4-Oxoheptanedioic acid: an orthogonal linker for solid-phase synthesis of base-sensitive oligonucleotides

Article abstract of DOI:10.1016/j.tetlet.2008.04.131

l,6-Dioxaspiro[4,4]nonane-2,7-dione has been found to react readily with alcohols in the presence of 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU). The 4-oxoheptanedioic acid tether obtained bears (l) a free carboxy group, which enables anchoring to aminoalkylated resins, and (2) a 4-oxobutanoate structural motif, which allows release of the target alcohol by a mild hydrazinium acetate treatment. To demonstrate the applicability of the procedure, nucleosides have been derivatized with this simple difunctional linker arm, anchored to a solid phase and subjected to synthesis and subsequent release of oligonucleotides bearing a base-sensitive biodegradable phosphate protection.

Full text of DOI:10.1016/j.tetlet.2008.04.131

Tetrahedron Letters 49 (2008) 4119–4121
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: http://www.elsevier.com/locate/tetlet
4-Oxoheptanedioic acid: an orthogonal linker for solid-phase synthesis
of base-sensitive oligonucleotides
*
Anna Leisvuori, Päivi Poijärvi-Virta, Pasi Virta , Harri Lönnberg
Department of Chemistry, University of Turku, Vatselankatu 2, FIN-20014 Turku, Finland
a r t i c l e i n f o
a b s t r a c t
Article history:
l,6-Dioxaspiro[4,4]nonane-2,7-dione has been found to react readily with alcohols in the presence of
1,8-diazabicyclo[5,4,0]undec-7-ene (DBU). The 4-oxoheptanedioic acid tether obtained bears (l) a free
carboxy group, which enables anchoring to aminoalkylated resins, and (2) a 4-oxobutanoate structural
motif, which allows release of the target alcohol by a mild hydrazinium acetate treatment. To demon-
strate the applicability of the procedure, nucleosides have been derivatized with this simple difunctional
linker arm, anchored to a solid phase and subjected to synthesis and subsequent release of oligonucleo-
tides bearing a base-sensitive biodegradable phosphate protection.
Received 3 March 2008
Revised 17 April 2008
Accepted 23 April 2008
Available online 27 April 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Synthesis of acid- and base-labile conjugates of oligonucleo-
tides, oligosaccharides or small molecular alcohols on a solid sup-
port may require utilization of an orthogonal linker which (1)
anchors the molecule via a hydroxyl group, (2) withstands various
chemical manipulations, but (3) may still be cleaved under mild
conditions. Antisense oligonucleotides bearing biodegradable
phosphate protections offer an illustrative example.1 The biode-
gradability is usually based on enzymatically hydrolyzable ester
linkages which do not withstand conventional ammonolytic
deprotection and, hence, an orthogonally cleavable linker and
orthogonally removable nucleobase protections have to be used.2
Unfortunately, there are few such linkers which are readily avail-
able. Multistep derivatization of the 30-terminal nucleoside has to
be carried out before attachment to the solid support. In addition,
the release is often slow or prone to side reactions. In some cases, a
base-labile hydroquinone-O,O-diacetic acid (‘Q-linker’)3 offers a
viable alternative, since its cleavage using potassium carbonate
in methanol is sufficiently fast to leave the ester functions intact.
The Q-linker is commercially available and compatible with the
methods generally applied to the attachment of the 30-terminal
nucleoside to solid supports. We herein describe a novel, easily
available difunctional handle, which meets the requirements of
orthogonality.
linkage, which enables release from the support by mild hydrazi-
nium acetate treatment. The latter reaction is used extensively
for removal of levulinoyl protections4 as 6-methyl-4,5-dihydropyr-
idazin-3(2H)-one,5 but surprisingly, a similar cyclative cleavage
has not been applied to linker chemistry. Hydrazinium acetate
treatment has only been used for release of oligosaccharides teth-
ered by a succinyl linker arm via the anomeric hydroxyl function,6
but such a release cannot be extended to succinates of non-ano-
meric hydroxyl groups. The applicability of the present 4-oxohep-
tanedioic acid linker arm has been demonstrated by the synthesis
of oligonucleotides bearing a base-sensitive biodegradable phos-
phate protection. The procedure for the derivatization and immo-
bilization of the 30-terminal nucleoside is exactly the same as
with the succinyl linker arm, but the cleavage may be performed
under conditions which leave ester functions intact. In contrast
to primary succinamides, which may undergo succinimide forma-
tion and, hence, result in a premature cleavage,7 the present linker
is stable in the presence of a strong non-nucleophilic Brønsted
base.
The reaction between 1,6-dioxaspiro[4,4] nonane-2,7-dione and
a nucleoside has been optimized with 50-O-DMTr-thymidine in the
presence of various base/solvent systems [DBU/DMF, DBU/dioxane,
4-dimethylaminopyridine (DMAP)/pyridine, DMAP/dioxane, NaH/
DMF]. Among these systems, DBU in dioxane turned out to be
the method of choice giving the desired tethered nucleoside 2a8
in 75% yield (Scheme 1). The reaction occurred readily also in the
presence of NaH in DMF, but this treatment cannot be applied to
derivatization of nucleosides having an exocyclic amino group.
The other organic bases tested (DMAP and pyridine) turned out
to be ineffective for the reaction. The prefabricated handle 2a
may be anchored to a long chain alkylamine controlled pore glass
The linker precursor, viz. 1,6-dioxaspiro[4,4]nonane-2,7-dione,
is a cheap commercially available reagent, which reacts readily
with alcohols in the presence of the strong organic base, DBU.
The resulting tether bears (1) a free carboxy function, which allows
anchoring of the prefabricated terminal unit to the support via an
amide linkage and (2) a keto group at the 4 position to the ester
* Corresponding author. Tel.: +358 2 333 6777.
E-mail address: pasi.virta@utu.fi (P. Virta).
(LCAA-CPG, 0.10 mmol gꢀ1
) in a conventional manner using
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.04.131

