Designed thiazole orange nucleotides for the synthesis of single labelled oligonucleotides that fluoresce upon matched hybridization

Article abstract of DOI:10.1039/c000697a

Probe molecules that enable the detection of specific DNA sequences are used in diagnostic and basic research. Most methods rely on the specificity of hybridization reactions, which complicates the detection of single base mutations at low temperature. Significant efforts have been devoted to the development of oligonucleotides that allow discrimination of single base mutations at temperatures where both the match and the mismatch probe-target complexes coexist. Oligonucleotides that contain environmentally sensitive fluorescence dyes such as thiazole orange (TO) provide single nucleotide specific fluorescence. However, most previously reported dye-DNA conjugates showed only little if any difference between the fluorescence of the single and the double stranded state. Here, we introduce a TO-containing acyclic nucleotide, which is coupled during automated oligonucleotide synthesis and provides for the desired fluorescence-up properties. The study reveals the conjugation mode as the most important issue. We show a design that leads to low fluorescence of the unbound probe (background) yet permits TO to adopt fluorescent binding modes after the probe-target complex has formed. In these probes, TO replaces a canonical nucleobase. Of note, the fluorescence of the "TO-base" remains low when a base mismatch is positioned in immediate vicinity.

Full text of DOI:10.1039/c000697a

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Designed thiazole orange nucleotides for the synthesis of single labelled
oligonucleotides that fluoresce upon matched hybridization†
Lucas Bethge, Ishwar Singh and Oliver Seitz*
Received 15th January 2010, Accepted 12th February 2010
First published as an Advance Article on the web 23rd March 2010
DOI: 10.1039/c000697a
Probe molecules that enable the detection of specific DNA sequences are used in diagnostic and basic
research. Most methods rely on the specificity of hybridization reactions, which complicates the
detection of single base mutations at low temperature. Significant efforts have been devoted to the
development of oligonucleotides that allow discrimination of single base mutations at temperatures
where both the match and the mismatch probe–target complexes coexist. Oligonucleotides that contain
environmentally sensitive fluorescence dyes such as thiazole orange (TO) provide single nucleotide
specific fluorescence. However, most previously reported dye–DNA conjugates showed only little if any
difference between the fluorescence of the single and the double stranded state. Here, we introduce a
TO-containing acyclic nucleotide, which is coupled during automated oligonucleotide synthesis and
provides for the desired fluorescence-up properties. The study reveals the conjugation mode as the most
important issue. We show a design that leads to low fluorescence of the unbound probe (background)
yet permits TO to adopt fluorescent binding modes after the probe–target complex has formed. In these
probes, TO replaces a canonical nucleobase. Of note, the fluorescence of the “TO–base” remains low
when a base mismatch is positioned in immediate vicinity.
soluble dyes with DNA has been reported.10 In attempts to
develop fluorescence-up oligonucleotides, TO, oxazole yellow and
Introduction
ethidium dyes have been attached to oligonucleotides (Fig. 1).11–14
However, most of the reported single fluorophore-labelled DNA
conjugates showed only little if any difference between the
fluorescence of the single and the double stranded state.15 Here,
we introduce a new TO nucleotide building block, which provides
for the desired enhancements of fluorescence upon hybridization.
The TO building block can be used in automated synthesis of
oligonucleotides. Our study reveals the conjugation mode as the
most important issue in the design of single labelled fluorescence-
up probes. This issue has received little attention in previous
studies. We show a design that confers a significant reduction
of the (background) fluorescence of single stranded state while
maintaining the high fluorescence of the double stranded state.
Thus, the resulting oligonucleotide probes provide enhancements
of fluorescence upon hybridization. We demonstrate that the
fluorescence of the thiazole orange nucleotide responded to
the introduction of mismatched base pairs. This enabled the
discrimination of single base mutations even at temperatures
where both the match and the mismatch probe–target complexes
coexist.
Fluorescent nucleotides are invaluable tools for studies of DNA–
DNA and DNA–protein recognition processes.1,2 For example,
environmentally sensitive fluorochromes can be used to report
changes of local DNA structure.3,4 This property is of interest
in the field of nucleic acid diagnostics, in particular for the
analysis of single base mutations. Considerable efforts have been
devoted to the development of fluorogenic oligonucleotide probes
that allow real-time detection of nucleic acids.2,5 Most DNA-
detection methods rely on the interaction between two, terminally
appended dyes.6 The sequence specificity of these methods is
typically determined by the selectivity of the hybridization.
Oligonucleotide probes that contain a single fluorescent nucleotide
can offer an additional level of sequence discrimination that is
provided by the environmental sensitivity of the fluorophore.7,8
In order to provide for high sensitivity and specificity of DNA
detection, useful probes should have (a) low fluorescence in-
tensity in the single stranded state, (b) high fluorescence inten-
sity after hybridization with matched targets and, ideally, (c)
weak fluorescence after hybridization with (single) mismatched
targets.
The contact of dyes with nucleobases usually leads to quenching
of fluorescence. Phenanthridinium dyes and some cyanines are
notable exceptions.9 Thiazole orange (TO), oxazole yellow and
their dimeric forms are perhaps the most powerful DNA stains.
Up to 3200-fold enhancement of fluorescence upon binding of
Humboldt-Universita¨t zu Berlin, Department of Chemistry, Brook-Taylor-
Str. 2, Berlin, D-12489, Germany. E-mail: oliver.seitz@chemie.hu-berlin.de
† Electronic supplementary information (ESI) available: Experimental
details and characterisation data. See DOI: 10.1039/c000697a
Fig. 1 DNA conjugates of ethidium bromide and thiazole orange.
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Results and discussion
Design
DNA stains such as thiazole orange experience dramatic flu-
orescence enhancements upon binding to nucleic acids.9,10 In-
tercalation and minor groove binding of double helical DNA
are preferred binding motives and nucleic acid recognition is
predominantly driven by rather unselective stacking and ionic
interactions. Thus, single stranded DNA also provides opportuni-
ties for binding of thiazole orange. Such interactions are probably
facilitated in oligonucleotide conjugates due to proximity effects.
The accompanying increase of fluorescence of the unbound state
explains why it has been so difficult to develop single labelled
fluorescence-up probes.12–14 The challenge is to design a linkage
that prevents stacking of TO within the single stranded probe
conjugate yet permits intercalation (or minor groove binding) and,
hence, fluorescence after the probe–target complex has formed.
According to this analysis, tethering via flexible linkers shall
be avoided. We assumed that the steric hindrance provided by
the glycoside in the spacer-free TO nucleotide A (Fig. 2) would
confer the desired properties. Stacking in the single stranded state
would be hindered. In the double helix, TO should occupy the
position of a canonical nucleobase. This may enforce stacking and
fluorescence may occur.
Scheme 1 Synthesis of (R)-glycidol(TO) (1). (i) 1. MgBr2, CH2Cl2, 0 ◦C,
2. lepidine (5), RT, 2 d, 77%; (ii) DMTrCl, imidazole, DMF, RT, 12 h, 95%;
(iii) 8, NEt3, CH2Cl2, RT, 24 h, 98%.
in the base-promoted reaction of 7 with the benzothiazolium
compound 8.
The synthesis of (D)- and (L)-serinol-based nucleosides was
commenced from (D)- and (L)-serine, which were converted to the
DMTr-protected (D)- or (L)-serinol building blocks 9D and 9L
by applying procedures described in the literature (Scheme 2).20
The coupling of the carboxyalkylated TO dyes such as 11 was
surprisingly difficult. The coupling proceeded smoothly when the
alcohol in 9D/9L was protected as TBDMS ether in 10D/10L.
Subsequent TBAF treatment furnished the TO-nucleosides 2D,
2L, 3D, 3L, which were converted to the phosphoramidites 15–17
(Scheme 3) and used without further purification in automated
DNA synthesis.
Fig. 2 Structures of a theoretical desoxyribose–TO monomer and of the
desired acyclic TO-labelled DNA monomers.
Scheme 2 Synthesis of TO-labelled serinol monomers 2L, 2D, 3L and
3D. The scheme only depicts the synthesis of the enantiomerically pure
monomers derived from (L)-serine. Monomers derived from (D)-serine
were synthesised in the same procedure. (iv) TBDMSCl, NEt3, DMAP,
CH2Cl2, 0 ◦C, 16 h, 86%; (v) 11 or 12, PyBOP, NMM, PPTS, DMF, RT,
16 h, 56%, (vi) TBAF, THF, 2 h, RT, 22–33%.
The probe design is reminiscent of the recently disclosed FIT
probes (FIT = forced intercalation), in which TO is used as an
artificial base in peptide nucleic acids (PNA).7,16,17 However, syn-
thesis of ribose-linked TO–DNA conjugates is virtually impossible
because the glycosidic bond in the hypothetical nucleotide A
would not be strong enough to sustain the conditions of DNA
synthesis. We therefore decided to explore open chain analogues.
We considered the use of sterically confined (R)-glycidol backbone
in 1, which is known from glycerol nucleic acid (GNA) and
probably the smallest backbone capable of Watson–Crick base
pairing.18,19 The serinol backbone in 2 and 3 is known from
amino acid nucleic acids (AANA)20 and provides bioisosteric
internucleotide linkage while the flexibility of the chromophore–
backbone linkage is restricted by means of the peptide bond.
Scheme 3 Synthesis of phosphoramidites 15–17. (i) Dicyanoimidazole,
18, MeCN, 2 h, 65%; (ii) DIPEA, 19, CH2Cl2, full conversion.
