RETRACTED ARTICLE: Synthesis of Phosphorodiamidate Morpholino Oligonucleotides and Their Chimeras Using Phosphoramidite Chemistry

Article abstract of DOI:10.1021/jacs.6b08854

Phosphorodiamidate morpholinos (PMOs) and PMO-DNA chimeras have been prepared on DNA synthesizers using phosphoramidite chemistry. This was possible by first generating boranephosphoroamidate morpholino internucleotide linkages followed by oxidative subst

Full text of DOI:10.1021/jacs.6b08854

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Article
Synthesis of Phosphorodiamidate Morpholino Oligonucleotides
and Their Chimeras Using Phosphoramidite Chemistry
Sibasish Paul, and Marvin H. Caruthers
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b08854 • Publication Date (Web): 14 Nov 2016
Downloaded from http://pubs.acs.org on November 14, 2016
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Synthesis of Phosphorodiamidate Morpholino
Oligonucleotides and Their Chimeras Using
Phosphoramidite Chemistry
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Sibasish Paul and Marvin H. Caruthers*
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309 USA
ABSTRACT
Phosphorodiamidate morpholinos (PMOs) and PMO-DNA chimeras have been prepared on
DNA synthesizers using phosphoramidite chemistry. This was possible by first generating
boranephosphoroamidate morpholino internucleotide linkages followed by oxidative substitution
with four different amines: N,N-dimethylamine, N-methylamine, ammonia, and morpholine.
When compared to a natural DNA duplex, the amino modified PMO was found to have a higher
melting temperature with either complementary DNA or RNA whereas the remaining PMO
analogues having morpholino, dimethylamino, or N-methylamino phosphorodiamidate linkages
had melting temperatures that were either comparable or reduced. Additionally the N,N-
dimethylamino PMO-DNA chimeras were found to stimulate RNaseH1 activity. Treatment of
HeLa cells with fluorescently labeled PMO chimeras demonstrated that these analogues were
efficiently taken up by cells in the presence of a lipid transfection reagent. Because of the
simplistic synthesis procedures, various PMO analogues are now readily available and should
therefore open new pathways for research into the antisense, diagnostic, and nanotechnology
oligonucleotide fields.
INTRODUCTION
The ability to target genes with antisense oligonucleotides has been shown to be a useful
approach for studying biological pathways and for investigating possible treatments of various
diseases.1,2 A very promising antisense oligonucleotide (PMO with N,N-dimethylamino, 3,
Figure 1)3 having nonionic internucleotide linkages and morpholino replacing the deoxyribose of
DNA has been investigated for almost last three decades. PMOs are known to inhibit gene
expression,4 are resistant to cellular nucleases,5,6 and form duplexes with complementary RNA
that have binding affinities comparable to natural DNA/RNA duplexes.7 In biological studies,
gene expression in embryos of Zebrafish and Xenopus Laevis has been manipulated using
PMOs.8 The efficacy, sequence specificity, stability and low toxicity of PMOs has been verified
in several publications, mostly describing work in embryos.9 Therapeutic development of PMOs
is currently underway in on-going clinical trials for treatment of Duchenne muscular dystrophy
(DMD) and against the infection and spread of the hemorrhagic filovirus Marburg.10 Recent
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research has also demonstrated that PMOs may prove useful for studies in the nanotechnology11
and surface hybridization12 fields.
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9
Although there are many potential applications for these promising oligonucleotide
analogues, their use remains severely limited.13For example PMOs are uncharged and do not
form complexes with cell penetrating peptides (CPP) or lipids. Therefore simply mixing cells
and a PMO with a CPP or lipid fails to produce antisense effects.14 Consequently covalent
conjugation of a CPP to a PMO is required for transfection.12 Moreover morpholino nucleoside
monomers, as prepared using previous chemistries, cannot be incorporated into chimeric
oligonucleotides. This is because PMOs must be prepared in a 5’ to 3’direction on a polystyrene
resin16 (Scheme 1). This requirement is not compatible with standard methods for the chemical
synthesis of natural DNA/RNA or most analogues as they are prepared in a stepwise 3’ to 5’
direction.
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Figure 1. Chemical structures of DNA, RNA, N,N-dimethylamino PMOs and Boranephosphoroamidate morpholino
oligonucleotides.
The most commonly used approach for preparing PMOs involves, as a first step,
condensing
a
5’-hydroxyl-N-trityl-morpholino nucleoside with N,N-dimethylamino-
dichlorophosphoramidate in order to generate a N,N-dimethylamino-chlorophosphoramidate
synthon (5). Coupling with base yields the dimer attached to the resin (Scheme 1). Further
detritylation with an acid salt generates a product that can be elongated by repeating the
cycle.17,18,19 There are several recognized problems with this approach. For example
condensation yields are low (the recovered yield for a dithymidine morpholino was 45%) and
require long reaction times.18 Moreover, Sinha et. al. has claimed that the 5’-
chlorophosphoroamidate monomers are unstable.20 Additionally this approach requires special
procedures, techniques, and materials (e.g a swellable polystyrene resin, unique reaction vessels,
and the pyridinium salt of trifluoroacetic acid as the detritylating reagent) that are not readily
available to most laboratories. Recently a new method has been developed (Scheme 1) using H-
phosphonate chemistry where (6) was used for the synthesis of polythymidine PMOs and a
PMO-DNA chimera (conjugated in 3’-3’ fashion).20 There are also problems with this approach.
For example, because the phosphorus atom in H-phosphonates is electrophilic and lacks a lone
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pair of electrons, it is much more resistant towards oxidation under ambient conditions than most
P (III) compounds. Moreover coupling yields are low (77%) towards formation of the H-
phosphonatemorpholino dimer.20These limitations led us to explore using the phosphoramidite
methodology21 for the synthesis of various PMO derivatives.
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Scheme 1. Current approaches for the synthesis of N,N-dimethylamino PMOs.
In a recent publication, the stereoselective conversion of boranephosphonate DNA, in the
presence of iodine and amines, to phosphoramidate DNA was reported.22 Based upon these
results, we outline in this manuscript a general method for synthesizing various PMO analogues.
This new approach has several significant advantages. 1.) The method is orthogonal and
compatible with standard DNA/RNA synthesis procedures. 2.) For the first time, PMOs having
internucleotide linkages other than N,N-dimethylamino functionality can be synthesized. 3.)
Additionally and also for the first time, PMO-DNA chimeras can be synthesized where the
internucleotide linkages are 3’ to 5’. Because these chimeras are anionic, they were found to be
active with RNAse H1 and were taken up by cells using a standard lipid transfecting reagent.
RESULTS
Synthon Preparation
Our initial objective was to prepare phosphordiamidite synthons (Scheme 2, compounds
9, 14, 19, 24) that could be used to generate PMO analogues (Scheme 3). Synthesis of the
thymine morpholino monomer (8) followed literature protocols. 5’-O-Dimethoxytrityl
ribothymidine (7) was treated with sodium periodate and then ammonium biborate to afford the
dihydroxythymine morpholino monomer. Reduction of the hydroxyl groups was carried out
(without further purification) using sodiumcyanoborohydride under mild acidic condition to
yield the thymine morpholino monomer (8).23,24 The thymine synthon (9) was then prepared in
86%
yield
by
phosphitylation
of
(8)
with
2-cyanoethyl-N,N,N’,N’-
tetraisopropylphosphordiamidite and 4,5-dicyanoimidazole (DCI) in dichloromethane under an
argon atmosphere.23 Using a similar protocol, a thymine synthon having the O-methyl-N,N-
diisopropylphosphordiamidite was prepared; however, the solid phase coupling with this synthon
was found to generate a lower yield when compared to 9 ( 96% vs. 89 % respectively after the
first coupling step). Synthesis of the corresponding monomers of cytosine, adenine, and guanine
was initiated by first protecting the nucleoside amino groups with bis(tert-butyl)isobutylsilyl
(BIBS).25The standard amide protecting groups cannot be used with cytosine, adenine, and
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guanine because boronation, an essential part of the synthesis cycle, would reduce these amides
to the corresponding alkyl groups which are stable and cannot be removed following synthesis.
