Phthalimide–Oxy Derivatives for 3′- or 5′-Conjugation of Oligonucleotides by Oxime Ligation and Circularization of DNA by “Bis- or Tris-Click” Oxime Ligation

Article abstract of DOI:10.1002/ejoc.201701317

A solid support and two phosphoramidites exhibiting a phthalimide–oxy group were synthesized. First, after treatment with hydrazine, the resulting 5′- and 3′-oxyamine oligonucleotides were conjugated with aldehyde derivatives by oxime ligation. Second, ol

Full text of DOI:10.1002/ejoc.201701317

A Journal of
Accepted Article
Title: Phthalimide-oxy derivatives for 3'- or 5'-Conjugation of
Oligonucleotides by Oxime Ligation and Circularization of DNAs
by "Bis- or Tris-Click" Oxime Ligation
Authors: Albert Meyer, Jean-Jacques Vasseur, Pascal Dumy, and
Francois Morvan
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201701317
Link to VoR: http://dx.doi.org/10.1002/ejoc.201701317
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10.1002/ejoc.201701317
European Journal of Organic Chemistry
Phthalimide-oxy derivatives for 3'- or 5'-Conjugation of Oligonucleotides by Oxime Ligation
and Circularization of DNAs by "Bis- or Tris-Click" Oxime Ligation
Albert Meyer, Jean-Jacques Vasseur, Pascal Dumy and François Morvan*
Institut des Biomolécules Max Mousseron, IBMM, UMR 5247, Université Montpellier, CNRS,
ENSCM, 34095 Montpellier Cedex 5, France. Francois.morvan@umontpellier.fr
ABSTRACT
A solid support and two phosphoramidites exhibiting a phthalimide-oxy group were
synthesized. First, after treatment with hydrazine, the resulting 5’- or 3’-oxyamine
oligonucleotides were conjugated with aldehyde derivatives by oxime ligation. Second,
oligonucleotides exhibiting at each end an oxyamine were circularized by means of different
dialdehydes. The cyclic oligonucleotides of different lengths (9 to 31-mer) were rapidly
obtained without the need of a template. Finally, a bis-cyclic oligonucleotide was synthesized
starting form an oligonucleotide bearing three oxyamines which reacted with a trialdehyde
forming three oxime ligations.
Modified oligonucleotides
Graphical abstract
1
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10.1002/ejoc.201701317
European Journal of Organic Chemistry
R
O
R
N
O
H
ONH2
DNA
synthesis
Click
DMTr
NH2O
H
H
DMTr
O
H
O
O
N
O
O
O
N(iPr)2
Cne
P
O
NH
H
O
O
N
O
O
Bis-Click
ONH2
O
N
O
N
O
O
NH2O
O
O
O
ONH2
N
O
O
H
H
N
N
O
H
O
O
ONH2
Tris-Click
Novel phthalimide-oxy phosphoramidites and a solid support were used to obtain
oligonucleotides bearing one, two or three oxyamines for their subsequent conjugation, mono-
and di-circularization by oxime ligation with mono-, di- or trialdehydes.
INTRODUCTION
Cyclic DNA and RNA exhibit unusual chemical and biological properties in comparison with
linear ones, and have been evaluated for several biological applications such as antisense,
triplex, and diagnostic applications.[1-5] First cyclic DNAs have been synthesized in the late
1980s[1,6-9] and new synthesis of cyclic DNAs and RNAs have been are reported along the years.
Their synthesis was performed using enzymatic or chemical methods affording them with
phosphodiester,[1,10-13] phosphorothiolatediester,[14-17] disulfide[18-20] oxime[17,21] and more
recently with a triazole linker[22-29] As a rule cyclic oligonucleotides are synthesized thanks to
complementary chemical functions borne on both ends of the sequence. Alternatively, the same
chemical function could be introduced at each end and then the circularization performed with
a linker bearing twice the complementary function. This strategy was recently applied to form
cyclic oligonucleotides by Copper catalyzed Azide Alkyne Cycloaddition (CuAAC)[30-31]
starting from a linear strand exhibiting an alkyne function at each end, and a linker bearing two
azides.[26,28-29,32]
Herein, we report a method for the preparation of cyclic of oligonucleotides (9 to 31-mers) by
two oxime ligations. To this purpose, a new solid support and phosphoramidites with a
phthalimide-oxy group were prepared. Firstly, the solid support was used for the 3'-conjugation
of oligonucleotides. Secondly in combination with the phthalimide-oxy phosphoramidites,
2
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10.1002/ejoc.201701317
European Journal of Organic Chemistry
oligonucleotides with aminooxy function at both ends were synthesized. Their circularization
was performed by different dialdehydes leading to cyclic oligonucleotides with two oxime
linkages. As an extension of the scope of this strategy, a dumbbell structure was prepared
bearing an alkyne function in a loop for derivatization with an azide dansyl through a CuAAC.
RESULTS AND DISCUSSION
Conjugation of oligonucleotides through oxime mainly occurred by the introduction of an
aldehyde function into an oligonucleotide, either at the 3' or 5'-end, reacting then with the
aminooxy derivatives.[33] There are only few examples of phosphoramidite derivatives allowing
the introduction of the aminooxy in the oligonucleotides[21,34-36] Surprisingly, none solid support
has been reported, so far, to introduce an aminooxy function at the 3'-end of an oligonucleotide
for subsequent conjugation with an aldehyde derivative.
