An efficient method for synthesizing dimethylsulfonio-34S-propionate hydrochloride from 34S8

Article abstract of DOI:10.1002/jlcr.3696

Dimethylsulfoniopropionate (DMSP, (2-carboxyethyl)dimethylsulfonium) is a highly abundant compound in marine environments. As a precursor to the climatically active gas, dimethylsulfide (DMS), DMSP connects the marine and terrestrial sulfur cycles. However, the fate of DMSP in microbial biomass is not well understood as only a few studies have performed isotopic labeling experiments. A previously published method synthesized ³⁴S-labeled DMSP from ³⁴S₈, but the efficiency was only 26% and required five separate reactions, expensive reagents, and purification of the products of each reaction. In this study, a method of synthesizing ³⁴S-labeled DMSP from ³⁴S₈ is described. Improvements include elemental steps, inexpensive reagents, purification of only one intermediate, and less time to complete. The efficiency of this method is 65% and results in pure DMSP with more than 98% isotope enrichment as determined by ¹H-nuclear magnetic resonance (NMR) and gas chromatography–mass spectrometry (GC–MS).

Full text of DOI:10.1002/jlcr.3696

Received: 8 October 2018
DOI: 10.1002/jlcr.3696
Revised: 24 October 2018
Accepted: 8 November 2018
R E S E A R C H A R T I C L E
An efficient method for synthesizing dimethylsulfonio‐
34
34S‐propionate hydrochloride from S8
Joseph S. Wirth
| William B. Whitman
Department of Microbiology, University of
Georgia, Athens, Georgia, USA
Dimethylsulfoniopropionate (DMSP, (2‐carboxyethyl)dimethylsulfonium) is a
highly abundant compound in marine environments. As a precursor to the cli-
matically active gas, dimethylsulfide (DMS), DMSP connects the marine and
terrestrial sulfur cycles. However, the fate of DMSP in microbial biomass is
not well understood as only a few studies have performed isotopic labeling
experiments. A previously published method synthesized 34S‐labeled DMSP
from 34S8, but the efficiency was only 26% and required five separate reactions,
expensive reagents, and purification of the products of each reaction. In this
study, a method of synthesizing 34S‐labeled DMSP from 34S8 is described.
Improvements include elemental steps, inexpensive reagents, purification of
only one intermediate, and less time to complete. The efficiency of this
method is 65% and results in pure DMSP with more than 98% isotope
Correspondence
William B. Whitman, Department of
Microbiology, University of Georgia,
Biological Sciences Building, Room 527,
120 Cedar Street, Athens, GA 30602, GA,
USA.
Email: whitman@uga.edu
Funding information
National Science Foundation Dimensions
of Biodiversity, Grant/Award Number:
OCE‐1342694
1
enrichment as determined by H‐nuclear magnetic resonance (NMR) and gas
chromatography–mass spectrometry (GC–MS).
1 | INTRODUCTION
organisms, and it is estimated that up to 10% of the total
fixed carbon in the oceans is in the form of DMSP.4
Dimethylsulfoniopropionate (4, DMSP, (2‐carboxyethyl)
dimethylsulfonium) is a highly abundant compound in
marine surface waters. In the North Sea, the concentra-
tion of DMSP cycles seasonally from micromolar levels
in the summer to picomolar levels in the spring and
fall.1,2 The majority of marine DMSP comes from halo-
phytic plants and algae, where it is believed to regulate
osmotic pressure in addition to antioxidant, predator
deterrent, and/or cryoprotectant functions.2 There is also
evidence that at least 0.5% of marine bacteria are capable
of producing DMSP, but this contribution to the global
sulfur cycle is not yet fully understood.3 Concurrent with
its role as an osmoregulatory molecule, plants that pro-
duce the most DMSP are generally halotolerant and of
marine origin, with sugarcane being the only nonmarine
exception. During 35S‐labeling studies with bacterial cells,
approximately 15% of added DMSP accumulated
intracellularly but was not metabolized.2 Molar levels of
intracellular DMSP have been observed in some
Furthermore, DMSP released from phytoplankton
blooms can satisfy up to 15% of the microbial carbon
demand and 100% of the microbial sulfur demand.4
DMSP is the precursor for the majority of atmospheric
dimethylsulfide (3, DMS), which is a climatically active
gas and connects the marine and terrestrial sulfur cycles.5
It was previously believed that H2S was responsible for
the transfer of sulfur between marine and terrestrial envi-
ronments but the necessary atmospheric concentrations
were never detected and the surface layers of the ocean
are too oxidizing to sustain equilibrium with the atmo-
sphere.5 However, the concentration of DMS in marine
surface layers is sufficiently high, and DMS is resistant
to oxidation in the lower atmosphere.5 Its photooxidation
in the upper atmosphere produces sulfur species that can
be transferred to terrestrial environments via rain and
promote the formation of cloud‐condensation nuclei,
resulting in an increased albedo effect and global
cooling.2,4,6,7
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wileyonlinelibrary.com/journal/jlcr
J Label Compd Radiopharm. 2019;62:52–58.