Synthesis of building blocks and TO-DNA-conjugates
For the synthesis of the glycerol–TO nucleoside 1 (Scheme 1),
(S)-glycidol (4) was treated with MgBr2 and lepidine (5). The
Lewis acid-meditated ring opening furnished lepidinium salt 6,
which was subsequently converted in the presence of imidazole
into the DMTr-ether 7. The TO chromophore in 1 was established
The synthesis of TO-nucleotide-containing oligonucleotides
was performed by applying known protocols.21 The TO-labelled
phosphoramidites were coupled at 0.2 M (CH3CN) concentration
over a period of 30 minutes. Their coupling efficiency was equal
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Table 1 Melting temperatures (TM)a of duplexes of TO–DNA conjugates 15–19 or unmodified reference DNA 20 with complementary DNA 21–24
5¢-G C C G T A A A T A G C C G-3¢
5¢-G C C G T X Z X T A G C C G-3¢
3¢-C G G C A Y T Y A T C G G C-5¢
(20)
(21X-25X, X = A, T, C, G)
(Y = T, 26; A, 27; G, 28; C, 29)
Monomer Z
XY = AT
XY = TA
21T·27
XY = CG
21C·28
XY = GC
21G·29
dA
1
20·26
55 ◦
21A·26
54 ◦
22A·26
55 ◦
23A·26
51 ◦
24A·26
53 ◦
25A·26
52 ◦
C
C
56 ◦
C
62 ◦
C
62 ◦
C
2L
2D
3L
3D
22T·27
57 ◦
23T·27
53 ◦
24T·27
49 ◦
25T·27
52 ◦
22C·28
65 ◦
23C·28
61 ◦
24C·28
59 ◦
25C·28
62 ◦
22G·29
61 ◦
23G·29
59 ◦
24G·29
59 ◦
25G·29
59 ◦
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
a Recorded as denaturation curves. 20, 21X–25X, 26–29 1 mM in 10 mM NaH2PO4 buffer, 100 mM NaCl, pH = 7, 1 h degassed.
to commercially available canonical phosphoramidites. For the
release of 5¢-DMTr-oligonucleotides, the solid support was treated
with aqueous concentrated ammonia solution for 4 h at 55 ◦C. The
oligonucleotides were purified by HPLC prior to, as well as after,
acidolytic DMTr-cleavage and desalted by size exclusion filtration.
stability (22T·27 vs. 24T·27, DTM = 8 ◦C or 22C·28 vs. 24C·28,
DTM = 6 ◦C). By contrast, the (D)-backbone better tolerated the
linker extension. This may indicate that the (D)-serinol linkage in
duplex DNA provides TO with a higher degree of flexibility than
the (L)-form, which is in line with the lower stability of duplexes
that contained the TO-nucleotide 2D rather than 2L.
Thermal stability
Fluorescence measurements
The TO–DNA conjugates 21X–25X were hybridized with com-
plementary DNA 26–29 (Table 1). The thermal stability was
assessed by means of UV-monitored melt analyses. The sigmoid
shape of melting curves suggested cooperative base-pairing. The
replacement of the central adenine in 20 by TO removes a
Watson–Crick base pair. Of note, TO was found to compensate
not only for the loss of hydrogen bonding, but even for the
perturbations caused by the open chain ribose analogues. For
example, the (S)-glycerol(TO) containing duplex 21A·26 revealed
almost unchanged thermal stability compared with the unmod-
ified complex 20·26, although canonical (S)-glycerol nucleotides
destabilize duplex DNA by DTM = 8 ◦C.18 Moreover, it has been
reported that (D)- o◦r (L)-serinol(TO) nucleotides reduce duplex
stability by DTM = 9 C or 12 ◦C.20 In contrast, the TO nucleotides
in 22A·26 and 23A·26 showed little if any destabilization. The
extension of the linker that connects the TO with the (L)-serinol
backbone by one carbon atom resulted in decreases of duplex
The responsiveness of TO fluorescence was studied by measure-
ments of emission spectra prior to and after hybridization with
fully complementary DNA. Fig. 3 shows fluorescence spectra of
probes, which differ only in the backbone. A comparison of the
probes in the single stranded form revealed major differences.
The glycerol–TO in 21T and the (L)-serinol–TO nucleotide in
22T had low emission intensity. The (D)-serinol in single strands
23T and 25T as well as the (L)-serinol that carried the C3-linked
chromophore in 24T conferred significantly higher intensities
of TO fluorescence. The hybridization of probes 23T, 24T and
25T, all of which showed high single strand fluorescence, with
complementary DNA 27 was accompanied by decreases of TO
emission. Such decreases of fluorescence have previously been
observed for oligonucleotides in which TO was attached via a
flexible tether.12,22 From the 5 modifications studied, only the
(L)-serinol (22A–22G) conferred enhancements of fluorescence
Fig. 3 Fluorescence emission spectra of TO–DNA conjugates 21T–25T before (dashed line) and after (solid line) addition of fully complementary DNA
27 at 25 ◦C, lex = 495 nm, SlitEx = 5, SlitEm = 2.5, 21T–25T, 27 1 mM in phosphate buffer (see conditions in Table 1).
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upon matched hybridization. The general trend of the fluorescence
properties was retained in probes that contained different next
neighbours of TO (ESI, Tables S1 and S2†). Again, the (S)-glycidol
and the (D)-serinol failed in providing the enhancement of TO
emission upon matched hybridization.
higher than the fluorescence of TT-mismatched duplex 22T·27T
(Fig. 4B). The TG- and the TC-mismatch were discriminated
with specificity factors of D = 6.5 and D = 5.6, respectively.
This specificity was achieved at 25 ◦C, a temperature where both
matched and single mismatched probe–target complexes exist.
The discrimination was even higher when the hybridization was
The stacking of TO with nucleobases closes fluorescence decay
channels because torsions around the central methine bridge in the
excited state are restricted.17,23 Recent investigations have shown
that a tight TO–nucleobase contact provides options for depletion
of the TO excited state.4 This complicates the analysis of the
fluorescence of “TO–bases” in DNA. High hybridization-induced
fluorescence enhancements probably call for a subtle balance
between linker constraints required to prevent TO–nucleobase
stacking in the single stranded state, and linker flexibility to avoid
strong electronic coupling between TO and nucleobases in the dou-
ble stranded state. The sterically confined glycerol in 21T efficiently
reduced single strand fluorescence, but forced TO to adopt non-
fluorescent modes in the double strand. On the contrary, the linkers
3D in 24T and 3L in 25T were probably too flexible to limit single
strand fluorescence. High fluorescence was also observed for single
stranded 23T. This is in line with the melt analyses which suggested
that 23T confers more degrees of freedom than 22T. Only the
(L)-serinol with the short carboxymethyl spacer in 2L provided
the desired properties. The nucleotide apparently is sufficiently
constrained to reduce background fluorescence yet allows TO to
adopt fluorescent binding modes when in the double strand.
The conjugation mode enforces TO to respond to changes of lo-
cal architecture. Mismatched base-pairs “soften” the duplex struc-
ture. We assumed that the increase of available space introduced
by mismatched base-pairs would attenuate the fluorescence of the
“TO–nucleobase”. Indeed, the enhancements of TO fluorescence
upon mismatched hybridization of 22T are significantly lower
than the fluorescence enhancements upon matched hybridization
(Fig. 4A). The background-corrected specificity factor D is a
measure for the power of discrimination between matched and
mismatched targets (Fig. 4B). Most fluorescence-up hybridization
probes provide for D ª 1 at temperatures below the Tm of the
mismatched probe–target complex. In contrast, the background-
corrected fluorescence of the matched duplex 22T·27A was 9-fold
◦
performed at 50 C. Under these conditions the presence of the
matched target 27A resulted in a 30-fold higher background-
corrected fluorescence than the presence of the mismatched target
27C.
Detection of G12V mutation of human Ras
The findings discussed above are not restricted to 22T. The
(L)-serinol(TOQ2)-monomer was also incorporated into a probe
designed to recognize a segment of the human ras gene, carrying a
carcinogenic G12V mutation, caused by a single base exchange of
a G (wild type) against T (mutant). Hybridization of 30 with fully
complementary DNA 31Mu at 25 ◦C is signalled by enhancement
of the TO-fluorescence. Even at this temperature, which is far
below the Tm of the mismatched probe–target-complex, (Tm
=
59 ◦C) the presence of the matched complex can be distinguished
from the presence of the single mismatched complex (Fig. 5A). A
notable feature of probes such as 30, in which TO serves as base
surrogate, is the ability to signal the presence of complementary
target and to discriminate single mismatched targets over a broad
temperature range. Fig. 5B shows the temperature dependence
of the hybridization-induced enhancements of TO fluorescence,
as well as the background corrected discrimination of single
mismatched target 31WT. The fluorescence responsiveness was
highest at a temperature close to the Tm = 64 ◦C of the matched
probe–target duplex. This is in the range of typical annealing
temperatures used in quantitative PCR analysis. Moreover, the
fluorescence intensity of matched probe–target duplex 30·31Mu is
always higher than the intensity provided of the single mismatched
probe–target duplex 30·31WT. Thus, the mismatched target can
be discriminated at any temperature below 64 ◦C. This behaviour
stands in stark contrast to detection methods that solely draw
upon the specificity of probe-target recognition.
