC–MS analysis of the reaction mixture showed one
major peak of diethyl [1-13C]hexylmalonate and two minor peaks, correspond-
ing to diethyl di-[1,10-13C2]hexylmalonate, and unreacted diethylmalonate. The
reaction mixture diluted with 15 ml of water was extracted with ethyl ether.
After evaporation of solvent, the residue was suspended in 10 ml of 2.8 M
KOH solution in water. The reaction mixture was refluxed for 3 h until the
hydrolysis of diethyl hexylmalonate was completed, based on GC–MS
analysis. The reaction mixture was diluted with 10 ml of water and ethanol
formed from hydrolysis was completely removed by azeotropic distillation.
This prevents back esterification with ethanol during acidic decarboxylation of
hexylmalonate. The GC–MS assay of the reaction mixture showed that under
these conditions diethyl dihexylmalonate was not hydrolyzed. The unhydro-
lyzed side product was extracted from the alkaline solution with ethyl acetate.
The water phase was made acidic and hexylmalonate was extracted with
diethyl ether. The crude hexylmalonate was then suspended in 5 ml of dioxane
+10 ml of 12 N HCl and refluxed for 24 h. The cooled solution was extracted
with diethyl ether. The combined organic extracts were dried with anhydrous
sodium sulfate, filtered, and the solvent completely evaporated under reduced
pressure. GC–MS and NMR analysis of the residue showed 99% chemical
purity and 98% isotopic enrichment of the product. The yield of
[3-13C]octanoic acid was 63% based on [1-13C]hexylbromide.
[3,3,3-2H3]Propionic acid. This compound was prepared from [3,3,
3-2H3]methyl iodide and unlabeled diethylmalonate using the same procedure
as was used for the synthesis of [3-13C]octanoic acid. The chemical purity,
isotope enrichment and yield of the product were 99, 98 and 47%.
[1-13C]Docosanoic acid. This compound was prepared from diethyl [1,3-13C2]
malonate and 1-bromoeicosane in the same manner as [3-13C]octanoic acid.
The product was recrystallized from hexane. The chemical purity, isotope
enrichment and yield of the product were 98, 99 and 69%.
4-Phenyl[1,2-13C2]butyric acid. This compound was prepared from diethyl
[1,2,3-13C3]malonate and 2-bromoethylbenzene in the same manner as
[3-13C]octanoic acid. GC–MS monitoring of the orientation experiment with
unlabeled diethylmalonate revealed that during the nucleophilic alkylation
reaction, part of the 2-bromoethylbenzene underwent a partial elimination to
styrene. This is why in the subsequent synthesis, a slight excess of 2-
bromoethylbenzene (1:1.15) was used for the alkylation of diethyl
[1,2,3-13C3]malonate. 4-Phenyl[1,2-13C2]butyric acid was recrystallized from
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T. KASUMOV AND H. BRUNENGRABER
hexane. The chemical purity, isotope enrichment and yield of the product were
98, 99 and 54%.
[1,2,11,12-13C4]Dodecanedioic acid. This compound was prepared from 2
equivalent of diethyl [1,2,3-13C3]malonate and 1 equivalent of 1,8-diiodooc-
tane in the same manner as [3-13C]octanoic acid. The product was
recrystallized from hexane/ether. The chemical purity, isotope enrichment
and yield of the product were 98, 99 and 59%.
Conclusion
A convenient and efficient three-step procedure for the preparation of 13C and
2H-labeled carboxylic acids, which avoids the isolation of intermediates, has
been developed utilizing malonic ester methodology. The reaction sequence
can be easily monitored by GC–MS analysis. The wide availability of GC–MS
instrumentation allows this procedure to be applicable in most research labs
for the synthesis of labeled (and unlabeled) fatty acids and dicarboxylic acids.
Acknowledgements
This work was supported by grants from the NIH (National Institutes of
Health) (Road map grant R33DK070291) and the AHA (American Heart
Association) (0465221B).
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Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 171–176