New cyanine dyes as base surrogates in PNA: Forced intercalation probes (FIT-probes) for homogeneous SNP detection

Article abstract of DOI:10.1016/j.bmc.2006.12.044

Forced intercalation probes (FIT-probes) are nucleic acid probes, in which an intercalator cyanine dye such as thiazole orange (TO) serves as a replacement of a canonical nucleobase. These probes signal hybridization by showing strong increases of fluorescence. TO in FIT-probes responds to adjacent base mismatches by attenuation of fluorescence intensities at conditions where both matched and mismatched target DNA are bound. The interesting features of TO labeled FIT-probes posed the question whether the forced intercalation concept can be extended to other cyanine dyes of the thiazole orange family. Herein, we present the synthesis of three asymmetrical cyanine dyes and their incorporation into PNA-conjugates by means of both divergent and linear solid-phase synthesis. Melting analysis revealed that the DNA affinity of PNA probes remained high irrespective of the replacement of a nucleobase by the cyanines YO (oxazole yellow), MO or JO. Of the three new tested dye-PNA-conjugates, the YO-containing PNA has properties useful for homogeneous SNP detection. YO-PNA is demonstrated to signal the presence of fully complementary DNA by up to 20-fold enhancement of fluorescence. In addition, YO emission discriminates against single base mismatches by attenuation of fluorescence. Oxazole yellow (YO) as a base surrogate in PNA may prove useful in the multiplex detection of single base mutations at non-stringent conditions.

Full text of DOI:10.1016/j.bmc.2006.12.044

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 114–125
New cyanine dyes as base surrogates in PNA: Forced intercalation
probes (FIT-probes) for homogeneous SNP detection
Lucas Bethge, Dilip Venkatrao Jarikote and Oliver Seitz*
Humboldt-Universit a¨ t zu Berlin, Institut f u¨ r Chemie, Brook-Taylor-Str. 2, 12489 Berlin, Germany
Received 1 September 2006; accepted 22 December 2006
Available online 12 January 2007
Abstract—Forced intercalation probes (FIT-probes) are nucleic acid probes, in which an intercalator cyanine dye such as thiazole
orange (TO) serves as a replacement of a canonical nucleobase. These probes signal hybridization by showing strong increases of
fluorescence. TO in FIT-probes responds to adjacent base mismatches by attenuation of fluorescence intensities at conditions where
both matched and mismatched target DNA are bound. The interesting features of TO labeled FIT-probes posed the question
whether the forced intercalation concept can be extended to other cyanine dyes of the thiazole orange family. Herein, we present
the synthesis of three asymmetrical cyanine dyes and their incorporation into PNA-conjugates by means of both divergent and linear
solid-phase synthesis. Melting analysis revealed that the DNA affinity of PNA probes remained high irrespective of the replacement
of a nucleobase by the cyanines YO (oxazole yellow), MO or JO. Of the three new tested dye–PNA-conjugates, the YO-containing
PNA has properties useful for homogeneous SNP detection. YO–PNA is demonstrated to signal the presence of fully complemen-
tary DNA by up to 20-fold enhancement of fluorescence. In addition, YO emission discriminates against single base mismatches by
attenuation of fluorescence. Oxazole yellow (YO) as a base surrogate in PNA may prove useful in the multiplex detection of single
base mutations at non-stringent conditions.
ꢀ
2007 Elsevier Ltd. All rights reserved.
1. Introduction
Recently, we introduced the concept of forced intercala-
tion probes in homogeneous detection of single base
1
–5
mutations. In these peptide nucleic acid (PNA)-based
probes, a canonical nucleobase was replaced by an inter-
calator dye such as thiazole orange (TO, 1a). After bind-
ing of FIT-probes to target DNA, the dye is forced to
intercalate next to the envisioned mutation site
(
Fig. 1). We found that this mode of intercalation pro-
vided PNA-probes that responded to adjacent base mis-
matches by attenuation of fluorescence intensities.
Figure 1. Design principle of FIT-probes. An intercalator fluorophore
F) serves as a base surrogate and is forced to intercalate adjacent to
Fluorescence-based discrimination between matched
and single mismatched hybridization at non-stringent
conditions has been reported for a variety of approach-
es including base-discriminating fluorescent nucleo-
(
the expected mutation site. High fluorescence is only obtained upon
formation of matched duplexes.
6
–10
11–15
16
17–20
sides,
modified nucleic acids, intercalator-quenched fluores-
pyrene,
fluorene, and fluorescein
share features with another recently reported probe
7–30
2
1,22
23,24
2
cent probes,
and FRET-labeled ligation probes.
Also the use of phenanthridinium as an artificial DNA
technology: Light-up probes.
Both are comprised
of the fluorophore thiazole orange and PNA for recog-
nition of DNA or RNA. However, thiazole orange in
FIT-PNA is coupled as base surrogate, while the same
dye in Light-up probes hangs via a flexible tether. It is
this binding mode that confers the unique responsive-
2
5,26
base has been demonstrated.
The FIT-PNA probes
*
0
Corresponding author. E-mail: oliver.seitz@chemie.hu-berlin.de
968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.12.044

L. Bethge et al. / Bioorg. Med. Chem. 16 (2008) 114–125
115
5
ness of TO fluorescence. The property that distin-
guishes FIT-probes from Light-up probes and most
other probes is the combination of the abilities to (1)
signal hybridization by strong enhancements of fluores-
cence emission (up to 30-fold) and (2) distinguish
matched from single mismatched hybridization at con-
ditions where both matched and mismatched target
DNA are bound.
ii
O
tBu
3
4
5
N
NH2
N
N
N
H
iii, iv
S
NEt2
OH
S
O
tBu
2
N
NH2
N
H
The interesting results obtained with thiazole orange
containing PNA posed the question whether other cya-
nine dyes of the thiazole orange family would show sim-
ilar properties. Additional colors would find utility in
multiplex assays, in which one color reports the presence
of a specific DNA target, whereas the other color detects
its single base mutant. We decided to explore forced
intercalation of three additional asymmetrical cyanine
dyes (1b–d) originally developed for unspecific DNA
i
v
X
N
SH
X
Y
vi, vii
vi, vii
comp. Y
6
6
6
a
b
c
CH S
CH O
N
N
S
X
X
N
S
S
6d
O
3
1–33
N
N
staining.
Here, we present full detail of the dye syn-
OTs
OTs
theses and the incorporation into PNA conjugates by
linear and divergent solid-phase synthesis. The utility
of the new FIT-probes in homogeneous SNP detection
is evaluated.
8
a: (X=S) 8b: (X=O)
8c: (X=S) 8d: (X=O)
Br
viii
viii
N
COOH
2. Results
1
1
a (TO-CH COOH)
9
1c (MO-CH COOH)
2
2
b (YO-CH COOH)
1d (JO-CH COOH)
2
.1. Dye synthesis
2
2
Scheme 1. Reagents and conditions: (i) EtOCSSK, EtOH, reflux, 41%;
ii) pivaloyl chloride, NEt , CH Cl
, 0 ꢁC, 94%; (iii) 2 equiv BuLi,
THF, ꢀ50 ꢁC; (iv) tetraethylthiuram disulfide (TETD), THF, ꢀ50 ꢁC,
5%; (v) NaOH, MeOH, reflux, 37%; (vi) MeI, K CO
, DMF, 0 ꢁC,
72–96%; (vii) p-TsOMe, neat, 130 ꢁC, 60–90%; (viii) 1-(carboxymeth-
yl)-4-methyl-chinolinium bromide (9), NEt , CH Cl , 30–70%.
First, the cyanine dyes thiazole orange (TO in 1a), oxa-
zole yellow (YO in 1b), thiazolopyridine (MO in 1c), and
oxazolopyridine (JO in 1d) had to be equipped with
carboxymethyl groups in order to allow attachment to
PNA (Fig. 2). The required starting materials for the
synthesis of thiazole orange derivative 1a and oxazole
yellow derivative 1b, thio-benzothiazole (6a) and thio-
benzoxazole (6b), are readily available. Thio-thiazolo-
pyridine (6c) and thio-oxazolopyridine (6d) needed to
(
3
2
2
7
2
3
3
2
2
was allowed to react with pivaloyl chloride to provide
pivalamide (4), which was treated sequentially with
3
7
3
4,35
n-BuLi and tetraethylthiuram disulfide (TETD) to
yield 2-(pivalamido)pyridin-3-yl diethylcarbamodithio-
ate (5).
be synthesized from 2-aminopyridine
3
(3) and 3-hy-
(2), respectively (Scheme 1).
5,36
droxy-2-aminopyridine
First, 2 was converted to thio-oxazolopyridine (6d) upon
treatment with potassium ethyl xanthate in refluxing
EtOH. Thio-thiazolopyridine (6c) was synthesized by
making use of ortho lithiation. 2-Aminopyridine (3)
Finally, thio-thiazolopyridine (6c) was obtained by
treatment of 5 with NaOH in refluxing MeOH. Conver-
sion to the corresponding quaternary salts 8a–d was per-
formed by treating sequentially 6a–d first with MeI in
3
5,36
presence of K CO in DMF to yield 7a–d
2
ondly with methyl p-toluenesulfonate.
and sec-
The regiose-
3
N
S
N
O
38,39
lectivity of quaternization was confirmed by HMBC
NMR experiments. Quaternary thiazolium and oxazoli-
um salts 8a–d were then allowed to react with 1-carboxy-
N
O
N
O
OH
OH
methyl-4-chinolinium bromide (9) in the presence of
40
NEt to yield all four asymmetric cyanine dyes 1a–d.