In contrast, the BIBS group can readily be removed using fluoride. The reaction of 5′,3′,2′-tri-O-
acetylcytidine (10) with BIBS-OTf (Tf = triflate) in the presence of 2,6-lutidine gave 76% yield
of 11 after 2 h stirring at 60° C under an argon atmosphere. However the synthesis of 16 and 21
from 15 and 20 required much longer reaction times (3 days) with yields of 25-31% (16) and 74
% (21). As outlined in Scheme 2, these silylated ribonucleosides were then treated with
ammonium hydroxide to remove the acetyl protecting groups, converted to the 5’-
dimethoxytrityl compounds, and then to the morpholino derivatives (13, 18, and 23). These
compounds were used to generate the phosphordiamidite synthons 14, 19 and 24. (See the
supporting information and experimental Sections for details). For the synthesis of PMO-DNA
chimeras, 5’-O-dimethoxytrityl-2’-deoxyribonucleoside-3’-phosphoramidite synthons 26, 27 and
28 were prepared following a literature protocol25 whereas compound 25 was obtained from
commercially available sources.
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Scheme 2. Synthesis of BIBS protected morpholino phosphordimidite synthons. (i) Lutidine (4.0 eq.)/1,4-Dioxane;
(ii) 1.0 M NH3/MeOH; (iii) DMT-Cl (1.2 eq.)/Pyridine; (iv) NaIO4 (1.1 eq.)/(NH4)2B4O7 (1.1 eq.)/CH3OH; (v)
NaCNBH3 (2.0 eq.)/AcOH(2.0 eq.)/CH3OH; (vi) P(OCH2CH2CN)(NiPr2)2 (1.2 eq.)/4,5-Dicyanoimidazole (0.5
eq.)/CH2Cl2. DMTr: Dimethoxytrityl
Synthesis of N,N-Dimethylamino PMO Oligonucleotides.
Once these synthons were available, our next goal was to optimize the solid-phase
synthesis cycle for preparing boranephosphoroamidate morpholino derivatives and for
converting these compounds to the corresponding PMOs. The synthesis cycle is outlined in
Scheme 3 and Table S1 (Supporting Information). Prior to synthesis, the 5’-DMT group on the
2’-deoxyribothymidine linked to a polystyrene support was removed with 0.5% trifluoroacetic
acid in chloroform containing 10% trimethylphosphiteborane (TMPB).25 The 5’-unprotected-2’-
deoxyribonucleoside (A)26 was then allowed to react with 9, 14, 19 or 24 in anhydrous
acetonitrile containing 4,5-dicyanoimidazole (DCI) in order to generate a dimer having a
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phosphoramiditediester internucleotide linkage (B). The coupling wait time was 300s. Following
boronation, capping, and detritylation, repetition of this cycle generates the product ready for
further conversion to the PMOs.
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B*
5. Removal of cyanoethyl
1:1 Et3N:CH3CN
B
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DMTrO
O
O
O
N
P
6. Iodine/dimethylamine
7. Silyl deprotection
Tetrabutyl ammonium
fluoride
8. Clevage from support
Ammonia
N
N
P
HO
O
NMe2
O
N
T
x
O
O
T
9, 14, 19, 24
O
O
O
A
1. Condensation
(4,5-dicyanoimidazole)
F
Repeat
B*
B*
DMTrO
O
HO
O
N
N
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P
BH3
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P
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TMPB
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P
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N
N
O
BH3
O
E
O
B
2. Boronation
(BH3)
3. Capping
(Ac2O)
4. Deprotection
0.5% TFA/10% TMPB/CHCl3
B*
DMTrO
N
O
O
P
NMe2
O
N
P
O
T
BH3
O
T= Thymine
B= Thymine, cytosine, adenine, guanine
O
C
T
OH
O
N
O
D
Scheme 3. Synthesis cycle for the preparation of PMOs. F: A PMO having only N,N-dimethylamino-
phosphorodiamidate internucleotide linkages; “X” is a selected number of N,N-dimethylamino-PMO nucleotide
units; Compound A can be replaced by any of the four 2’-deoxyribonucleosides or morpholino nucleosides but the
cytosine, adenine, and guanine bases must be protected with the bis(tert-butyl)isobutylsilyl group.
Post synthesis, supports were washed with acetonitrile, treated with a 1:1 mixture of
triethylamine:acetonitrile for 600 s in order to remove the cyanoethyl groups from
internucleotide linkages, washed with acetonitrile and dichloromethane several times to remove
residual triethylamine, and dried. These polystyrene supports were next removed from columns
and placed in 1.5 mL screw-cap glass reaction vials. For conversion to the N,N-dimethylamino
PMOs, the morpholino boranephosphoroamidates were treated two hours with a solution of 0.05
M iodine and 2.0 M dimethylamine in tetrahydrofuran. The resins were repeatedly washed with
acetonitrile and oligonucleotides were next treated with a 1.0 M solution of tetrabutyl
ammonium fluoride (TBAF) in THF to remove the BIBS protecting groups. (Et3N.HF could not
be used for removing these silyl groups as this reagent hydrolyzed phophorodiamidate linkages).
When oxidative amination reactions were carried out after removal of silyl protecting groups,
amine substitution at the N-4 of cytosine was observed (Supporting information, Figure S1). This
problem was overcome by performing the oxidative amination reaction prior to desilylation.
Once the silyl groups were removed, oligonucleotides were cleaved from supports using 30%
ammonium hydroxide, polystyrene resins were removed using 0.2 ꢀm centrifugal filters, and
solutions were passed through Illustra NAP-25 columns to remove fluoride salts. An additional
purification step was performed with each PMO using an Amicon® Ultra-15 3K centrifugal filter
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device (to remove shorter failure sequences). The oligonucleotides were then used for further
characterization and other studies.
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Zhang et al. reported incorporation of uridine morpholino phosphoramidite (similar to 9
except B* = uracil) into siRNA sequences by means of 5-(ethylthio)-1H-tetrazole (ETT) and was
able to convert the resulting morpholino phosphoramidite internucleotide linkage to the
corresponding phosphoramidate morpholino linkage with aqueous iodine.23,27 However we found
that ETT (pKa = 4.3) activated both morpholine and N,N-diisopropylamine in compounds 9, 14,
19 and 24 (Scheme 2). As a consequence we observed very low yields of the final PMO products
following their protocol. This was because activation of morpholine followed by boronation and
iodine/dimethylamine oxidation leads to a capped PMO oligonucleotide having a 5’-N,N-
diisopropylamino-phosphorodiamidate (C) that cannot be further elongated (Scheme 3). In order
to further investigate this problem, several less acidic activators (tetrazole, saccharin-1-
methylimidazole, 4,5-dicyanoimidazole) were tested in attempts to identify one that would react
with N,N-diisopropylamine (pKa = 11.1) but minimally with morpholine (pKa = 8.3).28 Of the
activators we screened by synthesizing PMOs; having a sequence corresponding to ODN 1
((Table 1), 4,5-dicyanoimidazole (pKa =5.2, 0.12 M, with 300 s coupling time) proved to
generate the highest yield (67%) of the desired PMO with low levels of side-products
(Supporting Information, Figure S2). Since the morpholino phosphoramidite diester (B), as
synthesized following the coupling step, was somewhat unstable towards the capping solution
we carried out boronation in order to generate a P (IV) morpholino compound (D) prior to the
capping step. After boronation, the support was washed with acetonitrile, failure sequences
capped using acetic anhydride, and detritylation carried out using a solution of 10% TMBP and
0.5% TFA in chloroform (D was stable to a acidic detritylation conditions).
