Construction of a library of structurally diverse ribonucleopeptides with catalytic groups

Article abstract of DOI:10.1016/j.bmc.2017.02.007

Functional screening of structurally diverse libraries consisting of proteins or nucleic acids is an effective method to obtain receptors or aptamers with unique molecular recognition characteristics. However, further modification of these selected receptors to exert a newly desired function is still a challenging task. We have constructed a library of structurally diverse ribonucleopeptides (RNPs) that are modified with a catalytic group, in which the catalytic group aligns with various orientations against the ATP binding pocket of RNA subunit. As a proof-of-principle, the screening of the constructed RNP library for the catalytic reaction of ester hydrolysis was successfully carried out. The size of both the substrate-binding RNA library and the catalytic group modified peptide library are independently expandable, and thus, the size of RNPs library could be enlarged by a combination of these two subunits. We anticipate that the library of functionalized and structurally diverse RNPs would be expanded for various other catalytic reactions.

Full text of DOI:10.1016/j.bmc.2017.02.007

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Construction of a library of structurally diverse ribonucleopeptides with
catalytic groups
⇑
Tomoki Tamura, Shun Nakano, Eiji Nakata, Takashi Morii
Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Functional screening of structurally diverse libraries consisting of proteins or nucleic acids is an effective
method to obtain receptors or aptamers with unique molecular recognition characteristics. However, fur-
ther modification of these selected receptors to exert a newly desired function is still a challenging task.
We have constructed a library of structurally diverse ribonucleopeptides (RNPs) that are modified with a
catalytic group, in which the catalytic group aligns with various orientations against the ATP binding
pocket of RNA subunit. As a proof-of-principle, the screening of the constructed RNP library for the cat-
alytic reaction of ester hydrolysis was successfully carried out. The size of both the substrate-binding RNA
library and the catalytic group modified peptide library are independently expandable, and thus, the size
of RNPs library could be enlarged by a combination of these two subunits. We anticipate that the library
of functionalized and structurally diverse RNPs would be expanded for various other catalytic reactions.
Ó 2017 Published by Elsevier Ltd.
Received 11 January 2017
Revised 31 January 2017
Accepted 3 February 2017
Available online xxxx
Keywords:
Ribonucleopeptide
Receptor
Structurally diverse library
Catalytic activity
1. Introduction
These chemical or genetic methods require laborious tasks to gen-
erate a library with limited size. Therefore, an alternative method
Biomacromolecular receptors provide useful frameworks for
constructing sensors with high specificity for a particular ligand.
Efforts have been taken to construct biosensors through modifica-
tion of proteins and nucleic acids receptors with appropriate fluo-
rophores1–5 or artificial enzymes with a catalytic group to achieve
proximity effect between the substrate and the catalytic group.6,7
However, naturally occurring receptors do not always provide
the recognition characteristics for the molecule of interest, and
rational design of a receptor with desired specificity is still a chal-
lenging task. A library of RNA molecules that differ in their three-
dimensional structures by means of randomized nucleotide
sequences has been applied for the selection of receptors for the
target ligands.8–10 Although the library-based selection offers one
of the promising methods to obtain aptamers with the specific
recognition characteristics for the molecule of interest, further
modification of the selected aptamers to exert a newly desired
function is often difficult due to the lack of their structural infor-
mation. In addition, the original substrate binding characteristics
of the aptamer could be impaired by the introduction of a new
functional group. Synthetic functional groups were incorporated
to biomacromolecules in the library by modification of proteins
or nucleic acids via chemical modification or genetic mutation.11,12
is required to construct a large size library to increase the possibil-
ity of selecting a ‘‘hit” biomacromolecule with the function of
interest.
Ribonucleopeptide (RNP) is one of the appropriate scaffolds to
construct a library with structurally diverse RNA-peptide com-
plexes. We have reported a method to obtain ATP-binding RNP
receptors by in vitro selection of a library of RNP with randomized
RNA sequences.13 Because the RNA subunit works as a receptor for
the substrate, the selected ATP-binding RNP receptor was further
converted into a new RNP library by complexation of the RNA sub-
unit and a library of Rev peptides, in which a peptide loop with
randomized amino acid residues was incorporated at the N-termi-
nus of Rev peptide. An ATP-binding RNP receptor selected from this
peptide-based RNP library exerted higher ATP-binding specificity
than the original RNP (Fig. 1a).14 A fluorescent RNP library was also
constructed by combining the RNA subunit library and a library
containing fluorophore-modified Rev peptides (Fig. 1a).15–17 The
original substrate-binding ability of the RNP receptor was main-
tained in the selected RNP sensor even upon modification of the
Rev peptide by a fluorophore. By taking advantage of the noncova-
lent complex formation of RNP, combination of the RNA library and
the peptide library would dramatically increase the size of the RNP
library. Therefore, RNP is a good candidate of scaffolds to construct
a library of structurally diverse biomolecular assemblies contain-
ing a substrate-binding pocket and a synthetic functional group.