1
2
a
1b
2
3
TO-CH COOH
YO-CH COOH
2
.2. Synthesis of PNA building blocks
N
N
N
S
N
O
The use of Fmoc/Bhoc-protected PNA monomers in
automated solid-phase synthesis provides convenient ac-
cess to thiazole orange labeled PNA-oligomers. The
N
O
N
O
2
OH
MO-CH COOH
OH
JO-CH COOH
required fluorescent Fmoc-PNA-monomers were pre-
pared by coupling the asymmetric cyanine dyes TO (1a),
YO (1b), and JO (1c) to the known Fmoc/allyl-protected
1
2
c
1d
2
4
1
aminoethylglycine backbone module 10 (Scheme 2).
Figure 2. Structures of the synthesized cyanine dyes 1a–d.

116
L. Bethge et al. / Bioorg. Med. Chem. 16 (2008) 114–125
TFA in the presence of m-cresol, water, and cysteine
Dye
Cl
O
methyl ester as cation scavenger. The conjugates 19a–d
were purified by semi-preparative RP-HPLC. Analytical
RP-HPLC, MALDI-TOF mass spectroscopy, UV/vis-,
and fluorescence spectroscopy confirmed purity,
molecular masses, and integrity of the chromophores
H2
N
O
O
i
Fmoc
Fmoc
N
N
OR
N
OR
H
H
1
1
2b, Dye = YO, R = All, 33%
1
0, R = All
2d, Dye = JO, R = All, 42%
(
Table 1).
ii
1
1
3b,
Dye = YO, R = H, 96%
2.4. Hybridization experiments
3d, Dye = JO, R = H, 78%
The introduction of cyanine chromophores as base sur-
rogates into the double helix inevitably leads to the
omission of hydrogen bonding interactions. We have
previously shown that thiazole orange can provide com-
pensation for potential losses of duplex stability by
N
N
N
O
O
O
4
means of its extraordinary base stacking ability. In-
deed, thiazole orange has been found to ‘pair’ to any
of the four nucleobases with the strength of an AT base
pair. To evaluate the ability of the three new dyes to
maintain DNA affinity of the respective PNA-probes,
melting curves were recorded and TM values were deter-
mined (Table 2).
N
N
O
O
O
Fmoc
N
Fmoc
N
N
OR
N
OR
H
H
Fmoc-Aeg(YO)-OR
Fmoc-Aeg(JO)-OR
1
2b, R = All
12d, R = All
13d, R = H
All cyanine-modified PNA–DNA duplexes showed sig-
moid melting curves which suggested cooperative base
pairing. The most stable duplex with complementary
DNA 20 was formed by TO–PNA 19a. YO–PNA 19b
closely resembles TO–PNA 19a only differing by the
oxazole which replaces the thiazole ring system of 19a.
The TM of the YO–PNA–DNA duplex is lower by
1
3b, R = H
Scheme 2. Reagents and conditions: (i) 1b or 1d, PyBOP, PPTS,
NMM, DMF, 16 h, 25 ꢁC; (ii) Pd(PPh , PhNHCH , THF, 16 h,
5 ꢁC; [PyBOP, (benzotriazol-1-yloxy)-tripyrrolidinophosphonium
hexafluorophosphat; PPTS, pyridinium p-toluenesulfonate; NMM,
N-methylmorpholine].
3
)
4
3
2
DTM = 2 ꢁC than the T of the corresponding TO-con-
M
taining duplex. This result suggests that TO better base
stacks than YO, which is in line with the order of duplex
affinities determined for free chromophores. The com-
parison between MO- and JO-containing duplexes re-
vealed, again, that the thiazole ring in MO–PNA 19c
conferred higher duplex stability than the oxazole ring
The synthesis of the TO-containing monomer was previ-
2
43
ously described. The rather challenging coupling reac-
tions succeeded by using PyBOP as activation agent and
4
2
PPTS to increase the low solubility of cyanine dyes. Sub-
sequent deprotection by Pd -catalyzed allyl transfer to N-
0
methylaniline afforded the desired Fmoc-PNA-mono-
mers (13a, b, d).
in JO–PNA 19d (TM = 71 ꢁC vs T = 68 ꢁC). Neverthe-
M
less, it can be concluded that all studied cyanine base
surrogates support the mutual recognition between
FIT-PNA and DNA target. TO appears to provide the
strongest stacking interactions, whereas JO probably
causes the largest perturbation to the duplex.
2
.3. Solid-phase synthesis
Fluorescent dye–PNA-conjugates were synthesized
2
,5
using both linear and divergent strategies.
While
TO-, YO-, and JO-dyes were introduced as Fmoc-
PNA-monomers, MO-dye labeling was achieved in a
postsynthesis procedure. The fully protected PNA resin
The cyanine dyes YO, TO, JO, and MO have been
selected because of their ability to signal binding to
3
1–33
nucleic acids by enhancements of fluorescence.
We
1
4 was assembled first by using 4 equiv of commercially
available Fmoc/Bhoc-protected PNA-monomers and
.6 equiv of HCTU in presence of 6 equiv NMM using
assumed that binding of PNA-conjugates 19b–d to
DNA 20 would also result in fluorescence enhancement
as previously reported for TO–PNA 19a. Figure 3 shows
fluorescence spectra of PNA–dye-conjugates 19a–d be-
fore (dashed lines) and after addition (solid lines) of
complementary DNA 20. As previously reported, addi-
tion of DNA 20 to TO–PNA 19a led to strong increases
of fluorescence by a factor of 19 at 25 ꢁC. In contrast,
YO–PNA 19b responded to hybridization at room tem-
perature by rather modest 4-fold fluorescence enhance-
ments. Interestingly, fluorescence responsiveness of
both TO- and YO–PNA 19a and 19b, respectively,
was significantly higher at elevated temperature. For
example, YO-containing duplex fluoresced at 60 ꢁC with
18-fold higher intensity than single stranded 19b (as
opposed to only 4-fold at 25 ꢁC, Table 3). More than
3
NMP as solvent (Scheme 3). Fluorescent Fmoc-PNA-
monomers 13a, b, d were introduced using double
couplings to yield resins 15a, b, d. In preparing for the
on-resin introduction of the MO chromophore, back-
bone building block 11 was introduced (15c). Resins
1
onally protected resin 16c was treated with Pd(PPh ) in
the presence of BH ÆNHMe to liberate the secondary
5a–d were extended to resins 18a, b, d and 16c. Orthog-
3
4
3
2
amine in 17c. The subsequent coupling with MO–
CH COOH (1c) was accomplished by applying the pre-
2
viously optimized reagent combination PyBOP/PPTS to
2
yield protected full-length resin 18c. For the release of
conjugates 19a–d, PNA resins 18a–d were treated with

L. Bethge et al. / Bioorg. Med. Chem. 16 (2008) 114–125
117
Fmoc Gly
i
Fmoc aR t aR gR cR cR gR Gly
ii
14
iii
dye
O
O
O
O
O
O
O
Fmoc
N
aR t aR gR cR cR gR Gly
Fmoc
N
N
aR t aR gR cR cR gR Gly
15a, b, d
N
H
15c
N
H
i
O
O
Ac-N
Ac-N
gR cR cR gR t aR
aR t aR gR cR cR gR Gly
N
H
16c
17c
i
iv
gR cR cR gR t aR
H
N
aR t aR gR cR cR gR Gly
N
H
v
MO
dye
O
O
O
O
Ac-N
gR cR cR gR t aR
N
Ac-N
gR cR cR gR t aR
N
aR t aR gR cR cR gR Gly
N
H
aR t aR gR cR cR gR Gly
18a, b, d
N
H
18c
vi
vi
dye
O
O
Ac-N
gR cR cR t aR
N
aR t aR gR cR cR gR Gly CONH2
N
H
19a-d
N
N
N
S
N
O
N
N
O
S
N
N
N
N
O
O
O
O
O
O
O
N
O
O
OH
O
OH
Fmoc
N
Fmoc
N
Fmoc
N
Fmoc
OH
N
H
OH
N
H
OH
N
H
N
H
Fmoc-Aeg(TO)-OH (13a)
Fmoc-Aeg(YO)-OH (13b)
Fmoc-Aeg(JO)-OH (13d)
Fmoc-Aeg(Alloc)-OH (11)
MO-CH2COOH (1c)
B
O
NH2
a, B = Adenin
O
O
O
c, B = Cytosin
g, B = Guanin
t, B = Thymin
=
R =
N
N
O
O
O
Tentagel
H
Scheme 3. Synthesis of dye–PNA-conjugates 19a–d. Left side shows linear assembly strategy using fluorescent Fmoc-PNA-monomers 13a, b, d.
Right side shows divergent assembly strategy using orthogonally protected PNA-backbone module 11. Subsequent deallylation and postassembly
coupling of MO–CH
NMM, HCTU, NMP; 3—Ac
lutidine, DMF; (iii) 1—piperidine, DMF (1:4); 2—Fmoc-Aeg(Alloc)-OH (11), NMM, HCTU, NMP; 3—Ac
NHMe , CH Cl ; (v) MO–CH COOH (1c), PyBOP, PPTS, NMM, DMF; (vi) TFA, m-cresol, H O, HCysOMe.
2
COOH (1c) provide dye–PNA-conjugate 19c. Reagents and conditions: (i) 1—piperidine, DMF (1:4); 2—Fmoc-B(Bhoc)-OH,
O, lutidine, DMF; (ii) 1—piperidine, DMF (1:4); 2—Fmoc-Aeg(dye)-OH (13a, b, d), NMM, HCTU, NMP; 3—Ac O,
O, lutidine, DMF; (iv) Pd(PPh )ÆBH
2
2
2
3
3
,
2
2
2
2
2
Table 1. Synthesized PNA-conjugates 19a–d and obtained overall
yields after HPLC-purification
at 60 ꢁC were fluorescence enhancements (up to 3-fold)
as high as observed in TO- and YO–PNA 19a and 19b
(
Table 3).