WIRTH AND WHITMAN
53
Bacterial catabolism of DMSP proceeds through
one of two known pathways. It can either undergo
cleavage to form DMS and either acrylic acid or 3‐
hydroxypropionate or it can undergo demethylation to
form methylmercaptopropionic acid, which can further
be broken down into methanethiol, carbon dioxide, and
acetaldehyde.2 In both cases, the DMS and methanethiol
can be metabolized further and assimilated into biomass.
Because very few studies have performed isotope‐labeling
experiments with DMSP, the fate of DMSP in microbial
biomass is not well understood.8-11 DMSP hydrochloride
can be easily synthesized via a Michael addition of DMS
to acrylic acid under acidic conditions in methylene chlo-
ride.12 Unfortunately, DMS enriched with a sulfur isotope
is not commercially available, and the only commercially
available form of isotopically labeled sulfur suitable for
conversion to DMS is elemental sulfur (1, S8). Thus,
incorporation of a specific sulfur isotope requires a
synthetic pathway to convert S8 to DMSP.
version of the methylene blue assay was used to calculate
the amount of S2– synthesized from reaction (I) and the
amount of Na2S used in reaction (II) (Scheme 1). The
methylene blue assays were performed twice on a 10–5
dilution of each solution of Na2S.13-16 All reported effi-
ciencies are relative to the amount of sulfur used.
1
DMSP was analyzed via H‐nuclear magnetic reso-
nance (NMR) by Dr Dongtao Cui (Chemical Sciences
Magnetic Resonance Facility, University of Georgia).
DMSP (5 mg) was dissolved in 600 μL of D2O, and
NMR spectra were obtained on a Bruker AVANCE III
HD NMR spectrometer at a frequency of 400 MHz.
NMR spectra were aligned by shifting the D2O peak to
the reference point of 4.790 ppm.17
DMS formed from DMSP by alkaline hydrolysis was
analyzed via gas chromatography–mass spectrometry
(GC–MS) at the Proteomics and Mass Spectrometry
Facility (University of Georgia) with a modified version
of the protocol described by Niki et al. (2004).18 A 5‐mL
serum vial was charged with 4, 4A, or 4B (6 mg) dissolved
in 100‐μL water and crimp sealed with a teflon‐coated
butyl rubber stopper, and the headspace was flushed with
N2 for 10 minutes. A syringe was used to add 100 μL of 4
M NaOH (aq), and the vial was incubated at 30°C for
1 hour to convert 4, 4A, or 4B to equimolar amounts of
3, 3A, or 3B, respectively.19 500 μL of the headspace was
applied to the injection port (heated at 150°C) of the GC
2 | EXPERIMENTAL
Unless stated otherwise, all chemicals were purchased
from commercial sources with American Chemical
Society (ACS)‐grade purity or higher and were used with-
out further purification. Metallic sodium was provided by
Dr Robert Phillips (Department of Chemistry, University
of Georgia). The 34S8 and I13CH3 were purchased from
Sigma‐Aldrich (St. Louis, MO) with 99% atom enrich-
ment. The NH3 (l) was generated by dripping 30%
NH4OH (aq) onto NaOH pellets, drying the NH3 (g) by
passing it over KOH pellets, and condensing the NH3
(g) on a cold finger filled with dry ice and ethanol. Dry
HCl (g) was generated by dripping concentrated HCl
(aq) into concentrated H2SO4 and bubbling the resulting
HCl (g) through concentrated H2SO4. All glassware used
in the experiments was acid washed in 3% HCl (aq) for
24 hours to remove trace contaminants and then baked
at 180°C for 24 hours to degrade any remaining organic
compounds.
(HP‐5890A, Agilent) with
a splitless duration of
2.75 minutes and an EC‐5 (0.25‐mm ID × 30 m × 0.25‐μm
film thickness, Alltech) column. The carrier gas was He
with a head pressure capped at 12 psi. The GC oven
was programmed to rise from 50°C to 150°C at a rate of
15°C min–1. 3, 3A, and 3B were detected by a mass
spectrometer (HP‐5971A, Agilent) with an electron‐
ionization (EI) ion source running in scan mode (moni-
tored m/z range was 45‐67) with 12 scans per second
and a detector temperature of 150°C.
2.1 | (2‐Carboxyethyl)dimethylsulfonium‐
34S chloride
Because of the price of 34S8, S8 was used to determine
the efficiency of reaction (I), Na2S was used to determine
the combined efficiency of reactions (II) and (III), and
DMS was used to determine the efficiency of reaction
(III) (Scheme 1). Because of the presence of excess Na
and the potential for oxidation of Na2S, a modified
Na234S (2A) was synthesized as previously described.20,21
A 10‐mL serum vial containing a teflon‐coated stir bar
was charged with 34S8 (1A) (0.1071 g, 394 μmol) and
freshly shaved, hexane‐washed Na (0.1742 g, 7.577 mmol),
flushed with N2 for 1 hour, and then incubated at –78°C
under a slow stream of nitrogen for the duration of the
reaction. The vial was charged with approximately 8 mL
of NH3 (l), incubated with stirring until no yellow color
could be seen, and then stirred for an additional
30 minutes. The vial was then flushed with a steady
stream of N2 until all NH3 had evaporated, leaving
SCHEME 1 Synthesis of 4. Reagents: a, Na; b, NH3 (l); c, ICH3;
d, NaOH (aq); e, acrylic acid; f, CH2Cl2; g, HCl (g)

