Development of an efficient method for phosphorodiamidate bond formation by using inorganic salts

Article abstract of DOI:10.1016/j.bmcl.2011.12.021

Phosphorodiamidate morpholino oligonucleotides (PMOs) have been extensively applied in antisense strategies for gene regulation because of their high stability in serum and low toxicity. However, chain elongation of PMOs requires long reaction time becaus

Full text of DOI:10.1016/j.bmcl.2011.12.021

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1445–1447
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of an efficient method for phosphorodiamidate bond
formation by using inorganic salts
⇑
Taro Harakawa, Hirosuke Tsunoda, Akihiro Ohkubo, Kohji Seio, Mitsuo Sekine
Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsutacho, Midoriku, Yokohama 226-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Phosphorodiamidate morpholino oligonucleotides (PMOs) have been extensively applied in antisense
strategies for gene regulation because of their high stability in serum and low toxicity. However, chain
elongation of PMOs requires long reaction time because few efficient methods have been developed
for the formation of phosphorodiamidate bonds. In this Letter, we examined the effect of various addi-
tives to improve the reaction efficiency for formation of the phosphorodiamidate bond in the synthesis
of PMOs. The addition of certain inorganic salts to the reaction media was found to be more effective. Par-
ticularly, lithium bromide was the most effective reagent and led to considerable acceleration (ca. 10-fold
improvement).
Received 4 November 2011
Revised 29 November 2011
Accepted 3 December 2011
Available online 8 December 2011
Keywords:
Morpholino nucleic acid
Phosphorodiamidate bond
Inorganic salt catalysis
Ó 2011 Elsevier Ltd. All rights reserved.
Chemical modification plays an important role in improving the
properties of natural DNA and RNA molecules, including hybridiza-
tion affinity, base recognition, and nuclease resistance.1–4 Modifi-
cation of the internucleotidic phosphate group can significantly
increase the nuclease resistance of artificial oligonucleotides.5–7
In particular, phosphorodiamidate morpholino oligonucleotides
(PMOs),8 which have a unique skeleton containing morpholine
rings and phosphorodiamidate moieties shown in Figure 1, have
been extensively applied in antisense strategies9 for gene regula-
tion because of their high stability in serum and low toxicity.10 Re-
cently, several research groups found that antisense PMOs could
efficiently induce functional dystrophin protein expression via
exon skipping by restoring in-frame transcripts in Duchenne mus-
cular dystrophy.11–13
respectively) using Summerton’s method.14 To estimate the effi-
ciency of the condensation using the morpholino-T unit, the solu-
tion-phase synthesis of morpholino TT-dimer 6 was performed
under various conditions, as shown in Scheme 1 and Table 1. When
dimethylimidazolidinone (DMI) was used as the solvent (entry 1),
the condensation of morpholino-T derivative 5 with 1.1 equiv of T
unit 1 in the presence of 2.2 equiv of N-ethylmorpholine (NEM)
was completed within 40 min without any side reactions. How-
ever, the isolated yield was very low because it was difficult to sep-
arate target compound 6 and DMI because of high boiling point
Summerton et al. previously reported the formation of phosp-
horodiamidate bonds using 50-phosphorochloridate morpholino
units (Fig. 1b and c) on polymer supports.14 The PMO units could
be readily isolated by silica gel chromatography because of their
high chemical stability despite the presence of a P–Cl bond.
However, chain elongation of PMOs requires a long reaction time
because of their low reactivity. Because few efficient methods have
been developed for the formation of phosphorodiamidate bonds,
there is a need for further optimization of existing procedures. In
this study, we improved the condensation for both the solution-
and solid-phase syntheses of PMOs by varying the reaction solvent,
base, and additives.
We first synthesized the morpholino-T, morpholino-C, morpho-
lino-A, morpholino-G units (1, Fig. 1b, 2 Fig. 1c, 3, Fig. 1d, 4 Fig. 1e,
⇑
Figure 1. Structure of (a) PMO, (b) morpholino-T unit, (c) morpholino-C unit, (d)
morpholino-A unit and (e) morpholino-G unit, Tr: triphenyl methyl group.
Corresponding author. Tel.: +81 45 924 5706; fax: +81 45 924 5772.
E-mail address: msekine@bio.titech.ac.jp (M. Sekine).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.021

