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Oligodeoxynucleotides containing 2'-amino-LNA nucleotides as constrained morpholino phosphoramidate and phosphorodiamidate monomers.

15 Jul 2017-Bioorganic & Medicinal Chemistry Letters (Pergamon Press)-Vol. 27, Iss: 14, pp 3173-3176

TL;DR: Thermal denaturation studies showed that the novel 2'-amino-LNA-based morpholino monomers exert a destabilizing effects on duplexes formed with complementary DNA and RNA.
Abstract: Incorporation in a 2'→5' direction of a phosphorodiamidite 2'-amino-LNA-T nucleotide as the morpholino phosphoramidate and N,N-dimethylamino phosphorodiamidate monomers into six oligonucleotides is reported Thermal denaturation studies showed that the novel 2'-amino-LNA-based morpholino monomers exert a destabilizing effects on duplexes formed with complementary DNA and RNA
Topics: Phosphoramidate (65%), Oligonucleotide (55%), Morpholino (54%), Oligonucleotide synthesis (54%), Nucleotide (51%)

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University of Southern Denmark
Oligodeoxynucleotides containing 2'-amino-LNA nucleotides as constrained morpholino
phosphoramidate and phosphorodiamidate monomers
Kristensen, Kim Vejlegaard; Paul, Sibasish; Kosbar, Tamer; Wengel, Jesper; Caruthers,
Marvin H
Published in:
Bioorganic & Medicinal Chemistry Letters
DOI:
10.1016/j.bmcl.2017.05.023
Publication date:
2017
Document version:
Final published version
Citation for pulished version (APA):
Kristensen, K. V., Paul, S., Kosbar, T., Wengel, J., & Caruthers, M. H. (2017). Oligodeoxynucleotides containing
2'-amino-LNA nucleotides as constrained morpholino phosphoramidate and phosphorodiamidate monomers.
Bioorganic & Medicinal Chemistry Letters
,
27
(14), 3173–3176. https://doi.org/10.1016/j.bmcl.2017.05.023
Go to publication entry in University of Southern Denmark's Research Portal
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Download date: 29. May. 2022

