Synthesis and biological validation of N7-(4-chlorophenoxyethyl) substituted dinucleotide cap analogs for mRNA translation

Article abstract of DOI:10.1016/j.bmc.2013.05.041

Design, synthesis and biological validation of dinucleotide cap analogs, N⁷-(4-chlorophenoxyethyl)-G(5′)ppp(5′)G (5a) and N ⁷-(4-chlorophenoxyethyl)-m^3′-OG(5′) ppp(5′)G (5b) are reported. The effect of N⁷-(4- chlorophenoxyethyl) substitution on cap analogs has been evaluated with respect to its in vitro transcription by using T7 RNA polymerase capping efficiency, and translational activity. The gel shift assay indicates that the new cap analogs (5a, 5b) showed 77% and 76% capping efficiency respectively, whereas the standard cap analog, m⁷G(5′)ppp(5′)G has a capping efficiency of 63%. The capping efficiency experiment clearly demonstrates that the N⁷-modified analogs are good substrate for T7 RNA polymerase. It is noteworthy that the mRNA poly(A) capped with N⁷-(4- chlorophenoxyethyl)-m^3′-OG(5′)ppp(5′)G (5b) was translated ～1.64-fold more efficiently, while compound (5a) was translated ～0.72-fold less efficiently than mRNA capped with standard cap analog. The observed low translation activity for (5a) could be due to stability in the form of dinucleotide cap analogs. Based on the substrate compatability of the N⁷ modification in dinucleotide form, these new analogs may be used for structure function studies as well as protein production.

Full text of DOI:10.1016/j.bmc.2013.05.041

Bioorganic & Medicinal Chemistry 21 (2013) 4570–4574
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological validation of N7-(4-chlorophenoxyethyl)
substituted dinucleotide cap analogs for mRNA translation
⇑
Anilkumar R. Kore , Zejun Xiao, Mu Li
Organic Chemistry, Life Technologies Corporation, Bioorganic Chemistry Division, 2130 Woodward Street, Austin, TX 78744-1832, USA
a r t i c l e i n f o
a b s t r a c t
Design, synthesis and biological validation of dinucleotide cap analogs, N7-(4-chlorophenoxyethyl)-
Article history:
30 -O
G(50)ppp(50)G (5a) and N7-(4-chlorophenoxyethyl)-m G(50)ppp(50)G (5b) are reported. The effect of
Received 24 March 2013
Revised 8 May 2013
Accepted 17 May 2013
Available online 1 June 2013
N7-(4-chlorophenoxyethyl) substitution on cap analogs has been evaluated with respect to its in vitro
transcription by using T7 RNA polymerase capping efficiency, and translational activity. The gel shift
assay indicates that the new cap analogs (5a, 5b) showed 77% and 76% capping efficiency respectively,
whereas the standard cap analog, m7G(50)ppp(50)G has a capping efficiency of 63%. The capping efficiency
Keywords:
Cap analog
experiment clearly demonstrates that the N7-modified analogs are good substrate for T7 RNA polymer-
0
ase. It is noteworthy that the mRNA poly(A) capped with N7-(4-chlorophenoxyethyl)-m3 -OG(50)ppp(50)G
N7-Substituted dinucleotide cap analog
Capping efficiency
In vitro transcription
Translation efficiency
Luciferase activity
HeLa cells
(5b) was translated ꢀ1.64-fold more efficiently, while compound (5a) was translated ꢀ0.72-fold less effi-
ciently than mRNA capped with standard cap analog. The observed low translation activity for (5a) could
be due to stability in the form of dinucleotide cap analogs. Based on the substrate compatability of the N7
modification in dinucleotide form, these new analogs may be used for structure function studies as well
as protein production.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
stronger binding affinity to eIF4E than m7GMP, and served as com-
petitive inhibitors of capped mRNA translation.15,16 The N7 modified
M7G-capped mRNA plays an important role in eukaryotic mRNA
metabolism, suchas mRNAprocessing, splicing, localization, nuclear
transport, translation, and mRNA degradation pathways.1–8 The un-
ique cap molecular structure is necessary for the capped mRNA to
bind the protein eukaryotic initiation factor eIF4E vital for gene ex-
press and cell metabolism. The cap binding protein possesses undis-
puted role in initiation of mRNA translation and subsequent protein
synthesis. Specifically the cap binding protein eIF4E has become
favorable target for anti-cancer studies. The recent findings on
anti-cancer drug agents have focused on molecular design to in-
creasethe bindingaffinityofpotentialdrug candidates tothe protein
eIF4E.9–14 Potential drug candidates serve as inhibitors for the
capped mRNA in cancer cell to bind with eIF4E, which results in
interrupting abnormal cancerous cell proliferation and inducing
apoptosis.14 The capped mRNA, m7GpppN contains a positive charge
resulting from N7-methyl substitution, which delocalizes over aro-
matic bicyclic rings of guanosine moiety. The positive charge plays
an important role in stabilizing the cap structure, and thus, m7G-
capped mRNA results in increase in binding affinity of m7G-capped
mRNA to cap binding protein compared with G-capped mRNA with-
out positive charge. The N7-alkylation 50-GMP derivatives displayed
50-GMP derivatives are utilized as effective inhibitors for the binding
and interaction between m7G-capped mRNA and cap binding pro-
tein. N7-Substituted phenoxyethyl guanosine 50-monophosphate
derivatives have shown higher binding affinity for the protein eIF4E.