Synthesis of building blocks exhibiting a phthalimide-oxy group. Two phosphoramidites 3
and 6 and a solid support 8 bearing a phthalimide-oxy group were synthesized in order to
introduce aminooxy functions at the 5'- or/and 3'-end of an oligonucleotide. Compound 3 was
prepared in three steps (Scheme 1). Firstly, triethyleneglycol was monotosylated[37] and
[38]
secondly, the tosyl was substituted by hydroxyphthalimide in presence of K2CO3
under
microwaves assistance giving 2. Thirdly, compound 2 was phosphitylated with 2-cyanoethyl-
N,N-diisopropylchloro phosphoroamidite in presence of diisopropylethylamine affording the
phosphoramidite
derivative
3.
Similarly
4[39]
1-O-(4,4′-dimethoxytrityl)-2-(6-
was substituted with
bromohexyloxymethyl)-2-methyl-1,3-propanediol
hydroxyphthalimide giving 5 and then converted into its phosphoramidite derivative 6 or
anchored on a succinyl LCAA-CPG solid support 7 using N-(3-dimethl aminopropyl)-N'-
ethylcarbodiimide, as coupling agent, affording the solid support 8 (Scheme 2). A loading of
35 µmol/g was calculated by trityl assay.
Ts-Cl
HO
O
Ts
O
O
O
OH
O
OH
aq
NaOH
THF
1
O
K2CO3
DMF
N
OH
O
Cl
O
P
N
CN
O
O
CN
O
N
N
O
O
O
O
P
N
O
O
O
OH
O
3
DIEA
O
2
CH2Cl2
3
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10.1002/ejoc.201701317
European Journal of Organic Chemistry
Scheme 1. Synthesis of phthalimideoxy-triethyleneglycol phosphoramidite 3.
O
N
OH
DMTr
DMTr
O
O
O
O
O
OH
O
OH
K2CO3
DMF
N
O
Br
4
5
O
Cl
O
O
P
CN
Pyridine
Et3N
HO
N
N
H
O
EDC
DIEA
CH2Cl2
7
DMTr
DMTr
O
O
O
O
O
O
O
O
O
O
N
O
N
N
O
P
N
CN
H
O
O
O
6
8
Scheme 2. Synthesis of phosphoramidite 6 and solid support 8 exhibiting a phthalimide-oxy-
hexyl group.
The phosphoramidites 3 and 6 and the solid support 8 were validated on a hexathymidylate. On
one hand, a T6 was elongated starting from solid support 8 by standard phosphoramidite
chemistry (Scheme 3) and on the other hand a T6 was elongated from commercially available
thymidine solid support and 3 or 6 was coupled using 20 eq and a 60 sec coupling step (Scheme
4).
A two-step deprotection protocol was applied with first a 30 min treatment with hydrazinium
acetate in pyridine and second a 2 h aqueous ammonia treatment both at room temperature. [36]
After ammonia evaporation, 3'- and 5'-aminooxyhexyl-THME-T6 (11 and 23 respectively) were
analyzed by C18 reverse phase HPLC and characterized by MALDI-TOF mass spectrometry.
While MS showed only the formation of the expected aminooxy-T6 ([M-H]-, m/z = 2059.50),
the HPLC profile of 11 and 23 showed two main peaks and also small peaks before and after
them (Fig. 1). This unexpected behavior was investigated. As an aminooxy function reacts
rapidly and easily with any traces of aldehyde or ketone present in air or HPLC buffer, the
aminooxy oligonucleotides are usually treated with acetone before their HPLC analysis.[35] So,
aminooxy-T6 were treated with acetone to form the corresponding adducts 12 and 24. Both
crude HPLC profiles showed only one peak which was characterized by MS as the expected
oxime ([M-H]- = 2099.56) (Fig. 2). To explain this phenomenon, we can hypothesize that the
4
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10.1002/ejoc.201701317
European Journal of Organic Chemistry
aminooxy function forms some interaction with the oligonucleotide leading to a constraint
structure. Since the THME motif exhibits a chiral carbon, the two diastereoisomers are eluted
differently. In contrast, when the oxime function is formed with the acetone, there is no more
interaction and the difference between both diastereoisomers is no more visible by HPLC. We
have already observed such phenomenon with cyclic and linear oligonucleotides built with this
THME linkage.[25] For the aminooxy-EG3-T6 18 and the corresponding acetone capped
oligonucleotide 19, there is no chiral center and so a single peak was visualized (Fig. 1-2).
According to this fact, the HPLC profiles of aminooxy oligonucleotides, especially when a
THME motif is present, do not give a realistic view of their purity while MALDI-TOS MS does
even if this technique is not quantitative.