4120
A. Leisvuori et al. / Tetrahedron Letters 49 (2008) 4119–4121
DMTrO
DMTrO
B
B
O
O
O
O
i
+
O
O
O
OH
1a and 1b
O
R
O
ii
O
2a
3a
2b:
3b:
and
and
R = OH
R = HN
iii, iv
iii, v, iv
O
N
H
N
3a
3b
oligonucleotide
O
Thy
1-3a: B = Thy
*
TT
=
O
1-3b: B = AdeAlloc
4-6
O
O
O
O
PivO
O
O
P
O
S
vi
Thy
O
O
H
N
4 and 7:sequence= 5'-TTT TTT-3'
OEt
2
O
*
5
8:
TT
O
and sequence= 5'-TT
TT-3'
H
N
+
oligonucleotide
7-9
*
O P
6 and 9:sequence= 5'-CC TT ACA-3'
Scheme 1. Reagents and conditions: (i) dioxaspiro[4,4]nonane-2,7-dione (structure also shown, 2 equiv), DBU (2 equiv), dioxane, 2a: 75%, 2b: 74%; (ii) LCAA-CPG, 2a or 2b
(1.0 equiv), HOSu (1.0 equiv), DIC (1.0 equiv), pyridine, 15 h at 25 °C, 3a: 17 lmol gꢀ1, 3b: 19 lmol gꢀ1; (iii) phosphoramidite chemistry following the standard RNA coupling
protocol; (iv) (PPh3)4Pd0 (cat.), 5% phenylsilane in CH2Cl2 (v/v), N2 atm, 1 h at 25 °C; (v) 1.5% DBU in acetonitrile, 1 h at 25 °C; (vi) NH2NH2ꢁH2O/AcOH/pyridine (0.0124/1/4, v/
v/v), 1 h at 25 °C.
N-hydroxysuccinimide/diisopropylcarbodiimide (HOSu/DIC)-acti-
vation (3a, 17 lmol gꢀ1). The unreacted amino groups on the
80
support were capped using acetic anhydride. The more powerful
coupling reagents, N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]-
pyridin-1-ylmethylene]-N-methylmethan aminium hexafluoro-
phosphate N-oxide (HATU)9 and 1-[bis(dimethylamino)methy-
lene]-1H-benzotriazolium tetrafluoroborate 3-oxide (TBTU),10
were also tested. With these reagents an increased acylation effi-
ciency was obtained, but a high loading (40–60 lmol gꢀ1) was
found to retard the DBU-promoted b-elimination used for removal
of the cyanoethyl groups (cf. below).
60
40
20
0
The cleavage of the linker was attempted with various hydrazi-
nium acetate treatments for the release of support-bound DMTr-dT
(3a) and T6 (4). The progress of the release was monitored by RP
HPLC and UV spectroscopy following both the increased amount
of released T6 (7) in the mixture and the decreased DMTr cation
response on the support (3a). The best result was obtained with
a mixture of 50 mmol Lꢀ1 hydrazinium acetate (NH2NH2ꢁH2O/
AcOH/pyridine, 0.0124:1:/4, v/v/v, at 25 °C), which provided prac-
tically complete cleavage of the linker in 1 h (see ꢂ in Fig. 1). The
hydrazinium acetate concentration was only one-tenth of that gen-
erally used for the removal of the levulinoyl group.5 It should be
noted that a trace amount was released over a longer period. This
was attributed to the potential inhomogeneity of the resin. The
reaction occurred also in the absence of pyridine (50 mmol Lꢀ1
NH2NH2ꢁAcOH in water), but with a decreased rate (see N in Fig.
1). Interestingly, an equally slow cleavage rate was obtained in
50 mmol Lꢀ1 NH2NH2ꢁH2O in 0.1 mol Lꢀ1 aqueous triethylammo-
nium acetate. The advantage of these latter procedures is that
the cleavage mixtures may be subjected to RP HPLC without
workup.
The stability of the linker in a mixture of 1.5% DBU in dry ace-
tonitrile (1 h at 25 °C) was followed by the DMTr-cation assay of
3a and the solid-supported oligonucleotides (cf. 4–6). Upon this
treatment, which may be used for on-resin removal of, for exam-
ple, O-cyanoethyl and O-4-nitrophenylethyl protections,11 no
decrease of DMTr-loadings was observed.
The Q-linker and a photo-labile 5-(4-hydroxymethyl-6-meth-
oxy-3-nitrophenoxy)butanoyl linker had previously been used for
the solid phase synthesis of oligonucleotides bearing a biodegrad-
able 2,2-bis(ethoxycarbonyl) 3-(pivaloyloxy)propyl phosphate
0,0
0,5
1,0
1,5
2,0
0.5 1.0
1.5 2.0
cleavage time (h)
Figure 1. Cleavage of anchored T6 using different hydrazinium acetate treatments.
Notation: 50 mmol Lꢀ1 NH2NH2ꢁH2O in AcOH–pyridine (1:4, v/v), at 25 °C (ꢂ),
50 mmol Lꢀ1 NH2NH2ꢁAcOH in H2O (N).
protection.12 The applicability of the present linker for this purpose
was demonstrated by the synthesis of oligonucleotides 8 and 9
bearing the same protection. The required dimeric building block,
that is, 50-O-(4,40-dimethoxytrityl)-(R
P,SP-OP-[2,2-bis(ethoxycar-
bonyl)-3-(pivaloyloxy)propyl]-P-thiothymidylyl-(30,50)-thymidine
30-[O-(2-cyanoethyl)(N,N-diisopropyl)]phosphoramidite], incorpo-
*
rated into the oligonucleotides (cf. T T in Scheme 1, was prepared
as previously described.12 For the synthesis of the hetero oligonu-
cleotide (9), allyloxycarbonyl (Alloc) was used for exocyclic amino
group protection.13 The corresponding Alloc group protected cya-
noethyl phosphoramidites were prepared according to the litera-
ture.13 The N4-Alloc-50-O-DMTr-dA modified resin 3b was
prepared as described for dT (3a) above.7 The prefabricated handle
2b was obtained in a 74% yield and a loading of 19 lmol gꢀ1 was
obtained for the resin 3b. Due to the somewhat low coupling effi-
ciency of the dimeric building block, oligonucleotides 5 and 6 were
assembled following standard RNA coupling protocol. The Alloc
protections of the hetero oligonucleotide 6 were removed by
(PPh3)4Pd0 catalyzed treatment in a mixture of 5% phenylsilane