In particular, 4-chloro/bromophenoxyethyl analogues exhibited
IC50 values in nanomolar range, which is over 200-folds increase in
binding affinity to protein eIF4E compared with m7-GMP.14 These
results and our continuous interest to identify novel modified cap
analogsexhibitinghighertranslationalefficiencyandlongerhalf-life
for mRNA,17–19 promted us to synthesize N7-(4-chlorophenoxyeth-
yl) substituted dinucleotide form of cap analog and study its biolog-
ical application. Herein, we report the first example for the synthesis
of dinucleotide form of cap analog containing N7-(4-chlorophenoxy-
ethyl) moiety such as, N7-(4-chlorophenoxyethyl)-G(50)ppp(50)G
0
(5a) and N7-(4-chlorophenoxyethyl)-m3 -OG(50)ppp(50)G (5b) and
their biological evaluation was compared with the standard cap
analog.
2. Experimental
2.1. General
All commercial reagents and solvents are used as such without
further purification. Guanosine-50-diphosphate sodium salt and
⇑
Corresponding author. Tel.: +1 512 721 3589; fax: +1 512 651 0201.
E-mail address: anil.kore@lifetech.com (A.R. Kore).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.041

A. R. Kore et al. / Bioorg. Med. Chem. 21 (2013) 4570–4574
4571
4-chlorophenyl-2-bromoethyl ether were purchased from Sigma–
Aldrich. 1H NMR spectra were obtained in D2O on a Bruker
400 MHz and 31P NMR were acquired on a Bruker 162 MHz. Chem-
ical shifts are reported in ppm, and signals are described as s (sin-
glet), d (doublet), t (triplet), q (quartet), and m (multiplet). ESI
mass spectra were recorded on Applied Biosystems/Scies MDX
API 150 model and MALDI-TOF spectra were obtained on Applied
Biosystems Voyager DE-PRO model. The crude reaction mixture
was purified by using GE ÄKTA Purifier 900 (GE Healthcare, Piscat-
away, NJ, USA) with diethylaminoethyl (DEAE) Sepharose column,
and purity of the fractions was determined by Waters Alliance
2695 HPLC with 2996 PDA detector (Waters Corporation, Milford,
MA, USA) using Dionex DNAPac PA200 (4 x 250 mm) column with
elution solvent system of ammonium phosphate mono basic (pH
2.8, pH 3.7).
Gel shift assay for the capping efficiency was conducted by uti-
lizing MAXIscript T7 kit (Life Technologies Corporation), while IVT
reactions were performed with linearized AmbLuc poly(A) DNA
template with the use of MEGAcript™ kit (Life Technologies Corpo-
ration). Purifications of the RNA from these transcription reactions
were done by using the MEGAclear™ Kit (Life Technologies Corpo-
ration) and associated vendor’s protocol. Luciferase signals were
detected using FLUO star Omega multi-mode microplate reader
as per manufacturer’s protocol (BMG Labtech).
resulting solution was poured into a solution of sodium perchlo-
rate (0.040 g) in acetone (10 mL) and the resulting solid was sepa-
rated by centrifuge, and was repeated above precipitate procedure
once. The solid was washed with acetone (10 mL). The resulting
white solid was dried by vacuum to give pure N7-(4-chlorophen-
oxyethyl) G[50]PPP[50]G sodium salt (5a) (0.045 g, 45%). 1H NMR
(D2O, 400 MHz): d 7.92 (1H, s), 7.03 (2H, d, J = 8.8 Hz), 6.65 (2H,
d, J = 8.8 Hz), 5.82 (1H, d, J = 3.2 Hz), 5.68 (1H, d, J = 6.0 Hz), 4.58
(2H, t, J = 5.6 Hz), 4.44 (1H, t, J = 4.0 Hz), 4.39–4.31 (4H, m), 4.29
(2H, m), 4.22 (2H, m), . 4.17 (4H, m), 31P NMR (D2O, 162 MHz): d
ꢁ11.5 (2P, d, J = 9.4 Hz), d ꢁ23.1 (1P, t, J = 17.8 Hz). MS (m/z) 941
[MꢁH].
