Synthetic receptor and its association with CTD peptides

Article abstract of DOI:10.1016/j.tet.2012.01.024

In this study, a receptor for phosphorylated peptide was developed. The receptor contains Zn²⁺-dipicolylamine (Zn-Dpa) moiety at one end and guanidiniocarbonyl pyrrole at the other. The association between the receptor and CTD (carboxy-terminal

Full text of DOI:10.1016/j.tet.2012.01.024

Tetrahedron 68 (2012) 2357e2362
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthetic receptor and its association with CTD peptides
*
Saihui Zhang, Liang Han, Chuan-guang Li, Jun Wang, Wei Wang, Zhi Yuan , Xueping Gao
College of Chemistry, Nankai University, Tianjin 300071, China
a r t i c l e i n f o
a b s t r a c t
In this study, a receptor for phosphorylated peptide was developed. The receptor contains Zn2þedipi-
colylamine (ZneDpa) moiety at one end and guanidiniocarbonyl pyrrole at the other. The association
between the receptor and CTD (carboxy-terminal domain of RNA polymerase II) peptides with different
phosphorylated patterns was investigated by isothermal titration calorimetry and molecular docking,
and the receptor showed much higher affinity towards bis-phosphorylated CTD peptide with affinity
constant K¼1.09ꢀ105.
Article history:
Received 5 November 2011
Received in revised form 30 December 2011
Accepted 13 January 2012
Available online 18 January 2012
Keywords:
Artificial receptor
Ó 2012 Elsevier Ltd. All rights reserved.
Phosphorylated peptides
CTD peptides
Isothermal titration calorimetry
1. Introduction
(CTD) composed of up to 52 heptapeptide repeats (Tyr-Ser-Pro-Thr-
Ser-Pro-Ser), and this sequence is thought to be phosphorylated at
Phosphorylation is one of the most important post-translational
modifications of protein. It regulates many important biological
processes, such as the growth and differentiation of living cell.
Developing receptors, which can tightly associate with phosphor-
ylated peptides is not only necessary for the regulation of protein
phosphorylation but also for the disruption of proteineprotein in-
teraction, additionally, when conjugated with some signal groups,
the receptor can also find applications in sensors and imaging.1
Pioneering studies by Hamachi and co-workers provide funda-
mental insight into recognition of phosphorylated peptides using
artificial receptors,2,3 after that, Andreas grauer and co-workers
reported the binding of carboxylate side-chain-containing phos-
phorylated peptide using synthetic receptors,4 and all these re-
ceptors employ zinc(II) complexes as the phosphate binding
moieties. Schmuck and co-workers developed receptors based on
guanidiniocarbonyl pyrrole, because of the electro-withdrawing
nature of the carbonyl and the hydrogen-bond-donating role of
pyrrole, guanidiniocarbonyl pyrrole shows much higher affinity
towards carboxylate and phosphate groups in comparison with
simple guanidinium ion.5 Besides, there are also some important
fundamental researches in the field of artificial receptors for
peptides.6e17
several positions in vivo, predominantly at serines 2 and 5.18 CTD
regulates the activities of the polymerase by phosphorylation of
different patterns, and many important biological events are con-
sidered to be related to this process, also, some antitumour drugs
have been proved to take effect by regulating the phosphorylation
of CTD.19 Hence, developing receptors, which can effectively asso-
ciate with CTD is of great biological importance.
In this study, we synthesized a receptor bearing Zn2þedipico-
lylamine at one end and guanidiniocarbonyl pyrrole at the other
end, and investigated its association with CTD peptides with dif-
ferent phosphorylated patterns.