Oligodeoxynucleotides containing 2
0
-amino-LNA nucleotides as
constrained morpholino phosphoramidate and phosphorodiamidate
monomers
Kim Vejlegaard
a
, Sibasish Paul
b
, Tamer Kosbar
a,c
, Jesper Wengel
a,
, Marvin H. Caruthers
b
a
Biomolecular Nanoscale Engineering Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark
b
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80303, USA
c
Department of Chemistry, Faculty of Science, Damietta University, New Damietta 34517, Egypt
article info
Article history:
Received 23 March 2017
Revised 5 May 2017
Accepted 7 May 2017
Available online 17 May 2017
Keywords:
2
0
-Amino-LNA
Morpholino
Phosphordiamidite
Oligonucleotide synthesis
abstract
Incorporation in a 2
0
? 5
0
direction of a phosphorodiamidite 2
0
-amino-LNA-T nucleotide as the mor-
pholino phosphoramidate and N,N-dimethylamino phosphorodiamidate monomers into six oligonu-
cleotides is reported. Thermal denaturation studies showed that the novel 2
0
-amino-LNA-based
morpholino monomers exert a destabilizing effects on duplexes formed with complementary DNA and
RNA.
Ó 2017 Elsevier Ltd. All rights reserved.
Phosphorodiamidate morpholino oligonucleotides (PMOs) (1,
Fig. 1) are used as antisense tools in gene knockdown experi-
ments.
1
They act as steric blockers, do not degrade their target
RNA,
2
and are enzymatically stable.
3
Current investigations of
PMOs and conjugates thereof as therapeutics for amending splicing
defects ex vivo and in vivo are underway.
4–14
Recently the first
PMO-drug Eteplirsen/Exondys 51 was approved by the FDA for
treatment of Duchenne muscular dystrophy.
15
Methods to synthesize PMOs in a 5
0
? 3
0
direction (reverse-
direction compared to standard oligonucleotide synthesis) utilize
either an N-trityl-5
0
-chlorophosphorodimethylamidate
16,17
or an
N-trityl-5
0
-H-phosphonate
18
morpholino building block (2 and 3,
Fig. 1). Recently, we have developed a method for synthesizing
PMOs and PMO-DNA chimeras in 3
0
? 5
0
direction. This procedure
uses silyl-protected nucleobase morpholino building blocks and
oxidative substitution of a borane phosphonate oligonucleotide
intermediate.
19
Because of the current interest in PMOs, we decided to explore
other morpholino-based monomers including the 2
0
-amino-LNA
nucleotide (5, Fig. 1), a derivative of LNA (locked nucleic acid) (4,
Fig. 1) having a 2
0
N-4
0
C methylene bridge. LNA and 2
0
-amino-LNA
are conformationally locked nucleotides
20
of which the latter can
be considered a bicyclic morpholino-based ring system (5, Fig. 1).
When incorporated into oligonucleotides, the 2
0
-amino functional-
ity faces the minor groove of nucleic acid duplexes.
21
This site in
oligonucleotides therefore provides a convenient handle when N-
acylated and N-alkylated
21
for appending amino acids residues,
22
fluorescence probes,
23–26
nucleobases
27
and a piperizino group
28
while preserving the LNA-type high-affinity hybridization with
complementary DNA and RNA strands. A new synthon, 3
0
-O-ben-
zyl-2
0
-amino-LNA-T phosphorodiamidite, was used to prepare an
alternative morpholino analogue having a 2
0
-5
0
linkage (6 and 7,
Fig. 1) and the 3
0
-hydroxyl protected through a benzyl group.
Molecular modelling studies were performed in order to assess
the structure of 3
0
-O-benzyl-2
0
-amino-LNA-T phosphoramidate
monomer 6 in a DNA:DNA duplex. In order to complete this study,
a 9-mer duplex consisting of DNA:RNA [5
0
-d(CTGATATGC):5
0
-r
(GCAUAUCAG)] was downloaded from the protein data bank
(PDB entry pdb 1HG9),
29
the RNA strand was converted to DNA
and 3
0
-O-benzyl-2
0
-amino-LNA phosphoramidate monomer 6 was
inserted in d(CTGAXATGC) as monomer X. An AMBER
force field
in Macro Model 9.1 was used to generate representative low
energy structures.
This modelling study indicated that the 3
0
-O-benzyl-2
0
-amino-
LNA-T is locked into a 3
0
-endo (N-type) conformation (Fig. 2a) in
a manner similar to 2
0
-amino-LNA-T monomer 5 inserted into
the same duplex (Fig. 2b). Moreover both monomers 5 and 6
exhibited excellent stacking interactions with the neighboring
http://dx.doi.org/10.1016/j.bmcl.2017.05.023
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
Corresponding author.
E-mail address: jwe@sdu.dk (J. Wengel).
Bioorganic & Medicinal Chemistry Letters 27 (2017) 3173–3176
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl

