Synthesis of new dinucleotide mRNA Cap analogs containing 2, 6-diaminopurine moiety

Article abstract of DOI:10.2174/157017810791112388

The first example of the synthesis of cap analog derivatives having 2, 6-diaminopurine nucleoside has been described. The desired modified cap analogs m⁷G[5']ppp[5']^2-aminoA 6 and m₂ ^7,3'°G[5']ppp[5']^2-aminoA 7 were obtained from the coupling reaction of imidazolide salt of 2-amino adenosine [5'] monophosphate with its corresponding TEA salt of m7GDP in the presence of ZnCl₂ as a catalyst in 73% and 64% yield, respectively. The structures of new cap analogs were confirmed by LC/MS, ¹H and ³¹P NMR.

Full text of DOI:10.2174/157017810791112388

200
Letters in Organic Chemistry, 2010, 7, 200-202
Synthesis of New Dinucleotide mRNA Cap Analogs Containing 2,
6-Diaminopurine Moiety
Anilkumar R. Kore* and Irudaya Charles
Life Technologies Corporation, Bioorganic Chemistry Division, 2130 Woodward Street, Austin, TX 78744-1832, USA
Received November 25, 2009: Revised February 08, 2010: Accepted February 09, 2010
Abstract: The first example of the synthesis of cap analog derivatives having 2, 6-diaminopurine nucleoside has been
described. The desired modified cap analogs m7G[5ꢀ]ppp[5ꢀ]2-aminoA 6 and m27,3ꢀOG[5ꢀ]ppp[5ꢀ] 2-aminoA 7 were obtained
from the coupling reaction of imidazolide salt of 2-amino adenosine [5ꢀ] monophosphate with its corresponding TEA salt
of m7GDP in the presence of ZnCl2 as a catalyst in 73% and 64% yield, respectively. The structures of new cap analogs
were confirmed by LC/MS, 1H and 31P NMR.
Keywords: mRNA Cap analogs, penultimate nucleotide and 2, 6-diaminopurine.
INTRODUCTION
that the reverse capped mRNAs that are formed with
standard dinucleotide cap analog, rather than being neutral,
impeded the translation. Latter, it was shown that chemical
modification at the 2ꢀ-OH group of N7-methylguanosine also
behaved as ARCA analogs giving exclusively forward
capped mRNAs after transcription reaction [11]. Several
literature reports followed these findings that mainly discuss
the benefits as well as the tolerance of chemical modification
of N7-methylguanosine nucleotide and phosphate groups of
cap analogs and their significance in translational efficiency
[12-14].
All eukaryotic mRNAs that undergo cap-dependent
translation contain a unique cap structure of the form
m7[5ꢀ]Gppp[5ꢀ]N[pN]n at the 5ꢀ-end of mRNA that plays
several crucial roles in the lifecycle of mRNA [1] such as
protecting mRNA against nucleases [2], accurate and
efficient splicing [3], translation initiation [4] and mRNA
transportation from nucleus to cytoplasm [5]. Considering
these interesting biological roles of cap analog, Hata et al.
[6] reported the first synthesis of standard dinucleotide cap
analog, m7[5ꢀ]Gppp[5ꢀ]G. It was hopped that in vitro
transcription with this cap analog would yield a capped
mRNA that would mimic the biological functions of in vivo
synthesized capped mRNA. However, it was found that the
in vitro transcription gave a mixture containing ꢁ to ꢂ of
reverse capped mRNAs, in addition to forward capped
mRNAs [7]. It was reasoned that 3ꢀ-OH of both the
nucleotides of this cap analog act as a nucleophile during the
transcription initiation leading to the formation of
heterogeneous mixture. It is worth mentioning that only
forward oriented cap analogs were translated [8].
Furthermore, cytoplasmic reverse-capped U1 RNAs fail to
be imported into the nucleus and when reverse capped pre-
U1 RNA transcripts were injected into Xenopus laevis
nuclei, they were exported more slowly than natural
transcripts [7]. It was also observed that the presence of a
cap structure on mRNA has a strong stimulatory effect on
translation [4,9].
However, only limited amount of information is available
in the literature about the chemical modification tolerance of
the nucleoside that is present at the other end of N7-
methylguanosine of the dinucleotide cap analogs. In general,
the penultimate nucleotide (X) of capped mRNA
(m7G[5ꢀ]ppp[5ꢀ]Xp(Np)m….) can be any of the four naturally
occurring nucleotide (A, C, G and U) for successful
translation. However, the compatibility of this nucleoside
with polymerase enzyme determines the cap orientation and
yield of the capped mRNA during transcription reaction
which has direct impact on the capped mRNA translational
efficiency. The literature survey shows that N7-
methylguanosine [15,16], 2ꢀ-O-methylnucleosides (A, C, G
or U) [17,18], 2ꢀ-deoxyguanosine [19], N7,O2ꢀ-
dimethylguanosine [20], N6-methyladenosine [19] and
N6,N6,O2ꢀ-trimethyladenosine [21] are the chemically
modified bases substituted for the other end of N7-
methylguanosine of the dinucleotide cap analogs. In our
quest to understand the mechanism of cap-dependent
translation machineries in order to improve the translational
efficiency of capped mRNA, we proposed a design of new
cap analogs that would carry non-natural nucleotide, 2, 6-
diaminopurine, as a penultimate nucleotide of the capped
mRNA after transcription reaction. The choice of 2,6-
diaminopurine for this study was based on its compatibility
with transcription machineries [22] and superior biological
properties such as duplex hybridization, mismatch specificity
and nuclease stability [23]. The other interesting aspect of
incorporating 2,6-diaminopurine into RNA by in vitro
This led to the design of anti-reverse cap analogs
(ARCA) carrying a chemical modification at the 3ꢀ-OH
group of N7-methylguanosine [8,10]. As the name implies,
these cap analogs exclusively provide forward capped
mRNAs whose translational efficiencies were 2.6 fold higher
than standard cap analog. The in vitro transcription and
translation of ARCA analogs provided valuable information
*Address correspondence to this author at the Life Technologies
Corporation, Bioorganic Chemistry Division, 2130 Woodward Street,
Austin, Texas 78744-1832, USA; Tel: +1 512 721 3589; Fax: +1 512 651
0201; E-mail: anil.kore@lifetech.com
1570-1786/10 $55.00+.00
© 2010 Bentham Science Publishers Ltd.