2.2.5. Synthesis of 30-O-methylguanosine 50-monophosphate
(2b)
Synthesis of 30-O-methylguanosine 50-monophosphate (2b) was
accomplished by following literature reported monophosphoryla-
tion20 of 30-O-methylguanosine (1b) involving phosphorous oxy-
chloride (1).21
2.2.6. Synthesis of N7-(4-chlorophenoxyethyl)-30-O-methylgu-
anosine 50-monophosphate (3b)
To a stirred solution of 30-O-methylguanosine 50-monophos-
phate TEA salt (2b) (0.477 g, 1 mmol) in DMSO (4 mL) was added
4-chlorophenyl-2-bromoethyl ether (0.944 g, 4 mmol). The result-
ing reaction mixture was heated at 55 °C for 48 h, and quenched by
addition of water (50 mL). Aqueous layer was adjusted to pH 5.5
using 2.5 M NaOH, and washed with DCM (20 mL). The crude
mixture was purified by FPLC (DEAE Sepharose FF media) by
elution of linear gradient of 0–1 M TEAB. The fractions containing
pure product were pooled, evaporated, and dried to afford N7-
(4-chlorophenoxyethyl)-30-O-methylguanosine 50-monophosphate
as triethylammonium salt (3b) (0.391 g, 55%). 1H NMR (D2O,
400 MHz): 7.10 (2H, m), 6.78 (2H, m), 5.9 (1H, m), 4.9 (2H, s),
4.35–4.45 (2H, m), 4.1–4.2 (1H, m), 3.9–4.1 (2H, m), 3.39 (3H, s),
3.11 (4H, q, J = 7.2 Hz), 1.19 (6H, t, J = 7.2 Hz). 31P NMR (D2O,
162 MHz): d 0.57 (1P, s). MS (m/z) 530 [MꢁH].
2.2. Chemistry
2.2.1. Synthesis of Guanosine 50-monophosphate (2a)
Synthesis of Guanosine 50-monophosphate (2a) was accom-
plished by following the reported literature monophosphoryla-
tion20 of Guanosine (1a) involving phosphorous oxychloride.21
2.2.2. Synthesis of N7-(4-chlorophenoxyethyl)-guanosine 50-
monophosphate (3a)
To a mixture of guanosine-50-monophosphate (2a) (0.463 g,
1 mmol, 1 equiv) in DMSO (4 mL) at 25 °C was added 4-chloro-
phenyl-2-bromoethyl ether (0.944 g, 4 equiv). The reaction mix-
ture was stirred for 48 h at 55 °C, and was quenched by addition
of water (50 mL). Aqueous layer was adjusted to pH 5.5 by 2.5 M
NaOH, and washed with DCM (20 mL). The crude mixture was
purified by FPLC (DEAE Sepharose FF media) by elution of linear
gradient of 0–1 M TEAB, and the pure fractions with the prod-
uct were pooled, evaporated, and dried to give N7-(4-chlorophen-
oxyethyl) guanosine-50-monophosphate TEA salt (3a) (0.192 g,
27.5%).
0
2.2.7. Synthesis of N7-(4-chlorophenoxyethyl)-m3 -OG(50)
ppp(50)G (5b)
To a stirred solution of N7-(4-chlorophenoxyethyl)-30-O-methyl
50-GMP, TEA salt (3b) (0.072 g, 0.1 mmol) and Im-GDP (4) (0.048 g,
0.08 mmol) in 2 mL of dry DMF, zinc chloride (0.068 mg, 0.5 mmol)
was added under nitrogen atmosphere. The resulting reaction mix-
ture was stirred for 72 h at 4 °C, and was quenched by addition of a
solution of EDTA (0.054 g) in water (10 mL). Aqueous layer was ad-
justed to pH 5.5 using 2.5 M NaOH, and washed with DCM (20 mL).
The crude mixture was purified by FPLC (DEAE Sepharose FF med-
ia) by elution of linear gradient of 0–1 M TEAB. The fractions con-
2.2.3. Synthesis of guanosine 50-diphosphate imidazolide
(Im-GDP) (4)
Imidazolide derivative of guanosine 50-diphosphate (4) was
synthesized from commercially available sodium salt of 50-GDP
taining pure product were pooled, evaporated, and dried to afford
0
22
by following reported procedure elsewhere.
