Synthesis of phosphorodiamidate morpholino oligonucleotides by H-phosphonate method

Article abstract of DOI:10.1016/j.tetlet.2015.05.080

Synthesis of T-containing phosphorodiamidate morpholino oligomers (PMO) by H-phosphonate method on solid support has been reported for the first time. Initially, 5-mer then 15-mer of T-containing PMO using H-phosphonate-T monomer was synthesized on polystyrene resin bead. The phosphonamidate backbone was oxidized by iodine-dimethylamine mixture followed by the cleavage from solid support by aqueous ammonia to obtain 15-mer T-containing PMO.

Full text of DOI:10.1016/j.tetlet.2015.05.080

Accepted Manuscript
Synthesis of phosphorodiamidate morpholino oligonucleotides by H-phospho-
nate method
Jhuma Bhadra, Jayanta Kundu, Keshab Ch. Ghosh, Surajit Sinha
PII:
S0040-4039(15)00913-2
DOI:
http://dx.doi.org/10.1016/j.tetlet.2015.05.080
TETL 46352
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 February 2015
21 May 2015
22 May 2015
Please cite this article as: Bhadra, J., Kundu, J., Ghosh, K.C., Sinha, S., Synthesis of phosphorodiamidate morpholino
oligonucleotides by H-phosphonate method, Tetrahedron Letters (2015), doi: http://dx.doi.org/10.1016/j.tetlet.
2015.05.080
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Tetrahedron Letters
1
Tetrahedron Letters
journal homepage: www.els evier.com
Synthesis of phosphorodiamidate morpholino oligonucleotides by H-phosphonate
method
Jhuma Bhadra a, Jayanta Kundu a Keshab Ch. Ghosh a and Surajit Sinha a, ∗
a Department of Organic Chemistry, Indian Association for the cultivation of Science, Jadavpur, Kolkata-700 032, India
ARTICLE INFO
ABSTRACT
Article history:
Received
Synthesis of T-containing phosphorodiamidate morpholino oligomers (PMO) by H-phosphonate
method on solid support has been reported for the first time. Initially, 5-mer then 15-mer of T-
containing PMO using H-phosphonate-T monomer was synthesized on polystyrene resin bead.
The phosphonamidate backbone was oxidized by iodine-dimethylamine mixture followed by the
cleavage from solid support by aqueous ammonia to obtain 15-mer T-containing PMO.
Received in revised form
Accepted
Available online
Keywords:
2014 Elsevier Ltd. All rights reserved.
Morpholino oligomer
Nucleosides
H-Phosphonate
H-Phosphonate-T
Solid support
Phosphorodiamidate morpholino oligos (PMO), 11 (Scheme 1)
are routinely used for gene silencing as antisense reagents when
they are designed targeting 5´ – UTR of mRNA2 or pre-mRNA
splicing.3 The extraordinary stability, lack of net charge on the
internucleosidic linkage backbone, and total resistance to
metabolic degradation has made this class of molecules worthy of
consideration as potential therapeutics. Phosphorodiamidate
morpholino oligos have entered into clinical trial unusually fast
for the treatment of Duchenne muscular dystrophy and flaunted
impressive safety and pharmacokinetics records during Phase I/II
clinical trials.4 Solid phase synthesis of PMO is reported by Gene
Tools in their patent5 using N-tritylated 5´-activated
chlorophosphora-midate morpholino monomers to get the desired
length of PMO (25 mer) for antisense properties (Scheme 1). The
coupling time between two monomers was not efficient in the
presence of large excess of N-ethylmorpholine, accordingly,
Sekine, M. et. al. reported6 another method to improve the
coupling efficiency using LiBr as an additive and has shown its
application only for the synthesis of 2-mer PMO in both solution
and solid phase.
Scheme 1. Solid phase synthesis of phosphorodiamidate
morpholino oligonucleotides (PMO).
We have been working on the morpholino chemistry7 for the
past several years where we realized that the stability of activated
5´-chlorophosphoramidate morpholino monomer is poor in
solution particularly in the presence of base. Our goal was to
develop the solid supported synthesis of PMO using a chemistry
which is compatible to a DNA synthesizer where coupling time is
efficient and activated monomers are stable. H-phosphonate
chemistry is well known for DNA synthesis8 however, such
chemistry has not been explored in the synthesis of PMO.