Compound
PNA-sequence
Yield
Ac-N
CONH2
CONH2
CONH2
19a
19b
19c
19d
gccga AegðTOÞ atagccgGly
3.4%
7.2%
1.8%
7.2%
In real-time PCR analysis, fluorescent probes bind to
target DNA in the annealing phase between 50 and
70 ꢁC. To evaluate the usefulness of the synthesized cya-
nine–PNA conjugates for real-time PCR applications,
we examined the temperature dependence of fluores-
cence signaling. Figure 4 shows that the fluorescence
Ac-N
Ac-N
Ac-N
gccga AegðYOÞ atagccgGly
gccga AegðMOÞ atagccgGly
CONH2
gccga AegðJOÞ atagccgGly
3
0-fold fluorescence intensification was obtained for
enhancement F /F observed upon hybridization of
ds ss
hybridization of TO PNA 19a at 60 ꢁC. Cyanine–
PNA-conjugates 19c (MO) and 19d (JO) proved less effi-
cient in signaling of hybridization. Neither at 25 ꢁC nor
TO–PNA 19a reaches an optimum at elevated tempera-
tures. More than 30-fold intensification of TO fluores-
cence is obtained when hybridization is performed at

118
L. Bethge et al. / Bioorg. Med. Chem. 16 (2008) 114–125
Table 2. T
M
values of PNA–DNA duplexes containing cyanine dyes as
cent base mismatches. MO–PNA 19c and JO–PNA 19d
base surrogates
were not included in this study due to the insignificance
of the hybridization-induced fluorescence changes.
Fluorescence spectra of 19a and 19b were recorded be-
fore and after addition of single mismatched DNA
2
1A, G, C and fluorescence enhancements F /F at spe-
ds ss
cific wavelengths were determined (Fig. 5). Hybridiza-
tion of single mismatched DNAs 21 resulted in all
cases in lower fluorescence enhancements than hybrid-
ization with matched DNA 20.
X
TO
73
YO
71
MO
71
JO
68
T
M
(ꢁC)
Measured as denaturation curves at 1 lM concentration in a buffered
solution (100 mM NaCl, 10 mM NaH PO , pH 7).
2
4
The background-corrected match/mismatch discrimina-
tion D = (F (match) ꢀ F )/(F (mismatch) ꢀ F ) is the
ds
ss
ds
ss
5
5–70 ꢁC as opposed to only 18-fold fluorescence
important parameter in applications such as real-time
PCR analysis. The previously demonstrated ability of
enhancement at 25 ꢁC. Relative fluorescence rapidly
decreases at temperatures higher than T . The temper-
ature-dependence of hybridization-induced fluorescence
enhancement is even more pronounced for YO–PNA
M
1
9b. For example, 19b furnished only moderate 4-fold
Table 3. Fluorescence properties of duplexes formed by dye–PNA-
conjugates 19a–d and complementary DNA 20
fluorescence enhancement at 25 ꢁC, which is modest
when compared to the 20-fold intensification at 70 ꢁC.
These results are important. They suggest that both
TO- and YO-containing FIT-PNA may be useful at
the typical temperatures applied in real time PCR anal-
ysis. In contrast, MO–PNA 19c and JO–PNA 19d will
probably have limited utility due to their rather modest
fluorescence responsiveness.
19aÆ20
(TO)
19bÆ20
(YO)
19cÆ20
(MO)
19dÆ20
(JO)
F
F
ds/Fss (25 ꢁC)
ds/Fss (60 ꢁC)
19.4
31.0
525
4.3
18.5
498
1.9
2.7
564
0.9
2.3
538
k (em, nm)
Measurement conditions as specified in Figure 3. Fds/Fss = fluorescence
enhancement at k (em) of dye–PNA conjugates 19a–c upon hybrid-
ization to complementary DNA 20. Fss = fluorescence intensity of
PNA single strand, Fds = fluorescence intensity after addition of DNA
20.
We next investigated the responsiveness of the chro-
mophores in PNA-conjugates 19a and 19b toward adja-
a
7
6
4
3
1
5
0
5
0
5
0
19a · 20
b
250
200
150
100
50
19b · 20
0
505
605
705
480
580
680
Wavelength λ [nm]
Wavelength λ [nm]
c
19c · 20
d
125
100
75
50
25
0
19d · 20
1
1
50
20
9
6
3
0
0
0
0
530
630
730
510
610
710
Wavelength λ [nm]
Wavelength λ [nm]
Figure 3. Fluorescence spectra of PNA-conjugates 19a–d. Dashed lines: single stranded dye–PNA-conjugates, solid lines: PNA–DNA duplex. (a)
9aÆ20,19a; (b) 19bÆ20, 19b; (c) 19cÆ20, 19c; (d) 19dÆ20, 19d. Measurement conditions: 1 lM dye–PNA-conjugate and DNA in buffer as specified in
Table 2 at 25 ꢁC. Excitation: (a) 495 nm; (b) 467 nm; (c) 517 nm; (d) 498 nm.
1

L. Bethge et al. / Bioorg. Med. Chem. 16 (2008) 114–125
119
3
3
2
2
1
1
5
0
5
0
5
0
5
0
3. Conclusions
We have presented dye–PNA-conjugates in which the
cyanine dyes thiazole orange (TO, 1a), oxazole yellow
YO, 1b), thiazolopyridine (MO, 1c), and oxazolopyri-
(
dine (JO, 1d) served as replacement of a canonical nucle-
obase. The synthesis was performed by linear and
divergent solid-phase synthesis. Melting analysis re-
vealed that the introduction of cyanine base surrogates
into PNA–DNA duplexes is tolerated. The DNA affinity
of cyanine-modified PNA probes is not significantly
affected. The obtained results provided evidence that
the concept of forced intercalation probes (in which a
fluorescent base surrogate is forced to intercalate next
to the expected mutations site) is not restricted to the
use of the previously explored thiazole orange dye. We
found that PNA containing oxazole yellow (YO) as base
surrogate is also suited to report the presence of fully
complementary DNA by enhancements of fluorescence.
In addition, YO emission responded to adjacent base
mismatches by attenuated fluorescence emission at tem-
25
35
45
55
65
75
85
Temperature [°C]
Figure 4. Temperature dependent fluorescence enhancement Fds/Fss
upon hybridization of 19a–d with complementary DNA 20. Excitation
1
9a: 515 nm, 19b: 490 nm, 19c: 560 nm, 19d: 535 nm, Emission 19a:
5
31 nm, 19b: 504 nm, 19c: 570 nm, 19d: 545 nm.
peratures below the T . Both TO- and YO–PNA are
M
hence suited to discriminate matched from single mis-
matched target at non-stringent hybridization condi-
tions. Largest fluorescence enhancements are obtained
when hybridization is performed at elevated tempera-
tures typically applied in real-time PCR analysis. We
wish to note that YO and TO fluorescence spectra can
be resolved. It is thus possible to selectively excite/mon-
itor YO fluorescence (e.g., at 490/504 nm) with only little
crosstalk to TO fluorescence (515/531 nm). Future work
will concern the combined use of FIT-YO- and FIT-
TO–PNA in multiplexed real-time PCR analysis of sin-
gle base mutations.
Figure 5. Background-corrected match/mismatch discrimination
D = (Fds(match) ꢀ Fss)/(Fds(mismatch) ꢀ Fss) of dye–PNA-conjugates
1
9a,b after formation of match duplexes (Y = T) and mismatch
4. Experimental
duplexes (Y = C, G, A). (a) 25 ꢁC; (b) 60 ꢁC. Fss = fluorescence
intensity of PNA single strand, Fds = fluorescence intensity of double
strand formed after addition of DNA 21A, G, C and 20.
4.1. General procedures and materials
Manual solid-phase synthesis (glycine-loading of resin)
was performed by using 5 mL polyethylene syringe reac-
tors that are equipped with a fritted disk. Automated
linear solid-phase synthesis was performed by using an
Intavis ResPep parallel synthesizer equipped with
microscale columns for PNA synthesis. All column
chromatography was performed with SDS 60 ACC sili-
TO–PNA to discriminate matched from mismatched
hybridization at non-stringent conditions became appar-
ent also in this study. At 25 ꢁC all matched and mis-
matched duplexes formed. However, probe 19a was
still able to discriminate the a-T match from a-A and
a-G mismatches with 3-fold selectivity. Match/mismatch
discrimination was lower (D = 1.5) for the a-C mis-
match. Interestingly, this mismatch was easily discrimi-
nated (D = 4.2) by YO–PNA 19b. In contrast, YO–
PNA 19b proved less suited (D = 1.7) to discriminate
against the a-A mismatch. The single nucleotide specific-
ity of fluorescence signaling can be improved by
performing the hybridization at elevated temperature.
TM
ca gel using a B u¨ chi Sepacore flash chromatographer
and TLC with E. Merck Silica Gel 60 F254 plates. H
1
1
3
and C NMR spectra were recorded with Bruker
DPX 300 spectrometer. The signals of the residual pro-
tonated solvent (CDCl , CD OD or DMSO-d ) were
3
3
6
used as reference signals. Coupling constants are given
in Hertz. HPLC was performed with an Agilent 1100
series instrument using a Varian Polaris-C18 A 5l (PN
A 2000-250x046) at 55 ꢁC for analytical runs and a
Varian Polaris C18 A 5l (PN A 2000-250-100) for semi-
preparative runs. Eluents analytical: A (0.1% HCOOH
in water + 1% MeCN) and B (0.1% HCOOH in
MeCN + 1% water); semipreparative: A (0.1% TFA in
water + 1% MeCN) and B (0.1% TFA in MeCN + 1%
water) were used in a linear gradient with a flow
rate of 1 mL/min for analytical and 6 mL/min for
At 60 ꢁC, a temperature that was still below the T
M
of mismatched probe-target complexes, match/mis-
match discrimination increased to 4- to 6-fold for
TO–PNA 19a and up to 7-fold for YO–PNA 19b.