nucleobases and nucleobase positioning suitable for engaging into
Watson–Crick base pairs (Fig. 2a and b). It is also evident from the
molecular modelling that the 3
0
-O-benzyl group is protruding from
the duplex into the minor groove and covers part of the phospho-
rous backbone. Based upon this modelling study, the 3
0
-O-benzyl
and the locked morpholino scaffold seemed not to impede Wat-
son-Crick base pairing thus giving us an incentive to begin an
experimental evaluation of novel constrained morpholino mono-
mers 6 and 7 in the context of DNA and RNA oligomers. In this
manuscript, we describe the first synthesis procedures for incorpo-
ration of these monomers into oligonucleotides and present an
evaluation of their duplex stability.
The phosphorodiamidite 9 (Scheme 1) was obtained in 87%
yield from the 3
0
-O-benzyl derivative 8 using 2-cyanoethyl-N,N,
N
0
,N
0
-tetraisopropylphosphorodiamidite as phosphitylating
reagent and 4,5-dicyanoimidazole (DCI) as activator.
As outlined in Fig. 3, two generalized pathways were used with
synthon 9 in order to prepare oligonucleotides containing the
monomers 6 or 7. For the preparation of 6, the first step was treat-
ment with acid to remove the dimethoxytrityl group. Synthon 9
was then activated and coupled with the growing oligonucleotide
in order to generate the phosphoramidite internucleotide linkage.
Following oxidation and capping, the cycle can be repeated using
synthon 9 or the standard 2
0
-deoxynucleoside 3
0
-phospho-
ramidites. The synthesis of 7 required modification of this cycle.
The detritylation step had to be carried out with trimethylphos-
phite borane (TMPB) in the presence of acid. TMPB reacts with
the generated dimethoxytrityl cation and therefore prevents for-
mation of an internucleotide tritylphosphonate linkage. Following
coupling with 9, boronation and capping completed the synthesis
steps for one cycle. In order to evaluate the overall yield and the
extent of degradation, various coupling conditions were explored
and the results are shown in Fig. S3 and Table S1 (ESIy). Based upon
these results, we conclude that the optimal conditions were 0.10 M
tetrazole for each of two coupling rounds of 900 s each giving 80%
total stepwise coupling yield.
For the synthesis of PMO-DNA chimeras containing monomer 7,
the borane phosphonate intermediate was converted, through
oxidative substitution,
20
to the N,N-dimethylamino phosphorodi-
amidate (see Fig. S2, ESIy). This post-synthetic transformation con-
sisted of 1) detritylation, 2) removal of the cyanoethyl substituent
with MeCN:NEt
3
(1:1; v/v) (thereby also oxidizing P(III) to P(V)), 3)
oxidative substitution by reaction with I
2
and dimethylamine, and
4) cleavage from the solid support using sat. aqNH
3
. The reaction
steps 1–2 were performed on the column whereas steps 3–4 were
completed on the polystyrene-resin in glass-vials.
Using synthon 9 and the standard 2
0
-deoxynucleoside 3
0
-phos-
phoramidites, oligonucleotides ON2-ON5 and ON7-ON8 were syn-
thesized, characterized by LC-MS, and used to evaluate the effect of
the novel constrained morpholino monomers on duplex stability.
ON1-ON10 were hybridized to complementary DNA or RNA and
thermal denaturation experiments were conducted in a medium
salt buffer (Table 1). When ON2-ON5 were hybridized to DNA,
one incorporation of monomer 6 induced a drastic drop in T
m
value
of 14.5 °C for ON2 while no detectable transition above 5 °C was
observed for ON3 with four incorporations of monomer 6. Replac-
ing the phosphoramidate monomer 6 with the N,N-dimethylamino
phosphorodiamidate monomer 7 induced a significant but less
pronounced decrease in stability as one incorporation reduced
the T
m
value by 10.5 °C for ON4. This difference is most likely
due to less electrostatic charge repulsion, since monomer 7 does
not have a negative charge. With two incorporations of monomer
7 into ON5 no transition was observed. With complementary
RNA, no transition above 5 °C was observed for ON2-ON5.
The mixed-base sequences ON7 and ON8 show a similar pattern
with a significant drop in T
m
of -10 °C towards complementary
DNA and of -7 °C towards complementary RNA for one incorpora-
tion of monomer 6, and with three incorporations the drop in T
m
value was even more pronounced. Additionally, T
m
-measurements
were performed on two reference oligonucleotides containing 2
0
-
amino-LNA-T nucleotides incorporated in the 3
0
? 5
0
(standard
manner) with a phosphodiester backbone as monomer 5 (Fig. 1)
(ON9 and ON10). These measurements showed the expected large
increases in thermal stability in sharp contrast to the effects of the
two morpholino-based incorporation motifs introduced herein.
The decrease in thermal stability might in part be due to the
2
0
? 5
0
incorporation pattern of monomer 6 and 7 as strong desta-
bilization has previously been reported upon incorporation of one
O
N
O
PO
Cl
N
Tr
O
N
O
PO
O
H
Tr
Et
3
NH
23
O
N
O
PO N
1
O
O
T
O
O
4
O
O
T
NH
T
O
N
H
O
O
O
O
T
N
BnO
P
O N
O
O
T
N
BnO
PO O
6
A)
B)
7
P OO
5
O
P
OO
O
P
OO
B
BB
Fig. 1. A) Chemical structures of phosphorodiamidate morpholino oligonucleotides
(PMO, 1) and the morpholino building blocks (2 and 3) used to synthesize PMO in a
6
0
? 3
0
direction. B) Chemical structures of phosphordiester monomers of LNA, 4
and 2
0
-amino LNA, 5. Structure 6 shows the 2
0
-amino-LNA-T phosphoramidate
monomer and structure 7 the 2
0
-amino-LNA-T dimethylamino phosphorodiamidate
monomer. B = nucleobases and T = thymin-1-yl.
Fig. 2. Snapshots from molecular dynamics simulations of a 9-mer duplex [(5
0
-d
(CTGAXATGC):5
0
-d(GCATATCAG)] modified at the central position (X) with a) 3
0
-O-
benzyl-2-amino-LNA phosphoramidate monomer 6, and b) 2
0
-amino-LNA-T mono-
mer 5. See ESIy Fig. S7 for a larger display including a snapshot of the dinucleotide
structure showing more clearly the sugar pucker of the modified monomers 5 and
6.
O
DMTrO
T
1.2 eq.
0.5 eq.
RT, 5 hours, anh. CH
2
Cl
2
, 87% yield
9
P
O
NC
N
N
NH
BnO
O
DMTrO
T
N
BnO
P
O
N
NC
8
N
N
H
NC
NC
Scheme 1. Synthesis of the phosphorodiamidite 9. T = Thymin-1-yl.
3174 K. Vejlegaard et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 3173–3176