In this communication, we wish to report the synthesis of T-
morpholino H-phosphonate and its use in the synthesis of 15-mer
of T-containing PMO on solid support.
T-morpholino monomer 2 was synthesized using our previously
reported method.7a Monomer 2 was then converted to H-
phosphonate
3 according to the procedure reported for
nucleosides8 by treatment with PCl3 and 1,2,4-triazole followed
by quenching with triethylammonium bicarbonate buffer (TEAB,
pH 8.5)9 (Scheme 2). The compound was purified by silica gel
———
∗ Corresponding author. Tel.: +91-33-2473-4971; fax: +91-33-2473-2805; e-mail: ocss5@iacs.res.in

2
Tetrahedron Letters
column chromatography and characterized by spectroscopic The first linker was attached in 99 % yield. Similarly the second
techniques.
linker was attached to obtain 6 in 99 % yield. The N-Tr-T-
morpholino monomer was functionalized with succinic
2
anhydride to obtain the succinic ester 11. Compound 11 was
purified by silica gel column chromatography to obtain 66%
yield. 11 was then loaded on the functionalized polystyrene resin
beads 6 after MMTr deprotection using HOBt (5 equiv.), HBTU
(5 equiv.), NEM, 15 equiv.) in NMP (50 µL) and vortexed for 12
hrs. The unreacted amine of the beads was capped with acetyl
group by treatment with acetic anhydride. After trityl
deprotection, the loading yield was calculated and found to be 96
%. Free amine of 7 was used for coupling with H-phosphonate T
3. For the coupling, 50 µL solution of 3 (20 equiv.), p-nitrophenol
(20 equiv.), 2,4,6-trichlorophenol (20 equiv.) and pivaloyl
chloride (20 equiv.) in 1:1 mixture of pyridine-acetonitrile was
added to trityl deprotected resin 7 and vortexed for 10 min. The
resin was then washed with acetonitrile (4 ml) to eliminate the
excess reagents and capped with capping reagent (1 ml). The
resin was again washed with DCM (2 ml) and deblocked using
deblocking reagent (1.5 ml, 2–3 min). The first coupling of T
(dimer) was obtained in 77 % yield. It was then washed with 30
% pyridine-acetonitrile (2 ml) and the cycle was repeated to get
initially the desired 5 mer (TTTTT) with phosphonamidate
backbone. This oligomer was oxidized to PMO using 0.1 M I2/ 2
M Me2NH in THF (5 min vortexed). After that the resin was
washed with THF (2 ml) and water (2 ml). Finally PMO was
cleaved from the solid support using 33 % aqueous NH3 (0.5 ml)
at 40 oC for 12 hrs.10 NH4OH solution (liquid phase) was
lyophilized to obtain the desired PMO and the pentamer
phosphorodiamidate oligomer was characterized by ESI mass.
Employing the same protocol, we synthesized the longer 15-mer
T-containing oligomer 10. The lyophilized crude product was
dissolved in water and six volume excess chilled acetone was
added, mixed well and a precipitate was formed which was
centrifuged to get a pellet.
Scheme 2. Synthesis of H-phosphonate T monomer.
In order to perform the solid supported synthesis, Novasyn TG-
amino resin (2 mg, 0.52 µmol, 0.26 mmol/g, pore size 130µm)
was suspended in N-methyl-2-pyrrolidone (NMP) and allowed to
swell for 12 hrs. It was then washed five times with NMP. Then a
solution of MMTr protected amino caproic acid 4 (5 equiv.),
HOBT (5 equiv.), HBTU (5 equiv.), N-ethylmorpholine (NEM,
15 equiv.) in NMP (50 µL) was added. The resin was suspended
in this solution and vortexed for 12 hrs. The resin was then
washed with NMP to remove the excess starting material/reagents
and capped with a freshly prepared solution of 10% Ac2O and
10% DIPEA in NMP for 2 min with vortexing. Resin beads were
then washed with NMP. To evaluate the coupling yield, resin was
washed with 1.5 ml of CYPTFA6 (3-cyano pyridine 300 mg, TFA
100 uL, CF3CH2OH 3.5 ml, DCM 15 ml) for 2–3 min. All the
flow through resin was collected, diluted 10-fold with
concentrated methanesulphonic acid and the yield was calculated
based on the measurement of the absorbance of MMTr cation by
UV-VIS spectrophotometer (λ= 472 nm, ε = 51,100 cm-1 M-1).