⇑
Corresponding author.
E-mail address: t-morii@iae.kyoto-u.ac.jp (T. Morii).
http://dx.doi.org/10.1016/j.bmc.2017.02.007
0968-0896/Ó 2017 Published by Elsevier Ltd.
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T. Tamura et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
ester group was utilized to select the RNPs that could accelerate
the ester hydrolysis reaction by the proximity effect between the
ATP binding domain and the catalytic group on the peptide
(Fig. 1a). Screening of the RNP library was successfully performed
for the hydrolysis reaction of the ester derivative of adenosine to
obtain catalytic RNPs.
2. Results and discussion
2.1. Construction of a functionalized structurally diverse RNP library
A library of structurally diverse RNPs with various geometries
between the substrate binding pocket and the catalytic group
was constructed by combination of the RNA subunits of ATP-bind-
ing RNP and a library of Rev peptide modified with catalytic
groups. From the RNA-oriented RNP library, a series of ATP-binding
RNP receptors was obtained through in vitro selection15,17 with a
variation in the RNA sequences (Fig. 1a). The nucleotide sequence
of ATP-binding RNP consisted of three segments, the RRE, consen-
sus, and variable regions (Fig. 2). The consensus region of the ATP-
binding RNP receptors forms the binding pocket for the substrate
adenosine.15,17 The consensus and the RRE regions were connected
through the variable region that differ in various length and
sequences of the nucleotides. The structural diversity in the vari-
able region affords diverse structures of RNPs, which in turn exhi-
bits a variety of geometries between the ATP-binding pocket and
the Rev peptide that is bound to the RRE sequence.
The N-terminus of Rev peptide in RNP can be chemically mod-
ified without diminishing the binding affinity of Rev peptide to the
RRE sequence.14–17 Judging from the structure of Rev-RRE com-
plex,18 the N-terminus of Rev peptide in RNP should locate closer
to the binding-pocket than its C-terminus. Therefore, the N-termi-
nus of Rev peptide is a suitable position for the modification by a
catalytic group. N,N-Dimethyl-4-aminopyridine (dmap), 4,40-
dimethyl-2,20-bipyridine (bpy) and histidine (H) were chosen as
possible catalytic groups for the ester hydrolysis reaction. In addi-
tion, peptide linkers with various amino acid sequences were
introduced between the catalytic groups and the N-terminus of
Rev peptides. Four types of short peptide motifs were selected
from known RNP structures19–22 (Fig. 3) to achieve a structural
diversity (Fig. 4). The motif pa (Fig. 3b) traverse the phosphate
backbone as found in the eukaryotic ribosome structure (Fig. 3a),
the motif pb (Fig. 3d) snugly fits in the major groove of RNA duplex
in the Tav2b/siRNA complex (Fig. 3c), the motif pc (Fig. 3f) binds
along the phosphate backbone in the complex of Mss16p DEAD-
box helicase domain 2/RNA duplex (Fig. 3e), and the motif pd
(Fig. 3h) passes over the phosphate backbone from the major
groove to the minor groove as found in the small RNA methyltrans-
ferase HEN1 complex (Fig. 3g). Synthetic flexible linkers containing
multiple repeating sequences (GGS) were also incorporated
between the N-terminus of Rev peptide and the catalytic group
(pe and pf). Combination of the RNA subunit library and the pep-
tide subunit library afforded a structurally diverse RNP library with
catalytic group, in which the substrate ATP resided in various ori-
entation and distances to the catalytic group (Fig. 1b).
Fig. 1. (a) Schematic illustration of the stepwise molding of RNP receptors. The RNP
receptor was first selected by in vitro selection of the RNP library. The resulting RNP
receptor was converted to more selective RNP receptors by a suitable peptide loop
containing randomized amino acid residues14 or to fluorescent RNP sensors by
fluorophore modification.15–17 Also, an RNP library containing catalytic groups in
the peptide subunit could provide catalytic RNPs. (b) Combination of a structurally
diverse RNA library and a structurally diverse peptide library affords the construc-
tion of
a library of RNPs, in which the catalytic group aligns with various
orientations to the substrate-binding pocket.
In this study, we have constructed a library of structurally
diverse RNP receptors equipped with a synthetic functional group.
A series of RNA subunits obtained by the in vitro selection of ATP-
binding RNP receptors15,17 afforded a library of structurally diverse
RNAs consisting of the ATP-binding region and the RRE (Rev
Responsive Element) nucleotide sequence. A library of peptides
was constructed by modification of the Rev peptide with catalytic
groups through various peptide linkers (Fig. 1b). Combination of
these two types of libraries by the complex formation between
the RRE RNA and the Rev peptide generated a library of functional-
ized and structurally diverse RNPs, in which the catalytic group
would align with various orientations against the ATP binding
pocket of RNA subunits.