2. Results and discussion
Molecular structures of the receptor and the controls were
shown in Fig. 1a, and the detailed synthesis was provided in ex-
perimental section. Receptors 1, 2 and 3 were controls. Receptor 1
was Zn2þedipicolylamine (ZneDpa), which can selectively bound
phosphate group with moderate affinity in aqueous solution. Re-
ceptor 2 was guanidiniocarbonyl pyrrole (GCP), which can bind
oxygen-containing anions via electrostatic force and hydrogen
bond. Receptor 3, which does not have guanidine group, was pre-
cursor of receptor 4. Receptor 4 was the anticipant receptor with
ZneDpa at one end and GCP at the other end, a benzene was
employed as the linker, which is considered to provide the receptor
with rigidity as well as hydrophobic property.
In eukaryotes, transcription of mRNA is controlled by poly-
merase II. RNA polymerase II contains a carboxy-terminal domain
The association between the receptors and CTD peptides with
different phosphorylated patterns was investigated by isothermal
* Corresponding author. Tel.: þ86 22 23501164; fax: þ86 22 23503510; e-mail
address: zhiy@nankai.edu.cn (Z. Yuan).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2012.01.024

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S. Zhang et al. / Tetrahedron 68 (2012) 2357e2362
Fig. 1. (a) Molecular structures of the synthetic receptors, (b) showed the phosphoralated patterns of CTD.
titration calorimetry (ITC), and the results were summarized in
Table 1 ‘d’ Indicate that the reaction heat is too small to be accu-
rately fitted in Origin. For receptor 1 and 2, 20 mM receptor was
loaded in the syringe and 1 mM peptide was loaded in the cell. For
receptor 3 and 4, limited by their solubility, 1e2 mM peptide was
Y2p5pS was well fitted using ‘two set of sites’ mode, and thus have
two association constants, and K2 is much higher than K1, this is
because Y2p5pS has two phosphate groups and more negative
charge, the association of the first ZneDpa decreased the net
negative charge of the peptide, and thus reduced the electrostatic
attraction. The associations between ZneDpa and the phosphor-
ylated CTD peptides were all endothermic, which are considered
to be entropy-driven.
Receptor 2 is a simple GCP, which can bind oxygen-containing
anions through electrostatic and hydrogen-bonding interaction.5
In comparison with ZneDpa, the associations between GCP and
the peptides were much weaker, with all the association constants
below 100. Thus, GCP have no selectivity towards the peptides. The
association is exothermic.
loaded in the syringe and 80e100 mM receptor was loaded in the
cell. YS, Y2pS, Y5pS and Y2p5pS denote non-phosphorylated CTD
peptide, CTD peptide phosphorylated at 2 site, 5 site, and at both 2,5
site, respectively. AceY2p5pSeNH2 denotes Y2p5pS with the terminal
groups are protected.
Receptor 1 is ZneDpa, which has been extensively used as the
coordination moiety of receptors/sensors for the recognition of
biologically relevant phosphate compounds. From Table 1, we can
see that receptor 1 shows moderate affinity towards the phos-
phorylated CTD peptides, and the association constant is about
Receptor 3 is composed of ZneDpa, pyrrole and benzene
groups, thus, it could probably associate with the peptides through
multiple interactions (coordination, hydrophobic and hydrogen-
bond interactions). In comparison with simple ZneDpa, receptor
103, while the association between receptor
1 and non-