Scheme 3. Solid phase synthesis of PMO. Reagent and conditions: (i) 4, HOBT, HBTU, NEM, NMP; (ii) 3-Cyanopyridine,
TFA, TFE, DCM; (iii) 11, HOBT, HBTU, NEM, NMP; (iv) 3, para-nitrophenol, 2,4,6-trichlorophenol, pivaloyl chloride,
pyridine, acetonitrile; (v) 0.1 M I2 / 2 M Me2NH in THF; (vi) Aqueous NH3

Tetrahedron Letters
3
The resulting pellet containing product 10 was re-dissolved in
TTTTT, 12
TTTTT
PMO
water and transferred into a dialysis bag with a 600 MWCO
which was gently shaken in water for 12 hrs. The dialyzed
product 10 was lyophilized to get final 15mer PMO as a white
powder and was characterized by ESI mass. The yield was
measured by UV-VIS spectrophotometer at absorbance 263 nm
to obtain 0.27 µmol (from 0.52 µmol resin beads) (Scheme 2).
Except first coupling on solid support, the coupling yield from 3rd
to 15th mer was 98 to 100% using H-phosphonate method. To the
best of our knowledge, we are the first to report the synthesis of
PMO by H-phosphonate method. Gene Tools synthesis of PMO
using chlorophosphoramidate method is their patented
technology5 and PMO is only commercially available with them
for biological applications.
PMO
DMTr
O
T
H-Phosphonate
method
14
O
O
P
HO
O Et3NH
T
T
O
T
T
T
O
O
O
O
N
O
HO
N
P
N
P
O
O
ODMTr
O
N
N
HO
O
O
P
O
15-Mer PMO was characterized by ESI mass and analyzed by their
fragmentation pattern. In every peak of mass spectrum, there is a loss
of one N(Me)2 from the [M + 4Na]4+ (Table 1) which is supported by
the literature11 when compared with the analysis of ESI mass spectral
data of phosphoramidate.
O
P
n = 3
O
OH
Phosphoroamidate
Phosphodiester
Phosphorodiamidate
TTTTTtt-ODMTr-5', 13
Table 1. Positive-ion ESI-MS data of 15-mer PMO
Observed mass (%)a
1230.3219 (22)b
1186.2678 (22)
Mol. formula (M + Na)+
C178H281N59O74P14Na4
C176H275N58O74P14Na4
Entry
Scheme 4: Synthesis of PMO and PMO-DNA chimer using
H-phosphonate method
1
2
C174H269N57O74P14Na4
C172H263N56O74P14Na4
C170H257N55O74P14Na4
C168H251N54O74P14Na4
1142.1805 (32)
1098.1792 (40)
1054.0815 (53)
1010.0498 (55)
3
4
5
6
7
To check the purity of PMO, we used the C-18 column.
Interestingly, 5-mer PMO 12 was eluted in 5.75 min when 5 to
40% acetonitrile in 0.1 M NH4OAc, pH 7.2, 25 min was used as a
solvent gradient (Figure 1). To confirm the retention time with
the length of PMO, we then injected Gene Tools 25 mer PMO
(5’-GTGATTCGATTCAAGATGCCGTCTC-3’) as a control in
C-18 column under the same conditions where it was eluted in 30
min.
C166H245N53O74P14Na4
C164H239N52O74P14Na4
C162H233N51O74P14Na4
C160H227N50O74P14Na4
966.0043 (52)
921.9444 (68)
8
877.8767 (78)
833.8475 (95)
789.8019 (100)
745.7612 (98)
701.7156 (58)
9
10
11 C158H221N49O74P14Na4
12 C156H215N48O74P14Na4
C154H209N47O74P14Na4
C152H203N46O74P14Na4
C150H197N45O74P14Na4
13
14
15
657.6720 (57)
613.6161 (51)
am/z with relative abundance (%) in parentheses. Calculated mass for
C178H281N59O74P14Na4 1238.6489
According to Gene Tools, PMO is purified by precipitation
method which is good enough for biological applications.
However Tang et al12 purified the caged PMO by HPLC using
Dionex DNAPac column, though elution time is in below 4 min.