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Figure 2. Synthesis of a N,N-Dimethylamino PMO trimer (29). The LC profile from the LCMS analysis of the total
reaction mixture is shown.
Using the procedure outlined in Scheme 3, initially a trimer having only thymine
nucleoside bases was synthesized, converted to the N,N-dimethylamino PMO derivative with
iodine/N,N-dimethylamine, removed from the support with ammonia, and analyzed by LCMS.
The four major peaks as shown in the LC profile (Figure 2) have the mass spectra expected for
the four diastereomers of the product (calculated mass, 902.3, found 901.3). A 94% yield of
compound 29 was calculated when the combined areas of all peaks from the crude reaction
mixture were compared to the peak area for these diastereomers. In a similar experiment where a
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dimer was prepared, the first coupling yield was found to be 96% which is far better than other
reported procedures (Supporting Information, Figure S3).18,20
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N,N-Dimethylamino PMOs having oligothymidine (Table 1, ODNs 1 and 2) and all four
bases (Table 1, ODNs 3-8), have been synthesized using this approach and characterized by
LCMS. Because it has been established that PMOs 12–16 in length are effective inhibitors of
RNA expression, either via blockage of splicing or mRNA translation,29 we carried out the
synthesis of a 16-mer having all four bases (Table 1, ODN 8). As can be seen from the LCMS
chromatogram of the unpurified reaction mixture (Figure 3A), both the yield (10 A260 units
isolated) and mass analysis were satisfactory.
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Synthesis of PMO-DNA chimeras
For the synthesis of PMO-DNA chimeras, 4,5-dicyanoimidazole (0.12 M and 300 s
coupling time) and ETT (0.25 M and 180 s coupling time) were used respectively for the
morpholino phosphordiamidite synthons (9, 14, 19 or 24) and the 5’-dimethoxytrityl-2’-
deoxyribonucleoside-3’-phosphoramidites (25, 26, 27 or 28).29 Following condensation, a
morpholino phosphoramidite diester was converted to the P(IV) borane linkage and standard
oxidation with 0.02 M iodine in THF/water/pyridine was used to convert a phosphite triester to
the phosphate triester. These synthesis steps were repeated until the PMO-DNA chimeras of the
desired sequence/length were prepared. Table S1 (Supporting information) summarizes these
synthesis steps.
Because PMO-DNA chimeras are new to the scientific community and could prove to be
useful for various research projects, several were synthesized (Table 1, ODNs 9-16). Initially
these chimeras, as a series of 21mer oligothymidines and containing four N,N-dimethylamino
PMO linkages, were prepared. In these 21mers, the morpholino diamidate linkages were placed
adjacent to either the 5′ or 3′ ends, near the middle of the 21 mer, and at every third position
throughout the oligomer (Table 1; ODNs, 9, 10, 11, 12 and 13). For these chimeras, substitution
of borane with dimethylamine upon activation by iodine proceeded efficiently and analysis of the
crude reaction mixtures by LCMS (Supporting Information) revealed that the expected
phosphorodiamidate linkages were formed in near quantitative yields. These encouraging results
were followed by the synthesis of PMO-DNA chimeras containing all four nucleobases with
variable locations and number of PMO linkages (Table 1: ODNs, 14, 15 and 16). LCMS analysis
and 31P NMR of the reaction mixtures demonstrated that the expected PMO-DNA chimeras
were obtained in high yields (Figure 3B for ODN 16; Supporting Information for ODNs 14 and
15) with an average yield being 10-20 A260 units (from a 0.2 µM synthesis cycle). These
experiments also demonstrated that treatment with dimethylamine does not lead to measurable
cleavage of the succinate linkage and loss of product during synthesis.
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7
Mol wt
ODNs
NO
Cal
Obs
8
9
ODN 1
ODN 2
ODN 3
3214.00
6846.17
3829.36
3214.01
6848.14
3828.16
t
T*T*T*T*T*T *T*T*T*
T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T *T*T*T*
t
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
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60
C*C*C*T*T*T*T*T *T*T*T*
t
ODN 4
ODN 5
3901.44
3924.42
3893.41
3902.30
3925.26
3894.25
3938.28
5264.75
6430.15
A*A*A*T*T*T*T*T *T*T*T*
t
G*G*T*T*T*T*T*T *T*T*T*
t
ODN 6
ODN 7
ODN 8
ODN 9
G*A*C*T*T*T*T*T*T T T
t
t
* *
*
3937.44
5264.26
6430.17
A*T*G*T*G*C*T*G*C*T*A*
T*A*A*C*A*C*G*A*T*A*C*G*C*G*A*
t
T*T*T*T*tP tP tP tP tP tP tP tP tP tP tP tP tP tP tP tP t
ODN 10
ODN 11
ODN 12
6430.17
6430.15
tP tP tP tP tP tP tP tP tP tP tP tP tP tP tP tP
t
T*T*T*T*
6430.17
6534.17
6482.26
tP tP tP tP tP tP tP tP T*T*T*T*tp tP tP tP tP tP tP tP t
6430.14
6534.38
T*T*T*T*tp tP tP tP tP tP tP tP tP tP tp T T T*T*
t
* *
T
tP tp T tP tptpT tP tptpT tP tptpT tP tptpT
t
ODN 13
6482.17
*
*
*
*
*
*
tPaPgPtPtPgPaPgPtPgPaPtP
t
A*T*C*A*
ODN 14
ODN 15
ODN 16
5343.45
5343.07
4999.99
5052.16
5000.26
5052.26
G*T*A*G*tPcPcPtPgPcPaPaPtPgPaPt
T*G*A*tPcPgPcPtPgPcPaPaP
T*G*A*
t
Table 1. Mass analysis (LCMS) of PMO and PMO-DNA chimeras (ODNs). Oligonucleotides containing N,N-
dimethylamino-phosphorodiamidate (*) and phosphate (p) internucleotide linkages are shown. Morpholino and 2’-
deoxyribonucleosides are shown as upper and lower case letters respectively.
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Figure 3. (A) LCMS analysis of the total reaction mixture from the synthesis of ODN 8. The data shows the LC
trace and a zoomed in view of the base peak in the mass spectra. The base peak in each case was a -3 charged
species. Full mass spectra are provided in the supplementary section. (B) 31P NMR of the total reaction mixture
obtained from the synthesis of ODN 16 (400 MHz, 15000 scans). The 31P NMR peaks at 18.63 ppm and -1.2 ppm
correspond to the N,N-dimethylamino-phosphorodiamidate and phosphodiester linkages respectfully.
Solid Phase Synthesis of PMOs with Amino, N-Methylamino and Morpholino Linkages
From previous research,25 it is known that the boranephosphonate linkage can be
activated with iodine for displacement by a large number of nucleophiles. Therefore in addition
to testing this new synthetic route by synthesizing PMO analogues having the N,N-
dimethylamino-phosphorodiamidate linkage, we decided to investigate other amines, which
could be used in order to generate several new PMO-DNA derivatives. Initially an
oligothymidine 21 mer having four boranephosphoroamidate morpholino linkages near the
center of this oligomer was synthesized. The support containing this oligonucleotide was divided
into three samples that were treated with N-methylamine, ammonia, and morpholine under
iodine oxidation conditions and then purified using reverse phase column chromatography.