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WIRTH AND WHITMAN
behind a white and silvery powder composed of excess Na
and 2A. Dimethylsulfide‐34S (3A) was synthesized from
the resulting 2A as previously described.22 The vial con-
taining 2A was crimp sealed with a teflon‐coated butyl
rubber stopper, and its headspace was replaced with N2
and pressurized to 10 psi. The contents of the vial were
dissolved in 3 mL of an anaerobic stock solution of
1.5 M NaOH (aq), and the vial was incubated on ice for
5 minutes. A glass syringe was used to add ICH3
(470 μL, 1.0716 g, 7.550 mmol) to the vial, and the vial
was incubated at 4°C with vigorous stirring for 4 hours.
To stop the reaction, a syringe was used to add 2 mL of
3 M Na2S2O3 (aq), and the vial was incubated at 4°C with
vigorous stirring for 30 minutes. The vial was chilled to –
196°C in N2 (l) and connected to a receiving flask. The 3A
was distilled from the solution by cooling the receiving
flask in N2 (l) while warming the vial to 40°C for
2.5 hours. (2‐Carboxyethyl)dimethylsulfonium‐34S (4A)
was synthesized as described previously.12 The receiving
flask containing distilled 3A was immediately charged
with –80°C CH2Cl2 (12 mL). The receiving flask was
removed from the N2 (l) and was immediately charged
with acrylic acid (260 μL, 0.2733 g, 3.792 mmol). Immedi-
ately afterwards, the solution was stirred vigorously at
room temperature for 30 minutes while bubbling in dry
HCl (g). The solution was dried at 50°C under a vacuum
for 2.5 hours. The resulting white solids were washed
with CH2Cl2 to yield white crystals composed of pure
4A (0.3551 g, 2.0578 mmol, 65.3%).
SCHEME 2 Published method for the synthesis of 4A.23
Reported efficiencies: reaction (I), 99%; reaction (II), 92%; reaction
(III), 87%; reaction (IV), 63%; reaction (V), 51%. Reagents: a, KCN
(aq); b, acetonitrile; c, tert‐butyl 3‐bromopropionate; d, H2O; e,
THF; f, SmI2; g, KOH in methanol; h, ICH3; i, nitromethane; j,
trimethyloxonium tetrafluoroborate; k, trifluoroacetic acid. The
products of reactions (II), (III), and (IV) were purified by thin layer
chromatography (TLC). The product of reaction (V) was purified
with ion‐exchange chromatography
utilized here, the loss of volatile intermediates, namely,
H2S (from aqueous 2, 2A, 2B) and DMS (3, 3A, 3B), was
minimized by using a combination gas‐tight reaction ves-
sels and careful control of the temperature and pH. Loss
of H2S was reduced by using concentrated sodium
hydroxide solutions.24 Loss of DMS was reduced with
low temperatures as it is a liquid below 38°C and a solid
below –98°C. By taking advantage of these facts, a new
method of producing 4A was developed (Schemes 1 and
3). However, because 34S8 (1A) is quite expensive, the pro-
tocol was first optimized using S8 (1), and the reactions
were repeated multiple times to ensure reproducibility.
1 was first reduced to 2 via a Birch reduction20,21 with
Na in NH3 (l). Subsequent evaporation of the NH3 yielded
a white powder primarily composed of anhydrous 2 with
an efficiency of 78.0 7.1%. The conversion of 2 to 3 was
accomplished by the nucleophilic attack of S2– on the
methyl group of ICH3 under anaerobic and basic
conditions, and the resulting 3 was subsequently purified
via distillation.22 Finally, 3 was converted to 4 via
Michael addition to acrylic acid in CH2Cl2 with an
efficiency of 106.2 13.7%.12 The efficiency of the synthe-
sis and purification of 3 was not determined because
measurements of the amount of 3 required large dilutions
of the headspace, which proved to be inaccurate (data not
shown). However, the efficiency of the conversion of 2 to
4 was 75.5 7.4%.
2.2 | (2‐Carboxyethyl)di(methyl‐13C)
sulfonium‐34S chloride
Na234S (2A) was synthesized from 34S8 (1A) (0.1005 g,
370 μmol) and freshly shaved, hexane‐washed Na
(0.1564 g, 6.803 mmol) using the methods described
above. Di(methyl‐13C)sulfide‐34S (3B) was synthesized
from the resulting 2A and I13CH3 (450 μL, 1.0305 g,
7.210 mmol) using the method described above.
(2‐Carboxyethyl)di(methyl‐13C)sulfonium‐34S (4B) was
synthesized from the resulting 3B and acrylic acid
(250 μL, 0.2628 g, 3.646 mmol) using the method
described above. This yielded white crystals composed
of pure 4B (0.3307 g, 1.895 mmol, 64.0%).
Because the boiling point of ICH3 (43°C) is very close
to that of DMS (38°C), there was a potential for unreacted
ICH3 to codistill, which would lower the purity of the
3 | RESULTS AND DISCUSSION
A previous study employed a strategy for the synthesis of
4A that avoided the production of volatile intermediates,
but this approach required five separate reactions with
purification of each intermediate and only produced an
overall yield of 26% (Scheme 2).23 In the approach
2–
final product. However, S2O3 is capable of converting
ICH3 to nonvolatile compounds.25 Thus, Na2S2O3 was
added in excess to ensure that all unreacted ICH3 was
consumed prior to distillation.

