Fully automated synthesis of 13N-labeled nitrosothiols

Article abstract of DOI:10.1016/j.tetlet.2010.03.122

In the present Letter, a fast, automated, and reproducible method for the synthesis of S-[¹³N]nitrosothiols is reported. The labeling strategy is based on trapping [¹³ N] NO₂^- in an anion exchange resin. The reaction with thiols in acidic media led to the formation of the desired nitrosothiols in short reaction times (60 s) with excellent radiochemical conversions (from 48.7% to 74.5%). Final radiotracers were purified by HPLC. Good radiochemical yields (from 33.8% to 60.6%, decay corrected) and radiochemical purities (>99%) were obtained in all cases. Stability of the labeled compounds was checked.

Full text of DOI:10.1016/j.tetlet.2010.03.122

Tetrahedron Letters 51 (2010) 2990–2993
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Fully automated synthesis of 13N-labeled nitrosothiols
Vanessa Gómez-Vallejo a, Koichi Kato b, Iosu Oliden a, Javier Calvo c, Zuriñe Baz a, José I. Borrell d, Jordi Llop a,
*
a Radiochemistry Department, CIC biomaGUNE, Parque Tecnológico de San Sebastián, p° Miramón 182, 20009 San Sebastián, Spain
b Department of Molecular Probes, Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
c Mass Spectrometry Platform, CIC biomaGUNE, Parque Tecnológico de San Sebastián, p° Miramón 182, 20009 San Sebastián, Spain
d Grup d’Enginyeria Molecular, Institut Químic de Sarrià, Universitat Ramon Llull, Via Augusta 390, 08017 Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
In the present Letter, a fast, automated, and reproducible method for the synthesis of S-[13N]nitrosothiols
Article history:
ꢁ
is reported. The labeling strategy is based on trapping ½13NꢀNO2 in an anion exchange resin. The reaction
Received 23 February 2010
Revised 25 March 2010
Accepted 30 March 2010
Available online 3 April 2010
with thiols in acidic media led to the formation of the desired nitrosothiols in short reaction times (60 s)
with excellent radiochemical conversions (from 48.7% to 74.5%). Final radiotracers were purified by HPLC.
Good radiochemical yields (from 33.8% to 60.6%, decay corrected) and radiochemical purities (>99%) were
obtained in all cases. Stability of the labeled compounds was checked.
Keywords:
Nitrosothiols
Ó 2010 Elsevier Ltd. All rights reserved.
Positron emission tomography
Nitrogen-13
Nitric oxide (NO) is a key signaling molecule involved in the
regulation of many biological and physiological processes, such
as blood flow and pressure,1 neurotransmission2 and inflammation
and host defense.3,4 The importance of NO has promoted a lot of
interest in the development of drugs able to generate NO directly
and/or indirectly for therapeutic treatments. Such is the case of
organic nitrates, which have been used for many years to treat
patients with ischemic heart disease; however, they are known
to develop tolerance5 and consequently other NO donor drugs have
been developed during the last years. One example is S-nitrosothi-
ols, which have several advantages over other NO donors, because
they represent endogenous NO reservoirs while they do not induce
oxidative stress or vascular tolerance.6
Ph3P and Br2.8 In the current Letter, we present a general procedure
for the radiosynthesis of S-[13N]nitrosothiols by trapping ½13NꢀNO2
ꢁ
in an anion exchange resin and reacting with the appropriate thiol
in acidic conditions (Scheme 1). An automatic remote controlled
system to perform the production (synthesis, purification, and
formulation) of S-[13N]nitrosothiols has been designed and
implemented.9
In a typical experiment, 13N (30 mCi, 1.11 GBq) was produced in
an IBA Cyclone 18/9 cyclotron via the 16O(p, 13N nuclear reaction.
a)
The target, containing 1.75 mL of water (ultrapure, Type I water,
ISO 3696) was irradiated with 18 MeV protons at a beam current
of 20 lA for 1.5 min (integrated current of 0.5 lAh). The resulting
solution was collected in a 5 mL conic vial, loaded in a glass column
In spite of the potential applications in the clinical environment
of S-nitrosothiols, the exact mechanisms underlying the role devel-
oped by such kind of molecules have not been fully elucidated.
