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DAI ET AL.
metabolites are required.11 Ideally, for each target mole-
cule, the corresponding internal standard that shares the
same structure and only differs in the isotope labeling
should be provided to minimize potential errors caused
by matrix effect, ionization efficiency, and other factors.
However, only a few isotope labeled acylcarnitines are
commercially available, most of which contain simple
acyl structures without functional groups and are labeled
with 3 or 9 deuterium atoms. This imposes two problems
for acylcarnitine analyses. Firstly, many acylcarnitines,
especially those with complex acyl structures such as
3-hydroxyisovalerylcarnitine (C5OH), tetradecenoyl-
carnitine (C14:1), and glutarylcarnitine (C5DC), are
semi-quantitatively measured by comparing with an
internal standard with similar sizes. For instance, the
quantification of C5OH is based on the concentration of
C5-D9 (isovalerylcarnitine with nine deuterium atoms),
which is provided as the authentic internal standard of
C5. Secondly, because the newborn screening panel is a
flow injection assay that measures many different
acylcarnitines without chromatographic separation,
sometimes, it is difficult to find an appropriate internal
standard from commercial sources to avoid mass over-
lapping. To solve these issues, herein we report a general
strategy for the synthesis of acylcarnitines that not only
enables the preparation of acylcarnitines with complex
acyl structures but also allows the incorporation of iso-
topes to increase their mass by any number in the range
of 3 to 12 Da. As a result, the authentic internal standard
for any acylcarnitine of interest can be synthesized with
its mass being carefully chosen to avoid overlapping with
other endogenous acylcarnitines.12
on Figure 1), it is satisfactory to use their racemic coun-
terparts as internal standards during tandem mass spec-
trometry analysis. The carboxylate of compound 1 is
protected with a benzyl group by the action of thionyl
chloride to form compound 2, which subsequently reacts
with formaldehyde and sodium cyanoborohydride via
reductive amination to obtain the key intermediate com-
pound 3. Next, an organic acid of choice (compound 4)
can be coupled to the hydroxyl group of compound 3 with
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)
and 4-dimethylaminopyridine (DMAP) to form com-
pound 5. Compound 6 is synthesized by incubating com-
pound 5 and methyl iodide in the presence of sodium
bicarbonate, which is subsequently converted to the chlo-
ride salt by anion exchange resin. Finally, the benzyl
protecting group is removed by either catalytic hydroge-
nation or basic hydrolysis to obtain the expected
acylcarnitines. This strategy differs from early reports on
the synthesis of acylcarnitines, which relied on the direct
coupling of an carboxylic acid chloride to L-carnitine.
Although the classical methods were able to produce
desired acylcarnitines, L-carnitine as a salt has a low solu-
bility in dichloromethane that is an optimal solvent for
the formation of an ester. Therefore, the direct coupling
of the carboxylic acid chloride to L-carnitine usually takes
place in an appropriate acid,13–15 which is more difficult
to handle and often leads to the poor yield of the esters.
Our approach allows the coupling of the carboxylic acid
to the intermediate 3 in dichloromethane, making it eas-
ier for reaction treatment and resulting in consistent high
yield of the esters.
By adopting commercially available isotope labeled
formaldehyde, sodium cyanoborohydride, and methyl
iodide, a labeled acylcarnitine with a mass shift of 3 to
12 Da over its unlabeled counterpart can be readily syn-
thesized (Table 1), making it much easier to select
suitable internal standards for acylcarnitine profiling
analyses.
2 | RESULTS AND DISCUSSIONS
As shown in Scheme 1, the synthesis of acylcarnitines
starts from racemic 4-amino-3-hydroxybutanoic acid
(compound 1). Although biologically active acylcarnitines
are L-configured on position 2 (indicated by an asterisk
Using this newly developed strategy, we have success-
fully synthesized many isotope labeled acylcarnitines
SCHEME 1 Synthesis of
acylcarnitines. Reaction conditions:
(a) thionyl chloride, 70ꢀC; (b) MeOH,
AcOH, pH = 6.0; (c) EDC, DMAP;
(d) NaHCO3; (e) H2/pd-C;
(f) Na2CO3. An asterisk indicates a
racemic position. A percentage in
parentheses is the yield of that
product
Dai, Xiaojun
