Synthesis of s-pixyl derivatives for mass spectrometric applications

Article abstract of DOI:10.1055/s-2005-872703

Synthesis of novel S-pixyls based on the thioxanthyl skeleton is described. Thioxanthone derivatives were prepared by a regioselective intramolecular Friedel-Crafts acylation reaction and converted into S-pixyl derivatives by Grignard synthesis. S-Pixyl carbocations stabilised by electron donating groups on the thioxanthyl backbone produced exceptional mass spectra under (MA)LDI conditions (laser desorption ionisation, both with and without matrix), and can be detected down to femtomol levels. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-2005-872703

LETTER
2453
Synthesis of S-Pixyl Derivatives for Mass Spectrometric Applications
Synthesisof
S
a
-Pixyl
D
eri
f
vative
s
raz Khan,a Pablo L. Bernad,a Vladimir A. Korshun,b Edwin M. Southern,a Mikhail S. Shchepinov*a
a
Tridend Technologies (a Division of OGT) Hirsch Building, Begbroke Business & Science Park, Sandy Lane, Yarnton, Oxford,
OX5 1PF, UK
Fax +44(1865)841916; E-mail: misha@tridend.com
b
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, Moscow, 117 997, Russia
Received 7 July 2005
For our applications the S-pixyl mass tag released as a
cation can then be analysed by (MA)LDI analysis.
Abstract: Synthesis of novel S-pixyls based on the thioxanthyl
skeleton is described. Thioxanthone derivatives were prepared by a
regioselective intramolecular Friedel–Crafts acylation reaction and
converted into S-pixyl derivatives by Grignard synthesis. S-Pixyl
carbocations stabilised by electron donating groups on the thioxan-
thyl backbone produced exceptional mass spectra under (MA)LDI
conditions (laser desorption ionisation, both with and without
matrix), and can be detected down to femtomol levels.
We have identified the thioxanthone ketone 4 as a key in-
termediate. Derivatised forms of this ketone are not com-
mercially available. Attempts to prepare substituted
ketones via reported literatures routes proved to be diffi-
cult.7 A series of functionalised thioxanthen-9-ones were
prepared via regioselective intramolecular Friedel–Crafts
acylation reaction (Scheme 2).
Key words: S-pixyls, regioselective Friedel–Crafts acylation,
(MA)LDI analysis, functionalised thioxanthen-9-ones, 2-
aroylpyrene
O
O
S
R4
R3
R2
Cl
R1
S
We have been pursuing the synthesis of novel trityl labels
as new molecular tools for mass spectrometric (MS) ap-
plications in genomics1 and proteomics.2 Our results have
shown that trityl-based mass tags fly extremely well in the
positive mode of laser desorption ionisation (LDI) MS
with and without matrix, due to the formation of a highly
stabilised carbocation.3 The presence of ortho- or para-
alkyl or -alkoxy groups on the phenyl ring of the trityl im-
prove the LDI process by further stabilising the positive
charge.
We were particularly interested in S-pixyl4 derivatives
due to their ability to stabilise carbocations and the fact
that the sulfur atom can be oxidised to the corresponding
sulfoxide. The resulting S(O)-pixyl derivatives were then
found to be resistant to acid treatment and were not ion-
ised even in 50% H2SO4. The reverse deoxygenation of
the sulfoxide function in S(O)-pixyl derivatives can be
satisfactorily carried out according to literature
procedures5 (Scheme 1). S-Pixyls are used currently as
protecting groups in organic chemistry and can be cleaved
from organic molecules by acid or UV irradiation.6
R1
3
4
R2
R4
R3
Scheme 2
Similar approaches using acid-mediated cyclisations of o-
(phenylthio)benzoic acids are reported for the synthesis of
simple thioxanthen-9-one ketones. The regioselectivity is
poor giving a mixture of regioisomers that require separa-
tion.8
Intermediate 7a was prepared via a Newman–Kwart reac-
tion, O→S transposition starting from commercially
available methyl 2-hydroxy-4-methoxybenzoate
(Scheme 3).9,10
5
1. ClC(S)N(CH3)2, DABCO,
DMF, 93%
CO2Me
OH
CO2Me
2. 200 °C, neat, 98%
MeO
MeO
S
N
5
6
O
CO2H
1. KOH, MeOH, 60 °C
2. H+, 92%
MeO
SH
MCPBA
7a
RO
RO
Scheme 3
reduction
R1
S
R2
R1
S
R2
1
O
2
Compound 5 was treated with dimethylthiocarbamoyl
chloride in the presence of DABCO in DMF. The desired
O-aryl N,N-dimethylthiocarbamoyl derivative was then
thermally rearranged into the S-aryl N,N-dimethylthiocar-
bamate. Alkaline hydrolysis in methanol gave 78 g of 7a
in 92% yield. The Ullmann reaction was used to prepare
methoxyphenylthiobenzoic acid 911,12 as described in the
Scheme 1
SYNLETT 2005, No. 16, pp 2453–2456
Advanced online publication: 21.09.2005
DOI: 10.1055/s-2005-872703; Art ID: D18705ST
© Georg Thieme Verlag Stuttgart · New York
0
4
.1
0
.2
0
0
5

2454
Shchepinov, M. S.