From this perspective, a technique able to detect and quantify
the presence of S-nitrosothiols in vivo and at trace levels is highly
desirable. Positron emission tomography, a noninvasive imaging
technique, fulfills these requirements.
filled with cadmium10 and allowed to react for 40 s to obtain a vir-
ꢁ
tually ½13NꢀNO3 free solution, which was eluted through an anion
exchange cartridge (Sep-PakÒ Accell Plus QMA, Waters) to selec-
tively retain ½13NꢀNO2ꢁ. The column was further eluted with
distilled water (2 mL) and the QMA cartridge was dried with inert
gas for 15 s. An acidic solution of the adequate thiol (0.5 mL, see
precursors, Table 111,12) was loaded into the cartridge, reaction
was allowed to occur and then the reaction mixture was pushed
directly into a collection vial (no purification) or to the HPLC sys-
tem (when a purification step was needed).
In a recent work,7 we presented a fast and simple strategy for
ꢁ
the generation of the radioactive precursor ½13NꢀNO2 and its reac-
tion with glutathione in acidic media to yield S-[13N]nitrosogluta-
thione ([13N]GSNO). In continuation of our work, we used the same
radioactive precursor to synthesize N-[13N]nitrosamines with high
ꢁ
yields by trapping ½13NꢀNO2 in an anion exchange resin and per-
forming the reaction with secondary amines in the presence of
Scheme 1. Synthesis of S-[13N]nitrosothiols by trapping ½13NꢀNO2 in an anion
ꢁ
* Corresponding author. Tel.: +34 943 005 333; fax: +34 943 005 301.
E-mail address: jllop@cicbiomagune.es (J. Llop).
exchange resin and reacting with thiols in acidic conditions.
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.03.122

V. Gómez-Vallejo et al. / Tetrahedron Letters 51 (2010) 2990–2993
2991
ꢁ
and the amount of ½13NꢀNO2 obtained after the reduction step.
The percentage of radioactivity as S-[13N]nitrosothiol in the reac-
tion mixture was determined by HPLC.13 The presence of the de-
sired radiotracers was confirmed by co-elution with reference
compounds, which were prepared on site following the procedure
described by Wang et al.14
Table 1
Structures of the synthesized S-[13N]nitrosothiols and the precursors used
Precursor
Nitrosothiol
13N
S
O
1-Adamantanethiol
1
2
Our first optimization runs were carried out on the synthesis of
1. In our previous work,7 the synthesis of [13N]GSNO was achieved
by dissolving glutathione in aqueous hydrochloric acid, which was
13N
O
Thiophenol
S
ꢁ
reacted with ½13NꢀNO2 in a reaction vial. In the current case, water
was not an appropriate solvent due to the hydrophobic character of
1-adamantanethiol and, consequently, acetonitrile was used as a
solvent. First attempts were performed by using HCl (final concen-
tration = 1.0 M, prepared by adding the correct amount of 35%
aqueous HCl to the precursor solution in acetonitrile), but low
radiochemical conversion values were observed, irrespective of
the precursor concentration (0.5–2.0 mg/mL) and reaction time
(60–120 s). The radioactivity profiles registered during syntheses
13N
O
Cyclohexanethiol
1-Propanethiol
3
4
S
H3C
13N
S
O
13N
S
O
O
O
O
ꢁ
suggested a strong displacement of the ½13NꢀNO2 anion out of
5
6
L
-Glutathione
NH
the cartridge while the precursor solution was loaded, preventing
HO
NH
OH
ꢁ
direct contact between ½13NꢀNO2 and 1-adamantanethiol and pre-
NH2
O
cluding the reaction to occur.