WIRTH AND WHITMAN
55
SCHEME 3 Synthesis of 4A and 4B. Reagents: a, Na; b, NH3 (l); c, ICH3; d, NaOH (aq); e, acrylic acid; f, CH2Cl2; g, HCl (g); h, I13CH3
1
To verify the purity of compounds 4A and 4B, H‐
NMR was performed. The spectrum of 4A was nearly
identical to that of a 4 standard (Figure 1). However,
the spectrum for 4B was very different. The triplet at
approximately 3.5 ppm was split into a triplet of triplets
due to the isotopic coupling with the methyl‐13C atoms.
The isotopic coupling also split the singlet indicative of
the methyl protons into a doublet of doublets (Figure
1C). This complex splitting pattern has been observed in
[13C2] DMSO (((methyl‐13C)sulfinyl)methane‐13C) and is
FIGURE 1 1H‐ nuclear magnetic resonance (NMR) spectra. All spectra have been aligned by shifting the D2O peak to 4.790 ppm.17 Peak
heights have been adjusted for clarity. The molecule corresponding to each spectrum is shown inside the box. Peaks have been labeled with
1
1
their corresponding atoms. A, H‐NMR spectrum of 4 (400 MHz, D2O). H‐NMR δ 3.52 (t, 2H, CβH2, J = 6.9 Hz), δ 2.98 (t, 2H, CαH2,
J = 6.9 Hz), δ 2.92 (s, 6H, CH3). B, 1H‐NMR spectrum of 4A (400 MHz, D2O). 1H‐NMR δ 3.51 (t, 2H, CβH2, J = 6.9 Hz), δ 2.97 (t, 2H, CαH2,
J = 6.9 Hz), δ 2.92 (s, 3H). C, 1H‐NMR spectrum of 4B. 1H‐NMR δ 3.51 (tt, 2H, CβH2, J = 6.8, 2.9 Hz), δ 2.97 (t, 2H, CαH2, J = 6.9 Hz), δ 2.92
(dd, 6H, 13CH3, J = 145.7, 3.6 Hz)