DMTr
O
O
O
NH
HO-TTT TTT
O
O
O
O
O
DNA
Synthesis
O
O
O
N
With T
N
O
O
O
8
O
9
.H
NH2NH2 2O
AcOH
Pyridine
HO-TTT TTT
O
HO-TTT TTT
O
NH4OH
O
H2N
O
O
OH
11
O
O
10
H2N
O
O
H
O
H2O
AcONH4
DMF
HO-TTT TTT
O
HO-TTT TTT
O
13
NH4OH
N
O
O
OH
N
O
O
OH
14
12
Scheme 3. 3'-Modified hexathymidylates
5
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10.1002/ejoc.201701317
European Journal of Organic Chemistry
HO
O
DNA
Synthesis
DNA
With
O
O
TTT TTT
O
Synthesis
O
TTT TTT
O
DMTr-T
O
O
O
O
O
and
With T
3
and
T
6
O
N
16
15
N
21
O
O
O
O
O
.H
NH2NH2 2O
.H
NH2NH2 2O
AcOH
AcOH
Pyridine
Pyridine
HO
O
TTT TTT
O
O
TTT TTT
O
O
O
O
O
17
22
H2N
H2N
O
NH4OH
O
O
O
O
H
NH4OH
H
HO
O
13
13
DMF
O
TTT TTT OH
O
TTT TTT OH
DMF
HO
O
NH4OH
23
NH4OH
H2N
H2N
O
O
N
O
O
O-TTT TTT-OH
18
O
O
25
O
O
TTT TTT OH
O
13
DMF
HO
AcONH4
O
TTT TTT OH
N
O
TTT TTT OH
O
O
O
O
N
O
N
O
19
O
20
24
Scheme 4. 5'-Modified hexathymidylates
Figure 1. HPLC profiles (top) and MALDI-TOF MS (bottom) of crude aminooxy
hexathymidylates. Calculated [M-H]-, m/z = 2059.50 for 11 and 23 and 1989.36 for 18.
6
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10.1002/ejoc.201701317
European Journal of Organic Chemistry
Figure 2. HPLC profiles (top) and MALDI-TOF MS (bottom) of crude acetone-capped oxime
hexathymidylates. Calculated [M-H]-, m/z = 2099.56 for 12 and 24 and 2029.43 for 19.
The three supported 3'- and 5'-aminooxy T6 models were conjugated with pyrene aldehyde
affording, after déprotection, the expected 3’-conjugate 14 and 5’-conjugates 25 and 20
(schemes 3 and 4). They were eluted as two peaks in C18 reverse phase HPLC corresponding to
the E and Z isomers of the oxime as already reported (Fig. 3).[34] Each conjugate was
characterized by MALDI-TOF MS and showed a characteristic UV band at 363 nm
corresponding to the pyrene. We visualized the expected ion plus that corresponding to a
fragmentation at the nitrogen-oxygen bound of the oxime linkage [M - 227 Da] during the MS
analysis.[40]
7
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10.1002/ejoc.201701317
European Journal of Organic Chemistry
Figure 3. HPLC profiles (top) and MALDI-TOF MS (bottom) of crude pyrenyl oxime
hexathymidylates. * shows peaks of E/Z of pyrenyl conjugates. Calculated [M-H]-, m/z =
2271.75 for 14 and 25 and 2201.61 for 20.
These results showed that it is possible to introduce an aminooxy function at the 3' or 5' ends
of an oligonucleotide. The next step was to introduce an aminooxy function at both ends of
oligonucleotides to circularize them by means of a dialdehyde.
Oligonucleotide elongation and circularization
A first 9-mer oligonucleotide (CTT CTT AGT) was synthesis starting from solid support 8 by
standard phosphoramidite chemistry, with a 30 sec coupling time for regular nucleoside
phosphoramidites and 60 sec for phosphoramidite 3 to ensure a high coupling (Scheme 5). The
deprotection of the aminooxy functions was performed on the solid support using the standard
treatment with aqueous hydrazine in acetic acid pyridine for 30 min. Then an ammonia
treatment was applied to release and deprotect the linear 9-mer 27 exhibiting an aminooxy
function at each end. It was characterized by MALDI-TOF mass spectrometry and analyzed by
8
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10.1002/ejoc.201701317
European Journal of Organic Chemistry
C18 reverse phase HPLC. MALDI-TOF MS confirmed the synthesis of 27 with the presence of
shortmers and the HPLC profile displayed two mains peaks (Fig. S1).
The cyclization was performed with three different dialdehydes: glyoxal 28a, glutaraldehyde
28b and terephthalaldehyde 28c. The linear bis-aminooxy oligonucleotide 27 (100 nmol) was
dissolved in sodium acetate buffer pH 4.2 (10 µM) and 1.2 molar equivalent of dialdehyde was
added. After 30 min, the solution was purified by size exclusion chromatography (SEC) to
remove the small excess of dialdehyde and buffer. The efficiency of the cyclization affording
oligonucleotides 29a-c was monitored by MALDI-TOF MS and HPLC. (Fig. S2-S4) Whatever
the dialdehyde, the reaction gave only the expected cyclic compound. The three dialdehydes
exhibited the same reactivity.