2.2. Design and synthesis of an ester derivative of adenosine for facile
screening of catalytic RNPs
Spectroscopic monitoring of the p-nitrophenyl ester hydrolysis
is a rapid, sensitive and quantitative way to evaluate the catalytic
activity of biomacromolecules.23,24 The p-nitrophenyl ester has
been used as the reporter motif of substrate because it shows little
or no absorbance of around 400 nm in water but provide character-
istic absorbance of p-nitrophenolate upon hydrolysis of the
The functionalized and structurally diverse RNPs were expected
to exhibit the ATP-binding ability. An adenosine derivative with an
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T. Tamura et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
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Fig. 2. Nucleotide sequences of the RNA subunit of ATP-binding RNP receptor.15,17 Nucleotides in the consensus region forms the binding pocket to the substrate ATP. The red
squared regions indicate the variable region that determines the relative orientation of the ATP-binding pocket to the peptide subunit.
ester.23–25 For such measurements, it is important to optimize the
conditions for the detection with high S/N ratio to accurately iden-
tify the ‘‘hit” molecules. The signals resulting from the reaction in
the presence of RNPs should be distinguishable from that in the
absence of RNP. Generally, autohydrolysis of the p-nitrophenyl
ester is accelerated in the basic condition, which causes the
increase of the background noise. Though the reaction in acidic
condition is desirable to avoid the autohydrolysis of p-nitrophenyl
ester, the reaction in acidic condition results little or no significant
absorbance of p-nitrophenolate due to the high pKa value of the p-
nitrophenol (7.15 at 25 °C).26 The extinction coefficient at 400 nm
caused by aggregation or bubble formation in the wells of the plate
when compared to the UV absorbance detection. These character-
istics of umbelliferyl ester prompted us to utilize umbelliferone as
the reporter motif of the ester derivative of adenosine for screening
the catalytic activity of structurally diverse RNPs library (see
Scheme 1).
The ATP-binding RNP receptor binds to the adenine base of ATP
by forming the Hoogsteen U:A:U triple (Fig. S1) as has been deter-
mined by NMR measurements.17 Due to this recognition mecha-
nism, modification of the ribose moiety of an adenosine
derivative would not significantly reduce its affinity of RNP recep-
tors. We designed an umbelliferyl ester derivative of adenosine
(Umb-Ado) 7 as the substrate for hydrolysis reaction by struc-
turally diverse RNPs. For the synthesis of Umb-Ado 7, 20,30-iso-
propylideneadenosine28 2 was converted to an azide compound
3, then to 50-deoxy-50-amino-20,30-isopropylideneadenosine 4 upon
hydrogenation.29 Condensation of 4 with succinic acid anhydride
gave 50-calboxylic acid 5, which was further converted to the
umbelliferyl ester 6 by WSC. Deprotection of the ester 6 gave the
designed substrate Umb-Ado 7.
(e400) of p-nitrophenol at pH 6.2 is only 2800 (Table S1). Thus,
the accurate determination of the hydrolysis reaction is difficult
by the high-through-put UV measurement in plate reader. In con-
trast, the fluorescent emission of umbelliferone is detectable by
plate reader even in slightly acidic conditions because of its high
quantum yield (Uf = 0.79).27
In addition, the fluorescence intensity of an umbelliferyl ester is
quite low (Uf = 0.0011 for 7-acetoxycoumarin).27 The fluorescence
detection has an advantage in reducing the background noise
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T. Tamura et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Fig. 4. (a) Schematic illustrations of catalyst modified Rev peptide through linker
peptide. (b) Structures of catalytic groups modified to N-terminus of the peptide
subunit. (c) Amino acid sequences of Rev peptide derivatives. Short peptide motifs
(in orange, Fig. 3) are introduced through flexible linkers composed by G and S to
the N-terminus of Rev peptide (TRAQRRNRRRRWRERQRAAAAR). As the catalytic
groups for hydrolysis, dmap, bpy and histidine were modified at the N-terminus of
peptides.
taken together, the condition in a buffer containing 30 mM Bis-
Tris-HCl (pH 6.0), 100 mM NaCl, 5 mM MgCl2 and 0.005% Tween
20 was used for screening the hydrolytic activity of RNPs library
at 20 °C.
Fig. 3. Structural models of short peptide motifs derived from known RNP
structures. RNP structures of (a) eukaryotic ribosome (PDB ID: 3U5I),19 (c) Tav2b/
siRNA complex (PDB ID: 2ZI0),20 (e) Mss116p DEAD-box helicase domain 2/RNA
duplex (PDB ID: 4DB2),21 and (g) small RNA methyltransferase HEN1 (PDB ID:
3HTX)22 were used for the short peptide motifs (b) pa, (d) pb, (f) pc, and (h) pd,
respectively.
Formation of the 1:1 complex of An15 RNA (see Fig. 2) and the
dmap-pa1-Rev peptide (see Fig. 4) at 20 °C in this buffer was con-
firmed by gel shit assay using 8% native polyacrylamide gel elec-
trophoresis (PAGE) for several ratios of RNA to peptide (Fig. S3).