Fig. 5 Background-corrected fluorescence of probe targeting G12V
mutation of human Ras gene (A) 30·31Mu (match, solid line) vs. 30·31WT
(mismatch, dashed line) at 25 ◦C. (B) Temperature dependent fluorescence
enhancement (solid line) of 30 upon hybridization with 31Mu and
background corrected discrimination (dashed line) of 30·31Mu (match)
Fig. 4 Background-corrected (A) relative fluorescence at 25 ◦C and
(B) discrimination of single mismatched vs. fully complementary targets.
22T·27A (match) vs. 22T·27T, 22T·27G and 22T·27C (mismatch) at 25 ◦C
(black bars) and 50 ◦C (grey bars). Rel. Fl. Int. = (F - F0)/(Fma - F0), D =
(Fma - F0)/(Fmi - F0), (F0, fluorescence intensity of single strands; Fma
,
vs. 30·31WT (mismatch). lex = 495 nm, lem 525= 5 nm SlitEx = 5, SlitEm =
F
mi, fluorescence intensity of matched and mismatched duplexes).
5, 30, 31WT, 31WT 1 mM in phosphate buffer (see condition in Table 1).
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commercially available and not further purified prior to usage.
Column chromatography was performed with SDS 60 ACC
silica gel using a Bu¨chi SepacoreTM flash chromatographer and
TLC with E. Merck Silica Gel 60 F254 plates. Optical rotations
were measured at the sodium D-line with an Perkin Elmer 241
polarimeter using a 100 mm glass cuvette. [a]D values are given
in units of 10-1 deg cm2 g-1. 1H and 13C NMR spectra were
recorded with Bruker DPX 300 spectrometer. The signals of the
residual protonated solvent (CDCl3, CD3OD or CD3CN) were
used as reference signals. Coupling constants are given in Hz.
High resolution mass spectra were measured with a Hewlett-
Packard GCMS 5995-A (ESI+) spectrometer. Semi preparative
HPLC was carried out on a 1105 HPLC System from Gilson,
for analytical RP-HPLC a 1105 HPLC System of Gilson and a
Acquity UPLC System of Waters were used. A UV-detector at a
wavelength l = 260 nm and l = 520 nm was used for the detection.
Semi preparative separations were carried out by using a Polaris
C18 A 5m (PN A 2000-250 ¥ 100)-column of Varian (pore size
Conclusions
It was the aim of this study to develop single labelled oligonu-
cleotides that experience strong fluorescence enhancements only
upon matched hybridization. We and others introduced thiazole
orange as an artificial base in DNA.14 The presented data highlight
the central importance of the backbone and linker structure that
anchors the “thiazole orange base”. There are, to our surprise,
only few reports about the influence of linkers on the fluorescence
properties of dye–nucleotide conjugates.24 An analysis of the linker
properties required to achieve the desired fluorescence behaviour
of single labelled dye–DNA conjugates has not been reported.
Our results clearly indicate that Tm optimization25 is a poor
guideline for the design of single labelled fluorescence-up probes
(exemplified by the high Tm of weakly fluorescent duplexes that
contain the glycidol-TO-DNA 21T).
The careful linker optimization presented in this study may
enable a generic design of TO labelled DNA oligonucleotides that
gain fluorescence upon hybridization. Such properties have until
recently only been provided by TO-labelled peptide nucleic acids
(PNA) such as the Light-Up probes26 and FIT-PNA.4,7,17 Both
probes capitalize on the low affinity of TO for PNA, which helps
reduce the fluorescence of the unbound state (= background).
Despite the complications that arise from the intrinsically higher
single strand fluorescence, DNA-based TO probes offer oppor-
tunities difficult to access by the PNA backbone. The DNA
backbone is amenable to cellular delivery by means of lipofection,
which may facilitate the detection of DNA and RNA in live cells.
DNA is easier to synthesize and enzymatic methods allow further
modification.
In summary, we synthesised the first DNA conjugates, in
which thiazole orange served as an artificial base that signals
hybridization by enhancements of fluorescence. The measured
data shows that our TO–DNA conjugates proved responsive
to adjacent base mismatches. This suggests applications in the
analysis of single base mutations. The study revealed the critical
influence of the backbone and the linker that connects the
“thiazole orange base” with the oligonucleotide. However, we
wish to note that changes of the anchor position may allow
for further improvements. This investigation was focused on
N-linked TO conjugates. Attachment of TO through one of
the adjacent quinoline carbon atoms would change the angle
in which TO penetrates the base stack, which may have an
impact on the fluorescence intensity achievable through matched
hybridization. Our analysis suggests that hybridization-induced
fluorescence enhancements can probably only be obtained when
the backbone/linker structure prevent intramolecular thiazole
orange–nucleobase stacking in the single stranded state yet confers
sufficient flexibility to allow thiazole orange to adopt fluorescent
binding modes in the double stranded state. Ongoing work is
directed to the homogeneous detection of RNA in biological
samples.
◦
-1
˚
220 A) at a flow rate of 4 mL min at 55 C. Analytical HPLC
was carried out by using a XBridge C18 5 m (250 ¥ 046)-column
of Waters (pore size 130 A) at a flow rate of 1 mL min at 55 ◦C
-1
˚
or a BEH 130 C18 1.7 mm (2.1 ¥ 50)-column of Waters (pore
size 130 A) at a flow rate of 1 mL min at 55 ◦C. As mobile
-1
˚
phase a binary mixture of A (0.1 M TEAA buffer, pH = 7, aq.)
and B (acetonitrile) was used. MALDI-TOF mass spectra were
measured on a Voyager-DETM Pro Biospectrometry Workstation
of PerSeptive Biosystems. As matrix was used a solution of 1 part
of a solution of 20 mg 2¢,4¢,6¢-trihydroxyacetophenone in 1 mL
MeCN and 1 part of a solution of 50 mg diammonium citrate in
1 mL water was. The sample concentration was calculated by using
oligo calculation at www.proligo.com and e(TO) = 6600 L mol-1.
UV-Vis measurements were performed with a Varian Cary 100,
fluorescence emission spectra were recorded on a Varian Cary
Eclipse using 10 mm UV quartz cuvettes.
(R)-2-Lepidinium-propane-1,3-diol bromide (6)
To a cooled (0 ◦C) solution of (S)-glycidol (4), (1.00 g,13.5 mmol,
0.9 mL) in 23 mL CH2Cl2–THF (1 : 2, v/v) under argon was
added MgBr2 (4.20 g, 23.0 mmol). After 15 min TLC indi-
cated full conversion of the starting material. The reaction was
stopped by addition of 3 mL of aqueous NH4Cl solution (10%).
The resulting mixture was extracted with CH2Cl2. The organic
phase was dried over MgSO4 and filtered. The volatiles were
removed under reduced pressure yielding 1.51 g (9.74 mmol)
(S)-3-bromopropane-1,2-diol (72%). The crude material was used
without further purification. To the bromodiol was added lepidine
(5) (2.81 g, 19.6 mmol) and the mixture was stirred for 2 days at
70 ◦C. The reaction product precipitated upon addition of CH2Cl2.
The residue was washed with cyclohexane and ether and was dried
at reduced pressure. Yield: 2.23 g (7.48 mmol, 77%), brown solid,
C13H16BrNO2 (298.18 g mol-1). Rf 0.75 (CH2Cl2–MeOH, 75 : 25,
v/v). [a]D = 8.9 (c = 0.10, MeOH). HRMS (m/z) calculated:
218.1176 [C13H16NO2]+, found: 218.1174.1H-NMR (CD3OD): d =
9.19 (d, J = 6.0 Hz, 1H), 8.62-8.58 (m, 2H), 8.30-8.24 (m, 1H),
8.10-8.00 (m, 2H), 5.50 (dd, J = 14.1 Hz and 2.7 Hz, 1H), 4.84
(dd, J = 14.2 Hz and 9.4 Hz, 1H), 4.20-4.10 (m, 1H), 3.84 (dd, J =
11.2 Hz and 4.7 Hz, 1H), 3.74 (dd, J = 11.2 Hz and 6.4 Hz, 1H),
Experimental section
Solvents (CH2Cl2, THF) were dried by using the solvent pu-
rification system SPS 800 of MBraun. Dry DMF (H2O <
0.01%) was purchased from Fluka. Aqueous solutions were made
of water of Milli-Q-Pore purity. Other used compounds were
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3.33-3.32 (m, 1H), 3.099 (s, 1H); 13C-NMR (CD3OD): d = 160.9,
(4H, m, DMT), 7.20-7.38 (7H, m, DMT), 7.48 (4H, m, DMT).
13C-NMR (CDCl3): d = -5.5 (SiMe2), 18.1 (SitBu), 25.8 (SitBu),
53.1 (CH), 55.1 (DMT-OCH3), 65.2 (CH2), 65.2 (CH2), 85.7
(DMT-Cq), 112.9 (4DMT-ArCH), 126.6 (1DMT-ArCH), 127.7
(2DMT-ArCH), 128.1 (2DMT-ArCH), 130.0 (4DMT-ArCH),
136.2 (2DMT-ArCq), 145.1 (DMT-ArCq), 158.3 (2DMT-ArCq).
150.3, 136.4, 130.5, 128.3, 123.4, 120.3, 71.0, 64.7, 61.5, 20.4.