OAc
O
This strong displacement was attributed to (i) the ionic strength
of chloride anion and (ii) the presence of water in the precursor
solution, both factors contributing to increase eluotropic power.
Trifluoroacetic acid (TFA) was considered a suitable candidate to
overcome this problem due to the lower ionic strength of the tri-
fluoroacetate anion and the possibility to prepare the precursor
solution in anhydrous conditions. In order to get an experimental
OAc
2-(N-Acetyl-D-
penicillamido)-2-
deoxy-1,3,4,6-tetra-
O-acetyl-b-
OAc
OAc
HN
D-
S
glucopyranose
N
O
NH
13
O
Ac
ꢁ
proof for the suitability of TFA, ½13NꢀNO2 (1 mCi) was loaded in a
During optimization runs, reaction time and acid concentration
were modified to optimize radiochemical conversion, calculated as
the ratio between the amount of radiotracer before purification
glass column (5 mm id, column length = 18 cm) prefilled with
anion exchange resin; the column was rinsed with TFA (0.5 mL,
1 M solution in acetonitrile) or HCl (0.5 mL, 1 M in acetonitrile,
Figure 1. Distribution of radioactivity (½13NꢀNO2ꢁ) in the column (5 mm id, column length = 18 cm) prefilled with anion exchange resin; Solid lines show radioactivity
distribution before elution. Dotted lines show distribution after elution with: (A) 0.5 mL of 1 M TFA solution in acetonitrile, (B) 0.5 mL of 1 M HCl solution in acetonitrile
prepared from 35% aqueous HCl solution, (C) 0.5 mL of 1 M TFA solution in water, and (D) 0.5 mL of 1 M HCl solution in water.

2992
V. Gómez-Vallejo et al. / Tetrahedron Letters 51 (2010) 2990–2993
Table 2
Experimental conditions, radiochemical conversion, radiochemical yield, radiochemical purity and specific radioactivity for the preparation of 13N-labeled nitrosothiols
Compound
Acid
Radiochemical conversiona,c,e (%)
Radiochemical yieldb,c,e (%)
Radiochemical purityd,e (%)
Specific radioactivitye,f (MBq/
lmol)
1
2
3
4
5
6
TFA
TFA
TFA
TFA
HCl
TFA
72.5 3.9
21.9 9.9
60.3 5.5
74.5 2.1
48.7 6.3
66.2 5.8
53.3 2.9
—
52.3 1.6
60.6 2.1
33.8 3.1
59.5 4.8
99.8 0.1
—
99.8 0.1
99.6 0.2
99.2 0.2
99.1 0.7
5900 230
—
6450 225
7210 340
185 25
8200 410
a
b
c
d
e
f
ꢁ
Calculated as the ratio between the amount of radiotracer before purification and the amount of ½13NꢀNO2 obtained after the reduction step.
Calculated as the ratio between the amount of radiotracer after purification and the amount of 13N generated in the cyclotron.
Decay corrected values.
Calculated from chromatographic profiles after purification.
AVG STDV (range).
End of synthesis.
Table 3
Stability of 13N-labeled nitrosothiols
Compound
RCPa (%) t = 12 min
RCPa (%) t = 24 min
RCPa (%) t = 36 min
RCPa (%) t = 48 min
1b
3b
4b
5b
6b
98.4 0.2
99.3 0.5
98.8 0.4
98.7 0.1
98.4 0.5
97.7 0.6
99.0 0.8
98.8 0.6
98.2 0.3
97.9 0.8
97.6 0.6
98.7 1.2
98.5 0.2
98.0 0.6
97.2 0.3
97.4 0.4
98.5 1.0
98.4 0.4
98.0 0.3
97.1 0.5
a
Radiochemical purity, calculated from chromatographic profiles.