phosphorylated CTD peptide is too weak to be detected. The as-
sociation between receptor 1 and bis-phosphorylated CTD peptide

S. Zhang et al. / Tetrahedron 68 (2012) 2357e2362
2359
Table 1
Stoichiometry (n), binding constant (K, Mꢁ1), enthalpy (
D
H, cal molꢁ1) and entropy ( S, cal molꢁ1 Kꢁ1) for the association between the receptors and the CTD peptides at 25 ꢂ
D C
Receptor
Peptide
2pS
Y5p
na
K
D
H
DS
1
1
1
Y
1.0
1.0
1.0
1.0
392ꢃ46.1
435ꢃ42.5
4.62 E3ꢃ0.76 E3
4.74 E3ꢃ0.80 E3
2.89 E3ꢃ0.58 E3
1.39 E3ꢃ1.46 E3
27.2
28.1
26.4
16.8
d
S
Y2p5p
S
4.93 E3ꢃ1.29E3
448ꢃ211
1
2
2
2
2
3
3
3
3
4
YS
d
d
Y2p
Y5p
S
S
1.0
1.0
1.0
1.0
1.0
1.0
1.0
20.8ꢃ1.03
ꢁ7.55 E3ꢃ0.51 E3
ꢁ9.46 E3ꢃ0.43 E3
ꢁ2.9 E4ꢃ0.56 E3
ꢁ9.56 E3ꢃ0.36 E3
1.8 E3ꢃ0.13 E3
1.7 E3ꢃ0.15 E3
4.1 E3ꢃ0.36 E3
d
ꢁ24.4
ꢁ25.7
ꢁ23.1
ꢁ26.1
24.9
22.6
22.5
d
23.4ꢃ1.05
Y2p5p
YS
S
S
25.5ꢃ0.31
20.5ꢃ0.83
Y2p
Y5p
S
S
1.48 E3ꢃ0.73 E3
5.57 E3ꢃ0.59 E3
2.04 E4ꢃ0.27 E4
d
Y2p5p
YS
Y2pS
0.45ꢃ 0.12
4.4ꢃ1.6
2.13 E4ꢃ0.88 E3
917ꢃ74.9
447.8ꢃ88.6
21.3
14.7
25.9
19.4
29.6
27.8
d
917ꢃ74.9
4
4
Y5p
S
1.02ꢃ0.42
1.65ꢃ1.10
0.09ꢃ0.06
0.19ꢃ0.05
2.98 E4ꢃ0.47 E3
3.13 E3ꢃ0.15 E3
1.09 E5ꢃ0.81 E4
5.3 E3ꢃ0.12 E3
d
2.68 E3ꢃ1.11 E3
1.39 E3ꢃ70.7
2.20 E3ꢃ0.42 E3
9.2 E3ꢃ0.44 E3
d
Y2p5p
YS
S
4
4
AceY2p5pSeNH2
0.14ꢃ0.06
0.34þ0.11
4.74 E4ꢃ0.14 E3
2.33 E3ꢃ0.78 E3
1.15 E4ꢃ56.85
1.14 E4ꢃ0.33 E3
24.0
60.2
a
For receptor 1, 2 and 3, the stoichiometry was fixed to 1.0, and the data plots were well fitted to the corresponding model. For receptor 4, the data plots were fitted without
fixing stoichiometry.
3 shows increased affinity towards all the phosphorylated CTD
peptides, this could be reasonably ascribed to the additional force
provided by benzene and pyrrole, but its association with non-
phosphorylated peptide YS is still weak, and can not be mea-
sured. Additionally, receptor 3 shows higher affinity towards Y5p
S
(Ka¼5.57 E3) in comparison with Y2pS (Ka¼1.48 E3), and this could
probably because that the phosphate of Y5pS is closer to C-ter-
minal carboxylate thus has more local net negative charge than
phosphate of Y2pS.
Receptor 4 is the anticipant receptor, which contains ZneDpa,
benzene and GCP, and is expected to associate phosphorylated CTD
peptide with higher affinity. All the associations between receptor 4
and the phosphorylated CTD peptides were endothermic (typical
ITC titration curve of receptor 4 to Y2p5pS is shown in Fig. 2), in-
dicating the entropy-driven nature of the association. In compari-
son with receptor 1, 2 and 3, receptor 4 shows much higher affinity
towards bis-phosphorylated CTD peptide Y2p5pS with association
constant of about 105. This indicates that GCP and ZneDpa are
synergistically involved in the association between receptor 4 and
Y2p5pS.
The association between receptor 4 and Y2p5pS were further
investigated by molecular docking. The results in Fig. 3 show that:
ZneDpa coordination group will associate with phosphate on 5-
Ser, and this is independent of the initial position of ZneDpa, in-
dicating the higher affinity of ZneDpa towards 5-Ser. This is
consistent with the higher affinity (measured by ITC) of receptor 3
towards Y5pS in comparison with Y2pS. Additionally, it can be
observed in Fig. 3 that GCP on receptor 4 is far from either the
terminal carboxylate or phosphate on 2-Ser, indicating no direct
association occurs between GCP and these two groups, further-
more, no hydrogen bond was found between GCP and the peptide
in Autodock, thus, GCP probably only provides electrostatic force
for the association.