Further enhancements of sequence specificity are
feasible at stringent conditions, at temperatures above
the TM of mismatched probe-target complexes (data
not shown).

120
L. Bethge et al. / Bioorg. Med. Chem. 16 (2008) 114–125
semipreparative HPLC. High-resolution mass spectra
were measured with a Hewlett-Packard GCMS 5995-A
as a pale red solid (2.18 g, 37 %). TLC R = 0.35
f
1
(CHCl /MeOH/AcOH 5:3:2). H NMR (DMSO-d6/
3
(
ESI+) spectrometer. MALDI-TOF mass spectra were
TFA) d ppm: 3.87 (3H, s, CH ), 5.45 (2H, s, CH ),
3
2
TM
recorded with a Voyager-DE Pro Biospectrometry
Workstation of PerSeptive Biosystems. DNA was pur-
chased from MWG-Biotech in HPSF quality. Fmoc/
Bhoc-protected PNA monomers were purchased from
Applied Biosystems. Water was purified with a Milli-
Q Ultra Pure Water Purification System (Millipore
Corp.). Solvents were dried and distilled following stan-
6.30 (1H, s, CH), 7.39 (1H, d, J = 7.6, ArH), 7.47 (1H,
d, J = 7.6, ArH), 7.66 (2H, m, 2ArH), 7.80 (2H, m,
2ArH), 7.90 (2H, m, 2ArH), 8.37 (1H, d, J = 7.4,
1
3
ArH), 8.74 (1H, d, J = 7.4, ArH). C NMR (DMSO-
d /TFA) d ppm: 30.6 (CH ), 54.4 (CH ), 74.8 (CH),
6
3
2
ꢂ
108.6, 110.8, 110.9, 117.5 (4ArC), 122.8 (ArC ), 124.5,
125.9, 125.9, 126.3 (4· ArC), 131.2 (ArC ), 133.2
q
q
dard procedures if needed. Dry DMF (H O < 0.01%)
2
(ArC), 137.8 (ArC ), 144.0 (ArC), 146.0, 150.1, 161.6,
q
þ
was purchased from Fluka. All other used compounds
were commercially available and not further purified
prior to usage.
168.7 (4· ArC ). HR-MS: m/z cacld for C H N O
q
20 17
2
3
+
333.1234, found 333.1231 [M] .
4
.2.3. Carboxymethylated thiazolopyridine (1c). Follow-
4
.2. Fluorescence spectrometry
ing the procedure of 1a were 875 mg (2.37 mmol) 4-
methyl-2-methylthio-thiazolopyridinium tosylate (7c),
670 mg (2.37 mmol) 1-(carboxymethyl)-4-methyl-chino-
linium bromide (9), and 480 mg (4.74 mmol, 659 lL) tri-
ethylamine in 50 mL CH Cl allowed to react. The
Fluorescence spectra were recorded by using a Varian
Cary Eclipse spectrometer. Measurements were carried
out in fluorescence quartz cuvettes (4· 10 mm) at
2
2
1
lM concentration in a buffered solution (100 mM
crude product was recrystallized from MeOH to yield
1c as a rusty brown solid (305 mg, 30 %). H NMR
1
NaCl, 10 mM NaH PO , pH 7). Excitation: TO:
95 nm; YO: 467 nm; MO: 517 nm; JO: 498 nm; Sli-
2
4
4
(DMSO-d /TFA) d ppm: 4.21 (3H, s, CH ), 5.28 (2H,
6
3
tEx = 5, SlitEm = 2.5. After addition of DNA and dye–
PNA-conjugates, solutions were heated to 95 ꢁC before
cooling to 25 or 60 ꢁC. Spectra were recorded after
s, CH ), 6.84 (1H, s, CH), 7.14 (1H, dd, J = 6.4,
J2 = 7.6, ArH), 7.46 (1H, m, ArH), 7.57 (1H, d,
J = 8.3, ArH), 7.73 (1H, m, ArH), 8.01 (1H, d, J = 7.5,
2
1
1
3
5
min at 25 or 60 ꢁC, thus obtaining constant emission.
ArH), 8.35 (4H, m, 4ArH). C NMR (DMSO-d6/
TFA) d ppm: 42.6 (CH ), 54.0 (CH ), 94.3 (CH),
3
2
4.2.1. Carboxymethylated thiazole orange (1a). To a
solution of 2.91 g (7.92 mmol) 3-methyl-2-(methylthio)-
benzothiazolium tosylate (8a) and 2.79 g (9.90 mmol)
109.1, 115.6, 116.8 (3· ArC), 122.6 (ArC ), 124.9,
q
125.5 (2· ArC), 131.7 (ArC ), 132.2, 133.9 (2· ArC),
q
138.0 (ArC ), 142.6, 146.4 (2· ArC), 146.4, 158.4 (2·
q
1
1
2
-(carboxymethyl)-4-methyl-chinolinium bromide (9) in
50 mL CH Cl was added 2.00 g (19.8 mmol,
.78 mL) triethylamine, which caused immediate change
ArC ), 169.1 (Cq), 172.7 (ArC ). HR-MS: m/z calcd
q
q
+
for C H N O S 350.0958, found 350.0961 [M] .
+
2
2
19 16
3
2
of color to red. The mixture was stirred under exclusion
of light at room temperature for 16 h. All volatiles were
removed under reduced pressure, the residue was dis-
4.2.4. Carboxymethylated oxazolopyridine (1d). Follow-
ing the procedure of 1a were 3.19 g (9.05 mmol) 4-meth-
yl-2-(methylthio)-oxazolopyridinium
tosylate
(8d),
solved in 600 mL refluxing MeOH, 1.5 L H O was
2
2.56 g (9.05 mmol) 1-(carboxymethyl)-4-methyl-chino-
linium bromide (9), and 1.83 g (18.1 mmol, 2.54 mL) tri-
ethylamine in 150 mL CH Cl allowed to react. The
added, and the resulting solution was stored at 4 ꢁC over
3
days. The resulting precipitate was collected by filtra-
2
2
tion and dried under reduced pressure to yield 1a as a
red solid (1.83 g, 54 %). TLC R = 0.37 (CHCl /
crude product was recrystallized from MeOH to yield
1d as a violet solid (2.61 g, 70%). TLC R = 0.29
f
3
f
1
1
(CHCl /MeOH/AcOH 5:3:2). H NMR (DMSO-d6/
MeOH/AcOH 5:3:2). H NMR (DMSO-d /TFA) d
6
3
ppm: 4.02 (3H, s, CH ), 5.45 (2H, s, CH ), 6.93 (1H,
TFA) d ppm: 4.12 (3H, s, CH ), 5.34 (2H, s, CH ),
3
2
3
2
s, CH), 7.32 (1H, d, J = 7.3, ArH), 7.42 (1H, t, J = 7.6,
ArH), 7.60 (1H, t, J = 7.5, ArH), 7.69 (1H, t, J = 7.7,
ArH), 7.78 (1H, d, J = 8.4, ArH), 7.83 (1H, d, J = 8.6,
ArH), 7.92 (1H, t, J = 8.4, ArH), 8.03 (1H, d, J = 7.7,
ArH), 8.49 (1H, d, J = 7.3, ArH), 8.75 (1H, d, J = 8.4,
6.47 (1H, s, CH), 7.31 (1H, t, J = 7.1, ArH), 7.55 (1H,
t, J = 7.6, ArH), 7.68 (1H, d, J = 8.6, ArH), 7.82 (1H,
t, J = 7.7, ArH), 8.19 (4H, m, 4ArH), 8.46 (1H, d,
1
3
J = 8.4 Hz, ArH). C NMR (DMSO-d /TFA) d ppm:
6
40.3 (CH ), 54.0 (CH ), 81.8 (CH), 108.5, 116.3, 117.1,
118.6 (4· ArC), 122.8 (ArC ), 125.0, 125.8, 132.6,
3
2
1
3
ArH). C NMR (DMSO-d /TFA) d ppm: 33.9 (CH ),
6
3
q
5
4.6 (CH ), 88.8 (CH), 107.4, 113.2, 117.64, 122.8
135.9 (ArC), 137.7 (ArC ), 142.6 (ArC), 145.1, 149.9,
q
2
(
1
4· ArC), 123.6, 124.1 (2· ArC ), 124.7, 125.6,
153.9 (3· ArC ), 169.0 (C ), 171.4 (ArC ). HR-MS: m/
q
q
q
þ
q
26.6, 128.2, 133.2 (5· ArC), 137.7, 140.3 (2· ArC ),
z calcd for C H N O
3
334.1186, found 334.1186
q
19 16
3
+
[M] .
1
m/z calcd for C H N O S 349.1005, found 349.1005
44.8 (ArC), 148.5, 160.8, 168.7 (3· ArC ). HR-MS:
q
+
2
0
17
2
2
+
M] .
[
4.2.5. 2-(Pivalamido)pyridine (4). To a solution of 4.71 g
50.0 mmol) 2-aminopyridine (3) and 6.35 g (62.5 mmol,
(
4.2.2. Carboxymethylated oxazole yellow (1b). Following
the procedure of 1a were 5.00 g (14.2 mmol) 3-methyl-2-
8.78 mL) triethylamine in 100 mL CH Cl at 0 ꢁC was
2
2
added a solution of 6.63 g (55.0 mmol) pivaloyl chloride
in 50 mL CH Cl dropwise over 2 h. After stirring for
(
(
methylthio)-benzoxazolium tosylate (8b), 5.00 g
17.7 mmol) 1-(carboxymethyl)-4-methyl-chinolinium
2
2
further 2 h, the reaction mixture was allowed to warm
to room temperature and stirred further for 60 min.