See Table 2 for radiochemical purity at end of synthesis.
b
prepared from 35% aqueous HCl solution), and the radioactivity
distribution within the column was measured by using a TLC plate
reader before and after rinsing (Fig. 1A and B). As expected, almost
well known that solid phase extraction purification methods are
highly desirable when dealing with short-lived isotopes. However,
with the aim of establishing a general procedure which could be
potentially extended to the preparation of other S-[13N]nitrosothi-
ols, semi-preparative HPLC15 was chosen as a purification method.
The fact that the reaction is carried out in a cartridge facilitates direct
loading of the mixture in the HPLC loop with minimal radioactivity
loss, while accurate adjustment of chromatographic conditions al-
lowed completion of purification in less than 6 min in all cases.
After collection of the desired fraction into a vial containing
2 mL of 0.5 mM EDTA aqueous solution, reformulation was carried
out (1–4 and 6) by trapping the radiotracer in a C-18 cartridge,
eluting with ethanol and reconstituting with physiologic saline
solution. In the case of 5, direct dilution of the collected fraction
and pH adjustment with phosphate buffer left the final solution
with ethanol concentration below 10%.
ꢁ
no displacement of the ½13NꢀNO2 anion was observed when TFA
was used (Fig. 1A), while displacement became significant when
the column was rinsed with HCl (Fig. 1B).
Optimization runs were then repeated for the synthesis of 1
using TFA instead of HCl. Radiochemical conversion values were
considerably higher but again no significant changes were ob-
served when precursor concentration (0.5–2.0 mg/mL) and reac-
tion time (60–120 s) were modified. A precursor concentration of
1 mg/mL and a reaction time of 60 s were considered to be ade-
quate for the preparation of S-[13N]nitrosothiols, and these exper-
imental conditions were adopted for the synthesis of 2, 3, 4, and 6.
In the particular case of 5, and with the aim of obtaining results
comparable to those reported previously,7 the precursor was dis-
solved in water and hydrochloric acid was used.
Final solutions were analyzed by means of HPLC.13 All unde-
ꢁ
As it can be seen in Table 2, radiochemical conversion values
were above 48% in all cases except for compound 2, where several
radioactive unidentified by-products were detected in the chro-
matographic profile. For compounds 1, 3, 4, and 6 values over
60% were reached. These results point out to the conclusion that
either the formation of aliphatic S-[13N]nitrosothiols is favored
with respect to the formation of aromatic ones or the latter are less
stable, undergoing fast decomposition. The relatively low radio-
chemical conversion obtained in the case of 5 (48.7% 6.3) was
first attributed to the small molar concentration of precursor.
However, the use of substantially higher precursor molar amounts
did not significantly increase radiochemical conversion while
might lower the efficiency of the later purification step. Careful
analysis of radioactivity profiles again suggested a strong displace-
sired radiochemical impurities (mainly unreacted ½13NꢀNO2 and
½
13NꢀNO3ꢁ) were successfully removed from the solution in all
cases (Table 2, radiochemical purity). No undesired peaks were de-
tected in the UV profile. Hence, concentration of precursor in the
final solutions was, according to the limit of detection of the ana-
lytical method, below 150, 60, 20, 1500, and 450 ng/mL for 1 and
3–6, respectively. The presence of labeled species (5 and 6) was
confirmed by UPLC-MS (ESI-TOF).16 This analytical technique was
not suitable for the identification of 1, 3, and 4. In this case, co-elu-
tion with reference compound in two different chromatographic
scenarios confirmed the presence of the desired labeled species.17
Average radiochemical yields (calculated as the ratio between
the amount of radiotracer after purification and the amount of 13N
generated in the cyclotron, decay corrected) for the preparation of
S-[13N]nitrosothiols were above 50% for 1, 3, 4, and 6 whilethey were
slightly lower in the case of 5 (Table 2) as expected according to
radiochemical conversions. Total synthesis time (including purifica-
tion) was less than 13 min in all cases and radiochemical purities
(determined by HPLC13) were above 99% for 1 and 3–6. In the case
of 2, radiochemical purity was under 85% immediately after
purification, pointing out to a fast decomposition of S-[13N]nitroso-
thiophenol. Specific activities (Table 2) were in the range 5900–
ꢁ
ment of the ½13NꢀNO2 anion while the precursor solution was
loaded; experiments performed to obtain information regarding
eluotropic power of aqueous HCl and TFA solutions showed that,
in this case, radioactivity displacement was very similar in both
scenarios (Fig. 1C and D) and the use of trifluoroacetic acid was
not considered an option to enhance radiochemical conversion.