This RNP showed almost quantitative complex formation with
one equivalent of peptide to RNA at the concentration of 5 mM. For-
mation of the ATP-binding RNP and Umb-Ado complex was next
analyzed by titration of a 5FAM-labeled Rev peptide (5FAM-
Rev)15 and An15 RNA complex with Umb-Ado (Fig. 6) and the esti-
mated dissociation constant (KD) was 2.6 0.2 mM in the above
mentioned conditions. This result indicated that the designed sub-
strate for the hydrolytic reaction, Umb-Ado, maintained the bind-
ing ability to the ATP-binding RNP receptor.15,17
2.3. Conditions for the screening of catalytic RNPs
Time-dependent spectral change of Umb-Ado alone was evalu-
ated at pH 6 (Fig. S2). Autohydrolysis of Umb-Ado was monitored
by the time-dependent fluorescence intensity change (kEx = 355 -
nm/kEm = 455 nm). Time course profiles of the background hydrol-
ysis reaction at 20 °C were monitored at different pH conditions to
determine the optimum conditions for screening the ester hydrol-
ysis activity of RNPs in buffers containing 100 mM NaCl, 5 mM
MgCl2, 0.005% Tween 20 as described previously for the ligand-
binding studies of ATP-binding RNP.15,17 Autohydrolysis of Umb-
Ado in the buffer at pH 7 resulted in a conversion yield of 80% after
three hours at 20 °C (Fig. 5). At pH 6, the conversion yield was 10%
after three hours. Almost no hydrolysis of Umb-Ado was observed
at pH 5 as judged from the emission intensity, but the quantum
yield for the fluorescence emission of umbelliferone was too low
to determine the accurate concentration at pH 5. These results
2.4. Screening of the structurally diverse RNP library for the
identification of catalytic RNPs
Screening of the library of structurally diverse RNPs with the
catalytic group was first performed by using roughly purified RNAs
and peptides in a high-through-put manner. RNA was purified with
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T. Tamura et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
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Scheme 1. Synthetic scheme of umbelliferyl ester of adenosine (Umb-Ado 7). Reaction conditions: (i) TsOH, acetone; (ii) dppa, DBU, 15-crown 5-ether, NaN3, 1,4-dioxane;
(iii) H2, Pd/C, MeOH; (iv) succinic anhydride, pyridine; (v) WSCIꢂHCl, umbelliferone, DMAP, CH2Cl2; (vi) 50% TFA/H2O.
Fig. 5. Time course plots of the autohydrolysis reaction of Umb-Ado in sodium
phosphate buffer, pH 7 (circle), Bis-Tris-HCl buffer, pH 6 (square), and acetate
buffer, pH 5 (triangle) at 20 °C.
Fig. 7. (a) Time course plots of the intensity of the sample containing Umb-Ado
with (circle) or without (triangle) An15/dmap-pa1-Rev. (b) Time course plots of ln
([Umb-Ado]) of the sample containing Umb-Ado with (circle) or without (triangle)
An15/dmap-pa1-Rev. All samples were measured in the buffer containing 30 mM
Bis-Tris-HCl (pH 6.0), 100 mM NaCl, 5 mM MgCl2, and 0.005% Tween 20 at 20 °C.
for the second screening, in which the RNA and the peptide compo-
nents were purified by denaturing PAGE and HPLC, respectively. In
the second screening, RNP with An15 RNA and dmap-pa1-Rev
showed the highest acceleration of the hydrolysis (Fig. 7a). An15
RNA or dmap-pa1-Rev by itself showed no acceleration of the ester
hydrolysis reaction (Fig. S5), indicating that the RNP complex for-
mation by An15 and dmap-pa1-Rev was necessary to accelerate
the reaction. The proximity effect between Umb-Ado in the bind-
ing pocket of An15 and the dmap group of dmap-pa1-Rev upon
the RNP complex formation likely accounts for the observed accel-
eration of the ester hydrolysis. To support this, hydrolysis of 7-ace-
toxycoumarin, an umbelliferyl ester without the adenosine moiety,
did not show any acceleration in the presence of An15/dmap-pa1-
Fig. 6. A titration curve for the relative fluorescence intensity changes of An15/
5FAM-Rev with increasing concentrations of Umb-Ado in the buffer containing
30 mM Bis-Tris-HCl (pH 6.0) with 100 mM NaCl, 5 mM MgCl2, and 0.005% Tween 20
at 20 °C.
size-exclusion chromatography after the transcription reaction,
and each peptide was purified only by the diethyl ether extraction
after deprotection and cleavage from the resin. In total, 609 RNPs
were obtained by the combination of 29 RNAs and 21 peptides
(Table S2). Each RNP of the library was mixed with Umb-Ado in
the screening buffer, then time course changes of the fluorescence
emission intensity at 455 nm was monitored. Progress of the ester
hydrolysis was estimated by using a standard curve of the umbel-
liferon emission (Fig. S4). 17 RNPs showed acceleration of the reac-
tion rate over 1.2-fold higher than that in the condition without
RNP in the first screening (Table S2). These RNPs were selected
Rev RNP (Fig. S6). The pseudo-first order kinetic constants (kobs
)
for the hydrolysis of Umb-Ado in the presence and absence of
An15/dmap-pa1-Rev were 4.0 and 3.4 ꢀ 10ꢁ4 minꢁ1, respectively.