(R)-1-DMT-2-Lepidinium-propane-3-ol bromide (7)
To a solution of (R)-3-lepidinium-propane-1,3-diol bromide (6)
(900 mg, 3.02 mmol) in 30 mL DMF was added dimethoxy-
tritylchloride (1.80 g, 5.13 mmol) and imidazole (665 mg,
9.66 mmol). After 12 h, 2 mL methanol was added. The
volatiles were removed under reduced pressure. The residue was
purified by flash column chromatography (CH2Cl2–MeOH–NEt3,
92.5 : 7 : 0.5, v/v/v). Yield: 1.14 g (1.90 mmol, 63%) brown solid,
C34H34BrNO4 (599.17 g mol-1). Rf 0.30 (CH2Cl2–MeOH, 90 : 10,
v/v). 1H-NMR (CD3OD): d = 9.31 (d, J = 6 Hz, 1H), 8.33-8.23
(m, 2H), 7.98-7.84 (m, 2H), 7.63-7.59 (m, 2H), 7.38-7.35 (m, 2H),
7.28-7.14 (m, 6H), 6.77 (d, J = 8.8 Hz, 4H), 5.30 (dd, J = 13.8 Hz
and 2.5 Hz, 1H), 4.99-4.92 (m, 1H), 4.19-4.16 (m, 1H), 3.69 (s,
6H), 3.51-3.46 (m, 1H), 3.26-3.20 (m, 1H), 2.86 (s, 3H). 13C-NMR
(CD3OD): d = 158.6, 148.8, 144.2, 137.4, 135.4, 135.3, 134.7,
129.8, 129.3, 128.0, 127.9, 127.6, 126.3, 122.4, 121.2, 118.8, 113.4,
86.9, 68.4, 64.7, 60.4, 55.2, 46.1, 46.1, 20.4.
(S)-1-DMT-3-TBDMS-Serinol (10L)
As described for 10D, (S)-3-DMT-serinol (9L) (2.64 g, 6.71 mmol)
in 50 mL CH2Cl2 was reacted with triethylamine (781 mg, 1.08 mL,
7.72 mmol), dimethylaminopyridine (94.3 mg, 0.772 mmol)
and tert-butyldimethylsilyl chloride (1.11 g, 7.38 mmol). Yield:
2.11 g (4.16 mmol, 43% d. Th.), colorless syrup, C30H41NO4Si
(507.74 g mol-1). Rf 0.75 (CH2Cl2–MeOH–NEt3, 100 : 10 : 0.1,
v/v/v). [a]D = -1.7 (c = 1.00, CH3CN). 1H-NMR (CDCl3):
d = 0.05 (6H, m, SiMe2), 0.88 (9H, s, SitBu), 3.06 (2H, m, CH,
CH2), 3.17 (1H, m, CH2), 3.57 (1H, dd, J1 = 5.2 Hz, J2
=
9.8 Hz, CH2), 3.68 (1H, dd, J1 = 4.4 Hz, J2 = 9.9 Hz, CH2),
3.81 (6H, s, DMT-OCH3), 6.85 (4H, m, DMT), 7.20-7.37 (7H, m,
DMT), 7.47 (4H, m, DMT). 13C-NMR (CDCl3): d = -5.5 (SiMe2),
18.2 (SitBu), 25.8 (SitBu), 53.1 (CH), 55.1 (DMT-OCH3), 65.3
(CH2), 65.3 (CH2), 85.7 (DMT-Cq), 113.0 (4DMT-ArCH), 126.6
(1DMT-ArCH), 127.7 (2DMT-ArCH), 128.1 (2DMT-ArCH),
130.0 (4DMT-ArCH), 136.2 (2DMT-ArCq), 145.1 (DMT-ArCq),
158.3 (2DMT-ArCq).
(R)-1-DMT-Glycerol(TO) (1)
To a solution of compound (7) (1.14 g, 1.90 mmol) and 2-
methyl-methylthiobenzothiazolium tosylate (8) in CH2Cl2 was
added triethylamine (486 mg, 670 mL, 4.80 mmol). After 24 h
the volatiles were removed under reduced pressure and the residue
was purified by flash column chromatography (CH2Cl2–MeOH–
NEt3, 97.5 : 2 : 0.5, v/v/v). Yield: 1.39 g (1.86 mmol, 98%), red
solid, C42H39BrN2O4S (747.74 g mol-1). [a]D = -1.5 (c = 0.05,
CH3CN). HRMS (m/z) calculated: 667.2625 [C42H39N2O4S]+,
found: 667.2625. 1H-NMR (CDCl3/NEt3): d = 8.45 (d, J = 7.2 Hz,
1H), 8.39 (d, J = 7.5 Hz, 1H), 7.83 (d, J = 8.4 Hz, 2H), 7.53-7.11
(m, 15H), 6.93 (d, J = 7.1 Hz, 1H), 6.74 (dd, J = 8.9 Hz and
2.0 Hz, 4H), 6.48 (s, 1H), 5.14 (d, J = 7.4 Hz, 1H), 4.79-4.73 (m,
1H), 4.61-4.54 (m, 1H), 4.13 (s, 1H), 3.79 (s, 3H), 3.69 (s, 6H),
3.43-3.39 (m, 1H), 3.28-3.22 (m, 1H); 13C-NMR (CDCl3): d =
159.4, 158.4. 148.9, 144.9, 144.6, 140.0, 137.4, 135.6, 135.5, 132.8,
129.9, 128.1, 127.9, 126.9, 126.8, 125.5, 124.4, 123.9, 122.2, 117.6,
112.1, 108.1, 87.9, 86.6, 68.6, 64.7, 57.2, 55.2, 46.2, 34.4, 8.8.
(R)-1-DMT-3-TBDMS-Serinol(TOQ1) (13D)
In a volume of 30 mL DMF, thiazole orange (TOQ1, 11) (1.30 g,
3.02 mmol) was suspended by applying ultrasound over a period
of 2 min. Subsequently, PyBOP (1.56 g, 3.02 mmol) and N-
methylmorpholine (305 mg, 332 mL, 101.15 mmol) were added.
The mixture was stirred for 2 min, whereupon the red suspension
considerably cleared up. Subsequently, PPTS (759 mg, 3.02 mmol)
was added. The mixture was again stirred for 2 min. To the clear
solution was added 1.02 g (2.01 mmol) (R)-1-DMT-3-TBDMS-
serinol (10D) in 10 mL DMF. The mixture was stirred for 16 h.
The volatiles were removed under reduced pressure. The residue
was suspended in 100 mL CH2Cl2. After filtration, the residue
was discarded and the filtrate was washed with 100 mL saturated
aqueous NaHCO3 solution, twice with 100 mL water and dried
over MgSO4. The solids were removed by filtration and the
volatiles were removed under reduced pressure. The residue was
further purified by flash column chromatography. Yield: 1.03 g
(1.13 mmol, 56%), red solid, C50H56BrN3O5SSi (919.05 g mol-1).
(R)-1-DMT-3-TBDMS-Serinol (10D)
To a solution of (S)-3-DMT-serinol (9D) (2.64 g, 6.71 mmol) in
50 mL CH2Cl2 was added at 0 ◦C triethylamine (781 mg, 1.08 mL,
7.72 mmol), dimethylaminopyridine (94.3 mg, 0.772 mmol) and
tert-butyldimethylsilyl chloride (1.11 g, 7.38 mmol). After 16 h
the reaction mixture was washed with 50 mL saturated aqueous
NaHCO3 solution, 2 times with 50 mL water and once with
50 mL saturated aqueous NaCl solution. The organic phase was
dried over MgSO4. The solids were removed by filtration and
the volatiles removed under reduced pressure. The residue was
further purified by flash column chromatography. Yield: 2.92 g
(5.75 mmol, 86%), colorless syrup, C30H41NO4Si (507.74 g mol-1).
Rf 0.75 (CH2Cl2–MeOH–NEt3, 100 : 10 : 0.1, v/v/v). [a]D = 1.9
(c = 1.00, CH3CN). 1H-NMR (CDCl3): d = 0.05 (6H, m, SiMe2),
0.89 (9H, s, SitBu), 3.06 (2H, m, CH, CH2), 3.17 (1H, m, CH2),
3.59 (1H, dd, J1 = 5.2 Hz, J2 = 9.8 Hz, CH2), 3.68 (1H, dd,
J1 = 4.4 Hz, J2 = 9.9 Hz, CH2), 3.80 (6H, s, DMT-OCH3), 6.85
Rf 0.75 (CH2Cl2–MeOH00NEt3, 89.5 : 10 : 0.5, v/v/v). [a]D
=
-2.7 (c = 0.05, CH3CN). HRMS (m/z) calculated: 838.3704
[C50H56N3O5SSi]+, found: 838.3697.1H-NMR (CD3CN): d = 0.01
(3H, s, SiMe2), 0.02 (3H, s, SiMe2) 0.83 (9H, s, SitBu), 3.10
(1H, dd, J1 = 5.4 Hz, J2 = 9.1 Hz, CHH¢), 3.21 (1H, dd, J1 =
5.7 Hz, J2 = 9.0 Hz, CHH¢), 3.62 (3H, s, TO-CH3), 3.73 (8H, m,
2DMT-OCH3, CH2), 4.10 (1H, m, CH), 4.90 (2H, s, TO-CH2),
6.33 (1H, s, TO-CH), 6.83 (4H, m, 4DMT), 6.89 (1H, m, TO),
7.03 (1H, m, TO), 7.10-7.50 (14H, m, 9DMT, 5TO), 7.57 (1H, m,
TO), 7.86 (1H, m, TO), 8.17 (1H, m, TO). 13C-NMR (CD3CN):
d = -5.3 (SiMe2), -5.3 (SiMe2), 18.7 (SitBu), 26.1 (SitBu), 34.6
(TO-CH3), 52.6 (CH), 55.8 (DMT-OCH3), 57.2 (TO-CH2), 62.7
(2CH2), 86.8 (DMT-Cq), 89.5 (TO-CH), 108.5 (TO-Ar-CH), 113.7
2444 | Org. Biomol. Chem., 2010, 8, 2439–2448
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(TO-Ar-CH), 113.9 (4DMT-ArCH), 118.0 (TO-Ar-CH), 123.3
(TO-Ar-CH), 124.7 (TO-Ar-CH), 125.3 (TO-Ar–Cq), 125.7 (TO-
Ar–Cq), 126.0 (TO-Ar-CH), 127.5 (TO-Ar-CH), 127.7 (1DMT-
ArCH), 128.7 (2DMT-ArCH), 128.9 (2DMT-ArCH), 129.1 (TO-
Ar-CH), 130.9 (2DMT-ArCH), 130.9 (2DMT-ArCH), 134.0 (TO-
Ar-CH), 136.8 (DMT-ArCq), 137.0 (DMT-ArCq), 138.6 (TO-Ar–
Cq), 141.1 (TO-Ar–Cq), 145.5 (TO-Ar-CH), 146.1 (DMT-Ar–Cq),
149.6 (TO-Ar–Cq), 159.5 (2DMT-ArCq), 161.9 (TO-Ar–Cq), 166.1
(TO-Cq).
