Synthesis of [15N]-cholamine bromide hydrobromide

Article abstract of DOI:10.1002/jlcr.3598

[¹⁵N]-Cholamine is an isotope tag for metabolomics research, because it possesses 2 important properties: an NMR active isotope and a permanent charge for MS sensitivity. Here, we present a scalable synthesis of [¹⁵N]-cholamine.

Full text of DOI:10.1002/jlcr.3598

Synthesis of [15N]-Cholamine Bromide Hydrobromide
Judy Szeto, Remy Lemoine, Rosary Nguyen, Lori L. Olson, and
Mary J. Tanga
SRI International, Biosciences Division
333 Ravenswood Ave, Menlo Park, CA 94025 USA
Keywords: [ N]-Cholamine bromide hydrobromide, smart isotope
tag, chemo-selective tag, carboxylic acid metabolites
Abstract (250 words max)
[ N]-Cholamine is an isotope tag for metabolomics research,
because it possesses two important properties: an NMR active
isotope and a permanent charge for MS sensitivity. Here we
present a scalable synthesis of [ N]-cholamine.
Introduction
The field of metabolomics seeks to characterize and quantify
small molecules found in a biological system. This task is
simplified if two analytical methods can be used in tandem,
such as exploiting the combined strengths of NMR (nuclear
magnetic resonance) and MS (mass spectrometry) for the
identification of unknown metabolites. Stable isotope
labelling is a strategy that increases the reliability of
identification. [ N]-Cholamine (2-([ N]-amino)-N,N,N-
trimethylethanaminium) has been shown to be a promising tag
for NMR- and MS-based profiling of metabolites containing
carboxylic acid moieties. Carboxylic acids represent one of
the four major functional groups in the metabolome, along with
amines, alcohols, and carbonyls. These four groups cover 95%
of the 8,000 endogenous human metabolites in the Human
Metabolome Database.
Results and discussion
There is only one reported synthesis of [ N]-cholamine as its
chloride hydrochloride salt as shown in Scheme 1. The
literature sequence involves the reaction of [ N]-phthalimide
potassium salt (1*) with (2-bromoethyl)trimethylammonium
bromide (2), followed by a two-step deprotection of the
phthalimido group. The product is obtained by precipitation
followed by several washes to remove the phthalic acid
byproduct of the hydrolysis reaction.
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Scheme 1. Reported synthesis of [ N]-cholamine chloride
hydrochloride (* indicates the position of the N label).
To support the NIH Common Fund’s Metabolomics Program, a
larger quantity of [ N]-cholamine was needed. We first
considered the scalability of the published procedure.
Notably, yields were not included in the report. Using
unlabeled phthalimide potassium (1), we were not able to
scale-up the literature procedures in a satisfactory manner.
The limitations of the reported process were two-fold. First,
the reaction of phthalimide potassium salt (1) with (2-
bromoethyl)trimethylammonium bromide (2) proceeded in a modest
45% yield. We attributed the low yield to the decomposition of
the alkylating reagent and the elimination of the
trimethylammonium group in the alkylated product. Both
compounds are susceptible to a Hofmann elimination reaction.
Second, the hydrolysis of the phthalimido group proceeded as
expected however, isolation of pure product was challenging.
Removal of the phthalic acid byproduct from the desired
product could not be done efficiently. Most of the target
compound was washed away along with the impurity.
This led us to explore an alternative method that would be
amenable to the production of [ N]-cholamine on large scale.
Exploratory experiments were first run using unlabeled
starting material. Scheme 2 describes the final iteration of
the synthesis that we developed and the scale at which the
reactions were ran.
Scheme 2. New synthesis of cholamine as its bromide
hydrobromide salt.
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The issue with the decomposition of the alkylating agent in
the literature procedure was solved by using 1,2-dibromoethane
(6) instead of (2-bromoethyl)trimethylammonium bromide (2).
Precipitation led to the isolation of the alkylated product 7
in 85% yield. The trimethylammonium group was then introduced
by displacing the bromide of compound 7 with trimethylamine.
Our new two-step synthesis of intermediate 3 from phthalimide
potassium (1) was more efficient than the one-step method
described previously (71% vs. 45%), and 18 g of 3 was
synthesized by this new protocol.