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T. Tamura et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
The kobs in the presence of RNP was 1.2-fold higher than that in the
absence of RNP (Fig. 7b).
tion were constructed by PCR amplification to add the promoter for
T7 RNA polymerase using PrimeSTAR DNA polymerase (TaKaRa)
with 50-DNA primer (50-TCTAATACGACTCACTATAGGTCTGGGC
GCA-30: T7 RNA promoter is underlined) and 30-DNA (50-
GGCCTGTACCGTC-30). RNA transcription was performed using an
T7-ScribeTM Standard RNA IVT Kit (CELLSCRIPTTM) for 3 h at
37 °C, according to the supplier’s recommended protocols. The
resulting RNA was extracted with phenol/chloroform, then precip-
itated with ethanol, and pelleted by centrifugation. The size-exclu-
sion with Micro Bio-Spin 6 column (Bio-Rad) was performed after
dissolving the RNA pellet in TE buffer. For the first screening, the
RNA was used without further purification. For the second screen-
ing, the RNA purified by denaturing polyacrylamide gel elec-
trophoresis and eluted. Concentrations of RNAs were determined
by UV spectroscopy.
3. Conclusion
A library of structurally diverse RNPs with catalytic group was
successfully prepared by a combination of an RNA library that
has a binding pocket for adenosine derivatives with a variety of
overall structures and a Rev peptide library that contained catalytic
group through various peptide linkers. The noncovalent complex
formation of RNA and the Rev peptide derivative enables the facile
expansion of the RNPs library size. An umbelliferone ester of ade-
nosine derivative Umb-Ado was useful for the fluorescence detec-
tion of ester hydrolysis reaction even in the acidic condition. The
umbelliferone ester is a sensitive alternative for the p-nitrophenol
esters widely used as the substrate for hydrolytic reactions espe-
cially in acidic conditions. The functionalized and structurally
diverse RNPs library was screened for the ester hydrolysis of
Umb-Ado. An RNP of An15 RNA and dmap-pa1-Rev, one of the
RNPs selected by the screening, clearly showed significant acceler-
ation of the hydrolysis of Umb-Ado. It is noteworthy that catalytic
RNPs albeit with moderate activity were selected from the small
size library, less than 1000, in this study. Because the numbers of
ATP-binding RNA and/or the Rev peptide with catalytic group are
readily increased, the size of RNP library would be expanded con-
veniently by the combination of these two subunits. Alternatively,
introduction of random mutation to RNA of the selected RNP
would afford a focused library for further selection of catalytic
RNP. The functionalized and structurally diverse RNPs library
would be quite useful for exploring catalytic RNPs not only for
the ester hydrolysis but also for various other chemical reactions.
4.2. Synthesis of the catalyst-modified peptide library
The peptides were synthesized as follows. Fmoc-NH-SAL-PEG
resin was placed in a dry flask, and a sufficient amount of DMF
was added to soak the resin; this mixture was allowed to swell
for 30 min. The resin was loaded onto an automated peptide syn-
thesizer (Liberty; CEM/PSSM-8; Shimadzu), and the subsequent
synthesis was performed according to Fmoc chemistry protocols
using protected Fmoc-amino acids and HBTU. Unnatural catalytic
groups (3-(Methyl-4-pyridylamino)propionic acid, 40-Methyl[2,20-
bipyridine]-4-butanoic acid) were directly coupled to N-terminus
deprotected Rev peptides on the resin in DMF containing WSCꢂHCl.
Acetylated histidine was introduced by usual Fmoc chemistry pro-
tocol and acetylation. The synthesized peptides were used as crude
for the first screening and purified by HPLC before carrying out the
second screening.