or few 2
0
? 5
0
linked 3
0
-deoxy monomers into DNA duplexes.
30
Notably, a 3
0
-deoxy derivative of monomer 6 has been
incorporated into DNA and morpholino oligomers, but melting
temperatures were reported only for the latter which makes direct
comparison with our data (Table 1) impossible.
31
In conclusion the 3
0
-O-benzyl-2
0
-amino-LNA-T phosphorodi-
amide 9 was successfully synthesized and incorporated in a 2
0
?
5
0
direction into six oligonucleotides as either one or three phos-
phoramidate monomer(s) 6 (ON2, ON3, ON7, and ON8) or, through
a borane phosphonate intermediate, as one or two N,N-dimethy-
lamino phosphorodiamidate monomer(s) 7 (ON4 and ON5) giving
chimeric PMO-DNA oligomers. Thermal denaturation experiments
revealed a significant decreasing effect on duplex stability of these
monomers which may at least in part be explained by interference
of 3
0
-O-benzyl group on the hydration of the phosphorus backbone.
A further development could be to select a removable protecting
group for the 3
0
-hydroxy function or to use a 3
0
-O-methyl sub-
stituent as a sterically less demanding group.
Acknowledgements
The VILLUM FONDEN is thanked for funding The Biomolecular
Nanoscale Engineering Center (BioNEC), a VILLUM center of excel-
lence, grant number VKR022710. Joan Hansen and Tina Grubbe are
thanked for technical assistance.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.05.
023.
O
DMTrO
T
N
BnO
P
O
N
NC
1) Detritylation
A) Phosphoramidite backbone:
3% TCA/CH
2
Cl
2
or
B) Borane phosphonate intermediate
10:0.5:89.5 of TMPB:TFA:CHCl
3
v/v/v
O
DMTrO
B
O
O
HO
B
O
P
O
O
B
O
3) A) Oxidation
or
B) Boronation
4) Capping
2) Coupling
0.1 M Tetrazole 2
× 900 s
O
DMTrO
T
N
BnO
R: =O (A)
or BH
3
(
B
)
Followed by MeOH wash
9
Repeat
O
NC
P
O
O
B
O
O
DMTrO
T
N
BnO
O
NC
R
A
5A) Detritylation
6A) Cleavage from support
32% NH
4
OH
P
O
O
B
O
O
O
T
N
BnO
O O
5B) Detritylation
6B) Cyanoethyl removal
1:1 NEt
3
:MeCN v/v
7B) Oxidative substitution
Iodine + 0.1 M HNMe
2
8B) Cleavage from support
32% NH
4
OH
,
55 °C
TMPB =
P
OMe
OMe
MeO
BH
3
B
P
O
O
B
O
O
O
T
N
BnO
O N
Chimeric PMO-DNA with monomer
7
Oligomer with monomer 6
Fig. 3. Synthetic cycle for incorporation of the phosphorodiamidite 9 with the optimal coupling condition of 0.1 M tetrazole for two rounds of 900 s. Route A enables the
formation of phosphoramidate monomer 6 using standard detritylation, oxidation with aqueous iodine and cleavage from resin using sat. aqNH
3
. Route B enables the
formation of chimeric PMO-DNA oligomers containing N,N-dimethylamino phosphorodiamidate monomer 7 using the trityl-scavenger mixture TMPB:CF
3
COOH:CHCl
3
10:0.5:89.5 v/v/v, boronation using 0.05 M BH
3
in THF and the post-synthetic transformation followed by cleavage from resin using sat. aqNH
3
. B = Nucleobases and
T = Thymin-1-yl monomer.
Table 1
Thermal denaturation studies for synthesized oligonucleotides
a.
Sequence T
m
-values in medium salt buffer
Complementary DNA Complementary RNA
5
0
-TTT-TTT-TTT-T-3
0
ON1 19.5 °C 13.5 °C
5
0
-TTT-TT6-TTT-T-3
0
ON2 5.0 °C(14.5 °C) <5.0 °C
5
0
-TTT-666-6TT-T-3
0
ON3 <5.0 °C <5.0 °C
5
0
-TTT-TT7-TTT-T-3 ON4 9.0 °C(10.5 °C) <5.0 °C
5
0
-TTT-T77-TTT-T-3
0
ON5 <5.0 °C <5.0 °C
5
0
-GTG-ATA-TGC-3
0
ON6 31 °C 27.5 °C
5
0
-GTG-A6A-TGC-3
0
ON7 20.5 °C(10 °C) 20.5 °C(7 °C)
5
0
-G6G-A6A-6GC-3
0
ON8 5.0 °C(26 °C) 6.0 °C(21.5 °C)
5
0
-GTG-A5A-TGC-3
0
ON9 34.5 °C (+3.5 °C) 35.5 °C (+8 °C)
5
0
-G5G-A5A-5GC-3
0
ON10 40.0 °C (+9 °C) 48.5 °C (+21 °C)
a
Thermal denaturation temperatures of DNA-DNA and DNA-RNA duplexes
measured as the average of the maxima of the first derivatives of the melting curves
(A
260
versus temperature) from two independent melting temperature determi-
nation with a deviation <0.5 °C. Numbers in brackets are
D
T
m
values measured in
degrees Celsius as the difference in T
m
-values between modified and unmodified
duplexes. The experiments were recorded in medium salt buffer (6.1 mM sodium
phosphate, 100 mM sodium chloride, 0.1 mM EDTA, pH 7.0 using 1.5 M concen-
trations of the two complementary strands (assuming identical extinction coeffi-
cients for all modified and unmodified oligonucleotides); 5 =2
0
-amino-LNA-T
phosphodiester monomer, 6 =2
0
-amino-LNA-T phosphoramidate, 7=2
0
-amino-
LNA-T N,N-dimethylamino phosphorodiamidate, T = thymin-1-yl monomer, C =
cytosine-1-yl monomer, A = adenine-9-yl monomer and G = guanine-9-yl
monomer.
K. Vejlegaard et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 3173–3176
3175