DMTr
O
O
O
O
N
O
O
8
O
SPOS with
T
A, G, C,
and
3
O
O
P
O
Cne
CTT CTTAGT
N
O
O
O
O
P
O
O
O
O
Cne
O
O
O
O
O
N
O
O
26
O
2.H
NH NH 2O:AcOH:Pyridine
1)
2
NH OH
2)
4
O-
O
P
H2N
O
O
O
O
O
O
CTT CTTAGT
O
P
O-
27
O
O
OH
H2N
O
O
H
O
NH4+AcO-
O
O
O
H
4.2
pH
or
H
or
H
H
O
H
28a
28b
28c
O-
O
P
O
P
O
O
O-
O
O
CTT CTTAGT
O
O
OH
O
N
L
O
N
O
29a-c
=
or
L
Ø,
a
c
b
Scheme 5. Circularization of oligonucleotides by bis-oxime ligation
9
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10.1002/ejoc.201701317
European Journal of Organic Chemistry
Table 1. Sequences and MALDI-TOF mass spectrometry data of the linear and circular
oligonucleotides
oligonucleotides
Sequences
5'—3'
[M-H]-
Calcd
[M-H]-
Found
[a]
NH2O-EG3 CTT CTT AGT ~C6ONH2
Circular 27 with glyoxal
3203.24
3225.25
3267.33
3301.35
6932.64
3203.35
3225.80
3266.66
3301.89
6932.33
27
29a
Circular 27 with glutaraldehyde
Circular 27 with terephthalaldehyde
29b
29c
NH2O-EG3 TTT CCG CGT TGG ATT
AGC TTT ~C6ONH2
21-mer linear
S3121mer
Circularized with terephthalaldehyde
7030.75
9981.63
7029.92
9982.11
21-mer circular
S3321mer
NH2O-EG3 TTT GAT TAC AGC CGG
TGT ACG ACC CTT TTT ~C6ONH2
Circularized with terephthalaldehyde
31-mer linear
S3131mer
10079.73 10080.56
31-mer circular
S3331mer
NH2O-EG3 GAC GCT AAT C-TTTT -G
ATT AGC GTC ~C6ONH2
7857.26
7937.39
7955.36
8106.64
7858.04
7938.86
7957.36
No hit
24-mer linear 31
[b]cap24-mer linear
32
Me2C=NO-EG3 GAC GCT AAT C-TTTT
-G ATT AGC GTC ~C6ON=CMe2
Circularized with terephthalaldehyde
24-mer circular
dumbbell 33
Dansyl 23-mer
linear 39
NH2O-EG3 GAC GCT AAT C-TXTT -G
ATT AGC GTC ~C6ONH2 with X = linker
with dansyl
Me2C=NO-EG3 GAC GCT AAT C-TXTT
-G ATT AGC GTC ~C6ON=CMe2 with X
= linker with dansyl
8186.77
8204.74
8186.17
8206.00
Cap Dansyl 23-
mer linear 40
Circularized with terephthalaldehyde
Dansyl 23-mer
circular dumbbell
41
10
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10.1002/ejoc.201701317
European Journal of Organic Chemistry
NH2OC6~ GAC GCT AAT CTT CTA
NH2OC6~ GCT GAA CAT TAG TCC
~C6ONH2
10031.87 10037.30
30-mer linear 43
Me2C=NOC6~ GAC GCT AAT CTT CTA 10152.06 10152.49
Me2C=NOC6 GCT GAA CAT TAG TCC
~C6ON=CMe2
Cap30-mer linear
44
Circularized with Benzene-1,3,5-
tricarboxaldehyde
10139.97 10139.82
Bi-cyclic 30-mer
45
[a]~C6ONH2 or NH2OC6~ correspond to HOCH2C(CH2OH)(CH3)-CH2O(CH2)6-ONH2
conjugated to the oligonucleotide through a phosphodiester linkage at the 3’- and 5’-end
respectively, [b]Cap means that aminooxy functions were capped with acetone.
The circularization of longer oligonucleotides (21, 24 and 31-mer) was performed with the
terephthalaldehyde (Schemes 6 and S1). The 24-mer 31 is able to form a hairpin with a T4 loop
while the two others (S31_21mer, S31_31mer) are not complementary. The synthesis of the linear
oligonucleotides was performed as described above. The circularization was checked after 60
min by MALDI-TOF MS showing the full cyclization affording 33 S3321mer and S3331mer (Table
1, Fig. S5-S8). Even for long oligonucleotides it was not necessary to use a template for their
circularization. Each of them was purified by C18 reverse phase HPLC.
After the elongation and deprotection of the 24-mer forming an hairpin structure 31, one part
was capped with acetone to further study the melting temperature of the linear hairpin 32 versus
the circular hairpin or dumbbell structure 33 (Scheme 6, Fig. S9-S11).
11
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10.1002/ejoc.201701317
European Journal of Organic Chemistry
O
P
O
Cne
O
P
O
O
GAC GCT AAT C TTTT G ATT AGC GTC
O
O Cne
O
O
SPOS with
and
O
O
8
T
3
A, G, C,
O
N
O
O
O
N
O
O
O
O
30
.H
NH2NH2 2O:AcOH:Pyridine
NH4OH
1)
2)
HO
-O
O
NH2
O
P
O
O
O
T
G ATT AGC GTC
C TAA TCG CAG
O
T
T
O-
NH2
T
P
O
O
O
O
O
H
H
O
24-mer linear
O
31
NH4+AcO-
O
4.2
pH
H
2O
28c
HO
HO
-O
P
-O
O
N
O
N
O
O
P
O
O
H
O
O
O
T
T
G ATT AGC GTC
C TAA TCG CAG
O
G ATT AGC GTC
C TAA TCG CAG
O
T
T
T
T
O-
N
T
O-
N
T
O
P
P
O
O
O
O
H
O
O
O
O
Cap24-mer
24-mer circular dumbbell
O
O
33
32
Scheme 6. Synthesis of capped 24-mer and 24-mer circular dumbbell.