86.8 (DMT-Cq), 89.5 (TO-CH), 108.6 (TO-Ar-CH), 113.7 (TO-
Ar-CH), 113.9 (4DMT-ArCH), 123.5 (TO-Ar-CH), 124.8 (TO-
Ar–Cq), 125.4 (TO-Ar–Cq), 125.8 (TO-Ar-CH), 126.1 (TO-Ar-
CH), 127.6 (TO-Ar-CH), 127.7 (1DMT-ArCH), 128.8 (2DMT-
ArCH), 128.9 (2DMT-ArCH), 129.2 (TO-Ar-CH), 130.9 (4DMT-
ArCH), 134.1 (TO-Ar-CH), 136.9 (DMT-ArCq), 137.0 (DMT-
ArCq), 138.8 (TO-Ar–Cq), 141.3 (TO-Ar–Cq), 145.6 (TO-Ar-CH),
146.0 (DMT-Ar–Cq), 149.8 (TO-Ar–Cq), 159.5 (2DMT-ArCq),
162.0 (TO-Ar–Cq), 166.3 (TO-Cq).
(S)-1-DMT-3-TBDMS-Serinol(TOQ1) (13L)
(S)-1-DMT-Serinol(TOQ1) (2L)
As described for 13D, thiazole orange (TOQ1, 11) (1.30 g,
3.02 mmol) was reacted with PyBOP (1.56 g, 3.02 mmol), N-
methylmorpholine (305 mg, 332 mL, 101.15 mmol), PPTS (759 mg,
3.02 mmol) and 1.02 g (2.01 mmol) (R)-1-DMT-3-TBDMS-
serinol (10L). Yield: 1.22 g (1.33 mmol, 66% d. Th.), red solid,
C50H56BrN3O5SSi (919.05 g mol-1). Rf 0.75 (CH2Cl2–MeOH–
NEt3, 89.5 : 10 : 0.5, v/v/v). [a]D = 2.5 (c = 0.05, CH3CN). HRMS
(m/z) calculated: 838.3704 [C50H56N3O5SSi]+, found: 838.3705.
1H-NMR (CD3CN): d = 0.00 (3H, s, SiMe2), 0.00 (3H, s, SiMe2)
0.81 (9H, s, SitBu), 3.11 (1H, dd, J1 = 5.9 Hz, J2 = 9.1 Hz,
CHH¢), 3.21 (1H, dd, J1 = 5.3 Hz, J2 = 9.1 Hz, CHH¢), 3.66-3.77
(11H, m, TO-CH3, 2DMT-OCH3, CH2), 4.08 (1H, m, CH), 5.06
(2H, s, TO-CH2), 6.51 (1H, s, TO-CH), 6.81 (4H, m, 4DMT), 7.04
(1H, m, TO), 7.20-7.30 (8H, m, 7DMT, TO), 7.38-7.55 (7H, m,
2DMT, 5TO), 7.70 (1H, m, TO), 7.99 (1H, m, TO), 8.31 (1H,
m, TO). 13C-NMR (CD3CN): d = -5.3 (SiMe2), -5.2 (SiMe2),
18.7 (SitBu), 26.2 (SitBu), 34.4 (TO-CH3), 52.5 (CH), 55.8 (DMT-
OCH3), 57.1 (TO-CH2), 62.6 (CH2), 62.7 (CH2), 86.8 (DMT-
Cq), 89.3 (TO-CH), 108.3 (TO-Ar-CH), 113.6 (TO-Ar-CH), 113.9
(4DMT-ArCH), 117.6 (TO-Ar-CH), 123.2 (TO-Ar-CH), 124.5
(TO-Ar-CH), 125.2 (TO-Ar–Cq), 125.6 (TO-Ar–Cq), 125.8 (TO-
Ar-CH), 127.5(TO-Ar-CH), 127.7 (1DMT-ArCH), 128.8 (2DMT-
ArCH), 128.8 (2DMT-ArCH), 129.1 (TO-Ar-CH), 130.9 (4DMT-
ArCH), 133.9 (TO-Ar-CH), 136.8 (DMT-ArCq), 136.9 (DMT-
ArCq), 138.4 (TO-Ar–Cq), 140.9 (TO-Ar–Cq), 145.3 (TO-Ar-CH),
146.0 (DMT-Ar–Cq), 149.2 (TO-Ar–Cq), 158.3 (2DMT-ArCq),
161.5 (TO-Ar–Cq), 166.0 (TO-Cq).
As described for 2D, (R)-1-DMT-3-TBDMS-serinol(TOQ1) (13D)
(276 mg, 0.300 mmol) was reacted with 600 mL of a 1 M solution of
TBAF in THF (0.600 mmol). Yield: 181 g (0.225 mmol, 75%), red
solid, C44H42BrN3O5S (804.79 g mol-1). Rf 0.62 (CH2Cl2–MeOH–
NEt3, 89.5 : 10 : 0.5, v/v/v). [a]D = 4.9 (c = 0.05, CH3CN). HRMS
(m/z) calculated: 724.2840 [C44H42N3O5S]+, found: 724.2847. 1H-
NMR (CD3CN): d = 3.12 (2H, m, CH2), 3.67 (2H, m, CH2),
3.73 (6H, m, 2DMT-OCH3), 3.77 (3H, s, TO-CH3), 4.09 (1H, m,
CH), 5.03 (2H, s, TO-CH2), 6.56 (1H, s, TO-CH), 6.81 (4H, m,
4DMT), 7.10 (1H, d, J = 7.0, TO), 7.20-7.60 (17H, m, 9DMT,
7TO, 1NH), 7.75 (1H, d, J = 7.8, TO), 8.01 (1H, d, J = 7.8, TO),
8.35 (1H, d, J = 8.1, TO). 13C-NMR (CD3CN): d = 34.6 (TO-
CH3), 52.9 (CH), 55.8 (2DMT-OCH3), 57.3 (TO-CH2), 62.2 (CH2),
63.2 (CH2), 86.8 (DMT-Cq), 89.5 (TO-CH), 108.5 (TO-Ar-CH),
113.7 (TO-Ar-CH), 113.9 (4DMT-ArCH), 123.4 (TO-Ar-CH),
124.8 (TO-Ar–Cq), 125.3 (TO-Ar–Cq), 125.7 (TO-Ar-CH), 126.0
(TO-Ar-CH), 127.5 (TO-Ar-CH), 127.7 (1DMT-ArCH), 128.7
(2DMT-ArCH), 128.9 (2DMT-ArCH), 129.2 (TO-Ar-CH), 130.9
(4DMT-ArCH), 134.1 (TO-Ar-CH), 136.8 (DMT-ArCq), 136.9
(DMT-ArCq), 138.7 (TO-Ar–Cq), 141.2 (TO-Ar–Cq), 145.6 (TO-
Ar-CH), 146.0 (DMT-Ar–Cq), 149.7 (TO-Ar–Cq), 159.5 (2DMT-
ArCq), 161.9 (TO-Ar–Cq), 166.3 (TO-Cq).
(R)-1-DMT-3-TBDMS-Serinol(TOQ2) (14D)
In a volume of 15 mL DMF thiazole orange (TOQ2, 12) (844 mg,
1.77 mmol) was suspended by applying ultrasound over a period
of 2 min. Subsequently, PyBOP (921 mg, 1.77 mmol) and N-
methylmorpholine (716 mg, 659 mL, 7.08 mmol) were added. To
the clear solution was added (R)-1-DMT-3-TBDMS-serinol (10D)
(450 mg, 0.886 mmol) in 5 mL DMF. The mixture was stirred for
16 h. The volatiles were removed under reduced pressure. The
residue was suspended in 100 mL CH2Cl2. After filtration, the
residue was discarded and the filtrate was washed with 100 mL
saturated aqueous NaHCO3 solution, twice with 100 mL water
and dried over MgSO4. The solids were removed by filtration
and the volatiles were removed under reduced pressure. The
residue was further purified by flash column chromatography.
