S(O)-pixyl protecting group as efficient mass-tag

Article abstract of DOI:10.1039/b504913j

We report herein the design, preparation and first applications of novel trityl tags with adjustable stability, efficient as protecting groups or MS analytes. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b504913j

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COMMUNICATION
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S(O)-Pixyl protecting group as efficient mass-tag{
Pablo L. Bernad, Jr,a Safraz Khan,a Vladimir A. Korshun,b Edwin M. Southernc and Mikhail S. Shchepinov*a
Received (in Cambridge, UK) 11th April 2005, Accepted 16th May 2005
First published as an Advance Article on the web 9th June 2005
DOI: 10.1039/b504913j
Trityls based on the 9-arylthioxanthenyl skeleton (S-pixyl)6 5c
We report herein the design, preparation and first applications
proved to be suitable candidates.{ S-pixyl cations are notably
stable and, as it happens with the triphenylmethyl series, can be
further stabilised by the addition of methoxy substituents to the
position 3 and/or 6 of the thioxanthenyl moiety,§ as was confirmed
by pKR+ measurements (Table 1).
of novel trityl tags with adjustable stability, efficient as
protecting groups or MS analytes.
Mass spectrometry (MS) plays a major role in the study of the
genome1 and the proteome2 but there is still room for improve-
ment. In particular, methods that increase sensitivity and dynamic
range would represent a significant advance. In this context,
chemical tagging strategies have been developed to overcome the
limits of analysing native molecular species.3
The properties of the S-pixyl derivatives were dramatically
modified when the sulfur atom was oxidised to the corresponding
sulfoxide (Scheme 2, Table 1). Resulting S(O)-pixyl compounds
were resistant to acid treatment and were not ionised even in 50%
H2SO4. The reverse deoxygenation of the sulfoxide function in
S(O)-pixyl derivatives was satisfactorily carried out according to
literature procedures.7
As a result of their stability as carbocations, triarylmethyl
derivatives (trityls) produce exceptional mass spectra under
(MA)LDI conditions (laser desorption ionisation, both with and
without matrix), and can be detected down to attomole levels.4
Additionally, trityls show a remarkable structural malleability. For
instance, bifunctional trityls have been used in areas such as
bioconjugation, cross-linking, optics etc.4 We aimed to combine
these properties to give rise to a range of new molecular tools with
applications in MS analysis.
When used as labels for genome analysis, trityl mass-tags are
typically attached to oligonucleotides through an ether bond. A
wide range of masses is accessible from a precursor 1 by treating a
NHS (N-hydroxy succinimide) activated side-chain with different
amines. In use, tags are released by acid or UV irradiation as
cations 3 for MS analysis (Scheme 1).5
Scheme 2 Interconversion of S-pixyls.
Table 1 pKR+ of S-pixyl cations
a
pKR+
R1
R2
R3
S-Px
S(O)-Px
H
H
OMe
OMe
a
H
H
H
OMe
Me
4a
4b
4c
4d
20.2
0.2
2.8
5a
5b
5c
5d
,215
#211
#212
OMe
Me
Me
4.5
27.1
pKR+ determined spectrophotochemically.8
Encouraged by our initial findings, we moved on to the
preparation of bifunctional S-pixyl mass-tags, equipped with a
NHS activated side-chain and a phosphoramidite reactive centre
to attach the tag to oligonucleotides.
Scheme 1 Trityl-based mass tags.
We have shown that the more stable a trityl cation is, the better
its detection in MS. However, ether bonds linking trityl tags that
produce the most stable cations are too labile to survive post-
labelling protocols. An ideal trityl mass-tag should be stable as a
protecting group (trityl ether) but, upon request, generate a stable
cation. We show here that these antagonist characteristics can be
combined into a single trityl structure, stable as a protecting group
but, also, capable of generating efficient mass tags either upon
chemical treatment or under LDI MS conditions.
S-pixyls 6, containing a carboxylic acid protected as an ortho
ester,9 were prepared by the reaction of the lithium derivative of 7
with 3-methoxythioxanthen-9-ones 8 (Scheme 3). The ortho esters
were then converted into the NHS activated esters with good yield.
The selective tritylation of the primary hydroxy group of 1,3-
butanediol was completed via the tetrafluoroborate salt of
compounds 9.10 In situ treatment with MCPBA yielded the stable
version of the mass-tags, S(O)-pixyls 10. Phosphitylation of the
secondary hydroxyl group yielded the desired functionalised tags
11. The phosphoramidite function did not show compatibility
problems with the presence of the sulfoxide in the molecule.