To further understand the interaction between receptor 4 and
Y2p5pS, a terminal groups-protected bis-phosphorylated CTD pep-
tide, i.e., AceY2p5pSeNH2, was introduced. ITC data showed that
Fig. 2. ITC data for titration of 80 m
M receptor 4 with 1 mM Y2p5pS at 25 ꢂC.
the association between receptor
4
and AceY2p5pSeNH2
actively involved in the association. Therefore, receptor 4 tend to
associate with C-terminal bis-phosphorylated CTD peptide, i.e.,
Y2p5pS, in comparison with other CTD peptides. The schematic
(K¼4.74ꢀ104) is much weaker in comparison with Y2p5p
S
(K¼1.09ꢀ105), indicating the terminal carboxylate group is also

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S. Zhang et al. / Tetrahedron 68 (2012) 2357e2362
Fig. 3. Molecular docking between receptor 4 and Y2p5pS with different initial fitting modes: (a) phosphate on 2-Ser was fitted to ZneDpa before blind docking, (b) phosphate on 5-
Ser was fitted to ZneDpa before blind docking, (c) no fitting was performed before blind docking. The final association between phosphate on Y2p5pS and ZneDpa on receptor 4 are
highlighted, and all the three figures show that the ZneDpa will associate with phosphate on 5-Ser independent of the initial fitting mode.
illustration of the association between receptor 4 and Y2p5pS is
shown in Fig. 4.
Advantage MS. Isothermal titration calorimetry measurement was
conducted using VP-ITC.
4.3. Synthesis of the receptors
The schematic illustration of the synthesis of the receptors was
shown in Fig. 5.
Preparation of receptor 1: Receptor 1 can be easily obtained by
dissolving certain amount of dipicolylamine by Zn(NO3)2 methanol
solution.
Preparation of receptor 2: Receptor 2 was prepared as reported
by Schmuck’s group.5
Preparation of receptor 3: 4-(Bromomethyl)benzoyl chloride
(10.2 g), 4.0 g methyl 1H-pyrrole-2-carboxylate and 8.72 g ZnCl2
were dissolved in 100 mL CHCl3, and the reaction was kept at 50 ꢂC
for 72 h under N2 atmosphere. Then the mixture was filtrated, and
the solvent was removed under reduced pressure, and a red solid
was obtained. This raw product was further purified by flash
chromatography (silica, CH2Cl2/CH3COOCH2CH3¼30:1), the first
fraction was collected, and compound 3 was obtained as a white
Fig. 4. Schematic illustration of the association between receptor 4 and Y2p5pS.
3. Conclusions
solid (yield: 3.24 g, 31.4%). 1H NMR (400 MHz, CDCl3):
d
¼3.93 (s,
3H), 4.53 (s, 1.3H), 4.65 (s, 0.7H), 6.83 (d, 1H), 6.93 (d, 1H), 7.52 (d,
2H), 7.90 (t, 2H), 10.04 (br, 1H).
We have synthesized receptor based on guanidiniocarbonyl
pyrrole and Zn2þedipicolylamine groups, and investigated its as-
sociation with CTD peptides with different phosphorylated pat-
terns. The receptor showed much higher affinity towards C-
terminal bis-phosphorylated CTD peptide Y2p5pS in comparison
with other forms of CTD peptides as revealed by isothermal titra-
tion calorimetry. In combination with molecular docking, the as-
sociation between the receptor and peptide was well elucidated,
and the results indicate that the coordination occurs between
ZneDpa and phosphate on 5-Ser, while the C-terminal carboxylate
are synergistically involved in the association via electrostatic
interaction.
