Optimization of anti-proliferative activity using a screening approach with a series of bis-heterocyclic G-quadruplex ligands

Article abstract of DOI:10.1016/j.bmcl.2013.07.057

Using a phenotypic screening and SAR optimization approach, a phenyl-bis-oxazole derivative has been identified with anti-proliferative activity, optimized with the use of a panel of cancer cell lines. The lead compound was synthesized by means of a short and effective two-step synthesis using Pd-catalyzed direct arylation. The compound stabilizes several quadruplex DNA sequences including a human telomeric DNA and one from the promoter of the HSP90 gene, although the structure-activity relationships of the series are not obviously related to the quadruplex binding.

Full text of DOI:10.1016/j.bmcl.2013.07.057

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5351–5355
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Optimization of anti-proliferative activity using a screening approach
with a series of bis-heterocyclic G-quadruplex ligands
⇑
Stephan A. Ohnmacht, Cristina Ciancimino, Giulia Vignaroli, Mekala Gunaratnam, Stephen Neidle
UCL School of Pharmacy, University College London, WC1N 1AX London, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Using a phenotypic screening and SAR optimization approach, a phenyl-bis-oxazole derivative has been
identified with anti-proliferative activity, optimized with the use of a panel of cancer cell lines. The lead
compound was synthesized by means of a short and effective two-step synthesis using Pd-catalyzed
direct arylation. The compound stabilizes several quadruplex DNA sequences including a human telomer-
ic DNA and one from the promoter of the HSP90 gene, although the structure–activity relationships of the
series are not obviously related to the quadruplex binding.
Received 19 June 2013
Revised 18 July 2013
Accepted 24 July 2013
Available online 1 August 2013
Keywords:
Quadruplex
Ó 2013 Elsevier Ltd. All rights reserved.
Anti-proliferative
Phenotypic screening
Telomerase
Oxazoles
The targeting of G-tracts in genomic DNA that can fold into
higher-order quadruplex structures,1–3 with the aid of small mole-
cules,4 is emerging as a novel approach to cancer therapy, and may
well have applications in other diseases. This concept has recently
been validated by the direct visualization of quadruplex DNA in
human cells and by the finding that they can be stabilized by small
molecules.5 Many first- and second-generation quadruplex-bind-
ing and stabilizing ligands share common features of large planar
surface areas and pendant cationic side-chains. Some have high
(>103) selectivity for quadruplex over duplex DNA and have shown
selectivity for cancer cell types over normal cells. Few have pro-
gressed to in vivo evaluation in human cancers, in part because
of the perception that their features are insufficiently drug-like.
More recently, a number of families of non-polycyclic quadruplex
ligands have also been described, some of which have selectivity
for particular promoter quadruplexes,3 such as those encoded in
the c-MYC,6 c-KIT7 and HSP908 promoter sequences. A large num-
ber of such quadruplex DNA targets are likely to exist, very few of
which have yet been validated. This raises the challenge that even
if selectivity for a chosen quadruplex is achieved, there is currently
no straightforward approach that can be taken to ensure that such
a target is unique within quadruplex space, although it is highly
likely that particular quadruplexes can have unique sequences
and tertiary structures. An example of this is shown by the (two)
quadruplexes encoded in the c-KIT promoter.9 Telomeric
quadruplexes may also have unique features since the length of
the single-stranded telomeric DNA sequence is sufficient for sev-
eral consecutive quadruplexes to be formed from it.10
Optimization of biological activity for a given library of com-
pounds has for the most part utilized structure–activity data ob-
tained from quadruplex affinity measurements. Structure-based
molecular design using crystallographic or NMR information has
mostly focused on telomeric11 or c-MYC quadruplexes,12 simply
because of the lack of structural data on many other potential
quadruplex targets. We present here an alternative approach that
starts with a family of quadruplex binding compounds and opti-
mizes them by means of several cycles of phenotypic screening
for anti-proliferative activity in a panel of cancer cell lines. This
does not rely on the assumption of any particular quadruplex tar-
get, and we cannot discount the possibility that there may be alter-
native non-quadruplex targets involved.