{ Electronic supplementary information (ESI) available: Experimental
details. See http://www.rsc.org/suppdata/cc/b5/b504913j/
*misha@tridend.com
3466 | Chem. Commun., 2005, 3466–3468
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Table 2 Molecular weight of ions 14, 15 and 16 in Scheme 4
Compound
Y
X
R
MW
14a
14b
15a
15b
16a
16b
O
O
—
—
—
—
H
OMe
H
OMe
H
OMe
NHS
NHS
NHC4H10
NHC4H10
NHC5H12
NHC5H12
472.12
502.13
430.18
460.19
444.20
474.21
form; ions 15 and 16 are differentiated by the alkyl chain of the
amide function.
The acid stability of the 59 protecting group in oligonucleotides
13 was assessed using standard deblock solution (path A,
Scheme 4). Oligonucleotide 13a remained protected whereas
oligonucleotide 13b cleaved to yield 14b.
We then tested the chemical activation of the stable mass-tags.
After coupling 13 with butyl amine, CPG resins were treated with
a 0.1 M solution of SiCl4/TFA in dichloromethane producing an
instantaneous orange colour in the flow-through solution, charac-
teristic of the presence of the S-pixyl cation (path B, Scheme 4). The
generation of the S-pixyl cation could be explained by the initial
reduction of the sulfoxide function to the corresponding sulfide by
SiCl4 and subsequent generation of the S-pixyl cation under
reductive-acidolysis conditions.7 (MA)LDI analysis of the flow-
through solution confirmed the presence of tagged systems 15.
S(O)-pixyl tags were exposed to the conditions required for the
liberation of oligonucleotides and the deprotection of the
phosphate groups. Resins 13a,b were coupled with different
amines, depending on whether they were exposed to acid or not;
systems that were not treated with acid were reacted with
butylamine (path C, Scheme 4) and systems treated with acid
were reacted with pentylamine (path D, Scheme 4).
Oligonucleotides 17a,b were deprotected and cleaved off the resin
by treatment with ethanolamine. A fraction of the obtained
solutions were purified by HPLC, confirming the stability of the
S(O)-pixyl tags to the experimental conditions.
Scheme 3 Synthesis of S(O)-pixyl mass tags.
For evaluation as mass tags, S(O)-pixyls 11 were incorporated
into the 59-end of model oligonucleotides 12 using a standard
protocol on an automated DNA synthesiser (Scheme 4).
A series of tests and experiments were designed to evaluate the
scope of the novel S(O)-pixyl tags. We aimed to determine their
properties as protecting groups followed by the optimisation of
their use as mass tags. Oligonucleotides protected with NHS
functionalised S(O)-pixyl tags were treated with amines and so
different mass tags were generated. Table 2 lists the possible ions
shown in Scheme 4: ions 14 would retain the sulfoxide function
whereas ions 15 and 16 would be in the corresponding sulfide
Our efforts were then directed to finding the best conditions for
the MS analysis of labelled oligonucleotides 17a,b through the
incorporated S-pixyl mass tags. For the chemical activation of the
mass-tags, a range of reagents for the deoxygenation of sulfoxides
in aqueous media was evaluated, among them Na2S2O4, Na2S2O5
and HI.11 The (MA)LDI analysis of the different crude mixtures
revealed the presence of the tags 15 and 16, with cleaner spectra
when the analysis was carried out without matrix (under LDI
conditions). This fact confirms the exceptional ability of the
S-pixyl tags to produce MS spectra under LDI conditions, with no
other peaks detectable.
To our surprise, we found that the best analytical results were
obtained when the oligonucleotide solutions were directly spotted
in the MS plate, without any pre-treatment to release the tags and
analysed under LDI conditions, regardless of whether they had
been purified by HPLC, eliminating the possibility of false
positives due to left over tags.
Despite analysing the non-purified reaction mixtures in no-
matrix conditions, we obtained clean MS spectra containing
exclusively the signals of the S-pixyl mass-tags; peaks related to
oligonucleotides were not detected. This was a remarkable finding,
since it effectively eliminated the cleanup and sample preparation
Scheme 4 From the oligonucleotide synthesis to the mass spectrometer.
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steps prior to performing the MS analysis, which are regarded as
weaknesses in MALDI MS analyses.12
In this communication we have introduced the concept of trityls
with adjustable stability with applications in mass spectrometry.
Using the S-pixyl skeleton as a starting framework and taking
benefit from the redox chemistry and photochemistry of its sulfur
atom and the unique properties of the S-pixyl group as a whole,
we have prepared bifinctional trityl systems which have application
as mass tags.
For instance, Fig. 1 shows the MS spectra obtained under these
conditions of a mixture of oligonucleotides 17a (0.5 ml; 1e–4 mM),
obtained from 13a through paths C and D in Scheme 4. The spect-
rum shows the peaks corresponding to ions 15a and 16a, Table 2.
In addition, we believe the compounds we have described have
the potential to be useful in other areas, such as protecting
groups15 or safety-catch handles for solid phase chemistry.16
Investigations are being carried out in our laboratory to
delineate the scope and limitations of our system with adjustable
stability.