4. Materials and methods
4.3. Synthesis of 50-deoxy-50-(umbelliferyl butylate)amido-adenosine
(Umb-Ado 7)
PrimeSTAR HS DNA polymerase for PCR reactions was obtained
from TaKaRa Bio Inc. (Shiga, Japan). T7-ScribeTM Standard RNA
IVT Kit for RNA preparation was obtained from CELLSCRIPTTM
(Madison, USA). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
4.3.1. Synthesis of 20,30-O-isopropylideneadenosine 228
A mixture of adenosine (4.96 g, 18.56 mmol), p-toluenesulfonic
acid monohydrate (3.53 g, 18.56 mmol) and acetone (150 mL) was
stirred at room temperature in an open vessel. After 46 h from
starting reaction, 5.10 g of p-toluenesulfonic acid monohydrate
was added. After 2 h, 5.21 g of p-toluenesulfonic acid monohydrate
was added (total 13.94 g, 73.3 mmol). After 2 h from adding the
acid, the spot of compound 1 disappeared. The mixture turned into
a yellow solution. The reaction mixture was quenched by adding
12.3 mL of triethylamine (88.4 mmol, 4.73 eq.). The mixture was
evaporated and purified by column chromatography (chloroform/
methanol 20:1) to give compound 2 (4.50 g, 14.6 mmol, 79%). 1H
NMR (300 MHz, DMSO-d6) d 1.33 and 1.55 (2s, 6H, CMe2), 3.52–
3.56 (m, 2H, H50), 4.19–4.23 (m, 1H, H40), 4.95–4.98 (m, 1H, H
30), 5.25 (vbs, 1H, OH), 5.33–5.36 (m, 1H, H 20), 6.11–6.12 (m,1H,
H 10), 7.34 (bs, 2H, NH2), 8.15 (s, 1H, H2), 8.34 (s, 1H, H8);
ESI-TOF-MS for C13H17N5O4: (MNa+) calcd: 330.12, found: 330.03.
hydro-chloride (WSCIꢂHCl), N-
a
-Fmoc-protected amino acids,
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluo-
rophos-phate (HBTU), 1-hydroxybenzotriazole (HOBt), SP grade
N,N-dimethylformamide (DMF), trifluoroacetic acid (TFA) and
4-[(2,4-Dimethoxyphenyl)-N-(9-fluorenylmethoxy-carbonyl) ami-
nomethyl]phenoxy-acetamido polyethyleneglycol resin (Fmoc-
NH-SAL-PEG resin) were obtained from Watanabe Chemical Indus-
tries (Hiroshima, Japan). Dichloromethane (DCM), 1,8-diazabicyclo
[5,4,0]-7-undecene (DBU) and umbelliferon were purchased from
Nacalai Tesque (Kyoto, Japan). N,N-Diisopropylethylamine (DIEA),
HPLC-grade acetonitrile and N,N-dimethyl-4-aminopyridine
(dmap), and p-toluenesulfonic acid monohydrate were obtained
from Sigma-Aldrich (St. Louis, USA). Adenosine, 15-crown 5-ether
and diphenylphosphoryl azide (dppa) were purchased from Tokyo
Chemical Industry (Tokyo, Japan). Reversed-phase C18 columns
(4.6 ꢀ 150 mm, Ultron VX-ODS; Shinwa Chemical Industries,
Kyoto, Japan and 10 ꢀ 150 mm, 5C18-ARII; Nacalai Tesque, Kyoto,
Japan) were used for purification of peptides for preparative pur-
poses. Sodium azide, succinic anhydride, gel electrophoresis grade
acrylamide, bisacrylamide, phenol, thioanisol, 1,2-ethanditiol, 10%
palladium on carbon and distilled organic solvents were purchased
from Wako Chemicals (Tokyo, Japan).
4.3.2. Synthesis of 50-deoxy-50-azido-20,30-O-isopropylideneadenosine
329
Compound
2
(1.0 g, 3.25 mmol, 1.0 eq.), dppa (1.4 mL,
6.51 mmol, 2.0 eq) and DBU (1.47 mL, 9.77 mmol, 3.0 eq.) were dis-
solved in 1,4-dioxane 10 mL under nitrogen atmosphere. The mix-
ture was stirred at room temperature for 2 h followed by addition
of sodium azide (1.0 g, 16.3 mmol, 5.0 eq.) and 15-crown 5-ether
(6.5 mL, 33 mmol, 0.01 eq.). The mixture was refluxed for 1 h and fil-
tered. The filtrate was evaporated and purified by column chro-
matography (chloroform/methanol 29:1) to give the compound 3
(0.87 g, 2.6 mmol, 80%). 1H NMR (300 MHz, CDCl3): d 1.39 and
4.1. Preparation of the RNA subunit library
The plasmid DNA templates were prepared as described previ-
ously.13–17 The double-stranded DNA templates for RNA transcrip-
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T. Tamura et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
1.62 (2 s, 6H, CMe2), 3.52–3.64 (m, 2H, H50), 4.36–4.41 (m, 1H, H40),
5.06 (dd, 1H, J = 3.3, 6.3 Hz, H30), 5.46 (dd, 1H, J = 2.5, 6.5 Hz, 2H0),
5.89 (br, 2H, NH2), 6.11 (d, 1H, J = 2.5 Hz, H10), 7.93 (s, 1H, H8), 8.36
(s, 1H, H2). ESI-TOF-MS for C13H16N8O3: (MNa+) calcd: 355.12,
found: 355.08.