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Journal ArticleDOI
TL;DR: In cell-free and cultured-cell systems where one wishes to block the translation of a messenger RNA coding for a normal protein, RNase H-independent morpholino antisense oligos provide complete resistance to nucleases, generally good targeting predictability, generally high in-cell efficacy, excellent sequence specificity, and very preliminary results suggest they may exhibit little non-antisense activity.
Abstract: RNase H-competent phosphorothioates (S-DNAs) have dominated the antisense field in large part because they offer reasonable resistance to nucleases, they afford good efficacy in cell-free test systems, they can be targeted against sites throughout the RNA transcript of a gene, and they are widely available from commercial sources at modest prices. However, these merits are counterbalanced by significant limitations, including: degradation by nucleases, poor in-cell targeting predictability, low sequence specificity, and a variety of non-antisense activities. In cell-free and cultured-cell systems where one wishes to block the translation of a messenger RNA coding for a normal protein, RNase H-independent morpholino antisense oligos provide complete resistance to nucleases, generally good targeting predictability, generally high in-cell efficacy, excellent sequence specificity, and very preliminary results suggest they may exhibit little non-antisense activity.

660 citations


Journal ArticleDOI
TL;DR: The excellent resistance of Morpholino phosphorodiamidates to enzymatic attack indicates their suitability for in vivo use.
Abstract: Oligomers possessing the Morpholino phosphorodiamidate backbone were evaluated for resistance to a variety of enzymes and biologic fluids. A 25-mer was incubated with nucleases, proteases, esterase...

263 citations



Journal ArticleDOI
TL;DR: It is suggested that the change in electronic density at the brim of the minor groove, introduced by the LNA modification, is causing an alteration of the pseudorotational profile of the 3'-flanking nucleotide, thus shifting this sugar equilibrium toward N-type conformation.
Abstract: Locked nucleic acids (LNAs) containing one or more 2‘-O,4‘-C-methylene-linked bicyclic ribonucleoside monomers possess a number of the prerequisites of an effective antisense oligonucleotide, e.g. ...

244 citations


Journal ArticleDOI
TL;DR: Different approaches to describe how the first description of a loss-of-function phenotype in zebrafish should be accomplished are discussed, with a specific focus on how to describe the effects of morpholino side effects.
Abstract: Morpholino oligomers have been used widely and for many years in the zebrafish community to transiently knock down the function of target genes. It has often been difficult, however, to reliably discriminate between specific and non-specific effects, and thus generally accepted guidelines to control for morpholino side effects do not exist. In light of recent methodologies to generate mutant lines in virtually any zebrafish gene, we discuss these different approaches with a specific focus on how the first description of a loss-of-function phenotype in zebrafish should be accomplished.

155 citations


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20211
20191