To a solution of 3.22 g compound 3, 2.19 g 2, 20-dipicolylamine
and 0.67 g K2CO3 in 50 mL dry DMF, was added dropwise 1.66 g KI
in 10 mL DMF, and the reaction was kept at 40 ꢂC for 10 h under N2
atmosphere. After removal of the solvent, a brown oil-like product
was obtained. The product was dissolved in 100 mL CHCl3 and fil-
trated, and then was washed with 50 mL 1 N HCl, 50 mL 2 N NaOH
and 100 mL H2O, the organic phase was then dried with MgSO4.
After removal of the solvent, a brown solid was obtained, and was
further purified by flash chromatography (silica, CHCl3/
CH3OH¼30:1), the second fraction was collected and compound 4
was obtained as a red solid (yield: 2.25 g, 51%). 1H NMR (400 MHz,
CDCl3):
d
¼3.75 (s, 6H), 3.82 (s, 3H), 6.78 (d, 2H), 6.88 (d, 2H), 7.26 (t,
4. Experimental section
4.1. Materials
2H), 7.69 (m, 4H), 7.80 (m, 4H), 8.50 (d, 2H). EI-MS: m/e calcd for
C26H25N4O3 [M] 277.7, found 277.0.
Compound 4 (0.30 g) was dissolved in 24 mL CH3CN/THF (v/
v¼5:1), then 0.20 g Zn(NO3)2 in 11.96 mL CH3OH was added, after
the solution was further stirred for 0.5 h at room temperature, the
white precipitation was filtrated and washed with CH3CN. Re-
ceptor 3 (compound 5) was obtained, 1H NMR (400 MHz, DMSO):
Peptides with purity >98% were purchased from GL Biochem
(Shanghai) Ltd. All other reagents were commercially available
analytical grade and used as received.
d
¼3.70 (s, 2H), 3.97 (s, 6H), 4.10 (s, 1H), 4.14 (s, 1H), 4.32 (s, 1H),
4.2. Instrumentation
4.36 (s, 1H), 6.64 (d, 2H), 6.74 (d, 2H), 7.22 (d, 2H), 7.50 (m, 4H),
7.65 (m, 2H), 7.99 (m, 2H), 8.08 (d, 2H), 8.95 (d, 2H), 10.08 (s, 2H)
ppm, MALDI-TOF: m/e calcd for C26H26N5O6 Zn [M]þ 567.91, found
568.2.
NMR characterization was performed on VARIAN UNITY-plus
400, molecular mass were measured on Thermo Finnigan LCQ-

S. Zhang et al. / Tetrahedron 68 (2012) 2357e2362
2361
Fig. 5. Synthesis of the receptors.
Preparation of receptor 4: Compound 4 (1.32 g) and 0.63 g
LiOH$H2O were dissolved in 100 mL THF/H2O (v/v¼4:1), the solu-
tion was then stirred at room temperature for two days. After the
concentrated to 10 mL, the solution was acidified to pH¼3, and
yellow precipitation was formed and filtrated, after washed with
water, the solid was dried in vacuum and 1.02 g compound 6 was
12.38 (br s, 0.49H) ppm, ESI-MS: m/e calcd for C26H26N7O2 [MþH]þ
468.2142, found 468.2130.
Compound 8 (0.23 g) was dissolved in 20 mL CH3OH, then
0.15 g Zn(NO3)2 in 10 mL CH3OH was added, after stirred for 0.5 h
at room temperature, a red precipitation was formed, after washed
with CH3CN, receptor 4 (compound 9) was obtained, yield 93.0%.
MALDI-TOF: m/e calcd for C26H25N8O5Zn [MþH3O]þ 612.9, found
612.3.
obtained, yield 79.8%. 1H NMR (400 MHz, DMSO):
d
¼3.77 (s, 6H),
6.77 (s, 2H), 6.82 (s, 2H), 7.26 (t, 2H), 7.61 (m, 4H), 7.80 (m, 4H), 8.50
(d, 2H), 11.68 (br s, 0.1H), 12.35 (br s, 1H) ppm, ESI-MS: m/e calcd for
C25H23N4O3 [MþH]þ 427.17565, found 427.1764.