The reaction was then quenched by addition of
bromide (9), and 2.88 g (28.5 mmol, 4.00 mL) triethyl-
amine in 260 mL CH Cl allowed to react to yield 1b
2
2

L. Bethge et al. / Bioorg. Med. Chem. 16 (2008) 114–125
121
1
00 mL H O. The organic layer was separated, washed
2
acid. The resulting precipitate was collected by filtration,
washed with H O, and dried under reduced pressure to
with 50 mL sat. NaHCO solution (2·), dried (MgSO ),
and evaporated. The residue was recrystallized from
cyclohexane to yield 4 as a white crystalline solid
3
4
2
1
yield 6d as a brownish solid (2.85 g, 41%). H NMR
(DMSO-d ) d ppm: 3.37 (1H, br, NH), 7.27 (1H, dd,
6
(
2
7
8.38 g, 94 %). TLC R = 0.42 (cyclohexane/EtOAc
f
J1 = 5.2, J2 = 8.1, ArH), 7.87 (1H, dd, J1 = 8.1,
J = 1.3, ArH), 8.22 (1H, dd, J = 5.2, J = 1.2, ArH).
1
:1). H NMR (CDCl ) d ppm: 1.31 (9H, s, 3CH ),
3
3
2
3
1
2
1
.02 (H, m, ArH), 7.69 (1H, m, ArH), 8.12 (1H, s,
C NMR (DMSO-d ) d ppm: 117.0, 119.1 (2· ArC),
6
1
3
NH), 8.24 (2H, m, ArH). C NMR (CDCl ) d ppm:
2
ArC), 151.4 (ArC ), 177.1 (C ).
141.6 (ArC ), 144.1 (ArC), 146.9, 181.3 (2· ArC ).
3
q
q
7.4 (3CH ), 39.8 (C ), 113.9, 119.6, 138.5, 147.3 (4·
3
q
4.2.9. 2-(Methylthio)-benzothiazole (7a). To a solution of
6.7 g (100 mmol) 2-thiobenzothiazol (6a) in 250 mL
DMF was added 13.8 g (100 mmol) K CO and stirred
q
q
1
4
.2.6. 2-(pivalamido)pyridine-3-yl-diethylcarbamodithio-
2
3
ate (5). To a solution of 8.32 g (46.7 mmol) 2-(pivalam-
ido)pyridine (4) in 100 mL THF at ꢀ50 ꢁC was added
n-BuLi (2.5 M in hexane, 39.2 mL, 98.0 mmol) in two
equimolar portions dropwise over 5 min each. After
the addition was complete, the yellowish solution was
stirred for 30 min at ꢀ50 ꢁC, warmed to 0 ꢁC, and stir-
red for another 4 h at which time a yellow precipitate
appeared. The reaction mixture was cooled to ꢀ50 ꢁC,
a solution of 16.6 g (56.0 mmol) tetraethylthiuram disul-
fide (TETD) in 50 mL THF was added dropwise, and
the resulting mixture stirred for 30 min. Cooling was
over 15 min. To this mixture was added 16.9 g
(120 mmol, 7.42 mL) MeI in one portion. After stirring
for 1 h of 500 mL H O was added. The resulting precip-
2
itate was collected by filtration and dried under reduced
pressure to yield 7a as a white solid (16.3 g, 90%). H
1
NMR (CD OD) d ppm: 2.70 (3H, s, CH ), 7.24 (1H,
3
3
t, J = 7.7, ArH), 7.36 (1H, t, J = 7.7, ArH), 7.74 (2H,
1
3
m, 2ArH). C NMR (CD OD) d ppm: 16.1 (S–CH ),
3
3
122.0, 122.3, 125.4, 127.4 (4· ArC), 136.1, 154.4, 170.7
(3· ArC ).
q
abandoned and after stirring overnight 250 mL Et O
2
was added. The organic phase was washed with
4.2.10. 2-(Methylthio)-benzoxazole (7b). To a solution of
10.0 g (66.1 mmol) 2-thiobenzoxazole (6b) in 200 mL
DMF was added 139.1 g (66.1 mmol) K CO and stirred
3
were reextracted with 200 mL Et O. The combined
00 mL H O (2·) and the combined aqueous phases
2
2
3
over 15 min. To this mixture was added 10.3 g
(79.3 mmol, 4.52 mL) MeI in one portion. After stirring
for 1 h of 600 mL H O was added. The reaction mixture
2
organic phases were dried (MgSO ), evaporated, and
4
purified by flash column chromatography (100 % cyclo-
hexane ! 100 % EtOAc) to yield 5 as a white solid
2
was extracted with 200 mL EtOAc (3·). The combined
1
11.5 g, 75%). TLC R = 0.48 (EtOAc). H NMR
(
organic phases were washed with 100 mL H O (3·),
f
2
(
J = 7.1, CH ), 3.84 (2H, q, J = 7.1, CH ), 4.00 (2H, q,
CDCl ): d ppm: 1.27 (12H, m, 4CH ), 1.38 (3H, t,
brine (1·), dried (MgSO ), evaporated, and dried under
3
3
4
reduced pressure to yield 7b as a brown oil (9.53 g, 87%).
TLC R = 0.58 (cyclohexane/EtOAc 10:1).
3
2
1
J = 7.1, CH ), 7.14 (1H, dd, J = 4.8, J = 7.7, ArH),
2
H
2
1
f
7
NH), 8.60 (1H, dd, J = 1.8, J = 4.8, ArH). C NMR
.76 (1H, dd, J = 1.8, J = 7.7, ArH), 8.52 (1H, s,
NMR (CDCl ) d ppm: 2.78 (3H, s, CH ), 7.25
1
2
3
3
1
3
(1H, t, J = 7.2, ArH), 7.30 (1H, t, J = 7.1, ArH), 7.46
1
2
1
3
(
CDCl ) d ppm: 11.4, 12.7 (2· CH ), 27.3 (3CH ) 39.7
(1H, d, J = 7.8, ArH), 7.63 (1H, d, J = 7.6, ArH).
NMR (CDCl3) ppm: 14.5 (S–CH3), 109.8,
118.3, 123.7, 124.2 (4· ArC), 141.9, 151.9, 165.7
3· ArC ).
C
3
3
3
(
C ), 47.6, 49.9 (2· CH ), 120.4 (ArC ), 120.9, 145.7,
d
q
2
q
1
50.7 (3· ArC), 153.8 (ArC ), 176.2, 188.5 (2· C ).
q
q
(
q
4
.2.7. 2-Thio-thiazolopyridine (6c). A solution of 1.54 g
(
4.73 mmol) 2-(pivalamido)pyridine-3-yl-diethyl-carba-
modithioate (5) and 5.85 g (147 mmol) NaOH in
0 mL EtOH was refluxed over 4 h. After cooling to
4.2.11. 2-Methylthio-thiazolopyridine (7c). With the
experimental setup described for 7b were 1.61 g
3
(9.55 mmol)
2-thio-thiazolopyridine
(6c),
1.32 g
room temperature, 75 mL of 2 M hydrochloric acid
was added whereupon an off-white precipitate appeared.
The precipitate was collected by filtration, washed with
(9.55 mmol) K CO , and 1.49 g (10.5 mmol, 654 lL)
2
3
MeI in 100 mL DMF allowed to react to yield 7c as
an off-white solid (1.66 g, 96%). TLC R = 0.64 (EtOAc).
f
1
H O, and dried under reduced pressure to yield 6c as
2
H NMR (CD OD) d ppm: 2.80 (3H, s, S–CH ), 7.30
3
3
an off-white solid (340 mg, 43%). TLC R = 0.68
f
(1H, dd, J = 4.8, J = 8.0, ArH), 8.32 (1H, dd,
J1 = 1.5, J = 8.0, ArH), 8.48 (1H, dd, J1 = 1.4,
2
1 2
1
(
EtOAc). H NMR (DMSO-d ) d ppm: 7.30 (1H, dd,
6
1
3
J1 = 4.9, J2 = 7.9, ArH), 8.13 (1H, dd, J1 = 1.5,
J = 7.9, ArH), 8.36 (1H, dd, J = 1.5, J = 4.9, ArH),
J2 = 4.8, ArH). C NMR (CD OD) d ppm: 16.0 (S–
3
CH ), 120.5 (ArC), 130.5 (ArC ), 132.4, 148.2 (2·
2
1
2
3
q
1
3
1
4.30 (1H, s, SH). C NMR (DMSO-d ) d ppm: 119.5
ArC), 164.7, 176.1 (2· ArC ).
6
q
(
1
ArC), 124.1 (ArC ), 130.5, 146.7 (2· ArC), 153.7,
q
90.9 (2· ArC ).