Synthesis of New Dinucleotide mRNA Cap Analogs
Letters in Organic Chemistry, 2010, Vol. 7, No. 3
201
transcription is that it enhances the signal intensity of the
resultant RNA [23]. This unique property has been utilized
in DNA microarrays based genomic profiling, wherein the
expression levels of thousands of genes can be
simultaneously examined. Considering potential in vitro and
in vivo application of cap analogs [12,24], we believe that
the above additional characteristic properties of 2,6-
diaminopurine would further benefit the cap analog variants
of the type m7G[5ꢀ]ppp[5ꢀ]2-aminoA 6 and m27,3ꢀOG[5ꢀ]ppp[5ꢀ]2-
aminoA 7. Herein, we describe the first example of the
chemical synthesis of new cap molecules 6 and 7 from
commercially available starting materials.
DNA template, NTPs and reaction condition. This
preferential exclusive incorporation of penultimate
nucleotide leads to the formation of capped mRNA
containing N7-methylguanosine group at its 5ꢀ-end that is
essential for successful and efficient translation. This
prompted us to synthesize the ARCA variant of cap analog
m27,3ꢀOG[5ꢀ]ppp[5ꢀ]2-aminoA 7 (Scheme 1) that would alleviate
the polymerase using N7-methylguanosine nucleotide part for
transcription initiation. The precursor 5 was synthesized
from 3ꢀ-O-methylguanosine in four steps as reported in the
literature [17] that involves monophosphorylation of 3ꢀ-O-
methylguanosine with POCl3 and trimethyl phosphite to get
3ꢀ-O-methyl GMP followed by conversion into imidazolide
salt with imidazole, triphenylphosphine and aldrithiol which
was phosphorylated with (Et3NH)3PO4 in the presence of
zinc chloride as a catalyst. Regioselective methylation of 3ꢀ-
O-methyl GDP with dimethyl sulfate at pH 4.0 [25] followed
by chromatographic purification using TEAB buffer
furnished the required precursor 5 as a TEA salt. The final
coupling of the precursor 5 with imidazolide 3 in the
presence of zinc chloride in DMF provided the desired
m27,3ꢀOG[5ꢀ]ppp[5ꢀ]2-aminoA 7 in 64% yields [27].
RESULTS AND DISCUSSION
The synthesis of the cap analog m7G[5ꢀ]ppp[5ꢀ]2-aminoA 6
(Scheme 1) starts with 5ꢀ-phosphorylation of 2, 6-
diaminopurine under Yoshikawa condition, which provided
51% of 2-amino AMP 2. The further reaction of 2-amino
AMP 2 with aldrithiol, triphenylphosphine, and imidazole in
the presence of triethylamine provided the imidazolide salt
of 2-amino AMP 3 in 85% yields. The m7GDP 4 was
obtained from guanosine diphosphate (GDP) by
regioselective methylation with dimethyl sulfate at pH 4
[25]. The final coupling of imidazolide 3 and m7GDP 4 in
the presence of zinc chloride in DMF provided
m7G[5ꢀ]ppp[5ꢀ]2-aminoA 6 in 73% yields [26].
In summary, we have reported the synthesis of two new
cap
analogs
m7G[5ꢀ]ppp[5ꢀ]2-amino
A
6
and
m27,3ꢀOG[5ꢀ]ppp[5ꢀ]2-aminoA 7. We believe that these molecules
could promote cap-dependent translation efficiently and find
applications related to in vitro and in vivo protein production.
Further biological studies such as their capping efficiency,
substrate validation with T7 RNA polymerase, and
translation efficiency are in progress and the results will be
reported elsewhere.
During in vitro transcription, the dinucleotide anti-
reverse cap analogs direct the other end of N7-
methylguanosine to be as a first nucleotide for transcription
initiation. This would be followed by the addition of other
nucleotides which depends on other components such as
NH2
NH2
NH2
N
N
N
N
N
N
N
N
N
O
P
O
N
N
NH2
N
NH2
N
NH2
HO
-O
P
O
N
O
(i)
O
O
O
O-
O-
(ii)
HN(Et)3
OH
OH
OH
OH
OH
OH
1
2
3
O
N
NH2
CH3
O
CH3
(Et)3NH
N
N
N
HN
N
HN
O-
P
O
P
O
P
O
O
N
N
N
H2N
N
NH2
H2N
N
O
O
O-
O
P
O
O
P
O
O
O
O
O-
O-
O-
O-
O
(iii)
OR
OH
OH
OH
OR
OH
(Et)3NH
4. R = H
5. R = CH3
6. R = H
7. R = CH3
Scheme 1. Synthesis of m7G[5ꢀ]ppp[5ꢀ]2-aminoA 6 and m27,3ꢀOG[5ꢀ]ppp[5ꢀ]2-aminoA 7. (i) POCl3, (OMe)3PO, 0°C; (ii) aldrithiol, PPh3, imidazole,
Et3N, DMF, rt; (iii) imidazolide salt of 2-amino AMP 3, ZnCl2, DMF, rt. The final yield after purification of 6 was 73%, and for 7 it was
64%.