We have previously identified a series of para-disubstituted
phenyl-bis-triazoles (Fig. 1, compound I) as well as a focused li-
brary of disubstituted naphthyl-bis-triazoles (of which compound
CL67 (II) emerged as the lead quadruplex-binding compound tar-
geting the HIF-1
a a pro-
pathway,13 presumed to be via a HIF-1
moter quadruplex14). Separately we have shown that a series of
disubstituted phenyl bis-oxazole compounds has selectivity for
the (hitherto unreported) promoter quadruplexes in the HSP90
gene.8
This series of HSP90 promoter quadruplex-binding compounds
have to date shown significant broad-spectrum anti-proliferative
activity in a panel of human carcinoma cell lines, which includes
the MCF-7 breast cancer line. In addition, a closely-related small li-
brary of meta-disubstituted phenyl-bis-triazoles (III) has yielded
⇑
Corresponding author.
E-mail address: s.neidle@ucl.ac.uk (S. Neidle).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.07.057

5352
S. A. Ohnmacht et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5351–5355
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
I
II
III
CL67
NH
HN
HN
NH
NH
HN
O
O
O
O
O
O
Et2N
NEt2
R2N
NR2
NR2
R2N
Figure 1. Three bis-triazole frameworks all of which lack low
lM in vitro potency against MCF7 breast cancer cells.
no potent lead compound that fulfills the criteria of activity
towards MCF7 cells. As part of ongoing optimization studies of
compound CL67 (with an IC50 value of >25 lM in the MCF-7 breast
carcinoma cell line) we have recently discovered that small struc-
tural modifications influencing, among other factors, lipophilicity,
can lead to very large changes in biological profile. We describe
here the design, synthesis and biological evaluation of this
particular class of compounds and have found that a phenotypic
screening approach as a tool for SAR optimization is superior to
considerations of in vitro quadruplex binding.
N3
H2N
1. KI/CH3CN
2. tBuONO, HCl conc.
NH2
N
H
N
N
NaN3, THF
MeN
N
NMe
NMe
Br
Br
Cl
1. KI/CH3CN
NH2
N
H
N
N
N
N
N
N
N
N
N
N
N
clogP 5.30
N
clogP 4.92
'click'
clogP 1.84
clogP 1.47
(protonated)
(protonated)
NH
HN
HN
NH
2
1
N
N
N
N
N
N
N
N
N
N
N
N
N
N
O
O
O
O
O
O
clogP 7.40
clogP 6.22
'direct arylation'
clogP 3.95
clogP 2.76
(protonated)
(protonated)
HN
NH
HN
N
NH
N
N
N
4
3
O
O
N
N
N
N
N
N
Scheme 1. Synthetic pathway for the preparation of final bis-heterocyclic compounds 1–4.

S. A. Ohnmacht et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5351–5355
5353
Our synthetic efforts initially focused on a Cu[I]-catalyzed 1,3-
dipolar cycloaddition,15 which allowed us to gain access to com-
pounds 1 and 2. This approach was subsequently expanded to in-
nificant change in clogP values on account of the differences in
electronic character between the two rings. The fourth and final
round of cellular evaluation involved the phenyl analogue 4 of
compound 3, with a >1 unit lower clogP value. This is slightly
and consistently more potent in the cell lines of this panel. Several
shorter side-chain length meta-disubstituted phenyl bis-oxazoles
(5–8) have also been synthesized and similarly show potency
across the cancer cell line panel, including in MCF7 cells (Fig. 3, Ta-
clude
a
direct arylation reaction giving rapid access to
compounds 3 and 4. The Scheme highlights the synthetic approach
in more detail. Commercially available 1-(3-chloropropyl)-4-
methyl-piperazine was reacted in two separate reactions with
meta-phenylenediamine and 3-bromo-aniline, respectively. The
meta-halogenated analogue was isolated, purified over silica and
directly employed in a direct arylation reaction using pivalic acid
and a palladium catalyst to give final compounds 3 and 4.16 The
azide precursor utilized in the cycloaddition reaction was gener-
ated via the standard sodium azide/tBuONO protocol.17 Finally, a
click reaction was used to generate target compounds 1 and 2. A
number of end-groups and side-chain lengths have been explored,
but here the focus is on the N-methyl-piperazine substituted com-
pounds in view of earlier studies indicating that this is a favored
end-group.18
N
N
N
N
O
O
O
O
5
6
NH
HN
NH
HN
N
N
A 96 h sulforhodamine B (SRB) assay19 to evaluate the anti-pro-
Et2N
NEt2
liferative activity of 1 showed that IC50 values were above 25 lM
for all lines in a small panel of cancer cell lines (Table 2). A second
round of cellular evaluation examined the effects of modifying the
substitution pattern on the terminal phenyl ring system from para
to meta. This did not enhance potency, as judged by lower IC50 val-
ues in the MCF7 and A549 cell lines (compound 2), but a significant
decrease in IC50 value for the two renal cancer cell lines was ob-
served. This is in accord with previous findings for compound
CL67,13 which has highly selective activity in renal compared to a
number of other carcinoma cell lines and suggests that the meta
substitution may be a critical factor in this selectivity. As the initial
rationale for these compounds was to further develop a SAR study
on CL67, it was decided to alter the linking heterocyclic moiety
from a triazole to an oxazole ring, which would contribute to an
overall increase in lipophilicity and possibly also stability in partic-
ular, as the oxazoles are aryl-substituted at the 2-position.