4.2.12. 2-(Methylthio)-oxazolopyridine (7d). With the
experimental setup described for 7b were 2.50 g
q
4
5
1
.2.8. 2-Thio-oxazolopyridine (6d). To a solution of
.00 g (45.5 mmol) 2-amino-3-hydroxy-pyridine (2) in
00 mL EtOH (abs) under an atmosphere of Ar was
(16.4 mmol)
(16.4 mmol) K CO , and 2.80 g (19.7 mmol, 1.23 mL)
2-thio-oxazolopyridine
(6d),
2.26 g
2
3
MeI in 40 mL DMF allowed to react to yield 7d as a
1
added 15.0 g (93.8 mmol) potassium ethyl xanthate in
equimolar portions every 3 h. After cooling, volatiles
were removed under reduced pressure, the residue dis-
white solid (1.97 g, 72%). H NMR (CDCl ) d ppm:
3
5
2.78 (3H, s, S–CH ), 7.16 (1H, dd, J = 5.0, J = 8.1,
ArH), 7.68 (1H, dd, J = 8.0, J = 1.5, ArH), 8.43 (1H,
1 2
3
1
2
1
3
solved in H O and acidified to pH 5 with glacial acetic
2
dd, J = 5.0, J = 1.3, ArH). C NMR (CDCl ) d
1 2 3

122
L. Bethge et al. / Bioorg. Med. Chem. 16 (2008) 114–125
+
ppm: 14.7 (S–CH ), 117.2, 118.8 (2· ArC), 144.2 (ArC ),
2.78 (3H, s, S–CH ), 4.42 (3H, s, N –CH ), 6.99 (2H,
3 3
3
q
1
45.5 (ArC), 155.9, 170.5 (ArC ).
d, J = 8.0, 2ArH), 7.54 (2H, d, J = 8.1, 2ArH), 7.78
1H, dd, J = 8.3, J = 6.4, ArH), 8.44 (1H, d, J = 8.2,
ArH), 9.04 (1H, d, J = 6.4, ArH). C NMR (CDCl )
q
(
1
2
1
3
4
.2.13. 3-Methyl-2-(methylthio)-benzothiazolium tosylate
3
+
(
(
8a). To 5.82 g (32.1 mmol) 2-methylthiobenzothiazole
7a) was added 6.58 g (35.3 mmol, 5.33 mL) methyl
d ppm: 15.1 (S–CH ), 21.1 (CH ), 42.0 (N –CH ),
121.2, 125.6, 128.3 (3· ArC), 138.9 (ArC ), 141.0
(ArC), 143.5, 147.3, 150.2, 177.8 (4· ArC ).
3
3
3
q
p-toluenesulfonate and heated to 130 ꢁC for 1 h. After
cooling to 70 ꢁC, acetone was added until a white pre-
cipitate appeared. Reflux was maintained for another
30 min before cooling to room temperature. The precip-
itate was collected by filtration and dried under reduced
q
4.2.17. 1-(Carboxymethyl)-4-methyl-chinolinium bromide
(9). A solution of 11.5 g (80.0 mmol) lepidine and 13.9 g
(100 mmol) bromoacetic acid in 40 mL EtOAc was stir-
red for 3 days at room temperature. The resulting pre-
cipitate was collected by filtration and dried under
reduced pressure to yield 9 as an off-white solid
(11.9 g, 53 %). H NMR (CD OD) d ppm: 1.99 (3H, s,
CH ), 3.79 (2H, s, CH ), 6.95 (2H, t, J = 7.1, 2ArH),
7.13 (1H, t, J = 7.8, ArH), 7.22 (1H, d, J = 8.9, ArH),
7.48 (1H, d, J = 8.5, ArH), 8.15 (1H, d, J = 6.1, ArH).
pressure to yield 8a as a pale yellow solid. (9.98 g, 85%).
H NMR (CD OD) d ppm: 2.29 (3H, s, CH ), 3.06 (3H,
1
3
3
+
s, S–CH ), 4.08 (3H, s, N –CH ), 7.13 (2H, d, J = 8.1,
3
3
1
2ArH), 7.61 (2H, d, J = 8.2, ArH), 7.67 (1H, t, J = 7.7,
ArH), 7.79 (1H, t, J = 7.5, ArH), 8.01 (1H, d, J = 8.5,
3
3
2
1
ArH), 8.16 (1H, d, J = 8.1, ArH). C NMR (CD OD)
3
3
+
d ppm: 18.5 (S–CH ), 21.4 (CH ), 36.9 (N –CH ),
3
3
3
1
3
1
1
1
16.5, 124.7 (2· ArH), 126.9 (2ArH), 128.5 (ArH),
C NMR (CD OD) d ppm: 20.6 (CH ), 58.7 (CH ),
3 3 2
29.8 (2ArH), 129.9 (ArC ), 130.7 (ArH), 141.6, 143.7,
44.1, 183.2 (4· ArCq).
120.0, 123.9, 128.3 (3· ArC), 130.7 (ArC ), 131.2,
137.0 (2· ArC), 139.6 (ArC ), 150.6 (ArC), 162.5
q
q
q
(
ArC ), 168.5 (C ).
q q
4
.2.14. 3-Methyl-2-(methylthio)-benzoxazolium tosylate
(
(
8b). To 9.53 g (57.7 mmol) 2-methylthiobenzoxazole
7b) was added 19.6 g (105 mmol, 15.9 mL) methyl p-tol-
4.2.18. General procedure for the coupling of cyanine dyes
1b and d to Fmoc-Aeg-OAll (10). To 1.00 mmol of cya-
nine dye 1b or d in 11 mL dry DMF were added
572 mg (1.10 mmol) PyBOP, 111 mg (1.10 mmol,
120 lL) NMM, and 253 mg (1.00 mmol) PPTS under
an atmosphere of argon and exclusion of light. This sus-
pension was stirred for 2 min whereupon complete dis-
solution occurs. The solution is then added to a
solution of 417 mg (1.00 mmol) Fmoc-Aeg-OAll hydro-
chloride (10) and 111 mg (1.10 mmol, 120 lL) NMM in
9 mL dry DMF. The resulting mixture was stirred over
16h. The volatiles were removed under reduced pressure
and the residue treated with 20 mL CH Cl . The result-
uenesulfonate and heated to 130 ꢁC for 1 h. After cool-
ing to 70 ꢁC, cyclohexane was added. Reflux was
maintained for another 30 min before cooling to room
temperature. The liquid phase was discarded. The solid
phase was ground and recrystallized from acetonitrile to
yield 8b as off-white moisture-sensitive flakes (17.2 g,
8
1
5%). H NMR (CDCl ) d ppm: 2.82 (3H, s, CH ),
3
3
+
.06 (3H, s, S–CH ), 4.05 (3H, s, N –CH ), 7.01 (2H,
3
d, J = 7.9, 2ArH), 7.52 (4H, m, 4ArH), 7.67 (1H, d,
3
3
1
J = 8.1, ArH), 7.77 (1H, d, J = 7.7, ArH). C NMR
3
+
(
CDCl ) d ppm: 15.0 (S–CH ), 21.3 (CH ), 33.5 (N –
3 3 3
2
2
CH ), 112.1, 113.5 (2· ArC), 125.9 (2ArC), 127.4,
ing precipitate was filtered off and discarded. The filtrate
was purified by flash column chromatography (CH Cl /
0 % MeOH ! 2 % MeOH).
3
1
1
27.8 (2· ArC), 128.4 (2ArC), 131.5, 129.5, 142.3,
2
2
49.3, 171.1 (5· ArC ).
q
4
.2.15. 4-Methyl-2-methylthio-thiazolopyridinium tosyl-
4.2.19. Fmoc-Aeg(YO)-OAll (12b). Yellow solid
(253 mg, 33 %). TLC R = 0.58 (CH Cl /MeOH/
HCOOH 89.5:10:0.5). H NMR (DMSO-d ) (two rota-
ate (8c). To 510 mg (2.79 mmol) 2-methylthio-thiazolo-
pyridine (7c) was added 520 mg (2.79 mmol) methyl p-
toluenesulfonate and heated to 130 ꢁC for 1 h. After
cooling the mixture to 100 ꢁC, cyclohexane was added.
Reflux was maintained for another 30 min before cool-
ing to room temperature. The resulting precipitate was
f
2
2
1
6
mers) d ppm: 3.14 (1H, m, N–CH ), 3.41 (2H, m, N–
2
CH ), 3.63 (1H, m, N–CH ), 3.89 (3H, s, CH ), 4.15
(1.2H, s, Gly-CH ), 4.24 (1H, m, Fmoc-CH), 4.31
2
2
3
2
(0.8H, d, J = 6.7, Fmoc-CH ) 4.40 (1.2H, d, J = 6.3,
2
collected by filtration to yield 8c as an off-white solid
Fmoc-CH ), 4.52 (0.8H, s, Gly-CH ), 4.57 (1.2H, d,
2
J = 5.0, All–CH ), 4.77 (0.8H, d, J = 5.1, All–CH ),
2 2
2
1
(
CH ), 2.80 (3H, s, S–CH ), 4.51 (3H, s, N –CH ), 7.03
925 mg, 90 %). H NMR (CDCl ) d ppm: 2.27 (3H, s,
3
+
5.19 (0.6H, d, J = 10.7, All@CH ), 5.29 (0.6 H, d,
3
3
3
1
2
(
7
(
(
2H, d, J = 7.8, 2ArH), 7.63 (2H, d, J = 7.9, 2ArH),
.74 (1H, m, ArH), 9.07 (1H, d, J = 7.2, ArH), 9.16
J = 17.4, All@CH ), 5.31 (0.4H, d, J = 9.5, All@CH ),
1
2
1
2
5.43 (0.4H, d, J = 17.5, All@CH ), 5.50 (0.8H, s, CH ),
1
2
2
1
3
1H, d, J = 4.3, ArH). C NMR (CDCl ) d ppm: 16.7
5.70 (1.2H, s, CH ), 5.87 (0.6H, m, All–CH), 6.04 (0.4H,
3
2
+
S–CH ), 21.2 (CH ), 44.2 (N –CH ), 120.5 (ArC),
m, All–CH), 6.34 (1H, s, CH), 7.31 (2H, t, J = 7.1,
2Fmoc-ArH), 7.41 (3H, m, 2Fmoc-ArH, ArH), 7.50
(1H, t, J = 7.6 Hz, ArH), 7.66 (4H, m, 2Fmoc-ArH,
3
3
3
1
25.8 (2ArC), 128.5 (2ArC), 133.8, 139.4 (ArC ), 139.8
q
(
ArC), 143.0 (ArC ), 143.5 (ArC), 154.6, 184.8 (ArC ).