202 Letters in Organic Chemistry, 2010, Vol. 7, No. 3
Kore and Charles
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Phosphorylation of eIF4E attenuates its interaction with mRNA 5'
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Kore, A.R.; Shanmugasundaram, M. Synthesis and biological
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ACKNOWLEDGEMENT
We wish to thank Shirley A. Recipon and S. Muthian for
commenting and critical reading of this manuscript.
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Spectral Data for m7G[5ꢂ]ppp[5ꢂ]2-aminoA 6: H NMR (400 MHz,
D2O) ꢃ 7.86 (s, 1H), 5.74 (d, J = 3.3 Hz, 1H), 5.65 (d, J = 6.2 Hz,
1H), 4.52 (t, J = 5.8 Hz, 1H), 4.39 - 4.35 (m, 1H), 4.34 - 4.30 (m,
1H), 4.28 - 4.18 (m, 4H), 4.16 - 4.08 (m, 3H), 3.90 (s, 3H). 31P
NMR (162 MHz, D2O) ꢃ -10.37 (d, J = 19.3 Hz, 2P), -21.93 (t, J =
19.5 Hz, 1P). MS (m/z): 801 [M-H]-.
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Spectral Data for m27,3ꢂOG[5ꢂ]ppp[5ꢂ]2-aminoA 7: 1H NMR (400 MHz,
D2O) ꢃ 8.05 (s, 1H), 5.89 (d, J = 2.6 Hz, 1H), 5.80 (d, J = 6.1 Hz,
1H), 4.60 (t, J = 5.6 Hz, 1H), 4.48 - 4.40 (m, 3H), 4.40 - 4.33 (m,
2H), 4.32 - 4.24 (m, 3H), 4.12 (dd, J = 4.8, 2.7 Hz, 1H), 4.02 (s,
3H), 3.59 (s, 3H). 31P NMR (162 MHz, D2O) ꢃ -10.35 (d, J = 19.0
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