1446
T. Harakawa et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1445–1447
As shown in entry 12, in the presence of LiI, the reaction time
was decreased to 10 min, although the isolated yield of the desired
product decreased owing to the formation of some byproducts.
Compared with this result, Bu4NI was less effective, as shown in
entry 13. However, in the presence of LiBr, the condensation was
complete in 15 min without any side reactions, and target com-
pound 6 was quantitatively isolated (entry 15). In sharp contrast,
the addition of LiCl had little effect on the condensation (entry
14). These results indicate that the addition of inorganic salts (LiBr
and LiI) could induce the halogen-exchange reaction of compound
1, leading to a more reactive species.15 Moreover, it was found that
the addition of excess LiBr (10 equiv) resulted in considerable
acceleration (reaction time of only 3 min, ca. 10-fold improve-
ment), as shown in entry 16.
Scheme 1. Solution-phase synthesis of morpholino TT-dimer 6.
Table 1
Efficiency of condensation of the morpholino-T unit with morpholino T (5)
Entry Solvent Base (2.2 equiv) Additive (1.1 equiv or Time Isolated
10 equiv)
(min) yield (%)
1
2
3
4
5
6
7
8
9
10
DMI
THF
DMF
CH3CN NEM
CH3CN Lutidine
CH3CN DBU
NEM
NEM
NEM
None
None
None
None
None
None
40
120
2
40
10
40
30
>720
>720
720
40
88
84
96
82
17
95
54a
80a
89
CH3CN Diisopropylethylamine None
CH3CN NEM
CH3CN NEM
CH3CN NEM
DMAP (1.1 equiv)
HOBt (1.1 equiv)
4-Methoxypyridine
N-oxide (1.1 equiv)
1H-Tetrazole
11
CH3CN NEM
15
82
(1.1 equiv)
12
13
14
15
16
CH3CN NEM
CH3CN NEM
CH3CN NEM
CH3CN NEM
CH3CN NEM
LiI (1.1 equiv)
10
60
30
88
85
98
TBAI (1.1 equiv)
LiCl (1.1 equiv)
LiBr (1.1 equiv)
LiBr (10 equiv)
15 Quant.
Quant.
3
a
The reaction mixture was worked up after 720 min.
and similar polarity. As shown in entry 2, the condensation in THF
was completed in 120 min. On the other hand, when DMF was
used as the solvent, some byproducts were observed (entry 3),
although the reaction time was very short (within 2 min). As
shown in entry 4, the condensation in CH3CN was completed after
40 min without any side reactions, and target dimer 6 was isolated
in high yield (96%). These results indicate that CH3CN is more suit-
able than other solvents for the solution-phase synthesis of PMOs
considering the reaction time and isolated yield.
To decrease the reaction time, we varied the base from NEM to
lutidine, DBU, and diisopropylethylamine. Although the reaction
using lutidine was faster than that using NEM (10 min, entry 5),
the isolated yield was lower. In the condensation using DBU, the
isolated yield surprisingly decreased to 17% owing to accompany-
ing complicated side reactions (entry 6). As shown in entry 7, the
use of diisopropylethylamine gave a result similar to that using
NEM. Therefore, we chose NEM as a suitable base reagent for the
synthesis of PMOs.
Scheme 2. Synthesis of PMO dimers 11–14 on polymer supports.
Next, we examined the effects of various additives on the
condensation for the synthesis of PMOs. The addition of typical
nucleophilic catalysts, such as 4-dimethylaminopyridine (DMAP),
1-hydroxybenzotriazole (HOBt), and 4-methoxypyridine N-oxide,
markedly retarded the reaction, all of which required more than
720 min for completion (entries 8–10). On the other hand, the
condensation using 1H-tetrazole was completed in only 15 min
(entry 11); however, the isolated yield was lower than that for
the reaction without an additive. Therefore, we examined the
effects of another series of additives on the reaction rate. In these
examples, a series of inorganic lithium salts (LiX, X = I, Br, and Cl)
and a quaternary ammonium salt (Bu4NI) were employed.
Figure 2. Reaction efficiency for the synthesis of morpholino-T dimers 11 on
polymer supports.