q q
2
ArH), 7.80 (2H, m, 2ArH), 7.91 (4H, m, 2Fmoc-
ArH, 2ArH), 8.22 (1H, m, ArH), 8.75 (1H, d, J = 8.5,
4
.2.16. 4-Methyl-2-(methylthio)-oxazolopyridinium tosyl-
1
3
ate (8d). Following the procedure of 8a were 1.97 g
11.8 mmol) 2-(methylthio)-oxazolopyridine (7d) and
.20 g (11.8 mmol, 1.76 mL) methyl p-toluenesulfonate
allowed to react to yield 8d as a white solid (2.52 g,
ArH). C NMR (DMSO-d ) (two rotamers) d ppm:
6
(
2
30.5 (CH ), 37.0, 38.0 (2· N–CH ), 46.7 (Fmoc-CH),
3
2
47.0, 47.4 (2· N–CH ), 47.5, 48.1 (2· Gly-CH ), 54.4,
2
2
54.9 (2· CH ), 64.9 (All–CH ), 65.3, 65.5 (2· Fmoc-
2
2
1
6
1%). H NMR (CDCl ) d ppm: 2.24 (3H, s, CH ),
CH ), 65.8 (All–CH ), 74.6, 74.7 (2· CH), 108.7,
3
3
2
2

L. Bethge et al. / Bioorg. Med. Chem. 16 (2008) 114–125
123
1
1
10.8, 110.8, 117.6 (4· ArC), 117.8, 118.5 (2· All@CH ),
2Fmoc-ArH, 2ArH), 8.24 (1H, m, ArH), 8.73 (1H, d,
2
1
3
20.1 (Fmoc-ArC), 122.9 (ArC ), 124.4 (ArC), 125.0
J = 7.9, ArH). C NMR (DMSO-d ) (two rotamers) d
q
6
(
1
Fmoc-ArC), 125.7, 125.9, 126.2 (3· ArC), 127.0,
ppm: 30.5 (CH ), 37.9 (N–CH ), 46.7 (Fmoc-CH), 47.0,
47.2 (2· N–CH ), 47.9, 49.2 (2· Gly-CH ), 54.4, 55.0
3
2
27.6 (2· Fmoc-ArC), 131.2 (ArC ), 132.1, 132.2 (2·
q
2
2
All–CH), 133.9 (ArC), 138.0, 138.1 (2· ArC ), 140.7,
(CH ), 65.4, 65.5 (2· Fmoc-CH ), 74.6, 74.7 (2· CH),
q
2
2
1
1
1
1
40.7, 143.7, 143.8 (4· Fmoc-ArC ), 144.1 (ArC),
108.7, 110.8, 110.8, 117.7 (4· ArC), 120.1, 120.1 (2·
q
46.0 (ArC ), 150.1 (ArC), 156.1, 156.5 (2· Fmoc-C ),
Fmoc-ArC),122.9, 123 (2· ArC ), 124.5 (ArC), 125.1
q
q
q
61.5, 161.5 (2· ArC), 166.2, 166.5 (2· C ), 168.4,
(Fmoc-ArC), 125.8, 125.9, 126.2 (3· ArC), 127.0, 127.6,
q
69.3 (2· Gly-C ). HR-MS: m/z calcd for
127.6 (3· Fmoc-ArC), 131.3 (ArC ), 132.9, 133.0 (2·
q
q
4
6+
+
C H N O 695.2864, found 695.2860 [M] .
ArC), 138.1, 138.2 (2· ArC ), 140.7, 140.7, 143.8, 143.8
4
2
39
q
(
4· Fmoc-ArC ), 144.2, 144.5 (ArC), 146.1, 150.2 (2·
q
4
4
8
.2.20. Fmoc-Aeg(JO)-OAll (12d). Violet solid (326 mg,
%). TL1C R = 0.68 (CH Cl /MeOH/HCOOH
ArC ), 156.1, 156.5 (2· Fmoc-C ), 161.6, 161.6 (2· ArC ),
q
q
q
2
166.0, 166.4 (2· C ), 170.2, 171.1 (2· Gly-C ). HR-MS: m/z
f
2
2
q
q
þ
calcd for C H N O 655.2551, found 695.2543 [M] .
39 35 4
6
+
9.5:10:0.5). H NMR (DMSO-d ) (two rotamers) d
6
ppm: 3.17 (1H, m, N–CH ), 3.41 (2H, m, N–CH ),
2
2
3
4
.64 (1H, m, N–CH ), 4.18 (4H, m, CH , Gly-CH ),
2
.24 (1H, m, Fmoc-CH), 4.32 (0.8H, d, J = 6.8, Fmoc-
4.2.22. Fmoc-Aeg(JO)-OH (13d). Following the proce-
dure of 12b were 395 mg (0.509 mmol) Fmoc-Aeg(JO)-
OAll (12d), 54.5 mg (0.509 mmol, 55.7 lL) N-methylan-
3
2
CH ) 4.41 (1.2H, d, J = 6.6, Fmoc-CH ), 4.52 (0.8H, s,
2
2
Gly-CH ), 4.57 (1.2H, d, J = 5.3, All–CH ), 4.77
2
iline, and 29.4 mg (0.025 mmol) Pd(PPh ) in 40 mL dry
2
3 4
(
0.8H, d, J = 5.5, All–CH ), 5.20 (0.7H, dd, J = 1.2,
1
and degassed THF allowed to react o yield 13d as a vio-
let solid (291 mg, 78 %). TLC R = 0.23 (CH Cl /
MeOH/HCOOH 89.5:10:0.5). H NMR (DMSO-d6)
2
J = 10.5, All@CH ), 5.30 (1H, m, All@CH ), 5.43
(
(
2
2
2
f
2
2
1
1.3H, m, All@CH , CH ), 5.59 (1H, s, CH ), 5.88
2
2
2
0.6H, m, All–CH), 6.03 (0.4H, m, All–CH), 6.48 (1H,
(two rotamers) d ppm: 3.15 (1H, m, N–CH ), 3.39
2
m, CH), 7.30 (3H, m, 2Fmoc-ArH, ArH), 7.40 (2H, t,
J = 7.30, 2Fmoc-AcH), 7.54 (2H, m, 2ArH), 7.771
(2H, m, N–CH ), 3.59 (1H, m, N–CH ), 4.03 (1H, m,
Gly-CH ), 4.20 (3H, s, CH ), 4.25 (1H, m, Fmoc-CH),
2 3
2
2
(
2
3H, m, 2Fmoc-ArH, ArH), 7.88 (2H, d, J = 7.4,
Fmoc-ArH), 8.01 (1H, d, J = 7.5, ArH), 8.10 (1H, d,
J = 7.7, ArH), 8.24 (2H, m, 2ArH), 8.45 (1H, d,
4.30 (0.7H, d, J = 6.8, Fmoc-CH ) 4.39 (1.3H, d,
2
J = 6.9, Fmoc-CH ), 5.36 (1H, s, CH ), 5.57 (1H, s,
2
2
CH ), 6.54 (1H, m, CH), 7.32 (3H, m, 2Fmoc-ArH,
2
1
3
J = 8.5, ArH). C NMR (DMSO-d ) (two rotamers) d
ArH), 7.41 (2H, t, J = 7.8, 2Fmoc-ArH), 7.56 (2H, m,
2ArH), 7.68 (3H, m, 2Fmoc-ArH, ArH), 7.88 (2H, d,
J = 7.5, 2Fmoc-ArH), 8.05 (1H, d, J = 6.1, ArH), 8.15
(1H, d, J = 7.7, ArH), 8.29 (2H, m, 2ArH), 8.50 (1H,
6
ppm: 38.0 (N–CH ), 40.2 (CH ), 46.7 (Fmoc-CH),
7.0, 47.4 (2· N–CH ), 48.1, 49.1 (2· Gly-CH ), 53.9,
2
3
4
2
2
5
CH ), 65.8 (Al–CH ), 81.7 (CH), 108.5, 116.2, 117.3
4.5 (2· CH ), 64.9 (All–CH ), 65.3, 65.5 (2· Fmoc-
2
2
1
3
d, J = 8.5, ArH). C NMR (DMSO-d ) (two rotamers)
6
2
2
(
1
3· ArC), 117.8, 118.4 (2· All@CH ), 118.5 (ArC),
d ppm: 37.9 (N–CH ), 40.2 (CH ), 46.6 (Fmoc-CH),
2
2
3
20.1 (Fmoc-ArC), 122.8 (ArC ), 124.9 (ArC), 125.0
47.0, 47.2 (2· N–CH ), 47.8, 49.1 (2· Gly-CH ), 54.0,
q
2
2
(
Fmoc-ArC), 125.7 (ArC), 127.0, 127.6 (2· Fmoc-
54.5 (2· CH ), 65.3, 65.5 (2· Fmoc-CH ), 81.5 (CH),
2
2
ArC), 132.1, 132.2 (2· All–CH), 132.4, 135.8 (2·
108.5, 116.2, 117.4, 118.4 (4· ArC), 120.1 (Fmoc-
ArC), 138.0, 138.0 (2· ArC ), 140.7, 140.7 (2· Fmoc-
ArC), 122.9 (ArC ), 125.0 (ArC), 125.0 (Fmoc-ArC),
125.7 (ArC), 127.0, 127.5, 127.6 (3· Fmoc-ArC),
q
q
ArC ), 142.8 (ArC), 143.7, 143.8 (2· Fmoc-ArC ),
q
q
1
45.1 (ArC ), 150.0 (ArC), 153.7 (ArC ), 156.1, 156.5
132.4, 135.8 (2ArC), 138.0, 138.1 (2· ArC ), 140.6,
q
q
q
(
Gly-C ), 171.4 (ArC ). HR-MS: m/z calcd for
2· Fmoc-C ), 166.2, 166.5 (2· C ), 168.4, 169.4 (2·
140.7 (2· Fmoc-ArC ), 142.9, 143.1 (2· ArC), 143.7,
q
q
q
143.8 (2· Fmoc-ArC ), 145.2, 148.6, 150.1, 154.0 (4·
q
q
q
þ
+
C H N O 696.2817, found 696.2814 [M] .