After establishing the synthetic procedure, and due to the pres-
ence of the precursor and radioactive impurities in the reaction mix-
ture, the implementation of a purification step was essential. It is
8200 MBq/lmol for 1, 2, 4, and 6, and were significantly lower

V. Gómez-Vallejo et al. / Tetrahedron Letters 51 (2010) 2990–2993
2993
2. Grisham, M. B.; Jourd’Heuil, D.; Wink, D. A. Am. J. Physiol. 1999, 276, G-315–G-
321.
3. Al-Sa’doni, H. H.; Ferro, A. Mini-Rev. Med. Chem. 2005, 5, 247–254.
4. Khan, M.; Jatana, M.; Elango, C.; Paintlia, A. S.; Singh, A. K.; Singh, I. Nitric Oxide
2006, 15, 114–124.
5. Al-Sa’doni, H.; Ferro, A. Clin. Sci. 2000, 98, 507–520.
6. Jaworski, K.; Kinard, F.; Goldstein, D.; Holvoet, P.; Trouet, A.; Schneider, Y.-J.;
Remacle, C. Eur. J. Pharmacol. 2001, 425, 11–19.
7. Llop, J.; Gómez-Vallejo, V.; Bosque, M.; Quincoces, G.; Peñuelas, I. Appl. Radiat.
Isot. 2009, 67, 95–99.
8. Gómez-Vallejo, V.; Kato, K.; Hanyu, M.; Minegishi, K.; Borrell, J. I.; Llop, J. Bioorg.
Med. Chem. Lett. 2009, 19, 1913–1915.
9. The scheme of the automated system, the characteristics of the individual
components and details about synthetic steps are included as Supplementary
data.
(185 MBq/
l
mol) in the case of 5, probably due to the presence of
ꢁ
ꢁ
NO3 and/or NO2 as impurities in the hydrochloric acid solution.
Stability tests performed on 1 and 3–6 showed that, in all cases,
radiochemical purities after 48 min (from end of synthesis) were
above 97% (Table 3). For compound 2, radiochemical purity quickly
decreased with time (results not shown) confirming the instability
of S-[13N]nitrosothiophenol; different additives were assayed to
improve stability with poor results.
To the best of our knowledge, the synthesis of S-[13N]nitrosothi-
ols has been reported previously only by us7 and by Vavrek and
Mulholland,18 who investigated the formation of water-soluble S-
[
13N]nitrosothiols following a similar procedure. In that case, puri-
10. Cadmium (20 g., granular, 5–20 mesh) was introduced in a glass column
(10 mm id, 8 cm in length) and sequentially washed with 1 M HCl (2 ꢂ 20 mL),
distilled water (3 ꢂ 20 mL), 0.5 M aqueous CuSO4 solution (2 ꢂ 20 mL), 0.1 M
aqueous NH4Cl solution (2 ꢂ 20 mL), and distilled water (3 ꢂ 20 mL). The
column showed excellent reductive properties for up to 10 consecutive runs
within one day. Reconditioning on a daily basis ensured optimal performance
for up to 100 runs.
fication step was not performed and the chemical purity of final
radiotracers was not precisely reported.