LETTER
literature (Scheme 4). Friedel–Crafts acylation gave the (MA)LDI analysis of an equimolar solution of S-pixyls 18
desired ketone in 80–93% yields, excellent regioselectiv- and 19 (5 mL of 2.5 × 10–6 M solution in THF) in the ab-
ity was observed for the synthesis of 10 (R1 = H, sence (Figure 2) and in the presence of matrix (Figure 1),
R2 = OMe), 1-methoxy-9H-thioxanthen-9-one (11) was 2,5-dihydroxybenzoic acid was performed. These results
not isolated (Table 1).13
confirm that the presence of alkyl or alkoxy groups on the
phenyl ring improves the sensitivity in the positive mode
of laser desorption ionisation at femtomol concentrations,
thus suggesting the way to modulate the desorption prop-
erties of the mass-tags by changing the number of electron
donating substituents on the ring system.
CO2H
+
Cu, K2CO3, DMF
160 °C
R1
SH
I
R2
8a R2 = H
7a R1 = OMe
7b R1 = H
8b R2 = OMe
CO2H
O
R1
S
1. (COCl)2, cat. DMF,
CH2Cl2, 98%
S
OMe
R2
2. AlCl3, CH2Cl2, r.t.,
80–93%
10
9a R1 = OMe, R2 = H, 78%
9b R1 = H, R2 = OMe, 81%
Figure 1 Equimolar mix of S-pixyls 18 and 19 in the presence of
matrix
Scheme 4
Interestingly, Boyd et al. described the one-pot Friedel–
Crafts acylation of anisole with thiosalicyclic acid in the
presence of sulfuric acid and sulfonyl chloride, generating
3-methoxy-9H-thioxanthen-9-one in 44% yield.6a How-
ever, their 1H NMR and 13C NMR spectroscopic data were
not in full agreement with our data for 10. We suspected
that they had in fact prepared the wrong regioisomer 2-
methoxy-9H-thioxanthen-9-one (12, Table 1). We pre-
pared 12 via reaction of 7b with 4-iodoanisole, followed
by Friedel–Crafts acylation in 68% yield. Compound 12
1
gave H NMR and 13C NMR data in full agreement with
Figure 2 Equimolar mix of S-pixyls 18 and 19 in the absence of
matrix
their reported data.13
Thiosalicyclic acids 7a and 7b both yield an identical
ketone 10, the high regioselectivity observed for 10 can be
explained by the strong para-directing effect of the 3-
methoxy group of 9b. The versatility of this approach was
further extended towards the synthesis of functionalised
ketones 14–17, which were prepared in good overall
yields from the starting aryl acyl chlorides (Table 1).
We also describe the synthesis of a novel pyrene ketone
21, prepared from the reaction of 2-mercapto-4-methoxy-
benzoic acid with 1-bromopyrene, followed by a Friedel–
Crafts acylation reaction to give 20 in 80% yield. The
pyrene ketone was treated with 4-methoxyphenylmagne-
sium bromide to give the desired pyrene S-pixyl 22 in
39% yield (Scheme 6). This combination of a fluorophore
Compounds 16 and 17 are of particular importance since
they give rise to side chain derivatised mass tags for ge-
nomics and proteomics applications.
CO2H
1. (COCl)2, cat DMF,
O
To evaluate the properties of S-pixyls as mass-tags for
analysis by (MA)LDI, S-pixyls 18 and 19 were prepared
by the addition of 4-methylphenylmagnesium bromide to
thioxanthen-9-ones 10 and 13, respectively (Scheme 5).