56
WIRTH AND WHITMAN
due to the AX3A'X'3 spin system.22 On the basis of the iso-
of the m/z values at 62 and 64 indicated that there was a
more than or equal to 99% enrichment of the 34S atom
(Figure 3). For 4B, the peak at m/z equal to 66 could
not be directly compared with the peak at m/z equal to
62 because the three minor peaks corresponding to m/z
equal to 57 to 59 (Figure 2) were shifted to 61 to 63,
respectively (Figure 4). However, the peak at m/z equal
to 66 showed a 98.9% relative enrichment as compared
with the peak at m/z equal to 64. This value agreed with
the value determined by integrating the peaks in the
1H‐NMR spectrum, which qualitatively suggested that
13CH3 was enriched by more than or equal to 99% in 4B
as compared with 12CH3 (Figure 1C). Taken together, with
1
topic coupling observed in the H‐NMR, there was more
than or equal to 99% enrichment of the methyl‐13C atoms
1
in 4B. Furthermore, H‐NMR showed that the resulting
compounds were contaminated with less than 1% 3‐
hydroxypropionate.
In order to determine the enrichment of the 34S
atoms, GC–MS analyses were performed on DMS formed
from DMSP. Alkaline hydrolysis converted 4, 4A, and 4B
into equimolar amounts of 3, 3A, and 3B, respectively,19
which were then analyzed via GC–MS. The relative abun-
dance of the peaks at m/z 62, 64, and 66 was examined for
4, 4A, and 4B, respectively (Figures 2–4). For 4A, the ratio
FIGURE 2 Gas chromatography–mass
spectrometry (GC–MS) spectrum of
dimethylsulfide produced by the alkaline
hydrolysis of 4. Peaks with less than 0.1%
relative abundance were omitted from the
table
FIGURE 3 Gas chromatography–mass
spectrometry (GC–MS) spectrum of
dimethylsulfide produced by the alkaline
hydrolysis of 4A. Peaks with less than 0.1%
relative abundance were omitted from the
table
FIGURE 4 Gas chromatography–mass
spectrometry (GC‐MS) spectrum of
dimethylsulfide produced by the alkaline
hydrolysis of 4B. Peaks with less than 0.1%
relative abundance were omitted from the
table

WIRTH AND WHITMAN
57
the high atom enrichment of 4A, these data indicated that
it was very likely that there was a more than 98% enrich-
ment of the 13C‐ and 34S‐atoms in 4B (Figure 4).
ORCID
Joseph S. Wirth https://orcid.org/0000-0002-9750-2845
William B. Whitman
https://orcid.org/0000-0003-1229-
Because this method uses ICH3 and acrylic acid, it
allows for isotopic labeling at one or more of the atoms
in 4. Acrylic‐1‐13C acid, acrylic‐13C3 acid, I13CH3, and
ICD3 are commercially available and can be used in place
of unlabeled acrylic acid or ICH3, respectively.
Supporting this claim, 4B was synthesized from 1A and
I13CH3 with an overall yield of 64.0%. This indicated that
the use of 13C‐labeled reactants has little effect on the
overall efficiency and supports the claim that this syn-
thetic method facilitates complex labeling experiments
in DMSP‐utilizing organisms. Furthermore, the products
of reactions (I) and/or (II) (Scheme 3) could be applied
in other syntheses to generate a variety of 34S‐labeled
compounds.
0423
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