Compound 3 was generated using our previously-reported
approach8 using a direct arylation/C–H activation step to facilitate
the late-stage introduction of the polar side-chains (Scheme 1). The
third round of cellular evaluation using compound 3 showed a sig-
nificant improvement in anti-proliferative activity (Table 2), with
IC50 values in all the cell lines of between 1.2 and 2.1 lM, the sole
structural difference being two oxazole rings replacing two tria-
zoles. This is a conservative structural change yet results in a sig-
N
N
N
N
O
O
O
O
8
7
O
O
N
N
N
N
Figure 3. Expanded focused library of meta-disubstituted phenyl-bis-oxazoles (5–
8) with broad spectrum anti-proliferative activities.
Table 1
D
Tm Values (°C) for FRET analyses of compounds 1–8 at 1 and 5
with a series of quadruplex-forming DNA sequences: F21T, c-KIT2, HSP90A
lM concentrations
F21T
c-KIT2
5
HSP90A
5
dsDNA
l
M
1
5
1
1
1
5
1
2
3
4
5
6
7
8
<2
2.9
<2
<2
<2
<2
<2
<2
<10
<10
<10
20.1
24.7
13.3
25.9
n/a
<2
2.4
<2
<2
<2
<2
<2
<2
<10
<2
<10
<10
<10
25.6
n/a
19.5
27.7
n/a
0
0
0
0
0
0
0
2.0
0
0
0
0
6.8
0
5.3
n/a
<10
<10
18.3
17.0
13.6
20.3
n/a
5.7
2.5
<2
<2
<2
3.4
5.0
dsDNA represents duplex DNA.
Esds are from triplicate measurements and average 0.3 °C.
Table 2
Short-term anti-proliferative activity (IC50 values in
lM) for compounds 1–8, in a
cancer cell line panel, comprising MCF7 (breast), 786-O/RCC4 (renal), A549 (lung
cancer) and WI38 (fibroblast) cell lines, determined using a 96 h SRB assay
Short term IC50
MCF-7
786-O
RCC4
A549
WI38
1
2
3
4
5
6
7
8
>25
>25
>25
>25
>25
>25
>25
8.7 0.1
1.2 0.3
0.9 0.1
1.1 0.3
1.3 0.1
0.7 0.1
2.3 0.2
8.4 0.1
2.1 0.1
1.2 0.2
1.1 0.1
1.1 0.1
1.3 0.1
1.9 0.3
15.3 0.5
3.1 0.2
1.1 0.2
1.0 0.1
2.8 0.2
1.0 0.3
4.4 0.2
1.9 0.2
1.0 0.2
2.1 0.5
3.1 0.2
1.2 0.3
1.4 0.2
1.6 0.3
1.2 0.4
0.6 0.1
2.5 0.4
1.0 0.2
1.2 0.1
Figure 2. FRET melting curve for compound 4 with the 21-mer Hsp90A promoter
quadruplex sequence at different ligand concentrations. There is a significant shift
in melting temperature (Tm) for compound 4 at the 5 lM concentration, whereas
there is little shift at the lower concentrations.
Esds are from triplicate (or more) measurements. IC50 values shown as >25 lM are
all substantially above this value and were not determined with equivalent
accuracy.