Yield: 273 mg (0.274 mmol, 31%), red solid, C51H58F6N3O5PSSi
(998.14 g mol-1). Rf 0.73 (CH2Cl2–MeOH–NEt3, 89.5 : 10 : 0.5,
v/v/v). [a]D = -2.1 (c = 0.05, CH3CN). HRMS (m/z) calculated:
852.3861 [C51H58N3O5SSi]+, found: 852.3852.1H-NMR (CD3CN):
d = -0.11 (6H, m, SiMe2), 0.71 (9H, s, SitBu), 2.81 (2H, t, J =
5.9, CH2-CO-), 2.90 (1H, dd, J1 = 5.4, J2 = 9.1, CHH¢), 2.99
(1H, dd, J1 = 6.1 Hz, J2 = 9.1 Hz, CHH¢), 3.47 (1H, dd, J1 =
5.8, J2 = 10.2, CHH¢), 3.58 (1H, dd, J1 = 4.5 Hz, J2 = 10.2,
CHH¢), 3.70 (6H, m, 2DMT-OCH3), 3.82 (3H, s, TO-CH3), 4.06
(R)-1-DMT-Serinol(TOQ1) (2D)
To a solution of 800 mg (0.872 mmol) (R)-1-DMT-3-TBDMS-
serinol(TOQ1) (13D) in 15 mL THF under an argon atmosphere
was added 1.74 mL of a 1 M solution of TBAF in THF
(1.74 mmol). After 1 h 80 mL saturated aqueous NaHCO3 solution
was added. The resulting red precipitate was collected, washed
4 times with ethyl acetate and dried under reduced pressure.
Yield: 533 g (0.663 mmol, 76%), red solid, C44H42BrN3O5S
(804.79 g mol-1). Rf 0.62 (CH2Cl2–MeOH–NEt3, 89.5 : 10 : 0.5,
v/v/v). [a]D = -5.1 (c = 0.05, CH3CN). HRMS (m/z) calculated:
1
724.2840 [C44H42N3O5S]+, found: 724.2842. H-NMR (CD3CN):
d = 3.11 (2H, m, CH2), 3.67 (2H, m, CH2), 3.73 (6H, m, 2DMT-
OCH3), 3.77 (3H, s, TO-CH3), 4.09 (1H, m, CH), 5.03 (2H, s,
TO-CH2), 6.55 (1H, s, TO-CH), 6.81 (4H, m, 4DMT), 7.09 (1H,
d, J = 7.0, TO), 7.20-7.60 (17H, m, 9DMT, 7TO, 1NH), 7.75 (1H,
d, J = 7.8, TO), 8.01 (1H, d, J = 7.8, TO), 8.35 (1H, d, J =
8.0, TO). 13C-NMR (CD3CN): d = 34.6 (TO-CH3), 52.9 (CH),
55.9 (2DMT-OCH3), 57.3 (TO-CH2), 62.2 (CH2), 63.2 (CH2),
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(1H, m, CH), 4.73 (2H, s, TO-CH2-), 6.41 (1H, d, J = 8.8, TO),
6.62 (1H, s,–CH=), 6.74 (4H, m, 4DMT), 7.03 (1H, d, J = 7.3,
TO), 7.19 (8H, m, 7DMT, TO), 7.35 (3H, m, 2DTM, 1NH), 7.54
(2H, m. 2TO), 7.67 (1H, m, TO), 7.77 (1H, m, TO), 7.90 (2H,
m, 2TO), 8.22 (1H, d, J = 7.3, TO), 8.49 (1H, d, J = 8.6, TO).
13C-NMR (CD3CN): d = -5.4 (SiMe2), -5.4 (SiMe2), 18.6 (SitBu),
26.0 (SitBu), 34.5 (TO-CH3), 35.4 (CH2-CO-), 51.7 (TO-CH2-),
52.0 (CH), 55.8 (2DMT-OCH3), 62.9 (CH2), 63.1 (CH2), 86.6
(DMT-Cq), 89.0 (–CH=), 108.8 (TO-Ar-CH), 113.5 (TO-Ar-CH),
113.8 (4DMT-ArCH), 118.6 (TO-Ar-CH), 123.4 (TO-Ar-CH),
125.2 (TO-Ar–Cq), 125.4 (TO-Ar–Cq), 125.6 (TO-Ar-CH), 126.4
(TO-Ar-CH), 127.6 (1DMT-ArCH), 128.6 (DMT-ArCH), 128.9
(DMT-ArCH), 129.1 (TO-Ar-CH), 130.8 (2DMT-ArCH), 130.9
(2DMT-ArCH), 134.1 (TO-Ar-CH), 136.9 (DMT-ArCq), 137.0
(DMT-ArCq), 138.1 (TO-Ar–Cq), 141.4 (TO-Ar–Cq), 145.5 (TO-
Ar-CH), 146.1 (DMT-Ar–Cq), 150.1 (TO-Ar–Cq), 159.5 (DMT-
ArCq), 159.5 (DMT-ArCq), 161.7 (TO-Ar–Cq), 169.8 (TO-Ar–Cq).
78%), red solid, C45H44FN3O5S (757.91 g mol-1). Rf 0.44 (CH2Cl2–
MeOH–NEt3, 89.5 : 10 : 0.5, v/v/v). [a]D = -4.5 (c = 0.05,
CH3CN). HRMS (m/z) calculated: 738.2996 [C45H44N3O5S]+,
found: 738.2983. H-NMR (CD3CN): d = 2.80 (2H, m, CH2-
1
CO-), 2.88 (1H, dd, J1 = 5.2, J2 = 9.3, CHH¢), 2.97 (1H, dd,
J1 = 5.9 Hz, J2 = 9.2 Hz, CHH¢), 3.45 (2H, m, CH), 3.67 (6H,
m, 2DMT-OCH3), 3.73 (3H, s, TO-CH3), 4.06 (1H, m, CH), 4.66
(2H, s, TO-CH2-), 6.48 (1H, s,–CH=), 6.56 (1H, d, J = 8.9, TO),
6.70 (4H, m, 4DMT), 6.83 (1H, d, J = 7.3, TO), 7.15 (7H, m,
7DMT), 7.30 (4H, m, 2DMT, 1NH, 1TO), 7.38 (1H, m. TO),
7.47 (1H, m. TO), 7.58 (1H, m, TO), 7.66 (1H, m, TO), 7.79
(2H, m, 2TO), 8.13 (1H, d, J = 7.3, TO), 8.34 (1H, d, J = 8.7,
TO). 13C-NMR (CD3CN): d = 34.4 (TO-CH3), 35.5 (CH2-CO-),
51.6 (TO-CH2-), 52.4 (CH), 55.9 (2DMT-OCH3), 62.5 (CH2), 63.6
(CH2), 86.6 (DMT-Cq), 88.9 (–CH=), 108.8 (TO-Ar-CH), 113.5
(TO-Ar-CH), 113.9 (4DMT-ArCH), 118.5 (TO-Ar-CH), 123.3
(TO-Ar-CH), 125.2 (TO-Ar–Cq), 125.3 (TO-Ar–Cq), 125.6 (TO-
Ar-CH), 126.3 (TO-Ar-CH), 127.7 (DMT-ArCH), 127.7 (TO-
ArCH), 128.7 (2DMT-ArCH), 128.9 (2DMT-ArCH), 129.1 (TO-
Ar-CH), 130.8 (2DMT-ArCH), 130.9 (2DMT-ArCH), 134.1 (TO-
Ar-CH), 136.9 (2DMT-ArCq), 137.9 (TO-Ar–Cq), 141.2 (TO-Ar–
Cq), 145.2 (TO-Ar-CH), 146.0 (DMT-Ar–Cq), 149.8 (TO-Ar–Cq),
159.5 (DMT-ArCq), 159.5 (DMT-ArCq), 161.4 (TO-Ar–Cq), 170.6
(TO-Ar–Cq).
(S)-1-DMT-3-TBDMS-Serinol(TOQ2) (14L)
As described for 14D, thiazole orange (TOQ2, 12) (844 mg,
1.77 mmol) was reacted with PyBOP (921 mg, 1.77 mmol), N-
methylmorpholine (716 mg, 659 mL, 7.08 mmol) and (R)-1-DMT-
3-TBDMS-serinol (10L) (450 mg, 0.886 mmol). Yield: 280 mg
(0.281 mmol, 32%), red solid, C51H58F6N3O5PSSi (998.14 g mol-1).
Rf 0.73 (CH2Cl2–MeOH–NEt3, 89.5 : 10 : 0.5, v/v/v). [a]D
=
(S)-1-DMT-Serinol(TOQ2) (3L)
2.0 (c = 0.05, CH3CN). HRMS (m/z) calculated: 852.3861
[C51H58N3O5SSi]+, found: 852.3851. 1H-NMR (CD3CN): d =
-0.12 (6H, m, SiMe2), 0.70 (9H, s, SitBu), 2.80 (2H, t, J = 5.8,
CH2-CO-), 2.89 (1H, dd, J1 = 5.4, J2 = 9.1, CHH¢), 2.98 (1H, dd,
J1 = 6.1 Hz, J2 = 9.0 Hz, CHH¢), 3.47 (1H, dd, J1 = 5.8, J2 =
10.2, CHH¢), 3.58 (1H, dd, J1 = 4.4 Hz, J2 = 10.1, CHH¢), 3.69
(6H, m, 2DMT-OCH3), 3.79 (3H, s, TO-CH3), 4.06 (1H, m, CH),
4.71 (2H, s, TO-CH2-), 6.42 (1H, d, J = 8.9, TO), 6.59 (1H, s,–
CH=), 6.73 (4H, m, 4DMT), 6.98 (1H, d, J = 7.3, TO), 7.16 (8H,
m, 7DMT, TO), 7.33 (3H, m, 2DTM, 1NH), 7.46 (1H, m. TO),
7.53 (1H, m. TO), 7.65 (1H, m, TO), 7.74 (1H, m, TO), 7.87 (2H,
m, 2TO), 8.20 (1H, d, J = 7.3, TO), 8.46 (1H, d, J = 8.6, TO).