Then, a dumbbell structure with a fluorescent dye was circularized. To this end, we combined
two click reactions corresponding to the oxime ligation for circularization and copper catalyzed
azide alkyne cycloaddition (CuAAC) for the conjugation with dansyl propylazide 37. We
previously showed that both reactions are orthogonal under certain conditions.[41-42] The
oligonucleotide was synthesized from solid support 8 and the phosphoramidite compound 34
exhibiting a propargyl function was introduced in the loop. The sequence was finished with the
phosphoramidite 3 (Scheme 7). The CuAAC was performed on solid-supported oligonucleotide
36, with dansyl propylazide 37 using CuSO4 and sodium ascorbate under microwaves
assistance[43] affording 38. The phthalimide groups were removed by hydrazine treatment and
the dansyl linear oligonucleotide 39 was obtained after ammonia treatment. One part of it was
treated with acetone to form the capped linear entity 40. On another part, terephathalaldehyde
was added in ammonium acetate buffer leading to the circularized oligonucleotide 41 as
confirmed by MALDI-TOF MS and HPLC analyses. Pure 41 was isolated by HPLC (Fig. S12-
S14).
12
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10.1002/ejoc.201701317
European Journal of Organic Chemistry
O
DMTr
O
O
TTG ATT AGC GTC
O
P
O
Cne
O
O
O
SPOS with
O
P
SPOS
with
O
P
O
TTG ATT AGC GTC
O
O
O
Cne
O
O
8
T
A, G, C,
O
Cne
O
O
O
O
DMTr
O
O
N
O
SPOS with
and
N
O
O
P
O
O
T
3
A, G, C,
O
O
N
(iPr)2
Cne
34
35
N
H
O
S
N
O
P
O
Cne
N
O
O
O
P
O
Cne
GAC GCT AAT CT
O
O
S
O
O
P
O
GAC GCT AAT CT
O
P
O
O
O
O
O
N
N
HN
N3
O
O
O
O
O
O
N
O
37
O
O
Cne
O
O
O
Cne
TTG ATT AGC GTC
O
O
P
O
P
O
O
P
O Cne
O
P
TTG ATT AGC GTC
O
O
O
Cne
O
O
O
N
Cne
O
O
CuSO4
Na Ascorbate
2O MeOH
O
O
N
Cne
O
O
38
O
O
O
N
O
H
O
O
N
O
MW
O
O
36
O
O
.H
NH2NH2 2O:AcOH:Pyridine
NH4OH
1)
2)
O
H
O
HO
-O
P
O-
P
O
NH2
N
N
N
N
O
O
O
O
O
S
O
O
TTG ATT AGC GTC
TC TAA TCG CAG
HO
O
H
HN
O
O
O
28c
O
O-
NH2
N
-O
P
O
P
O
O-
P
O
N
P
O
N
N
N
O-
O
O
O
H
O
O
O
O
O
S
O
O
O
TTG ATT AGC GTC
TC TAA TCG CAG
O
HN
O
O
NH4+AcO-
39
O
O
O-
N
O
P
O
4.2
pH
P
O
NH4+AcO-
4.2
O-
O
O
O
H
O
pH
HO
O
41
-O
P
O-
P
O
N
N
N
N
N
O
O
O
O
O
S
O
O
TTG ATT AGC GTC
TC TAA TCG CAG
O
HN
O
O
O
O
O-
N
O
P
O
P
O
O-
O
O
O
40
O
Scheme 7. Synthesis of dansyl-23-mer dumbbell.
Melting temperatures (Tm) of capped linear 24-mer 32 and 24-mer circular dumbbell 33 as well
as the dansyl conjugates as linear 40 and cyclic 41 oligonucleotides were measured. For both
linear structures a Tm of 77 °C was found while the circular structures displayed a Tm superior
to 95°C. Thus the cyclization increases the Tm by more than 18°C.
Bicyclic DNA
Finally, to extend the scope of the cyclization by oxime ligation, a bicyclic oligonucleotide was
synthesized applying a triple oxime ligation. Starting from solid support 8 a first 15-mer
oligonucleotide sequence was elongated and then phosphoramidite 6 was coupled and a second
15-mer oligonucleotide was elongated and phosphoramidite 6 was coupled affording
construction 42 (Scheme 8). A two-step deprotection was performed leading to oligonucleotide
43 with three aminooxy functions (Fig S15). One part was treated with acetone and another part
was bi-cyclized using benzene-1,3,5-tricarboxaldehyde affording the tri-capped sequence 44
and the bi-cyclic oligonucleotide 45, respectively (Fig. 4). Both were purified by C18 HPLC and
characterized by MALDI-TOF MS (Fig. 4). For the linear structure 44 a single peak was
13
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10.1002/ejoc.201701317
European Journal of Organic Chemistry
obtained while for the bi-cyclic structure 45, HPLC profile shows three main peaks due to the
presence of the E and Z isomers for each oxime motif. For both, only the ions corresponding to
the expected linear and bi-cyclic oligonucleotides were observed by MS.
DMTr
O
O
O
O
N
O
O
8
O
and
6
SPOS with
T
A, G, C,
O
GCT GAA CAT TAG TCC
O
GAC GCT AAT CTT CTA
O
O
O
O
O
O
OH
O
N
O
O
N
O
O
O
O
O
N
O
O
42
2.H
NH OH
4
NH NH 2O:AcOH:Pyridine
1)
2)
2
O
GCT GAA CAT TAG TCC
O
GAC GCT AAT CTT CTA
O
O
OH
O
O
OH
O
NH2
O
O
NH2
NH2
O
43
O
H
NH4+AcO-
4.2
pH
O
O
O
H
H
O
O
GCT
CTA
O
H
O
GCT GAA CAT TAG TCC
O
CTT
GAA
GAC GCT AAT CTT CTA
O
AAT
O
OH
CAT
O
O
GCT
5'
TAG
GAC
O
OH
O
O
N
TCC
O
N
N
O
O
O
N
3'
O
HO
N
H
N
O
O
O
H
OH
44
45
Scheme 8. Synthesis of a bicyclic oligonucleotide
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10.1002/ejoc.201701317
European Journal of Organic Chemistry
Figure 4. HPLC profiles and MALDI-TOF MS of (top) acetone-capped linear oligonucleotide
44 and (bottom) bicyclic oligonucleotide 45.