N7-(4-chlorophenoxyethyl)-m3 -OG(50)ppp(50)G as triethylammo-
nium salt. The TEA salt was dissolved in nuclease free water
(2 mL) and poured into a solution of sodium perchlorate (40 mg)
in acetone (10 mL. The resulting solid was separated by centrifuga-
tion. The ion exchange procedure was repeated one more time. The
solid thus obtained was washed with acetone (10 mL) and dried
2.2.4. Synthesis of N7-(4-chlorophenoxyethyl) G(50)ppp(50)G (5a)
To a mixture of N7-(4-chlorophenoxyethyl) GMP (3a) (0.070 g,
0.1 mmol, 1 equiv) and GDP-imidazole (4) (0.048 g, 0.8 equiv) in
DMF (2 mL) at 25 °C was added ZnCl2 (0.068 g, 5 equiv). The reac-
tion mixture was stirred for 6 h at 25 °C, and was quenched by
addition of a solution of EDTA (0.054 g) in water (10 mL). Aqueous
layer was adjusted pH 5.5 by 2.5 M NaOH, and washed with DCM
(20 mL). The crude mixture was purified by FPLC (DEAE Sepharose
FF media) by elution of linear gradient of 0–1 M TEAB, and the pure
fractions with the product were pooled, evaporated, and dried to
give of N7-(4-chlorophenoxyethyl) G[50]PPP[50]G TEA salt . The
TEA salt was dissolved into 2 mL of nuclease free water, and the
under vacuum to afford pure N7-(4-chlorophenoxyethyl)-
0
m3 -OG(50)ppp(50)G (5b) as sodium salt (0.080 mg, 79%). 1H NMR
(D2O, 400 MHz): d 7.91 (1H, s), 6.99 (2H, dd, J = 6.8, 2.0 Hz), 6.62
(2H, dd, J = 6.8, 2.0 Hz), 5.79 (1H, d, J = 4 Hz), 5.67 (1H, d,
J = 6.0 Hz), 4.58 (2H, t, J = 5.2 Hz), 4.41 (1H, t, J = 4.0 Hz), 4.39–
4.25 (4H, m), 4.25–4.2 (1H, br), 4.20–4.10 (3H, br), 4.02 (1H, t,
J = 5.2 Hz), 3.39 (3H, s). 31P NMR (D2O, 162 MHz): d ꢁ11.5 (2P,
m), d ꢁ23.1 (1P, t, J = 19.4 Hz). MS (m/z) 955 [MꢁH].

4572
A. R. Kore et al. / Bioorg. Med. Chem. 21 (2013) 4570–4574
2.3. Biology
3. Results and discussions
All cellular eukaryotic mRNAs have at their 50 end a unique
structure (Fig. 1), known as a ‘cap’ which consists of a 7-methylgu-
anosine linked via a 50,50-triphosphate bridge to the first tran-
scribed nucleotide (m7GpppN, where N = G, A, C or U). The cap
plays a crucial role in several cellular processes such as it promotes
pre-mRNA splicing, enables transport of RNA from the nucleus to
the cytoplasm, protects mRNA from degradation by 50 exonucleas-
es and is required for efficient translation.1–8 During the initiation
step of translation, the cap is specifically recognized by eIF4E
(eukaryotic initiation factor 4E), the smallest subunit of a larger
complex, eIF4F, which also consists of a RNA helicase, eIF4A, and
a scaffolding protein, eIF4G.23
The prevailing method for generating capped mRNA using
in vitro transcription employs a preformed dinucleotide of the
form m7G(50)ppp(50)G (standard mCAP) in excess over regular G
in the transcription reaction, so that it is incorporated as the initial
base in preference to regular G.1-4 However, the drawback of stan-
dard mCAP analog is that the 30 OH of either the G or m7G can serve
as the initiating nucleophile for transcriptional elongation leading
to the synthesis of two isomeric RNAs of either forward or reverse
form in approximately equal proportions depending upon the ionic
conditions of the transcription reaction. The reverse form of
capped mRNAs that is, G[50]pppm7G[pN]n will not be recognized
during the translation process, with only forward orientated se-
quences that is, m7G[50]pppG[pN]n being translated.24 The finding
2.3.1. Gel shift capping assay
The capping efficiency of modified cap analogue N7-(4-chloro-
phenoxyethyl)-G(50)ppp(50)G (5a) and N7-(4-chlorophenoxyeth-
0
yl)-m3 -OG(50)ppp(50)G (5b) was compared with the standard cap
m7G(50)ppp(50)G. The pTri b-actin template was used in an
in vitro transcription reaction omitting pyrimidine nucleotides
which results in the termination of transcription after the first 7
coded all purine nucleotides. Syntheses of the capped and un-
capped oligoribonucleotides were performed by using the MAXI-
script™
manufacturer’s protocol. Typically, 20
tion mixture contained the following final concentrations of com-
ponents: linearized pTri b actin vector template, 25 ng/ l (0.5
kit
(Life
Technologies
Corporation)
following
ll of the transcription reac-
l
lg
total); ATP, 2 mM; GTP, 0.4 mM; standard cap or modified cap
(5a, 5b), 1.6 mM each in separate reaction; reaction buffer, 1X;
T7 RNA Polymerase-Plus™, 20 units/
mmol). In the control reaction, no cap analog was added. The tran-
scription reactions were incubated at 37 °C for 2 h. After that 10
of the reaction mixtures were applied to a 20% dPAGE gel. Radia-
tion in the gel bands of interest were quantified by a phosphorim-
ager (GE Healthcare).