Additionally mixed sequence PMO-DNA chimers having all four bases and amino-
phosphorodiamidate internucleotide linkages were synthesized where the positions for the
diamidate linkages were located at the 5’, 3’, and 5’/3’ termini of these chimeras. The mass
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analysis from LCMS and the sequences of PMOs are listed in Table 2. Yields were comparable
to those obtained for the N,N-dimethylamino PMO chimeras.
7
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9
Mol wt
ODNs
NO
10
11
12
13
14
15
16
17
18
19
20
21
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23
24
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26
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Cal
Obs
6318.02
6318.21
ODN 17
ODN 18
ODN 19
tPtPtPtPtPtPtPtPT*T*T*T*tPtPtPtPtPtPtPtPt
tPtPtPtPtPtPtPtPT⧫T⧫T⧫T⧫tPtPtPtPtPtPtPtPt
6374.32
6374.08
6598.20
5230.97
4887.89
4883.96
tPtPtPtPtPtPtPtPT•T•T•T•tPtPtPtPtPtPtPtPt
6598.57
5231.48
ODN 20
ODN 21
ODN 22
tPaPgPtPtPgPaPgPtPgPaPtP
t
A*T*C*A*
G*T*A*G*tPcPcPtPgPcPaPaPtPgPaPt
T*G*A*tPcPgPcPtPgPcPaPaP
4888.26
4884.26
t
T*G*A*
Table 2. LCMS Analysis of PMO-DNA Chimeras. ODN 17, 20, 21and 22:
*= NH2; ODN 18:⧫= NHMe; ODN 19:
•
= morpholine. Phosphate linkages are denoted by “p”. Morpholino and 2’-deoxyribonucleosides are noted as upper
and lower case letters respectively.
Melting Temperatures of PMO-DNA Chimeras
In order to assess the target binding ability of the modified PMO-DNA chimeras, duplex
hybridization studies were performed with 2’-deoxyoligothymidines having amino,
methylamino, morpholino, and dimethylamino-morpholino internucleotide linkages. PMOs 11,
17, 18 and 19 were mixed with 2’-deoxyribo or riboadenosineoligonucleotides 21 nucleotides in
length (1:1 ratio) in a buffer (1.0 M NaCl, 10 mM sodium phosphate, pH 7.1) at an overall
o
concentration of 1.0µM of duplex. The samples were denatured at 90 C and cooled to 15 C.
o
o
They were then heated at a rate of 1 C/min, and A260 versus time was recorded. Melting
temperatures were taken as the temperature of half dissociation and were obtained from the first
derivative plots (Table 3).
Melting temperature studies of RNA heteroduplexes with PMOs 11, 17, 18, and 19
showed that introduction of phosphorodiamidate internucleotide linkages stabilized the
PMO:RNA duplex relative to the unmodified duplex. The trend in increased stabilization was
amino>N-methylamino~N,N-dimethylamino>morpholino. When the PMO-DNA chimeras were
allowed to form duplexes with a 2’-deoxyriboadenosine 21mer, the order of stabilization (when
compared to the Tm of the unmodified DNA.DNA duplex) was amino>N-methylamino>N,N-
dimethylamino>morpholino; where the morpholino analogue was destabilizing.
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a o
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Tm ( C)
∆Tm ( C)
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DNA RNA
DNA RNA
Number
Modification
NMe2
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ODN 23
ODN 11
ODN 17
ODN 18
58.5
58.4
61.4
59.0
51.1
52.4
53.4
52.2
0.03
0.3
0.6
0.3
NH2
0.8
0.1
NHMe
0.1
57.4
51.4
-0.3
ODN 19
Table 3. Tm Results with PMO-DNA Chimeras. PMO-DNA chimeras (ODNs 11, 17, 18, and 19) and also
unmodified ODN 23, d(T)21, were mixed with complementary d(A)21 or r(A)21 and the melting temperatures
a
b
measured (Supplementary Section). Tm measurements represent an average of at least three experiments. ꢁTm
represents (Tm ODN-Tm unmodified control)/number of modifications).
RNase H1 Activity of Chimeric PMO-RNA Heteroduplexes
N,N-Dimethylamino PMO-DNA chimeras were tested for their ability to stimulate
RNase H1 activity. The test systems were composed of a 5′-O-fluorescein labeled RNA and
complementary N,N-dimethylamino PMO-chimeras. The analyzed PMOs were oligothymidine
14 mers with cap/gap sequences (ODNs 24-26, Table S2, Supporting Information) having N,N-
dimethylamino PMO linkages on either end and 3-7 phosphodiester linkages in the center of the
analogues. Controls were complementary DNA and 2′-O-methyl RNA (which activate and do
not stimulate RNase H1 activity respectively). All these cap/gap oligonucleotide analogues were
found to activate RNA hydrolysis (Figure 4).
Figure 4. E. coli RNase H1 degradation of RNA when Catalyzed by PMO-DNA Chimeras. 5′-O-Fluorescein
labeled r(A)14 was allowed to form duplexes with complementary ODNs 24-26. These duplexes were then treated
with E. coli RNase H1 for 12 h and analyzed by PAGE. Lane 1: 2’-O-methyl RNA/RNA; Lane 2: 2’-O-methyl
RNA/RNA/RNase H1; Lane 3: DNA/RNA; Lane 4: DNA/RNA/RNase H1; Lane 5: ODN 24/RNA; Lane 6: ODN
24/RNA/RNase HI; Lane 7: ODN 25/RNA; Lane 8: ODN 25/RNA/RNase H1; Lane 9: ODN 26/RNA; Lane 10:
ODN26/RNA/RNaseH1.ODN24:T*T*T*T*T*TPTPTPT*T*T*T*T*T;ODN25:T*T*T*T*TPTPTPTPTPT*T*T*T*T;
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ODN 26: T*T*T*TPTPTPTPTPTPTPT*T*T*T. Oligonucleotides containing N,N-dimethylaminophosphorodiamidate
(*)and phosphate (p) linkages are shown. Morpholino and 2’-deoxynucleosidesare noted by upper and lower case
letters respectively.
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Fluorescence Microscopy
Since uncharged PMOs cannot be delivered to cells using lipid based transfection
reagents, we chose to investigate whether these PMO-DNA chimeras could be taken up by cells
in the presence of Dharmafect 1, a commonly used siRNA transfection reagent. In order to test
transfection of these chimeras, a PMO-DNA chimera was synthesize (ODN 27, 5’-FL-
T*G*T*A*apapcpcpaptpgpaptpgptpgpcptpG*C*T*A*t; see Table 1 for a description of these
abbreviations) where the internal, normal nucleotides were flanked at the 5’- and 3’- ends with
N,N-dimethylamino PMO nucleotides. ODN 27 also contained a fluorescein dye (FL) joined by
a six-carbonlinker (Supporting Information; Figure S32) to ODN 27. HeLa cells were transfected
with ODN 27 (100 nM concentration) in the presence of DharmaFECT 1 with live cells
(Supporting Information) and fixed cells (Figure 5) imaged by fluorescent microscopy after 20 h
and 18 h incubation respectively. Transfection was dose dependent as an increased fluorescent
signal was observed when cells were incubated with 100 nM of ODN 27 (Supporting
Information, Figure S33). The fluorescence appears to be distributed primarily in the nucleus
although there is also evidence of cytoplasmic distribution without the common punctuated
structures found for many analogues.
