Diastereoselective Synthesis of Phosphorodiamidate Morpholino Dimers

Full text of DOI:10.1002/bkcs.11958

Communication
DOI: 10.1002/bkcs.11958
BULLETIN OF THE
S. J. Min et al.
KOREAN CHEMICAL SOCIETY
Diastereoselective Synthesis of Phosphorodiamidate Morpholino
Dimers
*
So Jeong Min, Na Young Kim, Hyun Young Chae, and Keun Ho Chun
Department of Chemistry, Soongsil University, Seoul 06978, Republic of Korea.
*E-mail: kchun@ssu.ac.kr
Received September 20, 2019, Accepted December 12, 2019
Keywords: PMO, PMO dimer, Diastereoselective synthesis, Lithium bromide
Phosphorodiamidate morpholino oligonucleotide (PMO) is
the PMO dimers based on the same concept. For this purpose,
experiments were conducted to find the optimum conditions by
changing the reaction constituents and parameters, such as the
Lewis acid catalyst, base, protecting group, temperature, and
solvent. The experiment was conducted in route (a) and route
(b) (Scheme 2), and the results are summarized in Table 1 and
Table 2. The stereoselectivity of the synthesized compounds
was confirmed by the 1H NMR analysis of the -N(CH3)2 group
of phosphoamide.
Table 1 shows the results of optimizing the reaction con-
ditions by route (a). As shown from the results, route (a) was
not generally high in terms of the yield and stereoselectivity.
When LiBr was used as the Lewis acid catalyst, the yield
was high, but the stereoselectivity was low. The use of other
metals (Hg, Co, Cu) yielded worse results (Table 1, entries
11–15), and the combination of 9-BBN and DBU delivered
a yield and stereoselectivity similar to those obtained using
LiBr (Table 1, entries 1–5) is probably because monomer
5 did not form a good chelate ion with the Lewis acid cata-
lyst. Tert-butyldiphenylsilyl (TBDPS) was used as the
protecting group for 60-OH, and it exhibited the best results,
while the free OH and Bn group showed worse results
(Table 1, entries 6–8). Even when the reaction temperature
and the base were changed, no improvements were obtained.
In route (b), monomer 9 was expected to show the same
chelating structure and activity as monomer 3, as shown in
Scheme 1. The protecting group of monomer 8 was fixed
one of the third generation antisense oligonucleotides (ASOs)
that have been developed as therapeutic agents for use in a
variety of fields.1 A chiral center is formed in the P atom; con-
sequently, stereoisomers are formed during the PMO polymer
formation (Figure 1). The relationship between the phosphate
configuration and the physiological activity of PMO is
unknown; however, stereoisomers are generally distinguished
in vivo and are expected to exhibit different pharmacological,
pharmacokinetic, and toxicological properties.2 The optimal
synthesis conditions required to control the stereoselectivity of
P-chiral ASOs have been extensively studied over the last few
decades, although most of the studies have focused on pho-
sphorothioate oligonucleotides (PTOs).3
In 2017, Iwamoto reported that it was possible to increase
the efficiency of ASOs by adjusting the stereochemistry of
PTO. In their study, the stereocontrolled oligonucleotide syn-
thesis of iterative capping and sulfurization (SOSICS) using
a monomer containing an oxazaphospholidine chiral auxil-
iary was developed to synthesize therapeutic PTOs with high
stereochemical purity. Using this to investigate the activity
between mipomersen and the stereopure components, they
found that the stereochemistry of the phosphorothioate back-
bone increased the efficiency of the mRNA cleavage in vitro
and the persistence of its effects in vivo.2
In 2012, Sinha succeeded in the stereoselective synthesis
of activated PMO monomers under the LiBr–DBU condi-
tion. It was reported that steric control was possible due to
the low lattice energy of Li+ and the formation of the Li
chelate ion with the N atom of the morpholine ring,
NMe2POCl2, and the acylated nucleobase (Scheme 1).4
In this study, we synthesized PMO monomers according to
the known Sinha’s method1,4,5 and stereochemically controlled
Scheme 1. Plausible mechanism for the LiBr–DBU-mediated
Figure 1. Diastereoisomeric structures of the PMO dimer.
activation.
Bull. Korean Chem. Soc. 2020
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BULLETIN OF THE
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Table 1. Results of reaction condition for route (a).