The separation of phthalic acid from the desired product was
solved by deprotecting the primary amino group using
hydrazine. Instead of phthalic acid, 1,3-dihydrophthalazine-
1,4-dione byproduct is formed. The latter compound was
insoluble under the reaction conditions and readily removed
from the reaction mixture by filtration. Concentration of the
filtrate gave pure cholamine bromide (8). The target bromide
hydrobromide 9 was formed by treatment of 8 with hydrogen
bromide (69% yield from 3). The overall yield of unlabeled
cholamine bromide hydrobromide (9) from phthalimide potassium
salt (1) was 49% with only simple filtration and trituration
for purification. The process was applied to the synthesis of
10.5 g of 9.
Following the process developed with unlabeled materials,
three similar scaled runs for the preparation of [ N]-
cholamine bromide hydrobromide (9*) were performed (Scheme 3).
Gratifyingly, using [ N]-phthalimide potassium salt (1*) in
the improved four-step synthesis of [ N]-cholamine proceeded
in good overall yield (52% 9* from 1*, 23.9 g, 10 g, and 11
g).
Scheme 3. Synthesis of cholamine as its bromide hydrobromide
salt (* indicates the position of the N label).
Conclusion
An efficient, scalable, and operationally straightforward
synthesis of [ N]-cholamine was developed, which is an
improvement over the previously described route. The
intermediates and final product were isolated by simple
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filtration and trituration, and the synthesis was demonstrated
for as much as 23.9 g of [ N]-cholamine bromide hydrobromide.
Experimental
General methods
All reactions were carried out using commercial reagents of
the highest grade and without further purification. [ N]-
Phthalimide potassium salt was purchased from Aldrich. NMR
spectra were measured on a Varian 400 MHz instrument.
Chemical shifts are reported in parts per million ()
downfield from tetramethylsilane. Elemental analysis was
performed by ASL Environmental. Liquid chromatography-mass
spectrometry (LCMS) data were obtained using a Thermo LCQ
Fleet and Finnigan Surveyor System with a SeQuat ZIC-pHILIC 5
µm polymeric (50 x 4.6 mm) column using 25 nM ammonium acetate
(pH 9) in water/acetonitrile (30/70, v/v) as the mobile phase
at a flow rate of 0.5 mL/min with a ESI positive ion mode,
full scale (50-300 m/z).
[ N]-2-(2-Bromoethyl)isoindoline-1,3-dione (7*)
In a dried round-bottom flask with a magnetic stir bar, [ N]-
phthalimide potassium salt (1*, 28 g, 0.151 mol) was dissolved
in 57 mL of N,N-dimethylformamide and the resulting solution
was cooled to 0 ˚ C under inert atmosphere. At 0 ˚ C, 1,2-
dibromoethane (6, 40 mL, 0.464 mol, 3.0 equiv.) was then added
to the reaction flask. The reaction mixture was allowed to
warm to room temperature and stirred for 48 h before being
quenched with water (50 mL). The reaction mixture was
extracted with ethyl acetate (3 x 100 mL). The combined
organic layers were then washed with water (2 x 50 mL)
followed by brine (50 mL) before being dried with sodium
sulfate and concentrated. The residue was dried under vacuum
to give intermediate 7 as a white solid (27.7 g, 71% yield). H
NMR (400 MHz, CDCl)  7.87–7.85 (m, 2H), 7.74–7.72 (m, 2H),
4.10 (td, J = 6.8, 1.1 Hz, 2H), 3.6 (td, J = 6.7, 2.3 Hz, 2H).