Figure 5. Visualization of ODN 27 transfection using fluorescence microscopy. ODN 27 (100 nM) in reduced
serum medium (Opti-MEM) was transfected into HeLa cells using DharmaFECT 1 followed by 18 h of incubation
at 37 °C. Cells were washed with PBS and fixed using buffered formalin solution. Nuclei of the cells were
counterstained with DAPI (blue). (A)/(D) Image showing fluorescein localizationof ODN 27. (B)/(E) Images
showing DAPI localization of nuclei in the same cells. (C). Image showing overlay of A and B. (F) Phase contrast
image of the cells shown in D.
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DISCUSSION
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9
In this manuscript we outline a new method for synthesizing PMO and PMO-DNA
chimeras. The method is very robust as oligomers containing phosphosphorodiamidate
internucleotide linkages can be prepared; using phosphoramidite chemistry, in high yield on
automated DNA synthesizers. The process begins by incorporating morpholino
phosphoramidites into DNA, boronation, and then oxidation with iodine in the presence of
various amines in order to form a diverse set of PMOs and PMO-DNA chimeras. The method
appears to be quite general and should lead to the synthesis of a large number of PMO linkages.
Unlike procedures that use chlorophosphoramidate synthons, the more reactive
phosphordiamidite synthons were used to synthesize these analogues in high yields. Selective
activation, via dicyanoimidazole, of the diisopropylamino component of the morpholino
phosphordiamidite during coupling was a key step for assembling these analogs.
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PMO and PMO-DNA chimeras were synthesized on a DNA synthesizer. This is because
both the morpholino phosphordiamidite and 2’-deoxyribonucleoside synthons were designed for
synthesis of oligonucleotides in a 3’ to 5’ direction. This is not the case with previously
developed chemistries where the N,N-dimethylamino PMO synthons are designed for
synthesizing these analogues in a 5’ to 3’ direction. Moreover, as this new approach builds upon
using a boranephosphoroamidate internucleotide linkage, the same intermediate through iodine
oxidation with an appropriate amine can be used to generate a large variety of PMO chimeras.
For example PMO-LNA, PMO-RNA, PMO-DNAphosphorothioate/phosphorodithioate and
PMO-DNA phosphonoacetate/phosphonoformatechimeras among others can be prepared.
Relative to their potential use in various biological and biochemical applications, these
PMO-DNA chimeras exhibited three possible advantages over several other analogues. For
example, aminomorpholino phosphorodiamidate derivatives were found to form a more stable
duplex with complementary DNA or RNA than either unmodified DNA/RNA or the standard
N,N-dimethylaminoPMO analogue (~1.75 times more stable per modification with RNA than
the N,N-dimethyl analogue, see Table 3). Additionally these PMO-DNA chimeras are active
with RNase H1. This new result is encouraging relative to the standard N,N-dimethylamino
PMO analogs where the completely substituted PMO is inactive with RNase H1. As has been
known for some time, several other analogs, such as methylphosphonates31 and
phosphonoacetates32 as cap/gap oligomers are also known to activate RNase H1 activity. The
observation that cap/gap N,N-dimethylamino PMO chimeras activate RNase H1 provides
another analog with different biochemical properties (for example the aminoamidate derivative
has enhanced stability toward duplex formation) that may prove useful for various applications
in biology. Thus in a manner as observed previously with LNA/phosphorothioate
oligonucleotides and due to the increased stabilization of these PMO-DNA chimeras with
complementary RNA (relative to the unmodified duplexes), this kind of cap/gap analogue may
decrease off-target effects because shorter single stranded antisense oligonucleotides can be
used.33Moreover these PMO-DNA chimeras can easily be transfected into cells using regular
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DNA based transfecting reagents which eliminates the problems associated with delivery of
PMOs by such procedures as microinjection,34 hybridization of PMOs with DNA and delivery
with ethoxylated polyethylenimine,35 or conjugation with either peptides36 or dendritic molecular
transporters.37
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CONCLUSIONS
This manuscript outlines a new, versatile method for synthesizing different amine
substituted morpholino oligonucleotides other than the classical N,N-dimethylamino PMO
analogue. The synthesis strategy is compatible with other current methodologies and high
yielding. Moreover chimeras containing unmodified DNA/RNA and other analogs of
DNA/RNA can also be prepared. Using this new chemistry, certain analogs form duplexes with
complementary DNA or RNA that are more stable than natural DNA or DNA/RNA complexes.
Also various PMO-DNA chimeras were shown to be active with RNase H1 and can be
transfected into cells using standard lipid reagents. On the basis of these preliminary biochemical
results, analogues of this type may prove quite useful for numerous applications, as they are
stable toward nucleases,38 and activate RNase H1.
EXPERIMENTAL SECTION
Chemical Synthesis. The general procedure for the synthesis of appropriately protected
synthons (9, 14, 19 or 24) is as follows: 5’-O-DMT-N-BIBS protected morpholino nucleosides
(8, 13, 18 or 23, see Supporting Information) were dried overnight in vacuum. They were
dissolved in anhydrous CH2Cl2 followed by addition of 1.2 equivalents of 2-cyanoethyl-
N,N,N’,N’-tetraisopropylphosphorodiamidite under argon. After adding 0.5 equivalent of 4,5-
dicyanoimidazole, the reaction was allowed to proceed with stirring for 5 h under an argon
atmosphere at room temperature. At this time, TLC indicated complete conversion of starting
material. Reaction mixtures were diluted with CH2Cl2, washed first with a 5% NaHCO3 solution
and then brine. The organic layers were dried over Na2SO4, filtered and evaporated to dryness.
The products were purified by silica gel column chromatography. The silica gel slurry was
prepared with the starting eluant mixture containing an additional 5% triethylamine. After
pouring the slurry, the column was washed with two column volumes of the starting
solvent mixture containing no triethylamine. Compounds 9, 14, 19 and 24 were purified using
a gradient of 3:7 ethylacetate:hexanes to 1:1 ethylacetate:hexanes.
Compound
9:
5’-O-Dimethoxytritylmorpholinothymine-3’-O-cyanoethyl-N,N-
1
diisopropylphosphordiamidite: Yield: 86%. 31P NMR (CD2Cl2) δ: 127.21, 126.08. H NMR
(CD2Cl2, 400 MHz) δ: 9.51 (1H, bs), 7.50-7.47 (2H, m), 7.38-7.25 (8H, m), 6.89-6.86 (4H, m),
5.78-5.75 (0.5 H, dd, J= 12.0, 4.0 Hz), 5.65-5.62 (0.5H, dd, J= 12.0, 4.0 Hz), 4.08-4.02 (1H, m),
3.99-3.86 (3H, m), 3.82 (6H, s), 3.65-3.52 (2H, m), 3.48-3.34 (1H, m), 3.31-3.27 (1H, m), 3.13-
3.09 (1H, m), 2.75-2.68 (2H, m), 2.53-2.47 (2H, m), 1.96 (3H, m), 1.25-1.18 (12H, m). 13C NMR
(CD2Cl2) δ: 164.02, 163.96, 158.66, 150.13, 144.95, 135.92, 135.90, 135.79, 135.68, 135.52,
129.99, 128.07, 127.77, 126.77, 117.89, 117.74, 113.05, 110.48, 110.39, 86.05, 80.54, 80.49,
80.20, 77.36, 77.21, 64.37, 60.09, 59.84, 55.20, 49.05, 48.83, 47.58, 47.06, 46.83, 45.87, 45.78,
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43.91, 43.74, 24.36, 24.29, 24.22, 24.20, 20.77, 20.33, 20.66, 12.29. ESI-MS (m/z): Calculated=
744.3526, found= 744.3497 (M+H)+.