CH2Cl2, 89%
MeO
S
2. AlCl3, CH2Cl2, 80%
MeO
S
21
20
O
O
O
OH
HO
MgBr
THF
MgBr
R1
S
R2
R1
S
R2
S
MeO
THF, 39%
10 R1 = H, R2 = OMe
13 R1 = OMe, R2 = OMe
18 R1 = H, R2 = OMe, 89%
19 R1 = OMe, R2 = OMe, 79%
22
Scheme 6
Scheme 5
Synlett 2005, No. 16, 2453–2456 © Thieme Stuttgart · New York

LETTER
Synthesis of S-Pixyl Derivatives
2455
and a mass tag in one molecule can potentially be used for
orthogonal detection by mass spectrometry and optical
detection methods.3c
References
(1) (a) Gut, I. G. Hum. Mut. 2004, 23, 437. (b) Meng, Z.;
Simmons-Willis, T. A.; Limbach, P. A. Biomol. Eng. 2004,
21, 1. (c) Tang, K.; Fu, D.; Kotter, S.; Cotter, R. J.; Cantor,
C. J.; Koster, H. Nucleic Acids Res. 1995, 23, 3126.
(2) Aebersold, R.; Mann, M. Nature (London) 2003, 422, 198.
(3) (a) Shchepinov, M. S.; Chalk, R.; Southern, E. M. Nucleic
Acids Symp. Ser. 1999, 42, 107. (b) Shchepinov, M. S.;
Chalk, R.; Southern, E. M. Tetrahedron 2000, 56, 2713.
(c) Shchepinov, M. S.; Korshun, V. A.; Egeland, R. D.;
Southern, E. M. Tetrahedron Lett. 2000, 41, 4943.
(d) Shchepinov, M. S.; Korshun, V. A. Chem. Soc. Rev.
2003, 32, 170.
Initial MS analysis of S-pixyl 22 gave the desired molec-
ular ion (Figure 3). Further work on the fluorescent and
MS properties of 22 is currently ongoing and will be
reported in due course.
(4) Balgobin, N.; Chattopadhyaya, J. B. Chem. Scr. 1982, 19,
143.
(5) (a) Samanen, J. M.; Brandeis, E. J. Org. Chem. 1988, 53,
561. (b) Kiso, Y.; Kimura, T.; Yoshida, M.; Shimokura, M.;
Akaji, K.; Mimoto, T. J. Chem. Soc., Chem. Commun. 1989,
1511.
Figure 3
(6) (a) Coleman, M. P.; Boyd, M. K. J. Org. Chem. 2002, 67,
7641. (b) Bernad, P. L.; Khan, S.; Korshun, V.; Southern, E.
M.; Shchepinov, M. S. Chem. Commun. 2005, 3466.
(7) (a) Archer, S.; Miller, K. J.; Rej, R.; Periana, C.; Fricker, L.
J. Med. Chem. 1982, 25, 220. (b) Watanabe, M.; Date, M.;
Tsukazaki, M.; Furukawa, S. Chem. Pharm. Bull. 1989, 37,
36.
(8) (a) Brindle, I. D.; Doyle, P. P. Can. J. Chem. 1983, 61, 1869.
(b) Protiva, M.Šedivý, Z.; Holubek, J.; Svátek, E.;
Metyšová, J.; Bartošová, M. Coll. Czech. Chem. Commun.
1982, 47, 3134.
(9) (a) Org. Synth. Coll. Vol. II; Blatt, A. H., Ed.; Wiley: New
York, 1943, 580. (b) Katz, L.; Karger, L. S.; Schroeder, W.;
Cohen, M. S. J. Org. Chem. 1953, 18, 1380. (c) Bennett, O.
F.; Johnson, J.; Tramondozzi, J. Org. Prep. Proced. Int.
1974, 6, 287.
(10) (a) Newman, M. S.; Karnes, H. A. J. Org. Chem. 1966, 31,
3980. (b) Kaji, A.; Araki, Y.; Miyazaki, K. Bull. Chem. Soc.
Jpn. 1971, 44, 1393. (c) Decollo, T. V.; Lees, W. J. J. Org.
Chem. 2001, 66, 4244.
In conclusion, we have synthesised several substituted
thioxanthone derivatives via a regioselective Friedel–
Crafts acylation reaction in gram quantities. We have also
reported the preparation of a new pyrene S-pixyl 22 for
MS applications. (MA)LDI analysis of S-pixyls 18 and 19
confirm that the presence of electron donating groups on
the thioxanthyl backbone improves the sensitivity in the
positive mode of laser desorption ionisation. Further work
is ongoing and the synthesis of bifunctional trityl deriva-
tives will be reported in due course.