Again Sekine et al6 purified the dimer PMO using reversed-phase
HPLC though solvent gradient was not mentioned. In order to
know the purity of our PMO, accordingly, we have synthesized
both 5-mer PMO and a PMO-DNA chimer of a sequence,
TTTTT 12 and TTTTTtt-ODMTr 13, respectively. First 5-mer
PMO was synthesized using our method.10 After, trityl
deprotection, H-phosphonate of T-deoxyribonucleoside 14 was
coupled using Froehler’s method8 to get a 7-mer of PMO-DNA
chimer 13 in which last two linkages were phosphoramidate and
phosphodiester (Scheme 4). The compound was cleaved from the
resin by 33% ammonium hydroxide solution at 40oC for 5 hr.
The material was lyophilized and washed with acetonitrile to get
a white powder. The 7-mer chimer 13 synthesis indicated that in
PMO synthesis, the sequence can be modified as per need.
Figure 1: HPLC Chromatogram of PMO-5 mer 12. C-18
analytical column (4.6 mm × 250 mm, 5 µm), run by
acetonitrile 5 to 40% in 0.1 M NH4OAc, 25 min, 1 mL/min. λ
= 260 nm. Retention time = 5.75 min.
In case of PMO-DNA chimer 13, the best possible HPLC
chromatogram was obtained when solvent gradient 20 to 50%
acetonitrile in 0.1 M NH4OAc buffer, pH 7.2, 30 min was used
(Figure 2).

4
Tetrahedron
5) (a) Summerton, J.; Weller, D. U.S. Patent 5,185,444, 1993; (b)
Weller, D. D.; Hassinger J. N. U.S. Patent 2009/0088562A1; (c)
Reeves, M. D.; Weller, D. D. U.S. Patent 2009/0131624A1.
6) Harakawa, T.; Tsunoda, H.; Ohkubo, A.; Seio, K.; Sekine, M.
Bioorg. Med. Chem. Lett. 2012, 22, 1445.
7) (a) Pattanayak, S.; Paul, S.; Nandi, B.; Sinha, S. Nucleos. Nucleot.
Nucl. 2012, 31, 763; (b) Sinha, S.; Pattanayak, S.; Paul, S.; Nandi,
B. WO2011018798A2; Inter. Pat: Appl, 2011; (c) Paul, S.; Nandi,
B.; Pattanayak, S.; Sinha, S. Tetrahedron Lett. 2012, 53, 4179; (d)
Pattanayak, S.; Sinha, S. Tetrahedron Lett. 2012, 53, 6714; (e)
Nandi, B.; Pattanayak, S.; Paul, S. and Sinha, S. Euro. J. Org.
Chem. 2013, 1271; (f) Pattanayak, S.; Khatra, H.; Saha, S. and
Sinha, S. RSC Advances 2014, 4, 1951; (g) Paul, S.; Pattanayak,
S.; Sinha, S. Tetrahedron Lett. 2014, 55, 1072; (h) Ouyang, X.;
Shestopalov, I. A.; Sinha, S.; Zheng, G.; Pitt, C. L. W.; Li, W. H.;
Olson, A. J.; Chen, J. K. J. Am. Chem. Soc. 2009, 131, 13255; (i)
Shestopalov, I. A.; Sinha, S.; Chen, J. K. Nat. Chem. Biol. 2007, 3,
650.
Figure 2: HPLC Chromatogram of 13. Flow rate 1 mL/min. λ
= 260 nm. Retention time = 2.45 min.
8) (a) Froehler, B. C.; Ng, P. G. and Matteucci, M. D. Nucl. Acid.
Res. 1986, 14, 5399; (b) Froehler, B. C. Tetrahedron Lett. 1986,
27, 5575; (c) Froehler, B. C.; Matteucci, M. D. Nucleos. Nucleot.
1987, 6, 287.
The peak at 5.913 min was the truncated 5-mer PMO 12 and the
major fraction at 2.45 min was liophilized and ESI mass
indicated the desired product 13. ESI mass analysis has shown
the masses of 13 with DMTr: m/z = 1235.29 (15%), calcd for
C99H137N23O40P6 (M + 2H)2+: 1236.9; 1195.54 (20%), calcd for
C95H125N21O40P6 (M – 2NMe2 + 2H)2+: 1192.84; 656.88 (100%),
calcd for C69H94N19O38P6 (M – 4NMe2 + 3H)3+: 660.81 and
469.54 (98%), calcd for C91H116N19O40P6 (M – 4NMe2 + 5H)5+:
460.12, respectively.