Lewis
Temp Ratio
Yield
5 (equiv.)a 6 (equiv.) acidd
Base f
Solvent
(ꢀC) (S):(R) (%)
1
1
1
LiBr
DBU
DBU
DBU
DBU
DBU
DBU
DBU
DBU
MC:ACN
ACN
0
3.0:1.0 66
2
1
1
LiBr
−20 2.3:1.0 70
−20 2.5:1.0 50
0
3
1
1
LiBre
9-BBN
9-BBN
9-BBN
LiBr
ACN
4
1
1
MC:ACN
mixture 10
5
1
1.2
1
MC:ACN −20 2.5:1.0 10
6
1.5b
1b
1c
1
MC:ACN
MC:ACN
MC:ACN
0
0
0
1.2:1.0
6.0
Scheme 2. Synthetic routes of the PMO dimer
7
1.5
1.5
1.2
1.2
1
mixture 14
mixture 28
8
LiBr
9
LiBr
DIPEA MC:ACN −25 1.0:2.2 14
10
11
12
13
14
15
1
LiBr
NEM
DBU
DBU
DBU
MC:ACN −25 1.0:1.0 53
ACN
diastereoselectivity in route (b). However, in this case,
2 equiv of the activated monomer (8) was required to
complete the reaction (Table 2, entry 8).
1
HgCl2
CoCl2
CuCl2
0
0
0
0
0
mixture 68
mixture 56
1.4:1.0 51
mixture 63
mixture 28
1
1
ACN
1
1
ACN
Considering the solvent effect, it was found that the
stereoselectivity decreased when only ACN was used. For
DMF, the reaction rate increased but the yield and stereo-
selectivity decreased. This may be because Li+ is solvated
by DMF (Table 2, entries 9–10).
1.5
1.5
1
BF3OEt2 DBU
TEB DBU
MC:ACN
MC:ACN
1
a 5b.
b 5a.
c 5c.
d 5.5 equiv.
e 7 equiv.
1
The H NMR data of the PMO dimers which have previ-
f 5.5 equiv.
ously been identified for stereochemical configuration were
reported. The R isomer has a tendency to show an upfield
1
peak of the –N(CH3)2 group in the H NMR. Based on this
Table 2. Results of the reaction conditions for route (b).
data, the major stereoisomer of 7b, synthesized by route
(b), is expected to mainly have the R-configuration, since
the proton peak of the –N(CH3)2 group of phosphoamide
appears mainly in the upfield.6
In conclusion, the PMO dimer was stereoselectively syn-
thesized using the Li chelate ion. In the synthesis of the
dimer, the target compound with good stereoselectivity and a
relatively high yield was obtained when t-BuOLi was used
as the base at −25 ꢀC via route (b). Although further studies
are required to improve the yield and identify the exact ste-
reochemical configuration of the synthesized material, our
studies offer significant value as the first attempt to control
the phosphate stereoselectivity of PMO dimers.
Lewis
Temp Ratio
Yield
8 (equiv.) 9 (equiv.) acid
Base
Solvent
(ꢀC)
(S):(R) (%)
1
2
3
4
5
6
7
8
9
10
1
1
1
1
1
1
1
2
2
2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1
LiBr
LiBr
LiBr
LiBr
LiBr
LiBr
LiBr
LiBr
LiBr
LiBr
DBU
DBU
DBU
DBU
DBU
MC:ACN
0
1.0:6.7
5.0
11
MC:ACN −20 1.0:4.6
MC:ACN −25 1.0:20
MC:ACN −30 1.0:8.6
MC:ACN −40 1.0:20
8.9
10
18
DIPEA MC:ACN −25 1.0:8.6
NEM MC:ACN −25 mixture
t-BuOLi MC:ACN −25 1.0:8.6
6.0
5.0
60
1
t-BuOLi DMF
−25 1.0:2.8
−25 1.0:1.8
7.0
31
1
t-BuOLi ACN
with TBDPS, and the synthesis of the PMO dimer (7b) was
performed under LiBr–DBU conditions at 0 ꢀC, and
improved selectivity was achieved over route (a) (Table 2,
entry 1). As the temperature decreased to −30 ~ −40 ꢀC, the
reaction rate decreased, although enhanced stereoselectivity
was achieved (Table 2, entries 2–5). The best data was
obtained when the PMO dimer was synthesized at −25 ꢀC
in LiBr–DBU, and it showed a 90.5% de. However, the
yield was still low at 8.9% (Table 2, entry 3).
Changing the base to NEM, DIPEA, and t-BuOLi did
not result in any remarkable improvement in the yield and
stereoselectivity in route (a) (Table 1, entries 9–10). When
DIPEA was used in route (b), the stereoselectivity was
improved; however, a low yield was obtained. NEM,
which is a relatively weak base, failed to deliver the
proper product (Table 2, entries 6–7). However, when a
strong base was used (t-BuOLi), the yield was signifi-
cantly increased up to 60% with affordable
Supporting Information. Additional supporting informa-
tion may be found online in the Supporting Information
section at the end of the article.
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