Ar-H), 7.79 (d, J = 9.5 Hz, 1H, Ar-H), 8.01 (s, 1H, H8), 8.30 (s, 1H,
H2), 8.82 (d, J = 7.0 Hz, 1H, NH); ESI-TOF-MS for C23H22N6O8:
(MH+) calcd: 511.16, found: 511.09.
4.4. Determination of the binding affinity of Umb-Ado to RNP by
fluorescence titrations
4.3.3. Synthesis of 50-deoxy-50-amino-20,30-O-isopropylideneadenosine
429
The fluorescence measurements were performed using 96-well
plates on a Wallac ARVOsx 1420 multilabel counter or Tecan infin-
ity pro for the determination of the binding affinity of Umb-Ado to
RNP. A binding solution (100 mL) containing 1 mM of fluorescent
RNP (An15/5FAM-Rev) in 30 mM Bis-Tris-HCl (pH 6.0), 100 mM
NaCl, 5 mM MgCl2, and 0.005% Tween 20 with indicated concentra-
tion of substrate was gently swirled for few minutes and allowed
to settle for 30 min at 20 °C. Emission spectra were measured with
an appropriate filter set for each fluorophore. Excitation and emis-
sion wavelengths for 5FAM-Rev were 485 and 535 nm, respec-
tively. The binding affinity of fluorescent RNP to Umb-Ado was
obtained by fitting the substrate titration data using the equation:
Compound 3 (1.0 g, 3.3 mmol) and 10% Pd/C (200 mg) in metha-
nol (50 mL) were stirred in room temperature under hydrogen
atmosphere. After 4 h, the reaction mixture was filtered. This
resulting mixture containing compound 4 was used in the next
step without further purification. 1H NMR (300 MHz, CDCl3) d
1.41 and 1.64 (2 s, 6H, CMe2), 3.01–3.07 (m, 2H, H50), 4.30–4.31
(m, 1H, H40), 5.05 (dd, J = 6.3, 3.3 Hz, 1H, H30), 5.48 (dd, J = 6.3,
3.0 Hz, 1H, H20), 6.12 (d, J = 3.0 Hz, 1H, H10), 8.18 (s, 1H, H2), 8.82
(s, 1H, H8); ESI-TOF-MS for C13H16N8O3: (MNa+) calcd: 307.14,
found: 307.11.
4.3.4. Synthesis of 50-deoxy-50-(300-carboxypropyl) amido-20,30-O-
isopropylideneadenosine 5
ꢀ
Fobs ¼ A ð½FRNPꢃT þ ½substrateꢃT þ KDÞ
2
Compound 4 (313 mg, 1.02 mmol) and succinic anhydride
(795 mg, 7.5 mmol) were stirred in distilled pyridine (20 mL)
under the nitrogen atmosphere at 0 °C. The temperature was
increased to room temperature over 30 min. After 12 h, the organic
layer was washed with hydrochloric acid, conc. sodium bicarbon-
ate, brine and then dried over anhydrous sodium sulfate to give
compound 5 (230 mg, 0.57 mmol, 56%). 1H NMR (300 MHz, CDCl3)
d 1.35 and 1.69 (2 s, 6H, CMe2), 2.78–2.84 (m, 4H, (CH2)2), 3.23 (d,
J = 14.7 Hz, 1H, H50), 3.64–3.76 (m, 2H, NH2), 4.20–4.28 (m, 1H,
H50), 4.52 (d, J = 2.1 Hz, 1H, H40), 4.78 (dd, J = 6.3, 1.8 Hz, 1H,
H30), 5.30 (m, 1H, H20), 5.78 (d, J = 5.4 Hz, 1H, H10), 7.87 (s, 1H,
H2), 8.31 (d, J = 9.6 Hz, 1H, NH), 8.38 (s, 1H, H8); ESI-TOF-MS for
ꢁðð½FRNPꢃT þ ½substrateꢃT þ KDÞ
o
1=2
ꢁ4½FRNPꢃT½substrateꢃTÞ
=2 ½FRNPꢃT
where A is the increase in fluorescence at saturating substrate con-
centrations (Fmax ꢁ Fmin), KD is the equilibrium dissociation con-
stant, and [FRNP]T and [substrate]T are the total concentrations of
fluorescent RNP and the substrate, respectively.
4.5. Screening of RNP library
Fluorescence measurements in 96-well plates were performed
on a Wallac ARVOsx 1420 multilabel counter. A solution (100 lL)
C
17H22N6O6: (MH+) calcd: 407.17, found: 407.09.
containing 5 mM of each combination of RNPs and 20 mM Umb-
Ado in reaction buffer containing 30 mM Bis-Tris-HCl (pH 6.0),
100 mM NaCl, 5 mM MgCl2, and 0.005% Tween 20 was gently
swirled for 30 s and the time-dependent emission intensity of
the umbelliferone, which is a product of the hydrolysis, was mea-
sured every 20 min for 3 h. Excitation and emission wavelengths
for umbelliferone were 355 and 455 nm, respectively. Concentra-
tion of the umbelliferone was estimated by using a standard curve.