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8
Compound
14:
N2-Di(tert-butyl)isobutylsilyl-5’-O-dimethoxytrityl-
9
31
morpholinocytosine-3’-O-cyanoethyl-N,N-diisopropylphosphordiamidite: Yield: 83%. P NMR
(CD2Cl2) δ: 126.32, 124.88.1H NMR (CD2Cl2, 400 MHz) δ: 7.58-7.55 (1H, m), 7.50-7.48 (2H,
m), 7.38-7.31 (6H, m), 7.28-7.24 (1H, m), 6.88-6.85 (4H, m), 5.79-5.76 (0.5H, dd, J= 12.0, 4.0
Hz), 5.63-5.59 (0.5H, dd, J= 12.0, 4.0 Hz), 4.54 (1H, bs), 4.04-3.87 (3H, m), 3.82 (6H, s), 3.74-
3.71 (0.5H, m), 3.65-3.45 (3H, m), 3.30-3.24 (1.5H, m), 3.14-3.10 (1H, m), 2.80-2.75 (1H, m),
2.71-2.68 (1H, m), 2.55-2.46 (1H, m), 2.38-2.31 (1H, m), 2.11-2.07 (1H, m), 1.78 (1H, bs), 1.25-
1.15 (31H, m), 1.04-1.02 (8H, m). 13C NMR (CD2Cl2) δ:168.14, 158.61, 154.46, 144.98, 140.62,
140.42, 135.98, 135.88, 130.03, 128.07, 127.75, 126.71, 117.96, 117.75, 113.02, 96.20, 96.09,
85.97, 81.68, 81.34, 81.23, 77.05, 77.00, 64.50, 60.21, 60.09, 59.85, 55.18, 49.63, 49.41, 47.98,
47.13, 46.18, 45.91, 45.83, 43.96, 43.84, 43.77, 28.58, 26.21, 26.07, 24.78, 24.38, 24.26, 24.17,
24.12, 20.98, 20.76, 20.71, 20.62, 20.31, 20.23. ESI-MS (m/z): Calculated= 927.5333, found=
927.5329 (M+H)+.
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12
13
14
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18
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Compound 19: N2-Di(tert-butyl)isobutylsilyl-5’-O-dimethoxytritylmorpholinoadenine-3’-
O-cyanoethyl-N,N-diisopropylphosphordiamidite: Yield: 79%. 31P NMR (CD2Cl2) δ: 127.96,
1
125.36. H NMR (CD2Cl2, 400 MHz): 8.38-8.37 (1H, d), 8.01-8.00 (1H, d), 7.51-7.48 (2H, m),
7.38-7.31 (6H, m), 7.28-7.24 (1H, m), 6.88-6.86 (4H, m), 5.93-5.90 (0.5H, dd, J= 12.0, 4.0 Hz),
5.82-5.79 (0.5H, dd, J= 12.0, 4.0 Hz), 5.39 (1H, bs), 4.14-4.10 (0.5H, m), 4.06-4.02 (0.5H, m),
4.0-3.90 (2H, m), 3.82 (6H, s), 3.71-3.63 (1H, m), 3.60-3.52 (2.5H, m), 3.42-3.37 (0.5H, m),
3.34-3.30 (1H, m), 3.17-3.13 (1H, m), 3.02-2.91 (1H, m), 2.78-2.75 (1H, m), 2.72-2.68 (1H, m),
2.66-2.59 (1H, m), 1.26-1.18 (31H, m), 1.10-1.08 (8H, m). 13C NMR (CD2Cl2) δ: 158.64,
158.11, 152.55, 148.92, 144.97, 137.76, 135.92, 135.80, 130.02, 128.06, 127.77, 126.74, 121.60,
117.79, 117.71, 113.04, 86.50, 80.50, 80.44, 80.34, 80.22, 76.94, 76.81, 64.39, 60.14, 59.91,
55.18, 50.16, 49.93, 48.49, 48.44, 47.35, 47.12, 46.08, 46.00, 43.95, 43.83, 43.71, 28.62, 26.28,
26.19, 24.80, 24.35, 24.29, 24.18, 21.04, 20.79, 20.36, 20.27. ESI-MS (m/z): Calculated=
951.5446, found= 951.5437 (M+H)+.
Compound
24:
N2,O6-bis[di(tert-butyl)isobutylsilyl]-5’-O-dimethoxytrityl-
morpholinoguanine-3’-O-cyanoethyl-N,N-diisopropylphosphoradiamidite: Yield: 82%. 31P NMR
(CD2Cl2) δ: 127.62, 126.99. 1H NMR (CD2Cl2, 400 MHz): 7.79-7.76 (1H, d), 7.49-7.45 (2H, m),
7.37-7.29 (6H, m), 7.27-7.23 (1H, m), 6.87-6.84 (4H, m), 5.76-5.73 (1H, dd, J= 12.0, 4.0 Hz),
5.67-5.64 (1H, dd, J= 12.0, 4.0 Hz), 4.55 (1H, s), 4.05-3.88 (3H, m), 3.82 (6H, s), 3.67-3.54 (3H,
m), 3.49-3.45 (1H, m), 3.32-3.23 (1H, m), 3.19-2.90 (2H, m), 2.71-2.68 (2H, m), 2.61-2.54 (1H,
m), 2.23-2.16 (1H, m), 2.13-2.06 (1H, m), 1.27-1.15 (49H, m), 1.06-0.97 (27H, m).13C NMR
(CD2Cl2) δ: 160.67, 159.38, 158.61, 154.33, 154.06, 144.96, 136.47, 135.90, 135.79, 130.00,
128.02, 127.76, 126.69, 117.58, 116.77, 113.03, 85.99, 80.69, 80.58, 79.55, 79.49, 76.95, 76.45,
76.36, 64.22, 60.20, 60.12, 59.88, 59.86, 55.16, 49.71, 49.47, 47.77, 47.68, 47.46, 47.26, 45.96,
45.89, 43.99, 43.88, 43.77, 43.65, 28.76, 28.07, 26.39, 26.31, 26.27, 24.87, 24.68, 24.44, 24.37,
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24.27, 24.19, 21.90, 21.37, 20.83, 20.76, 20.75, 20.69, 20.35, 20.32, 20.27, 20.24. ESI-MS (m/z):
Calculated= 1165.7198, found= 1165.7203 (M+H)+.
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Automated PMO Synthesis. Synthesis was carried out on an ABI 394 Synthesizer. All
syntheses were performed at a 0.2 ꢀmol scale using a 5′-DMTr-2′-deoxythymidine joined to a
low volume polystyrene solid support via a succinate linkage. For synthesis of morpholino
oligonucleotides, a standard 0.2 ꢀ mole synthesis cycle was used with an increased coupling time
of 300 s. A wash with methanol followed the detritylation step. All the phosphordiamidites (9,
14, 19 or 24; 0.1 M) were dissolved in anhydrous CH3CN. Detritylation was carried out using a
0.5% solution of TFA in anhydrous CHCl3 containing 10% TMPB. Solutions for boronation
(0.05 M BH3-THF complex in THF) were prepared fresh prior to use. Reagents for activation
(4,5-dicyanoimidazole) and capping were purchased from Glen Research. A stepwise description
of the synthesis cycle is described in Table S1 (Supporting Information). Deprotection was
carried out in two steps: the solid support linked boranephosphoroamidate morpholino
oligonucleotides were first treated with a 1:1 solution of triethylamine in acetonitrile for 10
minutes followed by extensive washing with acetonitrile. The resin was then dried using a flow
of argon and transferred to a glass vial. A solution of iodine (0.05M) and dimethyl amine in
THF (2.0 M) was added to the glass vial. For reactions with ammonia, iodine (0.05 M) was
dissolved in a solution of ammonia (2.0 M) in isopropanol and this solution was added to the
resin and stirred overnight. The glass vial was then placed on a mechanical shaker for 24 hrs.