13C-NMR (CD3CN): d = -5.4 (SiMe2), -5.4 (SiMe2), 18.6 (SitBu),
26.0 (SitBu), 34.5 (TO-CH3), 35.4 (CH2-CO-), 51.7 (TO-CH2-),
52.0 (CH), 55.8 (2DMT-OCH3), 62.9 (CH2), 63.1 (CH2), 86.6
(DMT-Cq), 89.0 (–CH=), 108.8 (TO-Ar-CH), 113.5 (TO-Ar-CH),
113.8 (4DMT-ArCH), 118.6 (TO-Ar-CH), 123.4 (TO-Ar-CH),
125.2 (TO-Ar–Cq), 125.4 (TO-Ar–Cq), 125.6 (TO-Ar-CH), 126.4
(TO-Ar-CH), 127.6 (1DMT-ArCH), 128.6 (DMT-ArCH), 128.9
(DMT-ArCH), 129.1 (TO-Ar-CH), 130.8 (2DMT-ArCH), 130.9
(2DMT-ArCH), 134.1 (TO-Ar-CH), 136.9 (DMT-ArCq), 137.0
(DMT-ArCq), 138.0 (TO-Ar–Cq), 141.3 (TO-Ar–Cq), 145.4 (TO-
Ar-CH), 146.1 (DMT-Ar–Cq), 150.0 (TO-Ar–Cq), 159.4 (DMT-
ArCq), 159.5 (DMT-ArCq), 161.6 (TO-Ar–Cq), 169.8 (TO-Ar–Cq).
As described for 3D, (R)-1-DMT-3-TBDMS-serinol(TOQ2) (14L)
(280 mg, 0.281 mmol) was reacted with TBAF·3H2O(183 mg,
0.580 mmol). Yield: 187 g (0.247 mmol, 88%), red solid,
C45H44FN3O5S (757.91 g mol-1). Rf 0.44 (CH2Cl2–MeOH–NEt3,
89.5 : 10 : 0.5, v/v/v). [a]D = 3.8 (c = 0.05, CH3CN). HRMS (m/z)
calculated: 738.2996 [C45H44N3O5S]+, found: 738.2986. 1H-NMR
(CD3CN): d = 2.80 (2H, m, CH2-CO-), 2.87 (1H, dd, J1 = 5.2, J2 =
9.3, CHH¢), 2.97 (1H, dd, J1 = 6.0 Hz, J2 = 9.3 Hz, CHH¢), 3.46
(2H, m, CH), 3.66 (6H, m, 2DMT-OCH3), 3.73 (3H, s, TO-CH3),
4.05 (1H, m, CH), 4.66 (2H, s, TO-CH2-), 6.48 (1H, s,–CH=),
6.56 (1H, d, J = 8.8, TO), 6.70 (4H, m, 4DMT), 6.83 (1H, d,
J = 7.3, TO), 7.15 (7H, m, 7DMT), 7.30 (4H, m, 2DMT, 1NH,
1TO), 7.39 (1H, m. TO), 7.47 (1H, m. TO), 7.58 (1H, m, TO), 7.66
(1H, m, TO), 7.79 (2H, m, 2TO), 8.13 (1H, d, J = 7.3, TO), 8.34
(1H, d, J = 8.7, TO). 13C-NMR (CD3CN): d = 34.4 (TO-CH3),
35.5 (CH2-CO-), 51.6 (TO-CH2-), 52.4 (CH), 55.8 (2DMT-OCH3),
62.5 (CH2), 63.6 (CH2), 86.6 (DMT-Cq), 88.9 (–CH=), 108.8
(TO-Ar-CH), 113.4 (TO-Ar-CH), 113.9 (4DMT-ArCH), 118.5
(TO-Ar-CH), 123.3 (TO-Ar-CH), 125.2 (TO-Ar–Cq), 125.3 (TO-
Ar–Cq), 125.6 (TO-Ar-CH), 126.3 (TO-Ar-CH), 127.7 (DMT-
ArCH), 127.7 (TO-ArCH), 128.7 (2DMT-ArCH), 128.9 (2DMT-
ArCH), 129.1 (TO-Ar-CH), 130.8 (2DMT-ArCH), 130.9 (2DMT-
ArCH), 134.1 (TO-Ar-CH), 136.9 (2DMT-ArCq), 137.9 (TO-Ar–
Cq), 141.2 (TO-Ar–Cq), 145.2 (TO-Ar-CH), 146.0 (DMT-Ar–Cq),
149.8 (TO-Ar–Cq), 159.4 (DMT-ArCq), 159.5 (DMT-ArCq), 161.4
(TO-Ar–Cq), 170.6 (TO-Ar–Cq).
(R)-1-DMT-Serinol(TOQ2) (3D)
To a solution of (R)-1-DMT-3-TBDMS-Serinol(TOQ2) (14D)
(273 mg, 0.274 mmol) in 15 mL THF under an argon atmosphere
was added TBAF·3H2O (179 mg, 0.566 mmol). After 1 h 80 mL
saturated aqueous NaHCO3 solution was added. The resulting
red precipitate was collected, washed 4 times with ethyl acetate
and dried under reduced pressure. Yield: 162 g (0.213 mmol,
(S)-1-DMT-Glycerol(TO)-2-(O-cyanoethyl-N,N-diisopropyl)-
phosphoamidite (15)
In an argon atmosphere, (R)-glycerol(TO) (666 mg, 0.891 mmol)
(1) was dissolved in dicyanoimidazole (0.25 M, 10.2 ml
2446 | Org. Biomol. Chem., 2010, 8, 2439–2448
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solution in acetonitrile) and 2-cyanoethyl-N,N,N¢,N¢-tetraisopro-
pylphosphorodiamidite (714 ml, 2.10 mmol) was added. After 2 h
the reaction was stopped by the addition of 1 ml MeOH. Sub-
sequently, 50 ml CH2Cl2 was added and the organic phase was
three times washed with 20 ml saturated aqueous NaHCO3
solution. The solvent was removed under reduced pressure and
the crude product was further purified by flash column chromatog-
raphy (CH2Cl2–MeOH–NEt3, 97.5 : 2 : 0.5, v/v/v). Yield: 546 mg
(0.576 mmol, 65%), red solid, C51H56BrN4O5PS (947.96 g mol-1).
in 1.2 mL dry acetonitrile, filtered and used without further
purification for automated DNA synthesis. Yield: quantitative,
red solid, C53H59BrN5O6PS (1005.0 g mol-1). Rf 0.53 (CH2Cl2–
MeOH–NEt3, 89.5 : 10 : 0.5, v/v/v).
DNA synthesis, workup, purification and characterization
The oligodeoxynucleotides were assembled by using an AB
Applied Biosystems Synthesyzer Model 3400 and phosphoramidite
methodology. CPGs were purchased from Applied Biosystems and
˚
Link Technologies (1 mmol, pore size 500 A) and DNA syntheses
(D)-1-DMT-Serinol(TOQ1)-3-(O-cyanoethyl-N,N-diisopropyl)-
reagents from Applied Biosystems and Roth (dry acetonitrile, 2%
dichloroacetic acid in CH2Cl2, 4% tetrazole in acetonitrile, acetic
anhydride in 2,6-lutidin–THF (1 : 1 : 8), 16% 1-methylimidazole
in THF, Iod in water–pyridine–THF (3 : 2 : 20 : 75)). The phos-
phoramidites dT-, dAPac-, dCAc-, dGiPr-Pac, dABz-, dCBz and dGDMF
were used following the manufacturers instructions (0.1 M dry
acetonitrile). The phosphoramidites 15, 16D, 16L, 17D and 17L
were used in 0.2 M solution. The synthesizer was programmed
to yield oligomers carrying the terminal DMT protective group
(“trityl-on”). After synthesis the resulting CPGs were treated with
1 mL of saturated aqueous NH4OH was for 4 h at RT, the
supernatant was collected and the volatiles were evaporated by
using a Uniequip Speed-vac Unijet II. The crude product was
further purified by RP-HPLC. Afterwards, DMT removal was
induced through the addition of 50% AcOH aqueous solution over
30 min. The crude product was again purified by RP-HPLC. The
resulting oligomers were concentrated and desalted using NAP-5
Sephadex columns of GE Healthcare or Amersham Biosciences.
Finally, the oligomers were freeze dried with a Christ LDC 1m
lyophilizer. The residues were dissolved in water (Milli-Q-Pore)
to reach a final concentration of 0.1 mM. Identity and purity was
determined by using analytical RP-HPLC or UPLC and MALDI-
TOF mass spectroscopy (see the ESI†).
phosphoamidite (16D)
In an argon atmosphere, (D)-serinol(TOQ1) (2D) (295 mg,
0.366 mmol) was dissolved in 10 mL dry CH2Cl2 and DIPEA
(283 mg, 383 mL, 2.20 mmol) was added, followed by 2-cyanoethyl-
N,N-diisopropyl-chlorophosphorodiamidite (390 mg, 1.65 mmol,
368 mL). After 1 h, the reaction was stopped by the addition of
10 mL saturated aqueous NaHCO3 solution. The organic phase
was separated, washed twice with saturated aqueous NaHCO3
solution, dried over MgSO4 and concentrated. The crude product
was dissolved in 1.8 mL dry acetonitrile, filtered and used
without further purification for automated DNA synthesis. Yield:
quantitative, red solid, C53H59BrN5O6PS (1005.0 g mol-1). Rf 0.65
(CH2Cl2–MeOH–NEt3, 89.5 : 10 : 0.5, v/v/v).