ll; and (a-
32P) ATP, 800 (Ci/
l
l
2.4. Transcription reaction
T7 RNA polymerase transcription was performed by using the
MEGAscript™ kit (Life Technologies Corporation). All transcription
that the synthesis of two ‘anti-reverse cap analogs’ (ARCAs) such as
7,30-O
G[50]ppp[50]G and m7,30dG[50]ppp[50]G are also exclusively
reactions were performed in a 20
final concentrations of components: linearized AmbLuc poly(A)
DNA, (1.0 g total); 1X reaction buffer; ATP, UTP, and CTP,
7.5 mM each; and 50 units/ l of T7 RNA polymerase. Additionally,
ll final volume at the following
m2
incorporated only in the forward orientation because of modifica-
tions at the 30 position of the N7-methylguanosine ribose.19,25,26
The biological activity of RNAs capped with N7-(4-chlorophenoxy-
l
l
ethyl)-G(50)ppp(50)G
(5a)
and
N7-(4-chlorophenoxyethyl)-
GTP was present at 7.5 mM in the no-cap control reaction; and
reactions with cap analog included GTP at 1.5 mM and the cap ana-
log (standard cap or modified cap 5a, 5b) at 6.0 mM. The transcrip-
tion reactions were incubated at 37 °C for 2 h. In order to hydrolyze
0
m3 -OG(50)ppp(50)G (5b) were compared with the standard
m7G(50)ppp(50)G cap analog with respect to incorporation effi-
ciency, in vitro transcription yield, and translational activity.
The reaction pathway leading to the formation of desired cap
analogues 5a and 5b for the biological testing is depicted in
Scheme 1. Generally preparation of N7-alkyl GMP involves reaction
between sodium salt of GMP and alkyl halide in organic solvents
such as DMSO and DMF.16 Alkylation of sodium salt of N7-GMP
invariably results in very low yields (ꢀ2%) of N7-alkyl GMP.14 We
presumed that the low yield of N7-alkylation 50-GMP results from
insoluble nature of nucleotide sodium salt in common organic sol-
vents. It is to be noted that the presence of hydrophobic counter
ion present in the nucleotide helps to solubilize in the organic
medium.7 Therefore, we tried the reaction using triethyl
the remaining plasmid DNA, 1 ll of turbo DNase was added and
the reaction mixture was further incubated at 37 °C for 15 min.
Purifications of the RNA from these transcription reactions were
done by using the MEGAclear™ Kit (Life Technologies Corporation)
as per manufacturer’s protocol. The transcription yield of N7-
(4-chlorophenoxyethyl)-G(50)ppp(50)G (5a) and N7-(4-chlorophen-
0
oxyethyl)-m3 -OG(50)ppp(50)G (5b) was comparable with the
standard cap.
2.5. Luciferase assay
The translational efficiency of dinucleotide cap N7-(4-chloro-
phenoxyethyl)-G(50)ppp(50)G (5a) and N7-(4-chlorophenoxyeth-
0
yl)-m3 -OG(50)ppp(50)G (5b) was determined by transfecting
O
O
N
50-capped mRNA generated from transcription reaction into Hela
cells. Cells were plated in 96-well plate one day before transfection
CH3
N
N
N
NH
NH2
HN
H2N
O
P
O
O
P
O
O
P
O
5'
O
N
O
O
O
with 8000 cells in 100
50-capped RNA was prepared by mixing 100 ng (2
0.2 l Lipofectamine 2000 transfection reagent by following man-
ll 10% FBS DMEM each well. A complex of
N
O
ll) of RNA, and
O
O
O
P
O
OCH3
OH
OH
l
O
O-
N
N
NH
NH2
ufacturer’s protocol (Life Technologies Corporation, Catalog num-
ber 11668027), and incubated for 20 min at room temperature.