CONCLUSION
The use of the phosphoramidites 3 and 6 and of the solid support 8 bearing a phthalimide-oxy
group allowed the conjugation of oligonucleotide at the 3' and 5'-extremities with aldehyde
derivatives through the formation of an oxime. Furthermore, we demonstrated that
oligonucleotides exhibiting aminooxy at both ends were efficiency circularized by means of
either aliphatic or aromatic dialdehyde by a bis-oxime ligation. Finally, a bis-cyclization can be
performed starting from an oligonucleotide exhibiting three aminooxy functions one at both
ends and one in the middle of the sequence. This bis-circularized was carried out by reaction of
a tri-aldehyde leading to a tris-oxime ligation. According to this strategy the multiple oxime
ligation is an effective method to make DNA-based objects.
EXPERIMENTAL PROCEDURE
15
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10.1002/ejoc.201701317
European Journal of Organic Chemistry
All commercial chemicals were reagent grade and were used without further purification. DNA
synthesis reagents and phosphoramidites are commercially available. Flash column
1
13
chromatography was performed on silica gel 60 (40-63 µm). H NMR (300-400 MHz), C
NMR spectra (100 MHz), and 31P NMR (121 MHz) were recorded in the stated solvent at room
temperature unless otherwise specified. The compounds 1[37] and 4[39] were prepared according
to literature protocols.
8-phthalimidooxy-3,6-dioxaoctanol 2. To a solution of N-hydroxyphthalimide (700 mg, 4
mmol) and 8-tosyloxy-3,6-dioxaoctanol 1[37] (610 mg, 2 mmol) in DMF (20 ml) K2CO3 (550
mg, 4 mmol) was added. The red mixture was heated under microwaves at 60 °C for 2 h. After
filtration, the DMF solution was evaporated. The dark residue was diluted with CH2Cl2 (100
ml) and washed by water (2 x 100 ml). The organic layer was dried over Na2SO4 and evaporated
under vacuum. The residue was purified by silica gel chromatography (0 to 40% acetone in
CH2Cl2), affording compound 2 (379 mg, 64 %) as a red oil. TLC (2.5 % MeOH in CH2Cl2,
twice elutions) Rf: 0.40, 1H-NMR (CDCl3, 300 MHz): δ 2.72 (sb, 1H, OH), 3.48-3.65 (m, 8H,
-O-CH2CH2-O-, -CH2-O-, -O-CH2-), 3.81-3.84 (m, 2H, CH2-OH), 4.32-4.35 (m, 2H, N-O-CH2-
), 7.70-7.80 (m, 4H, phth). 13C-NMR (CDCl3, 100 MHz): δ 61.8 (-CH2-OH), 69.2, 70.3, 70.8
(-O-CH2-CH2-O-), 72.5 (-CH2O-Phth), 123.5 (CAr), 128.9 (CAr, Cq), 134.5 (CAr), 163.5
(C=O). HRMS (ESI/Q-TOF) m/z calcd for C14H18NO6 [M+H]+, 296.1134 found = 296.1132.
8-phthalimidooxy-3,6-dioxaoctanol-[(2-cyanoethyl)-(N,N-diisopropyl)phosphoramidite] 3.
To the solution of 2 (650 mg, 2.2 mmol) in dry CH2Cl2 (20 ml) were added successively DIEA
(453 µl, 2.6 mmol) and 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (511 µl, 2.2
mmol). The mixture was stirred under magnetic stirring for 1 hour at room temperature. Then,
the reaction was quenched by addition of 500 µl of water and diluted with CH2Cl2 (150 ml).
The organic layer was washed with NaHCO3 (100 mL), dried over Na2SO4, and evaporated.
The crude product was purified by silica gel chromatography (0 to 60% AcOEt in cyclohexane
containing 4% Et3N) to afford compound 3 (823 mg, 76%) as a clear oil. TLC (AcOEt/Et3N,
9:1, v/v) Rf : 0.40, 1H-NMR (CDCl3, 400 MHz): δ 1.16 (t, 12H, J=6.4Hz, Isopropyl), 2.64 (t,
2H, J=6.5Hz, -CH2CN), 3.55-3.88 (m, 14H, -CH-,-CH2-OP, O-CH2-CH2-O, -O-CH2-), 4.35-
4.37 (m, 2H, phthN-O-CH2-), 7.72-7.84 (m, 4H, phth).13C-NMR (CDCl3, 100 MHz): δ 20.3 (-
CH2-CN), 24.6, 24.6, 24.6, 24.6, (CH3.), 43.1, 43.1, (-CH-, iPr), 58.5, 62.5, 69.3, 70.5, 70.8,
71.2 (-CH2-O-), 77.2 (PhthN-O-CH2-), 117.8 (-CN), 123.5, 123.5 (CAr), 129.0, 129.0 (CAr),
16
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10.1002/ejoc.201701317
European Journal of Organic Chemistry
134.4, 134.4 (CAr.), 163.4, 163.4 (-C=O). 31P-NMR (CDCl3, 162 MHz): δ 148.57ppm. HRMS
(ESI/Q-TOF) m/z calcd for C23H37N3O8P [M+H2O+H]+, 514.2318 found = 514.2321.