Pablo L. Bernad, Jr,a Safraz Khan,a Vladimir A. Korshun,b
Edwin M. Southernc and Mikhail S. Shchepinov*a
aTridend Technologies (a Division of OGT), Hirsch Building, Begbroke
Business & Science Park, Sandy Lane, Yarnton, Oxford, UK OX5 1PF.
E-mail: misha@tridend.com; Fax: +44(0)186 5842 116
bShemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-
Maklaya 16/10, Moscow, 117 997, Russia
cOxford Gene Technology (IP) Ltd, Hirsch Building, Begbroke Business
& Science Park, Sandy Lane, Yarnton, Oxford, UK OX5 1PF
Fig. 1 MS LDI spectrum of an equimolar mixture of oligonucleotides
17a (R 5 NHC4H10 and NHC5H12): only mass tags are detected.
These results show that the oxidised S(O)-pixyl tags are reduced
and cleaved by the laser in the mass spectrometer. A literature
review revealed that certain sulfoxides can be converted to their
corresponding sulfides by direct photolysis in the presence of
sensitizers,13 and that S-pixyl protected nucleosides can be
deprotected when irradiated at 300 nm.14 Thus, these two
processes occur simultaneously under LDI analytical conditons.
Confirming our previous findings, LDI MS analysis of an
equimolar mixture of oligonucleotides protected with mono-
methoxy S(O)-pixyl and dimethoxy S(O)-pixyl (oligonucleotides
17a and 17b through path C, Scheme 4) showed the correlation
between the stability of the corresponding S-pixyl cations and the
intensity of the peaks they yield in MS (Fig. 2, peaks
corresponding to ions 15a and 15b in Table 2). Despite the lower
acid stability of the dimethoxy S(O)-pixyl tags, they could be used
when higher degree of MS sensitivity is required.
Notes and references
{ Alternative approaches using different heteroatoms (N, S, Se) and
structures are included in the supplementary information.{
§ In recent work carried out in our laboratory, we have successfully
developed a novel synthetic route to 3-methoxythioxanthenone and 3,6-
dimethoxythioxanthenone, from which 3-methoxy and 3,6-dimethoxy
substituted S-pixyl derivatives are readily available. Submitted for
publication.
1 I. G. Gut, Hum. Mut., 2004, 23, 437; Z. Meng, T. A. Simmons-Willis
and P. A. Limbach, Biomol. Eng., 2004, 21, 1; K. Tang, D. Fu, S. Kotter,
R. J. Cotter, C. R. Cantor and H. Koster, Nucl. Acid Res., 1995, 23,
3126.
2 R. Aebersold and M. Mann, Nature, 2003, 422, 198.
3 A. Leitner and W. Lindner, J. Chromatogr., B, 2004, 813, 1.
4 M. S. Shchepinov and V. A. Korshun, Chem. Soc. Rev., 2003, 32,
170.
5 M. S. Shchepinov, R. Chalk and E. M. Southern, Tetrahedron, 2000, 56,
2713.
6 N. Balgobin and J. B. Chattopadhyaya, Chem. Scr., 1982, 19,
143.
7 J. M. Samanen and E. Brandeis, J. Org. Chem., 1988, 53, 561; Y. Kiso,
T. Kimura, M. Yoshida, M. Shimokura, K. Akaji and T. Mimoto,
J. Chem. Soc., Chem. Commun., 1989, 1511.
8 N. C. Deno, J. J. Jaruzelski and A. Schriesheim, J. Am. Chem. Soc.,
1955, 77, 3044.
9 E. J. Corey, M. Kang, M. C. Desai, A. K. Ghosh and I. N. Houpis,
J. Am. Chem. Soc., 1988, 110, 649.
10 C. Bleasdale, S. B. Ellwood and B. T. Golding, J. Chem. Soc., Perkin
Trans. 1, 1990, 803.
11 M. Madesclaire, Tetrahedron, 1988, 44, 6537 and references therein.
12 L. H. Cohen and A. I. Gusev, Anal. Bioanal Chem., 2002, 373, 571.
13 G. M. Gurria and G. H. Posner, J. Org. Chem., 1973, 13, 2419.
14 M. P. Coleman and M. K. Boyd, J. Org. Chem., 2002, 67, 7641.
15 M. Pa´tek and M. Lebl, Pept.: Chem., Struct. Biol., Proc. Am. Pept
Symp., 13th, 1994, 146.
Fig. 2 MS LDI spectrum of an equimolar mixture of oligonucleotides
17a and 17b; R 5 NHC4H10.
16 M. Pa´tek and M. Lebl, Biopolymers (Pept. Sci.), 1988, 47, 353.
3468 | Chem. Commun., 2005, 3466–3468
This journal is ß The Royal Society of Chemistry 2005