Table 1 Synthesis of Substituted Thioxanthen-9-ones
O
R4
R3
R2
R1
S
(11) (a) Lindley, J. Tetrahedron 1984, 40, 1433. (b) Kulkarni, N.
N.; Kulkarni, V. S.; Lele, S. R.; Hosangadi, B. D.
Tetrahedron 1988, 44, 5145. (c) Protiva, M.; Šindelář, K.;
Šediv, Z.; Holubek, J.; Bartošová, M. Collect. Czech. Chem.
Commun. 1981, 46, 1808. (d) Jílek, J.; Šindelář, K.;
Pomykáček, J.; Kmoníček, V.; Šedivý, Z.; Hrubantová, M.;
Holubek, J.; Svátek, E.; Ryska, M.; Koruna, I.; Valchář, M.;
Dlabač, A.; Metyšová, J.; Dlohožková, N.; Protiva, M.
Collect. Czech. Chem. Commun. 1989, 54, 3294.
(12) General Procedure for the Copper-Mediated Ullmann
Reaction, Selected Compounds 9a and 20.
Ketone R1
R2
R3
R4
H
Yield
(%)a
10
11
12
13
14
15
16
17
H
H
H
OMe
H
H
H
80–93
OMe 0b
H
OMe
H
H
68
OMe OMe
H
83–95
39c
35c
32
H
H
H
Me
H
H
H
To a solution of thiosalicylic acid (0.2 mol) in 300 mL of
DMF was added aryl halide (0.2 mol), K2CO3 (0.2 mol) and
Cu powder (0.2 equiv), the mixture was warmed to reflux.
After 16–24 h the mixture was cooled to r.t., filtered and
poured into 1 N HCl and then extracted with EtOAc (4 ×).
The combined organic phases were then washed with H2O
(4 ×), and dried over Na2SO4, filtered and concentrated in
vacuo. The product was dried under high vacuum and used
with out further purification (78–95%).
H
Me
H
(CH2)2CO2Me
H
OMe OMe
O(CH2)2CO2Et
H
60
a Isolated yields.
b Synthesis of 10, regioisomer 11 not obtained (AlCl3, CH2Cl2, r.t.).
c Obtained as a mixture, purified via recrystallisation.
Analytic data of 9b: 1H NMR (200 MHz, DMSO-d6):
d = 8.30–7.50 (d, J = 9.2 Hz, 1 H), 7.50–7.35 (dd, J = 9.6
Hz, 1 H), 7.30–7.18 (t, J = 7.5 Hz, 1 H), 7.15–7.00 (m, 3 H),
6.85–6.74 (d, J = 9.0 Hz, 1 H), 3.8 (s, 3 H). 13C NMR (400
MHz, DMSO-d6): d = 168.00, 160.99, 142.35, 134.16,
133.26, 131.79, 131.68, 128.51, 127.88, 125.59, 120.77,
115.98, 56.15. HRMS (ESI): m/z calcd for C14H12NaO3S
[M + Na+]: 283.0405; found: 283.0416.
Acknowledgment
We would like to thank Tim Claridge for assistance with NMR
spectra and Colin Sparrow for running all high accurate mass mea-
surements, CRL, Department of Chemistry, University of Oxford.
Synlett 2005, No. 16, 2453–2456 © Thieme Stuttgart · New York

2456
Shchepinov, M. S.
LETTER
Analytic data of 20: 1H NMR (400 MHz, DMSO-d6):
d = 8.50–8.30 (m, 8 H), 8.25–8.12 (t, J = 7.65 Hz, 1 H),
8.10–8.00 (d, J = 8.7 Hz, 1 H), 6.80–6.70 (d, J = 8.8 Hz, 1
H), 5.70–5.60 (d, J = 2.4 Hz, 1 H), 3.50–3.40 (br OH, 1 H),
3.35 (s, 3 H). 13C NMR (400 MHz, DMSO-d6): d = 167.95,
162.98, 145.59, 135.73, 134.73, 134.04, 133.23, 131.49,
131.15, 130.22, 129.75, 128.12, 127.74, 127.12, 126.76,
126.44, 125.48, 125.18, 124.34, 119.95, 113.49, 109.61.