2-(4,4’-Dimethoxytrityloxymethyl)-2-methyl-3-(6-phthalimidooxyhexyloxy)-propanol 5.
To a solution of 2-(4,4’-dimethoxytrityloxymethyl)-2-methyl-3-(6-bromohexyloxy)-propanol
4[39] (700 mg, 1.2 mmol) in DMF anhydrous (15 ml) was added N-hydroxyphthalimide (340
mg, 2 mmol) and K2CO3 (550 mg, 4 mmol). The red mixture was heated under microwaves for
60 min at 60°C. After filtration and concentration, the residue was diluted with CH2Cl2 (100
ml), washed with brine (2 × 70 mL), dried over Na2SO4, and evaporated. The crude product
was purified by silica gel column chromatography (10 to 30% AcOEt in cyclohexane) to afford
title compound 5 (460 mg, 57%) as a clear oil. TLC (cyclohexane/AcOEt 7:3 v/v) Rf : 0.30, 1H
NMR (CDCl3, 300 MHz): 0.89 (s, 3 H, CH3); 1.36-1.63 (m, 6 H, -CH2-), 1.73-1.83 (m, 2 H, -
CH2-), 2.87 (bs, 1H, OH), 3.09 (s, 2 H, -CH2-ODmtr), 3.40-3.56 (m, 6 H, -O-CH2-, -CH2-O-
CH2-), 3.77 (s, 6 H, OCH3), 4.19 (2H, -CH2-O-phtha, J = 6.6 Hz), 6.80-6.83 (m, 4 H, Dmtr),
7.17-7.45 (m, 9 H, Dmtr), 7.69-7.83 (m, 4 H, phth). 13C NMR (CDCl3, 100 MHz): δ 18.0 (CH3),
25.5, 25.9, 28.1, 29.5 (-CH2-), 40.8 (Cq), 55.2, 55.2 (-OCH3), 66.4, 69.6, 71.7, 76.1 (-CH2-O-
CH2-, HO-CH2-, -CH2-O-Dmtr), 78.5 (-CH2-O-phth), 85.9 (Cq), 113.0, 113.0, 113.0, 113.0
(CAr.Dmtr), 123.5, 123.5, 126.7, 126.7, 127.8, 127.8, 128.2, 128.2, 129.0, 130.2, 130.2, 130.2,
130.2 (CAr), 134.5, 134.5 (CAr.Phth), 136.2, 136.2, 145.1, 158.4, 158.4 (Cq, Dmtr), 163.7,
163.7 (CO). HRMS (ESI/Q-TOF) m/z calcd for C40H45N1O8Na [M+Na]+, 690.3034 found
690.3040.
2-(4,4’-Dimethoxytrityloxymethyl)-2-methyl-3-(6-phthalimidooxyhexyloxy)-propanol-
[(2-cyanoethyl)-N,N-diisopropyl-phosphoramidite] 6. To a solution of 5 (460 mg, 0.69
mmol) and diisopropylethylamine (180 µl, 1.0 mmol) in CH2Cl2, anhydrous 2-cyanoethyl-N,N-
diisopropylchlorophosphoramidite (154 µl, 0.69 mmol) was added. The mixture was stirred for
3 hours under magnetic stirring at room temperature. Then, the reaction was quenched by
addition of 500 µl of water and diluted with CH2Cl2 (100 ml). The organic layer was washed
with NaHCO3 (100 mL), dried over Na2SO4, and evaporated. The crude product was purified
by silica gel column chromatography (10 to 30% AcOEt in cyclohexane containing 4% Et3N)
to afford a compound 6 (370 mg, 62%) as a clear oil. TLC (cyclohexane/AcOEt/Et3N 7:2/1
v/v/v) Rf : 0.50. 1H NMR (CDCl3, 400 MHz) : 0.98 (d, 3 H, J = 9.5 Hz, CH3); 1.11–1.18 (m, 12
H, 2 CH(CH3)2), 1.36-1.42 (m, 4 H, -CH2-), 1.47-1.60 (m, 2 H, -CH2-), 1.76-1.83 (m, 2 H, -
CH2-), 2.54 (dt, 2H, J1 = 3.6 Hz, J2 = 6.5 Hz, CH2-CN), 2.98-3.05 (m, 2 H, -CH2-ODmtr), 3.34-
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European Journal of Organic Chemistry
3.75 (m, 10 H, -CH-, -O-CH2-, -CH2-O-CH2-), 3.8 (s, 6 H, OCH3), 4.21 (2H, -CH2-O-phth, J =
13
6.7 Hz), 6.81-6.84 (m, 4 H, Dmtr), 7.18-7.45 (m, 9 H, Dmtr), 7.74-7.85 (m, 4 H, phtha). C
NMR (CDCl3, 100 MHz) : δ 17.8 (CH3), 20.4 (-CH2-CN), 24.6, 24.6, 24.6, 24.6 (CH3,iPr), 25.5,
25.9, 28.1, 29.6 (-CH2-), 41.3 (Cq), 43.1, 43.1, (-CH-, iPr), 55.2, 55.2 (-OCH3), 58.3, 65.1, 66.5,
71.4, 73.4 (-O-CH2-), 78.5 (-CH2-O-pht), 85.4 (Cq), 112.9, 112.9, 112.9, 112.9, (CAr), 117.7
(-CN), 123.5, 123.5, 126.6, 126.6, 127.6, 127.6, 128.3, 128.3, 129.0, 130.2, 130.2, 130.2, 130.2,
134.4, 134.4, (CAr), 136.6, 136.6, 145.4, 158.3, 158.3 (Cq), 163.7, 163.7 (CO). 31P-NMR
(CDCl3, 162 MHz): δ 147.42-147.54 ppm. HRMS (ESI/Q-TOF) m/z calcd for C49H63N3O9P
[M+H]+, 868.4302 found 868.4296.