In the work we are presenting here, decay corrected radiochem-
ical yield for the synthesis of 5 (33.8 3.1%) is slightly higher than
the one reported in our previous work7 (24.2%). Radiochemical
purity is somewhat higher than in the previous work (99.2 0.2%
vs 96.4 0.6%) and the method has been extended to the prepara-
tion of non water-soluble S-[13N]nitrosothiols. Therefore, the gen-
eral procedure here reported should allow the preparation of a
wide range of S-[13N]nitrosothiols with high chemical and radio-
chemical purity.
11. Precursors for the radiosynthesis of S-[13N]nitrosothiols 1–5 were purchased
from Sigma–Aldrich and used without further purification. For the synthesis of
2-(N-acetyl-D-penicillamido)-2-deoxy-1,3,4,6-tetra-O-acetyl-b-D-glucopyranose
(precursor for 6), the methodology described in Ref. 12 was followed.
12. Butler, A. R.; Greig, I. R.; Megson, I. L. WO/1998/020015, 1998.
13. For analytical HPLC, an Agilent 1200 Series HPLC system with a multiple
wavelength detector (k = 220 nm) and a radiometric detector was used. A
Mediterranean SeaRP-18 column (5 lm, 150 mm, 4.6 mm) was used as
stationary phase. For compounds 1–4 and 6, water/methanol/acetonitrile
(10:15:75) was used as mobile phase at a flow rate of 1 mL/min. For compound
5, aqueous TFA solution/acetonitrile (95:5) was used at a flow rate of 1 mL/min.
14. Wang, K.; Hou, Y.; Zhang, W.; Ksebati, M. B.; Xian, M.; Cheng, J. P.; Wang, P. G.
Bioorg. Med. Chem. Lett. 1999, 9, 2897–2902.
Acknowledgments
The authors would like to thank Dr. Peñuelas (Nuclear Medicine
Department, Clínica Universitaria de Navarra) for developing and
transferring to our research group the original idea of synthesizing
S-[13N]nitrosothiols. The authors also thank the Departamento de
Industria, Comercio y Turismo of the Vasc Government for financial
support.
15. For semi-preparative HPLC, an isocratic pump (Knauer) with a radiometric
detector was used. A Mediterranean Sea18 column (5 lm, 250 mm, 10 mm)
was used as stationary phase. For compounds 1–4 and 6, water/ethanol/
acetonitrile (10:15:75) was used as mobile phase at a flow rate of 6 mL/min.
For compound 5, aqueous TFA solution/ethanol (85:15) was used at a flow rate
of 4 mL/min.
16. UPLC/MS analyses were performed using an AQUITY UPLC separation module
coupled to a LCT TOF Premier XE mass spectrometer (Waters, Manchester, UK).
An Acquity BEH C18 column (1.7 lm, 5 mm, 2.1 mm) was used as stationary
phase. The elution buffers were A (methanol and 0.1% formic acid) and B
(water and 0.1% formic acid). The column was eluted with a linear gradient
consisted of 95% A to 1% over 2.5 min, 1% over 2.5–3.5 min, returned to 95 for
0.5 min and kept for a further 1 min. Total run was 5 min, injection volume was
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2010.03.122.
5 lL and the flow rate 600 lL/min. Detection was performed in positive ion
mode in the range of 50–1000, with a scan time of 1 s and a delay time of 0.1 s
in centered mode. GSNO was detected as protonated molecule (m/z = 377.08,
retention time = 0.81 min) and RIG was detected as sodium adduct (m/z =
572.15, retention time = 1.78 min).
References and notes
17. Scenario 1: as reported in Ref. 13; scenario 2: as reported in Ref. 16.
18. Vavrek, M. T.; Mulholland, G. K. J. Labelled. Compd. Radiopharm. 1995, 37, 118.
1. Lundberg, J. O.; Weitzberg, E.; Gladwin, M. T. Nat. Rev. Drug Disc. 2008, 7, 156–
167.