The resulting complex was added onto cultured cells. The transfec-
ted plates were incubated at 37 °C. Cells were harvested and lysed
at 4, 8, 16, 24, 32, and 40 h post-transfection. Luciferase production
was measured with Dual-LightÒ Luciferase & b-Galactosidase Re-
porter Gene Assay System (Life Technologies Corporation, Catalog
number T1003). Luciferase signals were detected using FLUO star
Omega multi-mode microplate reader (BMG Labtech). The fold
induction of luciferase protein data was normalized to the no
cap, mRNA poly(A) transfection results (control reaction).
N
O
O
O
OCH3
O-
NH
O
P
O
N
O
O
O
3'
OCH3
etc.
Figure 1. The chemical structure of the 50 terminus of a capped mRNA.

A. R. Kore et al. / Bioorg. Med. Chem. 21 (2013) 4570–4574
4573
O
N
O
N
4-Chlorophenyl-2-
bromoethyl ether,
N
N
N
N
NH
NH2
NH
NH2
O
P
O
POCl3,
O
O
HO
DMSO, 55 oC, 36 h
O
O
(OMe)3PO, O 0
C
OR OH
HN(Et)3
OR OH
2a R = H
1a R = H
2b R = CH3
1b R = CH3
O
Cl
O
N
N
NH
NH2
O
P
N
O
P
O
O
O
O
O
N
O
N
O
NH
NH2
O
P
O
O
N
N
OH OH
N
O
4
OR OH
HN(Et)3
ZnCl2, DMF, 4°C
3a R = H
3b R = CH3
Cl
O
N
O
O
N
N
NH
NH2
N
N
HN
O
P
O
O
P
O
O
P
O
O
O
O
O
H2N
N
O
O
OH OH
OR
OH
5a R = H
5b R = CH3
30 -O
Scheme 1. Synthesis of N7-(4-chlorophenoxyethyl)-G(50)ppp(50)G (5a) and N7-(4-chlorophenoxyethyl)-m G(50)ppp(50)G (5b) cap analogues.
ammonium salt of guanosine 50-monophosphate 2a and 30-O-
methylguanosine 50-monophosphate 2b. Reactions of 2a and 2b
with 4-chlorophenyl-2-bromoethyl ether in DMSO led to signifi-
cantly improved yield of the corresponding N7-(4-chlorophenoxy-
ethyl) substituted GMP 3a and 3b in 28% and 59% yields,
respectively. Finally, the coupling reaction of N7-(4-chlorophen-
oxyethyl) substituted GMP 3a and 3b with Im-GDP (4) in the pres-
ence of anhydrous zinc chloride as a catalyst using DMF as a
solvent afforded the corresponding cap analogues 5a and 5b in
45% and 79% yields, respectively.
case of N7-(4-chlorophenoxyethyl)-substituted cap analogues (5a,
5b) over standard m7G(50)ppp(50)G cap analogue might be due to
increased activation of ribose 30-hydroxyl group by bulky N7-(4-
chlorophenoxyethyl) substitution over N7-methyl and 30-O-methyl
substitution on guanosine.
The ability of modified cap analogs (5a, 5b) to be incorporated
into 1.83 Kb mRNA was explored using AmbLuc poly(A) super-
coiled plasmid (Life Technologies Corporation). Linearized plasmid,
generated from the above plasmid by digestion with Blp 1 enzyme,
was used as a template for in vitro transcription. The reaction was
carried out in the presence of either modified cap analogs (5a, 5b),
or standard cap analog or no cap followed by the purification of the
transcribed mRNAs using the MEGAclear™ Kit. The transcripts
yield with T7 RNA polymerase indicates that modified cap analogs
(5a, 5b) is a good substrate similar to standard cap analog (Fig. 3).