2-(4,4’-Dimethoxytrityloxymethyl)-2-methyl-3-(6-phthalimidooxyhexyloxy)-propyl
(succinate-LCAA-CPG)
8.
Succinate-LCAA
CPG
7
(750
g),
2-(4,4’-
dimethoxytrityloxymethyl)-2-methyl-3-(6-bromohexyloxy)-propanol 5 (350 mg, 0.52 mmol),
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (460 mg, 2.4 mmol),
DMAP (60 mg, 0.5 mmol), and Et3N (220 µL, 1.5 mmol) were gently shaken in anhydrous
pyridine (6 mL) for 48 h at room temperature. Then, the solid support was filtered off, washed
with CH2Cl2, and dried. A capping step with standard Cap A and Cap B solutions (1:1, v/v) was
applied for 2 h, and the solid support was filtered off, washed with CH2Cl2, and dried. Trityl
assay indicated a loading of 35 µmol/g.
Synthesis of oligonucleotides. The oligonucleotides were synthesized at the 1μmol-scale on a
DNA synthesizer (ABI 394) by standard phosphoramidite chemistry. For the coupling step,
benzylmercaptotetrazole (BMT) was used as activator (0.3 M in anhydrous CH3CN), with a
coupling time of 30 s for standard nucleosides phosphoramidites (0.075 M in anhydrous
CH3CN) and of 60 s for phosphoramidites 3 and 6 (0.1 M in anhydrous CH3CN). The capping
step was performed with acetic anhydride using commercial solutions (Cap A:
Ac2O/pyridine/THF, 10:10:80, v/v/v and Cap B: 10% N-methylimidazole in THF) for 15 s.
Oxidation was performed for 15 s using 0.1 M I2, THF/pyridine/water 90:5:5. Detritylation was
performed with 3% trichloroacetic acid (TCA) in CH2Cl2 for 65 s.
General procedure for deprotection of solid-supported oligonucleotides: The CPG beads
bearing modified oligonucleotides were treated with a solution of NH2NH2.H2O/Pyridine/acetic
acid 0.124:4:1 v/v/v for 30 min at room temperature. The solution was withdrawn and the beads
were washed with pyridine, methanol and acetonitrile. The beads were transferred to a 4 mL
18
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10.1002/ejoc.201701317
European Journal of Organic Chemistry
screw top vial and treated with 2 mL of concentrated aqueous ammonia at 55°C for 5 h. For
each compound, the supernatant was withdrawn and evaporated to dryness. The residue was
dissolved in water for subsequent analysis and characterization.
Capping with acetone. To an aliquot of aminooxy oligonucleotide in water a drop of acetone
was added. After 15 min, the solution was concentrated and the resulting oligonucleotide was
analysed by HPLC and characterized by MS.
Protocol for circularization. Bis-aminooxy oligonucleotide (0.1 µmol) was dissolved in 1.0
mL of ammonium acetate buffer (400 mM pH 4.5) and terephthalate dialdehyde (0.12 µmol)
was added. The resulting mixture was stirred for 1h at rt. The crude was applied on a NAP10
column for desalting.
Protocol for bis-circularization. tris-aminooxy oligonucleotide (0.1 µmol) was dissolved in
1.0 mL of ammonium acetate buffer (400 mM pH 4.5) and benzene-1,3,5-tricarboxaldehyde
(0.12 µmol) was added. The resulting mixture was stirred for 1h at rt. The crude was applied
on a NAP10 column for desalting.
Protocol for CuAAC. Solid-supported oligonucleotide 0.5 µmol with CuSO4 0.2 µmol (5 µL
of freshly prepared 40 mM aqueous solution) sodium ascorbate 1 µmol (10 µL of 100 mM
aqueous solution), dansyl propyl azide 1 µmol, 150 µL of water and 150 µL of methanol were
added in a sealed tube and heated at 60 °C under microwaves assistance for 1 h. Beads were
filtered off and washed with water and methanol and dried.
Melting temperature: Tm values of oligonucleotides were measured at a concentration of 0.5
µM in 10 mM sodium cacodylate, 100 mM NaCl, pH 7 buffer. A linear gradient of temperature
of 0.5 °C/min was applied from 95°C to 60°C and 60°C to 95°C. UV absorbance was read at
260 nm.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the website at DOI:
NMR spectra for 2, 3, 5 and 6. HPLC and MALDI-TOF MS profiles.
19
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10.1002/ejoc.201701317
European Journal of Organic Chemistry
ACKNOWLEDGMENTS
We thank IBMM for financial support through AAP-IBMM2017 funding program. FM is
member of Inserm.
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