4.4. ITC measurement
Compound 6 (0.93 g), 1.14 g PyBOP and 2 mL NMM were dis-
solved in 30 mL dry DMF, after stirred at room temperature for 1 h,
0.35 g N-Boc-Guanidine was added, the reaction was kept at 40 ꢂC
for 24 h, then the solution was concentrated to 20 mL and poured
into 200 mL H2O under vigorous stirring, and a yellow precipitation
was formed. After vacuum dried, 0.9 g compound 7 was obtained,
All the peptides and the receptors were dissolved in 50 mM
HEPES (pH¼7.2). The titration contains an initial injection of 2
mL
followed by 26 injections of 10 L receptors. Heat of dilution was
m
substracted from titration data before curve-fitting in Origin.
yield 79.3%. 1H NMR (400 MHz, DMSO):
d
¼1.48 (s, 9H), 3.77 (s, 6H),
6.77 (s, 2H), 6.82 (s, 2H), 7.26 (t, 2H), 7.61 (m, 4H), 7.80 (m, 4H), 8.50
(d, 2H), 11.68 (br s, 0.1H), 12.35 (br s, 1H) ppm, ESI-MS: m/e calcd for
C25H23N4O3 [MþH]þ 568.267, found 568.2677.
4.5. Computational methodologies
Molecules of the receptors and the peptides were generated on
Silicon Graphic Indio workstation using Sybyl 7.3 software package,
and the conformations of the molecules were optimized and se-
lected according to the previously reported method.20 The re-
ceptors were then fitted to the peptides, typically, for one mode of
the fitting between receptor 4 and Y2p5pS: oxygen atom on the
phosphate of 2-Ser was fitted to Zn2þ of ZneDpa.
Compound 7 (0.60 g) was dissolved in 10 mL concentrated HCl,
and the solution was kept stirring for 1 h at room temperature.
After vacuum dried, 0.49 g compound 8 was obtained as a yellow
solid, yield 92.5%. 1H NMR (400 MHz, DMSO):
d
¼3.91 (s, 6H), 4.37
(s, 4H), 6.76 (s, 0.34H), 6.77 (m, 0.52H), 7.56 (d, 2H), 7.68 (m, 2H),
7.90 (t, 2H), 8.13 (d, 2H), 8.48 (m, 2H), 8.77 (br s, 3H), 8.85 (m, 1H),

2362
S. Zhang et al. / Tetrahedron 68 (2012) 2357e2362
6. Yoon, S. S.; Still, W. C. Tetrahedron 1995, 51, 567e578.
Subsequently, the fitted molecules were subjected to blind
7. Chen, C.-T.; Wagner, H.; Still, W. C. Science 1998, 279, 851e853.
8. Rensing, S.; Schrader, T. Org. Lett. 2002, 4, 2161e2164.
9. Zhang, T.; Edwards, N. Y.; Bonizzoni, M.; Anslyn, E. V. J. Am. Chem. Soc. 2009, 131,
11976e11984.
docking in Autock 4.0. The running parameters were according to
our previous work.20
10. Yoon, S. S.; Still, W. C. J. Am. Chem. Soc. 1993, 115, 823e824.
11. Torneiro, M.; Still, W. C. J. Am. Chem. Soc. 1995, 117, 5887e5888.
12. Schmuck, C.; Geiger, L. J. Am. Chem. Soc. 2004, 126, 8898e8899.
13. Rodriguez-Docampo, Z.; Pascu, S. I.; Kubik, S.; Otto, S. J. Am. Chem. Soc. 2006,
128, 11206e11210.
Acknowledgements
This work is supported by the National Natural Science Foun-
dation of China (20634030, 21104034).
ꢀ
14. Peczuh, M. W.; Hamilton, A. D.; Sanchez-Quesada, J.; de Mendoza, J.; Haack, T.;
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