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S. A. Ohnmacht et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5351–5355
Figure 4. Representative low-energy molecular models for complexes of compounds 3 (left, colored cyan) and 4 (right, colored purple) with the parallel human telomeric
quadruplex.21 Models were obtained by initial manual optimization based on previous crystal structures of bound complexes, followed by intermolecular energy calculations.
ble 2). This strongly suggests that side chain length is not critical
for enhanced cellular activity.
quadruplex stabilization in FRET assays. Only at high concentra-
tions (5 M of compounds 4–7) was there significant stabilization
in the FRET assay (accurate curve fitting for compound 8 was not
possible at 5 M, so no FRET data is available at this concentra-
l
An initial rationale for the development of these and other re-
lated ligands has been that they act by interacting with cellular
G-quadruplexes such as those in human telomeres or in various
promoter sequences of oncogenic genes. We have previously
shown that the para-substituted phenyl-bis-oxazole framework
has selectivity for a quadruplex encoded in the promoter of the
HSP90 gene. Data from a FRET assay20 (Table 1) shows that com-
pounds 1–3 do not stabilize a human telomeric quadruplex se-
quence (F21T). However compound 4 (as well as 5 and 7,
although some duplex DNA binding is evident for these two com-
pounds) does have a significant stabilizing effect on this quadru-
plex as well as on the HSP90 and c-KIT2 quadruplexes, albeit at
higher concentrations (Table 1). Compound 4 does not stabilize
double-stranded DNA in the range of concentrations used.
An initial molecular modeling examination of the potential
interactions between compounds 3 and 4 and the parallel human
telomeric quadruplex crystal structure21 has been undertaken
(Fig. 4). The parallel topology was chosen since it has been ob-
served in all ligand–telomeric quadruplex crystal structures deter-
mined to date.11 The study suggests that there are unlikely to be
major differences between the two complexes, although the smal-
ler size of the core groups in compound 4 may result in improved
stacking of the two oxazole groups with adjacent guanine rings.
This is in accord with the slightly superior stabilization observed
in the FRET assays with compounds 4–8 at high ligand concentra-
tions (Fig. 2). We cannot rule out, and at this stage, have not exam-
ined, alternative binding modes involving groove interactions,
although it is possible that they might explain the large differences
in biological responses between compounds 1, 2 and 3, 4. No rele-
vant experimental data on these alternative binding modes is cur-
rently available.
l
tion). This suggests that either the compounds act at other quadru-
plex loci that are yet to be determined, or that the compounds act
by non-quadruplex mechanisms. Regardless of quadruplex target,
we suggest that cell-based phenotypic screening can speedily opti-
mize anti-proliferative activity in these series. This may be an use-
ful approach to obtaining poly-targeted quadruplex-binding
compounds with high biological activity and good pharmacological
profiles. The compounds presented here have some structural
resemblance to kinase inhibitors, although there is no indication
that they interact with kinases—the related compound CL67 shows
no activity in a large kinase panel (Welsh et al., unpublished
observations).
Acknowledgments
S.A.O. and C.C. contributed equally to this work. We are grateful
to Cancer Research UK (Programme Grant No. C129/A4489 to S.N.).
The Novartis Institute of BioMedical Research, Boston, is thanked
for obtaining CL67 data in their kinase panel.
References and notes
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In summary this study has shown that in this series of com-
pounds the determinants of optimal anti-proliferative potency are:
1. The possession of a central phenyl ring.
2. Two attached phenyl-oxazole rings.
3. These should bemeta attached to the phenyl rings.
4. The length of the linker and nature of the terminal group is of
lesser importance.