The translational efficiency of modified cap analogs (5a, 5b) was
determined with respect to standard cap analog by transfecting
capped mRNA that was transcribed from AmbLuc poly(A) vector
with T7 RNA polymerase into HeLa cells. Cells were harvested
Next, the capping efficiency of the modified cap analogs (5a, 5b)
with respect to standard cap analog during transcription was mea-
sured by gel shift assay (Fig. 2). pTri b actin vector was used to gen-
erate transcripts of 6 nucleotides in length. For gel shift assay, the
reactions were performed in the presence of (a-
32P) ATP to inter-
nally label the transcript. Gel shift assay (Fig. 2) revealed 63% cap-
ping efficiency for standard cap analogue and 77%, 76% capping
efficiency for the modified cap analogues (5a, 5b). As can be seen
from Figure 2, N7-(4-chlorophenoxyethyl)-G(50)ppp(50)G (5a) and
0
N7-(4-chlorophenoxyethyl)-m3 -OG(50)ppp(50)G (5b) capped mRNA
migrates slower than the standard capped mRNA. This is essen-
tially due to additionally increased molecular weight and bulky
size of modified dinucleotide cap analogs (5a, 5b). Observed facile
incorporation of triphosphates and higher capping efficiency in the
Figure 3. T7 RNA polymerase transcription reaction yields (quantified at A260 nm
Figure 2. The capping efficiency of standard m7G(50)ppp(50)G and N7-(4-chloro-
by using Nanodrop) involving standard m7G(50)ppp(50)G cap and N7-(4-chloro-
30 -O
30 -O
phenoxyethyl)-G(50)ppp(50)G (5a), N7-(4-chlorophenoxyethyl)-m G(50)ppp(50)G
phenoxyethyl)-G(50)ppp(50)G (5a), N7-(4-chlorophenoxyethyl)-m G(50)ppp(50)G
(5b) cap analogues by gel shift assay.
(5b) cap analogues.

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A. R. Kore et al. / Bioorg. Med. Chem. 21 (2013) 4570–4574
0
20
18
16
14
12
10
8
m3 -OG(50)ppp(50)G (5b), that carry N7 guanosine modification is re-
Control, no Cap
ported. The new cap analogue (5b) generates exclusively forward-
oriented capped mRNA during in vitro transcription. The new mod-
ified dinucleotide cap analogs (5a, 5b) were shown to be a sub-
strate for T7 RNA polymerase. Modified analog (5b) shows
ꢀ1.64-fold higher translational activity compared to standard cap
analog that could be attributed to the presence of exclusive for-
ward capped transcripts, strong binding affinity to the cap binding
proteins and its increased cellular stability. Considering the biolog-
ical compatibility of N7 modified cap analogs (5a, 5b) for both tran-
scription and translational processes one can expect its application
in protein production, anti-cancer and gene therapy.
Standard mCap
N7-Modified Cap 5a
N7-Modified Cap 5b
6
4
2
0
0
5
10
15
20
25
30
35
40
Time , h
References and notes
Figure 4. Translation efficiency of 50-capped mRNA poly(A) from standard
1. Furuichi, Y.; Shatkin, A. J. Adv. Virus Res. 2000, 55, 135.
2. Sharp, P. A. Cell 1994, 77, 805.
3. Lewis, J. D.; Izaurralde, E. Eur. J. Biochem. 1997, 247, 461.
4. Izaurralde, E.; Stepinski, J.; Darzynkiewicz, E.; Mattaj, I. W. J. Cell Biol. 1992, 118,
1287.
5. Roads, R. E. Prog. Mol. Subcell. Biol. 1985, 9, 104.
6. Jemielity, J.; Kowalska, J.; Rydzik, M. R.; Darzynkiewicz, E. New J. Chem. 2010,
34, 829.
m7G(50)ppp(50)G cap and N7-(4-chlorophenoxyethyl)-G(50)ppp(50)G (5a), N7-(4-
0
chlorophenoxyethyl)-m3 -OG(50)ppp(50)G (5b) cap analogues. The standard mCAP,
and modified cap (5a, 5b), 50-capped in vitro transcribed poly(A) tailed luciferase
RNA(100 ng) was transfected into HeLa cells. The fold induction of luciferase
protein data was normalized to the control reaction that is, no cap, mRNA poly(A)
transfection results.
7. Kore, A. R.; Charles, I.; Shanmugasundaram, M.; Xiao, Z.; Conrad, R. C. Mini-Rev.
Org. Chem. 2008, 5, 179.
8. Gingras, A. C.; Raught, B.; Sonenberg, N. Annu. Rev. Biochem. 1999, 68, 913.
9. Ghosh, B.; Benyumov, A. O.; Ghosh, P.; Jia, Y.; Avdulov, S.; Dahlberg, P. S.;
Peterson, M.; Smith, K.; Polunovsky, V. A.; Bitterman, P. B.; Wagner, C. R. ACS
Chem. Biol. 2009, 4, 367.
and lysed at different time points of post-transfection and the pro-
tein production was measured in terms of luciferase activity. Lucif-
erase activity data was normalized to the control reaction wherein
uncapped mRNA was used. After 16 h of post-transfection, the
luciferase activity of mRNA containing N7-(4-chlorophenoxyeth-
10. Kerekatte, V.; Smiley, K.; Hu, B.; Smith, A.; Gelder, F.; DeBenedetti, A. Int. J.
Cancer 1995, 64, 27.