C NMR (100 MHz, CDCl)  167.8 (d, J = 18.4 Hz), 133.9 (d,
J = 22.9 Hz), 131.8 (d, J = 10.7 Hz), 123.4 (d, J =
27.4 Hz), 39.2 (d, J = 13.7 Hz), 28.1.
[ N]-2-(1,3-Dioxoisoindolin-2-yl)-N,N,N-trimethylethan-1-
aminium bromide (3*)
In a 420 mL sealed-tube flask (Chemglass) with a magnetic stir
bar, 2 M trimethylamine in tetrahydrofuran (218 mL, 0.436 mol,
4 equiv.) was added to intermediate 7* (27.7 g, 0.109 mol)
under inert atmosphere. The resulting solution was heated at
55 ˚ C for 24 h. The reaction mixture was then transferred to a
flask using dichloromethane and concentrated. The white solid
was then washed with dichloromethane (3 x 60 mL), filtered,
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and dried under vacuum to obtain 3* as a white solid (33 g,
97% yield). H NMR (400 MHz, CDCl)  7.78–7.74 (m, 2H), 7.73–
7.68 (m, 2H), 4.04 (t, J = 7.0 Hz, 2H), 3.54 (td, J = 7.2, 1.6
Hz, 2H), 3.11 (s, 9H). C NMR (100 MHz, CDCl)  169.6 (d, J
= 13.7 Hz), 135.0, 131.1 (d, J = 9.2 Hz), 123.6, 63.5,
53.2 (d, J = 4.6 Hz), 31.6 (d, J = 13.7 Hz).
[ N]-2-Amino-N,N,N-trimethylethanaminium bromide (8*)
In a round-bottom flask with a magnetic stir bar, intermediate
3* (17 g, 0.054 mol) was suspended in 271 mL of a 7:3 (v/v)
mixture of chloroform and ethanol, and the solution was cooled
to 0 ˚ C. Aqueous hydrazine monohydrate (64% (w/w), 6.2 mL,
0.081 mol, 1.5 equiv.) was added dropwise. After the addition,
the reaction became homogenous and was stirred at 50 ˚ C for 12
h with a reflux condenser under inert atmosphere. The white
precipitate that formed was removed by filtration and washed
with an 8.5:1.5 mixture (v/v) of chloroform and ethanol (3 x
50 mL). The filtrate was concentrated and dried under vacuum.
The residue was used in next step without purification or
characterization.
[ N]-2-Amino-N,N,N-trimethylethanaminium bromide, hydrobromide
salt ([ N]-Cholamine bromide hydrobromide salt) (9*)
Compound 8* (from process above, theoretical 0.054 mol) was
dissolved in 54 mL of deionized water and the solution was
cooled to 0 ˚ C. At 0 ˚ C, aqueous hydrobromic acid (48%, 13.7
mL) was added dropwise, and the resulting slurry was stirred
at 0 ˚ C for 1 h. The reaction mixture was evaporated and then
co-evaporated with a toluene and methanol mixture (4.5:0.5, 2
x 25 mL) at 45 ˚ C. When half of the solvent had been removed,
the white precipitate was filtered and then washed with water
(2 x 10 mL). The filtrate was further evaporated with toluene
and methanol mixture, and the residue was dried under vacuum.
The solid was triturated with minimal amounts of methanol (3 x
15 mL), filtered, and dried under vacuum to provide [ N]-
cholamine bromide hydrobromide salt (9*, 11 g, 76% yield). H
NMR (400 MHz, DO)  3.62–3.58 (m, 2H), 3.45–3.41 (m, 2H), 3.12
(s, 9H). C NMR, (100 MHz, DO)  61.3, 53.6, 32.9 (d, J =
7.7 Hz). MS, theory for CH N N: m/z = 104.12; found m/z =
104.11. Elemental analysis, calcd for CH BrN N: C, 22.66%; H,
6.09%; N, 10.95%; Br, 60.31%; found C, 22.50 ± 0.07%; H, 6.05
± 3.6%; N, 10.45 ± 0.1%; Br, 58.46 ± 3%.
Using the above procedures, three batches of [ N]-cholamine
bromide hydrobromide salt were prepared (44.9 g total).
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Acknowledgements
This project has been funded in whole or in part with Federal
funds from the National Heart, Lung, and Blood Institute,
National Institutes of Health, Department of Health and Human
Services, Contract No. HHSN268201300022C.
Conflict of Interest
The authors did not report any conflict of interest.
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Synthesis of [15N]-Cholamine Bromide Hydrobromide
Judy Szeto, Remy Lemoine, Rosary Nguyen, Lori L. Olson, and
Mary J. Tanga
SRI International, Biosciences Division
333 Ravenswood Ave, Menlo Park, CA 94025 USA
Graphical Abstract
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