4.3.5. Synthesis of 50-deoxy-50-(umbelliferyl butylate)amido-20,30-O-
isopropylideneadenosine 6
Compound 5 (28 mg, 0.07 mmol), and umbelliferone (57 mg,
0.35 mmol) were stirred in dichloromethane (15 mL) under nitro-
gen atmosphere at 0 °C. DMAP (0.86 mg, 0.007 mmol) and
WSCIꢂHCl (16 mg, 0.084 mmol) were added and the reaction tem-
perature was increased to room temperature over 30 min. After
24 h, the resulting solution was evaporated and purified by HPLC
(column: 5C18-AR-II 20 mm ꢀ 150 mm/mobile phase 5% acetic
acid with 24–48% acetonitrile) to give compound 6 (0.011 mmol,
16%). 1H NMR (300 MHz, CDCl3) d 1.18 and 1.58 (2 s, 6H, CMe2),
2.69–2.75 (m, 1H, H50), 2.86–2.95 (m, 2H, H80), 3.16–3.25 (m, 2H,
H90), 4.24–4.32 (m, 1H, H50), 4.51 (d, J = 1.9 Hz, 1H, H40), 4.78 (dd,
J = 6.2, 1.9 Hz, 1H, H30), 5.31 (t, J = 5.7 Hz, 1H, H20), 5.74–5.78 (b,
3H, H10 and NH2), 6.34 (d, J = 9.4 Hz, 1H, Ar-H), 7.06 (dd, J = 8.5,
2.2 Hz, 1H, Ar-H), 7.13 (d, J = 2.2 Hz, 1H, Ar-H), 7.44 (d, J = 8.5 Hz,
1H, Ar-H), 7.67 (d, J = 9.6 Hz, 1H, Ar-H), 7.84 (s, 1H, H8), 8.36 (s,
1H, H2), 8.79 (d, J = 9.3 Hz, 1H, NH); ESI-TOF-MS for C26H26N6O8:
(MH+) calcd: 551.18, found: 551.20.
4.6. Calculation of the pseudo-first order kinetics constant (kobs
)
The pseudo-first order kinetics constant (kobs) was obtained
from the slope of the log plot of the time-dependent concentration
of umbelliferone.
Acknowledgment
This work was supported in part by the Grants-in-Aid for Scien-
tific Research from the Ministry of Education, Culture, Sports,
Science and Technology, Japan to S.N. (No. 26810090) and T.M.
(25248038 & 15H01402). T.T. acknowledges the Japan Society for
the Promotion of Science for the fellowship (No. 15J09936).
4.3.6. Synthesis of 50-deoxy-50-(umbelliferyl butylate)amidoadenosine
(Umb-Ado) 7
A solution containing compound 6 (20 mg, 0.036 mmol) was
stirred in 50% TFA/H2O and after 48 h it was purified by HPLC (col-
umn: 5C18-AR-II 20 mm ꢀ 150 mm/mobile phase 5% acetic acid
with 24–48% acetonitrile) to give compound 7 (0.008 mmol,
A. Supplementary material
Supplementary data (pH dependence of extinction coefficient of
p-nitrophenol (Table S1), summary of the acceleration ratio for the
reaction rate (kobs) in the first screening (Table S2), binding mode
for the ATP-binding RNP receptor and adenosine (Fig. S1), time
dependent spectra of Umb-Ado in the buffer (Fig. S2), autoradio-
grams of the gel shift assay to confirm the complex formation
of RNP (Fig. S3), standard curve for the fluorescent intensity of
1
24%). H NMR (300 MHz, CDCl3) d 2.75–2.81 (m, 2H, H90), 2.95–
3.06 (m, 2H, H80), 3.34–3.38 (m, 3H, H40 and H50), 4.29 (d,
J = 2.57, 1H, H30), 4.75 (t, J = 6.2 Hz, 1H, H20), 5.81 (d, J = 6.6 Hz,
1H, H10), 6.42 (d, J = 9.4 Hz, 1H, Ar-H), 7.10 (dd, J = 8.4, 2.2 Hz,
1H, Ar-H), 7.17 (d, J = 1.8 Hz, 1H, Ar-H), 7.55 (d, J = 8.4 Hz, 1H,
Please cite this article in press as: Tamura T., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.007

8
T. Tamura et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
14. Sato S, Fukuda M, Hagihara M, Tanabe Y, Ohkubo K, Morii T. J Am Chem Soc.
umbelliferone (Fig. S4), time course plots for the intensity of
samples containing Umb-Ado (Fig. S5), time course plots for the
intensity of samples containing 7-acetoxycoumarin (Fig. S6), 1H
NMR spectra of compound 6 and 7 (Figs. S7 and S8) and HPLC
chromatogram of dmap-pa1-Rev (Fig. S9)) associated with this
article can be found, in the online version, at http://dx.doi.org/10.
1016/j.bmc.2017.02.007.
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