T. Harakawa et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1445–1447
1447
Figure 3. Reversed-phase HPLC profiles of the mixtures containing (a) PMO TT-dimer 11, (b) PMO TC-dimer 12, (c) PMO TA-dimer 13 and (d) PMO TG-dimer 14 after the 60-
min condensation on polymer supports.
In addition, we attempted to further improve the conditions for
the synthesis of PMOs using polymer supports. The condensation of
morpholino units 1–4 with morpholino T-loaded polystyrene was
performed in the presence of different bases and additives. Follow-
ing the condensation, which was performed under various condi-
tions, the Tr group was removed under mild acidic conditions,16
and target PMO dimers 11–14 were released from the resins by
treatment with 28% NH4OH or AMA17 for 1–12 h, as shown in
Scheme 2. The crude mixtures thus obtained were analyzed by re-
versed-phase HPLC, and the reaction efficiencies were evaluated
by the ratio of the peak area of target dimers 11–14 to the combined
areas of all peaks of PMO observed by HPLC. The light blue line in
Figure 2 shows the time course of the formation of dimer 11. The
efficiency of the condensation in CH3CN was unexpectedly low ow-
ing to aggregation of the resin. To avoid this problem, we changed
the solvent to DMI, which was previously reported as a suitable sol-
vent for solid-phase synthesis.14 As a result, the reaction efficiency
improved, as shown by the orange line in Figure 2. In addition, it
was found that the reaction efficiency was enhanced by the addi-
tion of 6 equiv of LiBr (light green line), consistent with the results
of the solution-phase synthesis mentioned above. However, the
addition of a large excess of LiBr (20 equiv) did not further acceler-
ate the reaction (purple line in Fig. 2).Figure 3a shows the reversed-
phase HPLC profile of the crude mixture obtained from the 60-min
condensation using 20 equiv of LiBr. Target dimer 11 was isolated
by reversed-phase HPLC in 45% yield and characterized by electro-
spray ionization mass spectrometry. Furthermore, we performed
the synthesis of other PMO dimers 12–14 using 20 equiv of LiBr
in DMI. These reaction efficiencies were as high as that in the syn-
thesis of TT-dimer 11, as shown in Figure 3b and c. The isolated
yields of these dimers 12–14 were 30%, 42%, and 46%, respectively.
In summary, we examined the effects of various additives to im-
prove the reactivity of intermediates involved in the condensation.
Catalysts that are typically used in oligonucleotide and peptide
syntheses and nucleophilic substitutions did not show an enhance-
ment of the reaction rate. Similarly, organic salts like TBAI showed
no improvement in the efficiency of the condensation. However,
the addition of certain inorganic salts to the reaction media was
found to be more effective. Particularly, lithium bromide was the
most effective reagent and led to considerable acceleration (ca.
10-fold improvement). The additive effect of LiBr was also ob-
served in the synthesis of PMO dimers 11–14 on polymer supports.
Further studies are now in progress.
Acknowledgments
This study was supported by a Grant-in-Aid for Scientific Re-
search from the Ministry of Education, Culture, Sports, Science
and Technology, Japan. This study was also partly supported by a
grant from the global COE project, Ministry of Education, Culture,
Sports, Science and Technology, Japan.
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