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have shown superior stabilization ability with several quadruplex
sequences (HSP90A and the human telomeric quadruplexes) yet
with comparable anti-proliferative abilities. Few of the bis-triazole
and oxazole derivatives presented here had any significant
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(4H, m, 4 ꢀ Ar–H), 8.19 (2H, t, J = 1.8 Hz, 2 ꢀ Ar–H), 8.46–8.48 (4H, m, 4 ꢀ Ar–
H). 13C NMR (100 MHz, CDCl3) d 148.2 (2 ꢀ CH), 143.2 (2 ꢀ CH), 138.0 (2 ꢀ CH),
133.6 (quat), 133.3 (quat), 131.0 (2 ꢀ CH), 128.8 (2 ꢀ quat), 128.1 (2 ꢀ quat),
125.0 (2 ꢀ CH), 124.3 (2 ꢀ quat), 118.6 (2 ꢀ quat), 118.2 (2 ꢀ CH), 118.1
(2 ꢀ CH), 110.7 (2 ꢀ CH), 54.4 (2 ꢀ CH2), 53.3 (4 ꢀ CH2), 50.7 (4 ꢀ CH2), 43.8
(2 ꢀ CH2), 41.7 (2 ꢀ CH2), 23.3 (2 ꢀ CH3). HRMS (ES+) calculated for C42H52N12
[M+1]+ 725.4516, found 725.4482.
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1000–4000 cells with 160
well of 96 well plate (Nunc, Denmark). After incubation for 24 h, the
compound to be tested was dissolved in 40 L of medium and was added in a
range of concentrations, and the cells incubated for 96 h. The medium was
then removed and the cells fixed by incubation with TCA (10%, Sigma–Aldrich,
UK) for 30 min at 4 °C. After removal of the TCA, the cells were washed with
deionised water five times and dried at 60 °C for 1 h. The cells were then
lL media (WI38: 6000/well) were seeded into each
a
l
15. Moorhouse, A. D.; Santos, A. M.; Gunaratnam, M.; Moore, M.; Neidle, S.; Moses,
J. E. J. Am. Chem. Soc. 2006, 128, 15972.
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3578.
incubated with sulforhodamine B (80
UK) for 15 min at rt. The SRB was removed, the wells washed with 1% acetic
acid (200 L), and dried at 60 °C for 1 h. Tris-base (100 L, 10 mM, Acros
Organics, UK) solution was added to each well, and the plates were gently
shaken for 5 min. The absorbance at 540 nm was measured with a plate reader
(Spectrostar Omega, BMG Labtech, Germany). The data were normalized to the
value of 100 for the control experiment (untreated cells), and the IC50 values
were obtained by interpolation from a plot with Origin (Version 7.0, OriginLab
Corp.), as the concentration leading to an absorbance intensity of 50%.
lL, 0.4% in 1% acetic acid, Acros Organics,
17. Direct arylation: 3,30-(5,50-(1,3-phenylene)bis(oxazole-5,2-diyl))bis(N-(3-(4-
methylpiperazine-1-yl)propyl)aniline) (3, 4). An oven dried microwave vial
was charged with Pd(OAc)2 (0.1 equiv), K2CO3 (3 equiv), ligand (2-
l
l
dicyclohexylphosphino-20,60-diisopropoxybiphenyl,
0.2 equiv),
oxazole
precursor (1 equiv) and arylbromide (2.2 equiv). The microwave vial was
capped, evacuated and backfilled with argon and then pivalic acid (0.4 equiv)
and toluene were added. The vial was heated at 110 °C for 72 h. After cooling to
room temperature, the reaction mixture was concentrated and purified using
silica gel. (400 MHz, CDCl3) d 1.26 (4H, s, 2 ꢀ CH2), 1.65 (4H, br s, 2 ꢀ CH2), 1.85
(4H, quintet, J = 6.3 Hz, 2 ꢀ CH2), 2.31 (6H, s, 2 ꢀ CH3), 2.51–2.55 (12H, m,
6 ꢀ CH2), 3.30 (4H, t, J = 6.3 Hz, 2 ꢀ CH2), 6.72 (2H, dd, J = 1.6, 8.1 Hz, 2 ꢀ Ar–
H), 7.29 (2H, t, J = 7.8 Hz, 2 ꢀ Ar–H), 7.35 (2H, t, J = 1.6 Hz, 2 ꢀ Ar–H), 7.46 (2H,
d, J = 7.8 Hz, 2 ꢀ Ar–H), 7.49–7.53 (2H, m, 2 ꢀ Ar–H), 7.69 (2H, dd, J = 1.5,
7.8 Hz, 2 ꢀ Ar–H), 8.01 (1H, br s, Ar–H). 13C NMR (100 MHz, CDCl3) d 162.1
(2 ꢀ quat), 150.4 (CH), 149.1 (2 ꢀ CH), 129.7 (2 ꢀ CH), 129.6 (2 ꢀ CH), 129.0
(2 ꢀ CH), 128.1 (2 ꢀ CH), 124.1 (2 ꢀ quat), 124.0 (2 ꢀ quat), 119.7 (2 ꢀ quat),
115.3 (CH), 115.2 (2 ꢀ CH), 109.8 (2 ꢀ CH), 57.2 (2 ꢀ CH2), 55.3 (4 ꢀ CH2), 53.2
(4 ꢀ CH2), 46.0 (2 ꢀ CH2), 43.5 (2 ꢀ CH2), 25.6 (2 ꢀ CH3). HRMS (ES+)
calculated for C40H50N8O2 [M+1]+ 675.4135, found 675.4125.