(L)-1-DMT-Serinol(TOQ1)-3-(O-cyanoethyl-N,N-diisopropyl)-
phosphoamidite (16L)
A solution of (L)-serinol(TOQ1) (2L) (345 mg, 0.427 mmol)
in 15 mL dry CH2Cl2 was allowed to react with DIPEA
(331 mg, 447 mL, 2.56 mmol) and 2-cyanoethyl-N,N-diisopropyl-
chlorophosphorodiamidite (454 mg, 1.92 mmol, 428 mL) as
described for 16D. The crude product was dissolved in 2.1 mL
dry acetonitrile, filtered and used without further purification
for automated DNA synthesis. Yield: quantitative, red solid,
C53H59BrN5O6PS (1005.0 g mol-1). Rf 0.64 (CH2Cl2–MeOH–NEt3,
89.5 : 10 : 0.5, v/v/v).
References
1 M. J. Rist and J. P. Marino, Curr. Org. Chem., 2002, 6, 775–793; C.
Hariharan, L. B. Bloom, S. A. Helquist, E. T. Kool and L. J. Reha-
Krantz, Biochemistry, 2006, 45, 2836–2844.
(D)-1-DMT-Serinol(TOQ2)-3-(O-cyanoethyl-N,N-diisopropyl)-
phosphoamidite (17D)
2 K. Nakatani, ChemBioChem, 2004, 5, 1623–1633.
3 G. Tamulaitis, M. Zaremba, R. H. Szczepanowski, M. Bochtler and V.
Siksnys, Nucleic Acids Res., 2007, 35, 4792–4799; A. S. Piatek, S. Tyagi,
A. C. Pol, A. Telenti, L. P. Miller, F. R. Kramer and D. Alland, Nat.
Biotechnol., 1998, 16, 359–363.
A solution of (D)-serinol(TOQ2) (3D) (180 mg, 0.237 mmol)
in 10 mL dry CH2Cl2 was allowed to react with DI-
PEA (153.6 mg, 207 mL, 1.19 mmol) and 2-cyanoethyl-N,N-
diisopropyl-chlorophosphorodiamidite (141 mg, 0.594 mmol,
134 mL) as described for 16D. The crude product was dissolved
in 1.2 mL dry acetonitrile, filtered and used without further
purification for automated DNA synthesis. Yield: quantitative,
red solid, C53H59BrN5O6PS (1005.0 g mol-1). Rf 0.53 (CH2Cl2–
MeOH–NEt3, 89.5 : 10 : 0.5, v/v/v).
4 D. V. Jarikote, N. Krebs, S. Tannert, B. Ro¨der and O. Seitz, Chem.–
Eur. J., 2007, 13, 300–10.
5 R. T. Ranasinghe and T. Brown, Chem. Commun., 2005, 5487–5502.
6 R. A. Cardullo, S. Agrawal, C. Flores, P. C. Zamecnik and D. E. Wolf,
Proc. Natl. Acad. Sci. U. S. A., 1988, 85, 8790–8794; P. M. Holland,
R. D. Abramson, R. Watson and D. H. Gelfand, Proc. Natl. Acad.
Sci. U. S. A., 1991, 88, 7276–7280; S. Tyagi and F. R. Kramer, Nat.
Biotechnol., 1996, 14, 303–308.
7 O. Ko¨hler, D. Venkatrao, D. V. Jarikote and O. Seitz, ChemBioChem,
2005, 6, 69; L. Bethge, D. V. Jarikote and O. Seitz, Bioorg. Med.
Chem., 2008, 16, 114; E. Socher, L. Bethge, A. Knoll, N. Jungnick,
A. Herrmann and O. Seitz, Angew. Chem., Int. Ed., 2008, 47, 9555.
8 A. Okamoto, K. Tainaka, Y. Ochi, K. Kanatani and I. Saito, Mol.
BioSyst., 2006, 2, 122–126; A. Okamoto, K. Kanatani and I. Saito,
J. Am. Chem. Soc., 2004, 126, 4820–4827; A. Okamoto, T. Ichiba and
I. Saito, J. Am. Chem. Soc., 2004, 126, 8364–8365.
(L)-1-DMT-Serinol(TOQ2)-3-(O-cyanoethyl-N,N-diisopropyl)-
phosphoamidite (17L)
A solution of (L)-serinol(TOQ2) (3L) (150 mg, 0.198 mmol)
in 10 mL dry CH2Cl2 was allowed to react with DI-
PEA (153.6 mg, 207 mL, 1.19 mmol) and 2-cyanoethyl-N,N-
diisopropyl-chlorophosphorodiamidite (141 mg, 0.594 mmol,
134 mL) as described for 16D. The crude product was dissolved
9 J. B. Lepecq and C. Paoletti, J. Mol. Biol., 1967, 27, 87; L. G. Lee, C. H.
Chen and L. A. Chiu, Cytometry, 1986, 7, 508–517.
10 H. S. Rye, S. Yue, D. E. Wemmer, M. A. Quesada, R. P. Haugland, R. A.
Mathies and A. N. Glazer, Nucleic Acids Res., 1992, 20, 2803–2812.
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 2439–2448 | 2447
©

View Article Online
11 T. Ishiguro, J. Saitoh, H. Yawata, M. Otsuka, T. Inoue and Y. Sugiura,
Nucleic Acids Res., 1996, 24, 4992–4997; K. Tanaka, Y. Yamamoto,
A. Kuzuya and M. Komiyama, Nucleosides, Nucleotides Nucleic Acids,
2008, 27, 1175–1185.
18 E. Larsen, K. Danel, A. A. H. Abdelaleem, P. Nielsen, J. Wengel and
E. B. Pedersen, Nucleosides, Nucleotides Nucleic Acids, 1995, 14, 1097;
P. Nielsen, L. H. Dreioe and J. Wengel, Bioorg. Med. Chem., 1995, 3.
19 L. L. Zhang, A. Peritz and E. Meggers, J. Am. Chem. Soc., 2005, 127,
4174.
20 K. S. Ramasamy and W. Seifert, Bioorg. Med. Chem. Lett., 1996, 6,
1799; R. Benhida, M. Devys, J. L. Fourrey, F. Lecubin and J. S. Sun,
Tetrahedron Lett., 1998, 39, 6167.
12 E. Privat and U. Asseline, Bioconjugate Chem., 2001, 12,
757.
13 U. Asseline, M. Chassignol, Y. Aubert and V. Roig, Org. Biomol. Chem.,
2006, 4, 1949; R. Huber, N. Amann and H. A. Wagenknecht, J. Org.
Chem., 2004, 69, 744–751.
14 F. Menacher, M. Rubner, S. Berndl and H. A. Wagenknecht, J. Org.
Chem., 2008, 73, 4263.
21 M. H. Caruthers, A. D. Barone, S. L. Beaucage, D. R. Dodds, E. F.
Fisher, L. J. McBride, M. Matteucci, Z. Stabinsky and J. Y. Tang,
Methods Enzymol., 1987, 154, 287–313.
15 A very recent example suggests that fluorescence-up probes can be
obtained when TO is attached to an acyclic DNA-monomer via at
the benzothiazole ring by means of a flexible linker. The flexibility of
the linkage also allowed for minor groove interaction of TO, which
was prevented at high salt concentrations. C. Prunkl, S. Berndl, C.
Wanninger-Weiss, J. Barbaric and H. A. Wagenknecht, Phys. Chem.
Chem. Phys., 2010, 12, 32–43.
16 O. Seitz, F. Bergmann and D. Heindl, Angew. Chem., Int. Ed., 1999,
38, 2203–2206; O. Ko¨hler, D. V. Jarikote, I. Singh, V. S. Parmar, E.
Weinhold and O. Seitz, Pure Appl. Chem., 2005, 77, 327.
17 E. Socher, D. V. Jarikote, A. Knoll, L. Ro¨glin, J. Burmeister and O.
Seitz, Anal. Biochem., 2008, 375, 318.
22 S. Berndl and H. A. Wagenknecht, Angew. Chem., Int. Ed., 2009, 48,
2418.
23 V. Karunakaran, J. L. F. Lustres, L. J. Zhao, N. P. Ernsting and O. Seitz,
J. Am. Chem. Soc., 2006, 128, 2954–2962.
24 M. Masuko, H. Ohtani, K. Ebata and A. Shimadzu, Nucleic Acids
Res., 1998, 26, 5409–5416; K. Fukui and K. Tanaka, Nucleic Acids
Res., 1996, 24, 3962–3967; D. V. Jarikote, O. Ko¨hler, E. Socher and O.
Seitz, Eur. J. Org. Chem., 2005, 3187.
25 J. Barbaric, C. Wanninger-Weiss and H. A. Wagenknecht, Eur. J. Org.
Chem., 2009, 364–370.
26 N. Svanvik, G. Westman, D. Y. Wang and M. Kubista, Anal. Biochem.,
2000, 281, 26–35.
2448 | Org. Biomol. Chem., 2010, 8, 2439–2448
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