For the synthesis of the methylamine and morpholinodiamidate derivatives, a 2.0 M solution of
each amine in THF was used.
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The vials containing resins were centrifuged at 4000 rpm and the supernatants removed
with a pipette. Subsequently resins were washed 4-5 times with 2 mL aliquots of acetonitrile,
shaken vigorously, placed in a centrifuge at 4000 rpm for 5 minutes and supernatants removed.
For amino modified morpholino derivatives, the ammonia solution in isopropanol was removed
under vacuum. These morpholino oligonucleotides were desilylated by overnight fluoride (1.0
mL of 1.0 M solution of tetrabutylammonium fluoride in THF) treatment. The resins were then
treated with 1 mL of 37% ammonium hydroxide in water for 1 h in order to remove the
product from the resins and the polystyrene resin beads were removed using a 0.2 µm
centrifugal filter.
The total volume of each solution was diluted to 2.5 mL by adding 0.5 mL Millipore
water and the PMOs were desalted using Illustra™ NAP™-25 Columns (GE Healthcare).
Initially these columns were equilibrated with 25.0 mL of water. After allowing water to
completely enter the gel beds by gravity flow, 2.5 mL of samples were loaded on to columns and
the purified samples were eluted using 3.5 mL Millipore water. Second step purifications were
carried out for oligonucleotides having more than 10 nucleotides by using Amicon® Ultra-4 3K
devices. Solutions that accumulated after Nap column purification (3.5 mL total volume) were
loaded on to Amicon devices and centrifuged at 4000xg for 30 minutes. Concentrated solutes
were washed with 3.0 mL X 2 of Milipore water in a similar fashion. The oligonucleotides were
collected and used for various experiments.
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Synthesis of a 5′-Fluorescein PMO-DNA Chimera. After completion of a DMTr-ON synthesis
of the boranephosphoroamidate morpholino chimera, the ODN was conjugated with 5'-Amino-
Modifier C6-TFA (Glen research) using a standard DNA synthesis cycle. After carrying out the
iodine oxidation reaction and desilylation of exocyclic amines, the oligonucleotide was purified
using reverse phase HPLC (Buffer A: triethylammonium bicarbonate, 0.05 M; Buffer B:
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acetonitrile; 0-100% B in 50 mins; 55 C; 4.0 mL/min flow rate). The purified ODN was
o
dissolved in 1 mL of a solution of 1:1 NH4OH: CH3NH2 and heated at 65 C for three hours to
remove the trifluroacetamido group. The reaction mixture was dried and dissolved in a buffer
(200 ꢀL) containing 20 mM sodium phosphate and 0.15 M NaCl (pH 8.0) and the concentration
was measured. A 20-fold molar excess of 5-(and 6-)carboxy-fluorescein succinimidyl ester
(Thermo Fisher Scientific) was dissolved in DMSO and added to the ODN solution. The reaction
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mixture was stirred at room temperature for 1 h followed by stirring at 4 C for a period of 3 h.
The reaction mixture was diluted using 300 ꢀL water and excess NHS-fluorescein was removed
using Illustra™ NAP™-5 Column.
E. coli RNase H1 hydrolysis of RNA heteroduplexes. Experiments with E. coli RNaseH1
(Promega) were performed using the conditions described by Dellinger et al.30 The reactions
were carried out using an assay buffer of 50 mM tris-HCl (pH 8.0), 20 mM KCl, 9mM MgCl2, 1
mM β-mercaptoethanol, and 250 µg/mL bovine serum albumin. An oligodeoxynucleotide or
modified oligodeoxynucleotide (200 pmol) and 5’-O-fluorescein labeled, complementary
oligoribonucleotide were added to the assay buffer (35 µL). Following the addition of E. coli
RNase H1 (3 units), reactions were carried out at 25° C over 12 h. The reaction mixtures were
diluted with an equal volume of 80% formamide gel loading buffer containing tracking dyes and
analyzed by polyacrylamide gel electrophoresis (20%, 19:1 cross-linking, 7 M urea). All
reactions were performed in triplicate. The developed gels were analyzed using a Molecular
Dynamics Typhoon Phosphorimager.
Lipid transfection as observed by microscope imaging. An ODN 27 stock solution was
diluted with 200 ꢀL OptiMEM to a final concentration of 0.1 ꢀM ODN. In a separate Eppendorf
Tube, 5.0 ꢀL DharmaFECT 1 was diluted with 200 ꢀL OptiMEM. The 200ꢀL solution of ODN
27 and the DhamaFECT 1 solution were mixed, equilibrated for 20 min, and 600 ꢀL OptiMEM
was added. HeLa cells were seeded at 0.3 × 106 cells/well on a coverslip placed in a six well
plate in DMEM medium containing 10% FBS and penstrep. After 24 hours, medium was
removed and the cells were washed twice (2.0 mL D-PBS/wash) before transfection at 80%
confluency. D-PBS was removed from the HeLa cells, and 1.0 mL of the transfection mixture
was added to each well. Cells were then incubated at 37 °C for 18 hours and washed twice (2.0
mL D-PBS/wash). Cells were covered with 1.0 mL of 10% neutral buffered formalin for 15
mins. The formalin solution was removed and the cells were covered with 3.0 mL DPBS for 10
min at RT. The coverslips were removed from the wells and mounted upside down on
coverslides using Flouromount-G with DAPI as mounting media and observed using an inverted
microscope (OlympusIX 81) equipped with a Hamamatsu C4742-95 CCD and CoolSNAP ES
digital camera (Photometrics).
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ASSOCIATED CONTENT
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Supporting Information
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Detailed synthetic procedures, characterization information and LCMS spectra of
synthesized oligomers are provided. This information is provided free of charge via the Internet
at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
marvin.caruthers@colorado.edu
ACKNOWLEDGEMENTS
We thank Dr. Douglas J. Dellinger (Agilent Technologies) for providing access to an
LCMS instrument and Dr. Subhadeep Roy for meaningful discussions. We thank Joe Dragavon
for fluorescent microscopy. The research was supported by the University of Colorado.
REFERENCES
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R63.
3. Summerton, J.; Weller, D. Antisense Nucleic Drug Dev. 1997, 7, 187-195.
4. Summerton, J. Biochim. Biophys. Acta. 1999, 1489, 141-158.
5. Hudziak, R. M.; Barofsky, E.; Barofsky, D. F.; Weller, D. L.; Huang, S. B.; Weller, D. D.
Antisense Nucleic Drug Dev. 1996, 6, 267-272.
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91, 1009-1018.
7. Summerton, J. E. In Morpholinos and Related Antisense Biomolecules; Janson, C. G.;
During, M. J., Eds.; Kluwer Academic/Plenum Publishers P.O. Box 17, 3300 AA
Dordrecht, the Netherlands 2003.
8. Ekker, S. C; Larson, J. D. Genesis, 2001, 30, 89-93.
9. www.gene-tools.com/morpholino-publication-database
10. www.sarepta.com
11. Paul, S.; Jana, S.; Bhadra, J.; Sinha, S. Chem. Commun., 2013, 49, 11278-11280.
12.Tercero, N.; Wang, K.; Gong, P.; Levicky, R. J. Am. Chem. Soc., 2009, 131, 4953-4961.