20. FRET DNA melting assays on compounds 1–8 were performed as previously
described (Guyen, B. Schultes, C. M. Hazel, P. Mann, J. Neidle, S. Org. Biomol.
Chem. 2004, 2, 981) using a fluorescence resonance energy transfer (FRET)
assay modified as a high-throughput screen in a 96-well format. The labelled
oligonucleotides had attached the donor fluorophore FAM: 6-
carboxyfluorescein
carboxytetramethyl-rhodamine. The FRET probe sequences were diluted
from stock to the correct concentration (400 nM) in 60 mM potassium
and
the
acceptor
fluorophore
TAMRA:
6-
a
cacodylate buffer (pH 7.4) and then annealed by heating to 95 °C for 10 min,
followed by cooling to room temperature in the heating block (3–3.5 h). The
compounds were stored as a 1 mM stock solution in 10% DMSO/90% 1 mmol
HCl; final solutions (at 2 ꢀ concentration) were prepared using 60 mM
potassium cacodylate buffer (pH 7.4). Relevant controls using BRACO-19 (in
addition to blank runs) were also performed to check for quality of DNA
samples (e.g., F21T). 96-Well plates (MJ Research, Waltham, MA) were
18. Click
reaction:
3,30-(4,40-(naphthalene-2,7-diyl)bis(1H-1,2,3-triazole-4,1-
diyl))bis(N-(3-(4-methylpiperazin-1-yl)-propyl) aniline) (1, 2). Dialkyne
(1 equiv) was dissolved in the appropriate volume of solvent (H2O:tBuOH
1:1), followed by the addition of required azide (3 equiv) and the catalytic
mixture of CuSO4 ꢀ 5H2O (0.05 equiv), sodium ascorbate (0.5 equiv) and
bathophenanthroline disulfonic acid disodium salt hydrate (0.1 equiv). The
reaction was performed with excess of the required amine (6 equiv). The
mixture was heated under microwave irradiation for 15 min at 120 °C. Crude
product was obtained following evaporation of solvents and subsequent C18
reversed phase semi-prep purification. 1H NMR (400 MHz, CDCl3) d 1.81 (4H,
quintet, J = 6.6 Hz, 2 ꢀ CH2), 2.45 (4H, t, J = 6.6 Hz, 2 ꢀ CH2), 2.58 (6H, s,
2 ꢀ CH3), 2.67 (8H, br s, 4 ꢀ CH2), 2.94 (8H, br s, 4 ꢀ CH2), 4.16 (4H, t, J = 7.3 Hz,
2 ꢀ CH2), 7.64–7.68 (2H, m, 2 ꢀ Ar–H), 7.75–7.77 (4H, m, 4 ꢀ Ar–H), 7.93–8.03
prepared by aliquoting 50
lL of the annealed DNA into each well, followed
by 50 L of the compound solutions. Measurements were made on a DNA
l
Engine Opticon (MJ Research) with excitation at 450–495 nm and detection at
515–545 nm. Fluorescence readings were taken at intervals of 0.5 °C in the
range 30–100 °C, with a constant temperature being maintained for 30 s prior
to each reading to ensure a stable value. Final analysis of the data was carried
out using
Northampton, MA). The advanced curve-fitting function in Origin 7.0 was
used for calculation of Tm values. Esds in Tm are 0.1 °C.
21. Parkinson, G. N.; Lee, M. P. H.; Neidle, S. Nature 2002, 417, 876.
a script written in the program Origin 7.0 (OriginLab Corp.,
D
D