HRMS (ESI): m/z calcd for C24H16NaO3S [M + Na+]:
407.0718; found: 407.0717.
product was purified by flash chromatography on silica gel,
hexane–EtOAc gradient elution to give the desired product
(80–95%).
Analytic data of 10: 1H NMR (400 MHz, CDCl3): d = 8.65–
8.58 (d, J = 8.1 Hz, 1 H), 8.55–8.50 (d, J = 9.0 Hz, 1 H),
7.60–7.40 (m, 3 H), 7.05–6.97 (d, J = 11.6 Hz, 1 H), 6.90 (d,
J = 2.4 Hz, 1 H), 3.90 (s, 3 H). 13C NMR (400 MHz, CDCl3):
d = 179.02, 162.48, 139.54, 136.89, 132.88, 131.92, 129.69,
129.27, 126.28, 125.71, 123.02, 115.08, 107.79, 55.66.
HRMS (ESI): m/z calcd for C14H11NaO2S [M + H]:
243.0480; found: 243.0473.
(13) General Procedure for the Intramolecular Friedel–
Crafts Acylation Reaction, Selected Compounds 10, 12,
13 and 21.
Analytic data of 12: 1H NMR (400 MHz, CDCl3): d = 8.65–
8.60 (d, J = 8.3 Hz, 1 H), 8.02 (d, J = 2.8 Hz, 1 H), 7.60–7.45
Preparation of Acid Chlorides.
(m, 4 H), 7.30–7.20 (d, J = 8.9 Hz, 1 H), 3.95 (s, 3 H). 13
NMR (400 MHz, CDCl3): d = 179.65, 158.35, 137.49,
131.99, 130.19, 129.84, 129.14, 128.55, 127.27, 126.06,
C
o-(Phenylthio)benzoic acid derivatives (0.1 mol), placed in a
dry 250 mL round-bottom flask under a positive pressure of
argon. Then, 100 mL of dry CH2Cl2 were added and the
reaction cooled to 0 °C. A few drops of DMF were added
followed by the dropwise addition of oxalyl chloride (1.5
equiv) with stirring. The reaction was stirred for 1 h or
longer until the suspension had completely dissolved. The
product was concentrated in vacuo and dried under high
vacuum to give the acid chloride, which was used without
further purification (90–100%).
125.97, 122.72, 110.29, 55.67. HRMS (ESI): m/z calcd for
C14H11NaO2S [M + H]: 243.0480; found: 243.0469.
Analytic data of 13: 1H NMR (400 MHz, CDCl3): d = 8.60–
8.50 (d, J = 4.5 Hz, 1 H), 7.05–6.98 (d, J = 5.1 Hz, 1 H), 6.90
(s, 1 H), 3.90 (s, 3 H). 13C NMR (400 MHz, CDCl3):
d = 178.30, 162.21, 139.12, 131.75, 123.03, 114.81, 107.98,
55.64. HRMS (ESI): m/z calcd for C15H13NaO3S [M + H]:
273.0585; found: 273.0576.
Intramolecular Friedel–Crafts Acylation.
Analytic data of 21: 1H NMR (200 MHz, CDCl3): d = 8.90
(s, 1 H), 8.70–8.60 (d, J = 8.9 Hz, 1 H), 8.59–8.48 (d, J = 9.3
Hz, 1 H), 8.35–7.90 (m, 6 H), 7.24–7.00 (m, 2 H), 4 (s, 3 H).
13C NMR (500 MHz, CDCl3): d = 180.14, 162.71, 138.72,
132.18, 131.98, 131.73, 131.01, 129.15, 128.44, 128.37,
127.79, 127.56, 126.79, 126.50, 126.21, 125.87, 125.78,
125.49, 124.20, 122.58, 122, 115.33, 108.53, 55.84. HRMS
(ESI): m/z calcd for C24H15O2S [M + H]: 267.0793; found:
267.0797.
The acid chloride (0.1 mol) was placed in a dry 250 mL
round-bottom flask under a positive pressure of argon. Then,
100 mL of dry CH2Cl2 were added and the reaction was
stirred at r.t., AlCl3 (1.5 equiv) was added slowly and the
reaction was stirred for 1–2 h. The reaction was quenched
with H2O and extracted with CH2Cl2 (2 ×). The combined
organic phases were washed with dilute NaHCO3 and dried
over Na2SO4, filtered and concentrated in vacuo. The
Synlett 2005, No. 16, 2453–2456 © Thieme Stuttgart · New York