ArC ), 156.1, 156.5 (2· Fmoc-C ), 166.2, 166.6 (2·
4
1
38
5
6
q
q
C ), 170.1, 171.1 (Gly-C ), 171.5 (ArC ). HR-MS: m/z
q
q
q
þ
calcd. for C H N O 656.2504, found 656.2497 [M] .
38 34 5
6
+
4
.2.21. Fmoc-Aeg(YO)-OH (13b). To 217 mg (0.279 mmol)
Fmoc-Aeg(YO)-OAll (12b) in 20 mL dry and degassed
THF were added 29.8 mg (0.279 mmol, 30.5 lL) N-methy-
laniline and 16.1 mg (0.014 mmol) Pd(PPh ) under an
4.3. Solid-phase synthesis of labeled PNA conjugates
3
4
atmosphere of Ar and exclusion of light. This solution
was stirred for 16 h. Afterward all volatiles were removed
and the residue was purified by flash column chromatogra-
phy (CH Cl , 0% MeOH, 0% HCOOH ! 4% MeOH,
4.3.1. Loading of Novagen TGR resin. The resin (250 mg,
0.29 lmol/g) was allowed to swell in 10 mL DMF for
30 min. For Fmoc removal the resin was twice treated
with 1.5 mLofDMF/piperidine (4:1, v/v) andsubsequent-
ly washed with DMF (5· 2 mL), CH Cl (5· 2 mL), and
2
2
0
.5% HCOOH) to yield 13b as a red solid (198 g, 96 %).
2
2
1
TLC R = 0.20 (CH Cl /MeOH/HCOOH 89.5:10:0.5). H
DMF (5· 2 mL). For preactivation PyBOP (130.1 mg,
250 lmol) and NMM (37.9 mg, 375 lmol) were added
to a solution of Fmoc-protected glycine (74.3 mg,
250 lmol) in DMF (1.5 mL). After 3 min, the mixture
was added to the resin. After 2.5 h, the resin was washed
with DMF (5· 2 mL), CH Cl (5· 2 mL), and DMF (5·
f
2
2
NMR (DMSO-d ) (two rotamers) d ppm: 3.16 (1H, m,
6
N–CH ), 3.40 (2H, m, N–CH ), 3.61 (1H, m, N–CH ),
2
2
2
3
Fmoc-CH), 4.30 (0.8H, d, J = 6.9, Fmoc-CH ) 4.39
.87 (3H, m, CH ), 4.03 (2H, s, Gly-CH ), 4.22 (1H, m,
3 2
2
(1.2H, d, J = 6.6, Fmoc-CH ), 5.47 (1H, s, CH ), 5.68
2 2
2
2
(1H, s, CH ), 6.30 (0.5H, s, CH), 6.32 (0.5H, s, CH), 7.31
2
2 mL). For capping, the resin was treated twice with
(
2H, t, J = 7.3, 2Fmoc-ArH), 7.40 (3H, m, 2Fmoc-ArH,
ArH), 7.49 (1H, t, J = 7.7, ArH), 7.67 (4H, m,
Fmoc-ArH, 2ArH), 7.78 (2H, m, 2ArH), 7.89 (4H, m,
1.5 mL of a solution of Ac O/pyridine (1:9, v/v). The resin
2
was washed with DMF (5· 2 mL) and CH Cl (5· 2 mL),
2
2
2
and finally dried under reduced pressure.

124
L. Bethge et al. / Bioorg. Med. Chem. 16 (2008) 114–125
4
.3.2. Linear solid-phase synthesis. Fmoc-glycine loaded
MALDI-TOF-MS (sinapinic acid): m/z calcd for
C167H202N O S : 4073, found 4073 [M(average)] .
83 42
+
+
resin (ca. 2 lmol) was allowed to swell in DMF
(
2 mL). After 30 min, the resin was transferred to a syn-
Ac-N
CONH2
thesizer reactor. The resin was washed (2· 200 lL
DMF).
4.3.10.
gccgta AegðYOÞ atagccgGly
(19b).
11.1 mg (ca. 2 lmol) of Fmoc-glycine loaded Novasyn
TGR Rink-Amide resin was used. OD260 = 19.1
4
.3.3. Fmoc cleavage. A solution of DMF/piperidine
(144 nmol, 7.2%); t = 10.79 min; MALDI-TOF-MS
R
þ
(4:1, v/v, 200 lL) was added to the resin. After 2 min,
the procedure was repeated. Finally the resin was
(sinapinic acid): m/z calcd for C167
found 4061 [M(average)] .
H
202
N
83O43 4060,
+
washed with DMF (7· 200 lL).
Ac-N
CONH2
4
.3.11.
gccgta AegðMOÞ atagccgGly
(19c).
4.3.4. Coupling of Fmoc-Bhoc-PNA-monomers. A preac-
tivation vessel was charged with a 0.6 M HCTU solution
in NMP (13.3 lL), a 4 M NMM solution in DMF
1.1 mg (ca. 2 lmol) of Fmoc-glycine loaded Novasyn
TGR Rink-Amide resin was used to first assemble resin
(15c), which was washed with dry and degassed CH Cl
2
2
(
4 lL), and a 0.2 M PNA monomer solution in NMP
40 lL). After 2 min, 40 lL of preactivation solution
(5· 1 mL) and treated with a solution of 12 lmol Me N-
2
(
was transferred to the resin. After 30 min, the resin
was washed with DMF (2· 200 lL).
HÆBH and 2 lmol Pd(PPh ) in 1 mL of dry and de-
3
3 4
gassed CH Cl for 10 min for Alloc removal. The resin
was washed consecutively with dry and degassed
CH Cl , dry DMF, and dry and degassed CH Cl (each
2 2
2
2
2
2
4.3.5. Coupling of Fmoc-Aeg(dye)-OH (12a,b,d) and
Fmoc-Aeg(Alloc)-OH (11). A preactivation vessel was
3· 1 mL), and the treatment with Me NHÆBH and
2
3
Pd(PPh ) is repeated for 20 min. Finally the resin was
3 4
charged with a 0.6 M HCTU solution in NMP
washed consecutively with CH Cl (2· 1 mL), DMF
2
2
(
13.3 lL), a 4 M NMM solution in DMF (4 lL), and
a 0.2 M solution of 13a, b, d or 11, and PPTS in NMP
40 lL). After 2 min, 40 lL of the preactivation solution
(3· 1 mL), dioxane/H O (2· 1 mL), MeOH (1· 1 mL),
2
DMF (3· 3 mL), and CH Cl (3· 3 mL). For labeling
2
2
(
with MO-dye 10 lmol of 1c was dissolved in dry
DMF (0.1 M) and 9.8 lmol PyBOP, 10 lmol PPTS,
and 12 lmol NMM were added and the resulting mix-
ture was shaken for 5 min at room temperature. With
this solution the resin was treated for 2 h. The procedure
was repeated three times. Finally the resin was washed
was transferred to the resin. After 60 min, the procedure
was repeated and finally the resin was washed with
DMF (2· 200 lL).
4.3.6. Capping. One hundred microliters of a solution of
Ac O/2,6-lutidine/DMF (5:6:89, v/v/v) was added to the
with DMF (5· 1 mL) and CH
Cl
(10· 1 mL). Libera-
2
2
2
resin. After 2 min, the resin was washed with DMF (2·
tion from the resin and deprotection was carried out
as described in the general methods. OD260 = 4.68
2
00 lL).
(35.3 nmol, 1.8 %); t = 11.11 min; MALDI-TOF-MS
(sinapinic acid): m/z calcd For C166
R
þ
4
.3.7. Cleavage. A solution of cysteine methyl ester
H
201
N
84
O
42
S
1
+
4077, found 4079 [M(average)] .
hydrochloride (7.5 mg, 45 lmol) in 1.5 mL of a solution
of TFA/m-cresol/H O (93:5:2, v/v/v) was passed through
2
Ac-N
CONH2
the dried resin in 30 min. The resin was washed with
TFA (1· 200 lL). The combined filtrates were concen-
trated in vacuo.
4.3.12.
gccgta AegðJOÞ atagccgGly
(19d).
11.1 mg (ca. 2 lmol) of Fmoc-glycine loaded Novasyn
TGR Rink-Amide resin was used. OD260 = 19.0
(
(
145 nmol, 7.2 %); t = 10.37 min; M ALDI-TOF-MS
R
þ
sinapinic acid): m/z calcd For C166H N O
201 84 43
4061,
4
.3.8. Purification. To the concentrated cleavage solu-
+
found 4062 [M(average)] .
tion was added cold diethyl ether. The precipitate was
collected by centrifugation and disposal of the superna-
tant. The residue was dissolved in water and precleaned
by using a water-equilibrated Sep-pak C18 cartridge.
Colored eluates obtained upon elution with MeCN/
ꢂ
References and notes
1
2
3
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1
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2
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ꢀ
1
ꢀ1
,
6
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8
9
. Saito, Y.; Hanawa, K.; Motegi, K.; Omoto, K.; Okamoto,
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Lmol , and e(JO) = 7200 Lmol
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Ac-N
CONH2
4
.3.9.
gccgta AegðTOÞ atagccgGly
(19a). Thir-
teen milligrams (ca. 4 lmol) of Fmoc-glycine loaded
Novasyn TGR Rink-Amide resin was used.
OD260 = 20.58 (151 nmol, 3.7%); tR = 12.31 min;
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