0
yl)-m3 -OG(50)ppp(50)G cap analog (5b) was almost ꢀ1.64-fold
higher than that of mRNA containing standard m7G(50)ppp(50)G
11. Wang, Y. K.; Chen, W.; Blair, D.; Pu, M.; Xu, Y.; Miller, S. J.; Redfield, A. G.;
Chiles, T. C.; Roberts, M. F. J. Am. Chem. Soc. 2008, 130, 7746.
12. Oyarzabal, J.; Zarich, N.; Albarran, M. I.; Palacios, I.; Urbano-Cuadrado, M.;
Mateos, G.; Reymundo, I.; Rabal, O.; Salgado, A.; Corrionero, A.; Fominaya, J.;
Pastor, J.; Bischoff, J. R. J. Med. Chem. 2010, 53, 6618.
13. Nathan, C. O.; Liu, L.; Li, B. D.; Abreo, F. W.; Nandy, I.; DeBenedetti, A. Oncogene
1997, 15, 579.
14. Chen, X.; Kopecky, D. J.; Mihalic, J.; Jeffries, S.; Min, X.; Heath, J.; Deignan, J.; Lai,
S.; Fu, Z.; Guimaraes, C.; Shen, S.; Li, S.; Johnstone, S.; Thibault, S.; Xu, H.;
Cardozo, M.; Shen, W.; Walker, N.; Kayser, F.; Wang, Z. J. Med. Chem. 2012, 55,
3837.
15. Darzynkiewicz, A.; Ekiel, I.; Lassota, P.; Tahara, S. M. Biochemistry 1987, 26,
4372.
16. Darzynkiewicz, A.; Stepinski, J.; Ekiel, I.; Goyer, C.; Sonenberg, N.; Temeriusz,
A.; Jin, Y.; Sijuwade, T.; Haber, D.; Tahara, S. M. Biochemistry 1989, 28, 4771.
17. Kore, A. R.; Shanmugasundaram, M.; Charles, I.; Cheng, A. M.; Barta, T. J. Bioorg.
Med. Chem. Lett. 2007, 17, 5295.
18. Kore, A. R.; Charles, I.; Shanmugasundaram, M. Chem. Lett. 2009, 38, 652.
19. Kore, A. R.; Shanmugasundaram, M.; Charles, I.; Vlassov, A. V.; Barta, T. J. J. Am.
Chem. Soc. 2009, 131, 6364.
20. Kore, A. R.; Shanmugasundaram, M. Bioorg. Med. Chem. Lett. 2008, 18, 880.
21. Kore, A. R.; Parmar, G.; Reddy, S. Nucleosides, Nucleotides, Nucleic Acids 2006, 25,
307.
cap
analog.
However,
the
N7-(4-chlorophenoxyethyl)-
G(50)ppp(50)G (5a) dinucleotide form of the cap analog showed
only ꢀ0.72-fold than the mRNA capped with standard cap analog.
The observed low translation activity for (5a) could be due to sta-
bility in the form of dinucleotide cap analogs. Both the longer life of
modified cap analog (5b) capped transcripts and the absence of
nonfunctional (reverse-capped) capped transcripts during transla-
tion could be responsible for the higher translational efficiency of
modified cap analog (5b) capped mRNA (Fig. 4). The outcome of
the effect of N7-substituted phenoxyethyl guanosine in the form
of dinucleotide form is quiet different from its N7-substituted than
phenoxyethyl guanosine 50-monophosphate form. The N7-substi-
tuted phenoxyethyl guanosine dinucleotide form (5a) and (5b)
are substrate for RNA polymerase and recognized by elF4E during
translation process. The resulting translation efficiency is slightly
lower for (5a), could be due to stability of the capped mRNA
complex.
22. Kore, A. R.; Charles, I. Bioorg. Med. Chem. 2010, 18, 8061.
23. Sonenberg, N. In Translational Control; Hershey, J. W. B., Mathews, M. B.,
Sonenberg, N., Eds.; CSHL Press: New York, 1996; p 245.
24. Pasquinelli, A. E.; Dahlberg, J. E.; Lund, E. RNA 1995, 1, 957.
25. Stepinski, J.; Waddell, C.; Stolarski, R.; Darzynkiewicz, E.; Rhoads, R. E. RNA
2001, 7, 1486.
4. Conclusion
In conclusion, the synthesis of novel cap analogs, N7-(4-chloro-
phenoxyethyl)-G(50)ppp(50)G (5a), N7-(4-chlorophenoxyethyl)-
26. Peng, Z. H.; Sharma, V.; Singleton, S. F.; Gershon, P. D. Org. Lett. 2002, 4, 161.