9) Preparation and characterisation of H-Phosphonate T 3: To a
stirred solution of PCl3 (10.34 mmol) and N-ethyl morpholine
(103 mmol) in dry DCM was added 1, 2, 4-triazole (35.04 mmol)
and stirred at room temperature for 30 min. DCM solution of 2
(2.06 mmol, dried by co-evaporation from CH3CN) was then
added dropwise at 0o C. The reaction mixture was stirred at 0o C
for 10 min (until TLC showed complete consumption of starting
material). The reaction mixture was poured into 80 ml of 1M
TEAB buffer (pH 8.57) and stirred for 5 min and then separated in
a separating funnel. The aqueous layer was extracted with DCM
(2 × 100 ml). The combined organic part was dried over Na2SO4
and concentrated under reduced pressure. Crude product was
purified by column chromatography in 100–200 mesh silica gel
packing with 2% Et3N – DCM and eluting with 2% Et3N/ 8%
MeOH–DCM. Yield: 73.34% (980 mg), Rf = 0.45 (10% MeOH–
DCM). IR (neat/CHCl3): ν 3444, 2979, 2677, 2493, 1699, 1471,
1392, 1259, 1217, 1054 cm-1. 1H NMR (500 MHz, CDCl3): δ 1.23
(9H, t, J = 7Hz), 1.29–1.39 (2H, m), 1.70 (3H, s), 2.96 (6H, q, J =
7Hz), 3.06 (1H, d, J = 11.5Hz), 3.23 (1H, d, J = 11Hz), 3.44 (1H,
q, J = 11Hz ), 3.73–3.80 (2H, m), 4.24–4.26 (1H, m), 5.74 (1H, d,
J = 359.5Hz), 6.02 (1H, d, J = 8.5Hz), 6.96 (1H, s), 7.07–7.08
(3H, m), 7.17–7.20 (6H, m), 7.36 (6H, br s), 8.03 (1H, s), 9.45
(1H, bs), 11.82 (1H, br s). 13C NMR (125 MHz, CDCl3): δ 9.4,
12.2, 45.6, 49.2, 51.7, 52.6, 64, 76.6, 80.1, 110.2, 126.3, 127.6,
128.99, 135.3, 146.5, 150, 163.99. 31P NMR (200 MHz, CDCl3): δ
In summary, the synthesis of H-phosphonate of T morpholino
monomer and its use in phosphorodiamidate morpholino
oligomer (PMO) synthesis on solid support has been shown for
the first time. This method is unique in comparison to Gene
Tools chlorophosphoramidate method because it can be
transferred to the DNA synthesizer where H-phosphonate
chemistry is already used for DNA synthesis. H-phosphonate T
morpholino monomer 3 is stable in solution and unlike
ribonucleoside H-phosphonate, 3 is not hygroscopic in nature.
The coupling was done in acetonitrile-pyridine solvent which is
normally used during DNA synthesis. Except Gene Tools
chlorophosphoramidate chemistry, this is a second method for
PMO synthesis. We are now exploring this method to the
synthesis of PMO with mixed sequences of A, G, C, T and will
be published in due course.
+
4.56 (PPh3 –4.802). HRMS (ESI) (M + H) calculated for
C35H46N4O6PH+ = 649.32 found 649.3147
10) Solid phase synthesis cycle: Morpholino H- Phosphonate (3) was
dried by co evaporating with dry CH3CN and dissolved in
anhydrous Pyridine/CH3CN (1/1). Synthesis was performed in
plastic frit using the following protocol.
Acknowledgments
S.S. thanks to CSIR, India, for financial support by a grant. J. B.
thankful to IACS and J. K. and K. C. G. thankful to CSIR for
their fellowships.
Synthesis Cycle (Scheme 2) (ii, iv)
1) Washing – dry CH3CN (2 ml)
2) Capping – 10% Ac2O- CH3CN / 10% Pyridine CH3CN (1:1,
1 ml).
3) Deblocking – CYPTFA (1.5 to 2 ml)
Supplementary data
4) Coupling – 3 (20 equiv), para-nitrophenol (20 equiv), 2,4,6-
trichlorophenol (20 equiv), pivaloyl chloride (20 equiv) in
CH3CN- Pyridine (1/1) (10 min)
Supplementary data associated with this article can be found, in
the online version, at
5) Repeated above steps 1- 4 until the desired oligonucleotide
sequence was completed.
6) Oxidization – 0.1 M I2 / 2 M Me2NH in THF for 5 min.
7) Deprotection – Aqueous NH3, 40oC, 12 hrs.
References and notes
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