13. Mangos, M. M.; Min, K. L.; Viazovkina, E.; Galarneau, A.; Elzagheid, M. I.; Parniak, M.
A.; Damha, M. J. J. Am. Chem. Soc. 2003, 125, 654-661.
14. Moulton, H. M.; Hase, M. C.; Smith, K. M.; Iversen, P. L. Antisense Nucleic Drug Dev.
2003,13, 31-43.
15. Järver, P.; Coursindel, T.; Andaloussi, S. EL.; Godfrey, C.; Wood, M. JA.; Gait, M.
Molecular Therapy Nucleic Acids, 2012, 1, e27/1-e27/17.
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16. For the morpholino derivatives, the numbering of the primary hydroxyl group should be
6’.In order to correlate with the numbering system commonly used in the nucleic acid
field, we marked it as 5’ for this manuscript.
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17. Summerton, J.; Weller, D. US Patent 5217866 A 19930608, 1993.
18. Harakawa, T.; Tsunoda, H.; Ohkubo, A.; Seio, K.; Sekine, M. Bioorg. Med. Chem. Lett.
2012, 22, 1445–1447.
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19. Paul, S.; Pattanayak,S.; Sinha, S. Tetrahedron Lett. 2014, 55, 1072–1076.
20. Bhadra, J.; Kundu, J.; Ghosh, K. C.; Sinha, S. Tetrahedron Lett. 2015, 56, 4565-4568.
21. Beaucage, S. L.; Caruthers, M. H. Tetrahedron Lett. 1981, 22, 1859-1862.
22. Paul, S; Roy, S.; Monfregola, L.; Shang, S.; Shoemaker, R.; Caruthers, M. H. J. Am.
Chem. Soc. 2015, 137, 3253−3264.
23. Zhang, N.; Tan, C.; Cai, P.; Jiang, Y.; Zhang, P.; Zhao, Y. Tetrahedron Lett. 2008, 49,
3570-3573.
24. Pattanayak, S.; Paul, S.; Nandi, B.; Sinha, S. Nucleo. Nucleot. Nucl. Acids 2012, 31, 763–
782.
25. Roy, S.; Olesiak, M.; Shang, S.; Caruthers, M. H. J. Am. Chem. Soc. 2013, 135, 6234-
6241.
26. The CPG support could not be used because this support is not compatible with fluoride
reagents that are required to removeN-silyl ether protecting groups from the bases.
27. Zhang, N.; Tan, C.; Cai, P.; Zhang, P.; Zhao,Y.; Jiang, Y. Bioorg. Med. Chem. 2009, 17,
2441–2446.
28. Perrin, D. D. In Dissociation Constants of Organic Bases in Aqueous
Solution,Butterworths Publishers, Halsbury House, 35, Chancery Lane,London, 1965,
pp. 41, 137, 141. The values given in brackets correspond to the pKas calculated in water
and are used as formal indicators of amine basicity.
29. The use of ETT for the synthesis of chimeras was possible because the
borane/morpholino P(IV) linkage was stable towards this activator.
30. Dellinger, D. J.; Sheehan, D. M.; Christensen, N. K.; Lindberg, J. G.; Caruthers, M. H. J.
Am. Chem. Soc. 2003, 125, 940 – 950.
31. Giles, R. V.; Tidd, D. M. Nucleic Acids Research 1992, 20, 763-770.
32. Sheehan, D. M.; Lunstad, B.; Yamada, C., M.; Stell, B. G.; Caruthers, M. H. Nucleic
Acids Res. 2003, 31, 4109-4118.
33. Obad, S.; dos Santos, C. O.; Petri, A.; Heidenblad, M.; Broom, O.; Ruse, C.; Fu, C.;
Lindow, M.; Stenvang, J.; Straarup, E. M.; Hansen, H. F.; KochT.; Pappin, D.; Hannon,
G. J.; Kauppinen, S. Nat. Genet. 2011, 43, 371-378.
34. Nasevicius, A.; Ekker, S. C. Nat. Genet. 2000, 26, 216-220.
35. Morcos, P. A. Genesis 2001, 30, 94-102.
36. Moulton, H. M.; Nelson, M. H.; Hatlevig, S. A.; Reddy, M.T.; Iversen, P. L.
Bioconjugate Chem. 2004, 15, 290-299.
37. Li, Y-F; Morcos, P. A. Bioconjugate Chem. 2008, 19, 1464-1470
38. Summerton J. J. Drug Discov.Develop.AndDeliv.2016, 3, 1019-1025.
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Table of Contents Figure
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Figure 2. Synthesis of a N,N-Dimethylamino PMO trimer (29). The LC profile from the LCMS analysis of the
total reaction mixture is shown.
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Figure 3. (A) LCMS analysis of the total reaction mixture from the synthesis of ODN 8. The data shows the
LC trace and a zoomed in view of the base peak in the mass spectra. The base peak in each case was a -3
charged species. Full mass spectra are provided in the supplementary section. (B) 31P NMR of the total
reaction mixture obtained from the synthesis of ODN 16 (400 MHz, 15000 scans). The 31P NMR peaks at
18.63 ppm and -1.2 ppm correspond to the N,N-dimethylamino-phosphorodiamidate and phosphodiester
linkages respectfully.
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Figure 4. E. coli RNase H1 degradation of RNA when Catalyzed by PMOꢀDNA Chimeras. 5′ꢀOꢀFluorescein
labele dr(A)14 was allowed to form duplexes with complementary ODNs 24ꢀ26. These duplexes were then
treated with E. coli RNase H1 for 12 h and analyzed by PAGE. Lane 1: 2’ꢀOꢀmethyl RNA/RNA; Lane 2: 2’ꢀOꢀ
methyl RNA/RNA/RNase H1; Lane 3: DNA/RNA; Lane 4: DNA/RNA/RNase H1; Lane 5: ODN 24/RNA; Lane 6:
ODN 24/RNA/RNase HI; Lane 7: ODN 25/RNA; Lane 8: ODN 25/RNA/RNase H1; Lane 9: ODN 26/RNA; Lane
10: ODN 26/RNA/RNase H1.
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Figure 5. Visualization of ODN 27 transfection using fluorescence microscopy. ODN 27 (100 nM) in reduced
serum medium (Opti-MEM) was transfected into HeLa cells using Dharmafect 1 followed by 18 h of
incubation at 37 °C. Cells were washed with PBS and fixed using buffered formalin solution. Nuclei of the
cells were counterstained with DAPI (blue). (A)/(D) Image showing fluorescein localizationof ODN
27. (B)/(E) Image showing DAPI localization of nuclei in the same cells. (C). Image showing overlay of
images A and B. Images. (F) Phase contrast image of the cells shown in D.
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Scheme 1. Current approaches for the synthesis of N,N-Dimethylamino PMOs.
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Scheme 2. Synthesis of BIBS protected Morpholino Phosphordimidite synthons. (i) Lutidine (4.0 eq.)/1,4-
Dioxane; (ii) 1.0 M NH3/MeOH; (iii) DMT-Cl (1.2 eq.)/Pyridine; (iv) NaIO4 (1.1 eq.)/(NH4)2B4O7 (1.1
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Dicyanoimidazole (0.5 eq.)/CH2Cl2.
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Scheme 3. Synthesis cycle for the preparation of PMOs.
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Table 1. Synthesis cycle for preparing PMOs and PMO-DNA chimeras.
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