A. Leisvuori et al. / Tetrahedron Letters 49 (2008) 4119–4121
4121
age may be performed with hydrazinium acetate, which leaves
esters intact. In addition, the present linker is orthogonal with
the protecting groups, which may be removed by b-elimination.
Acknowledgement
The financial support from the Academy of Finland is gratefully
acknowledged.
References and notes
1. Poijärvi-Virta, P.; Lönnberg, H. Curr. Med. Chem. 2006, 13, 3441–3465.
2. (a) Alvarez, K.; Vasseur, J.-J.; Beltran, T.; Imbach, J.-L. J. Org. Chem. 1999, 64,
6319–6328; (b) Spinelli, N.; Meyer, A.; Hayakawa, Y.; Imbach, J.-L.; Vasseur, J.-J.
Eur. J. Org. Chem. 2002, 49–56; (c) Guerlavais-Dagland, T.; Meyer, A.; Imbach, J.-
L.; Morvan, F. Eur. J. Org. Chem. 2003, 2327–2335.
3. Pon, R. T.; Yu, S. Nucleic Acid Res. 1997, 25, 3629–3635.
4. Hassner, A.; Strand, G.; Rubinstein, M.; Patchornik, A. J. Am. Chem. Soc. 1975, 97,
1614–1615.
5. van Boom, J. H.; Burgers, P. M. J. Tetrahedron Lett. 1976, 4875–4878.
6. Excoffier, G.; Gagnaire, D.; Utille, J.-P. Carbohydr. Res. 1975, 39, 368–373.
7. Brown, T.; Pritchard, C. E.; Turner, G.; Salisbury, S. A. J. Chem. Soc., Chem.
Commun. 1989, 891–893.
8. Procedure for the preparation of tethered nucleosides (2a and 2b): DMTr-dT (1a,
0.50 g, 0.92 mmol), 1.6-diooxaspiro[4.4]nonane-2.7-dione (0.30 g, 1.9 mmol)
and DBU (0.28 mL, 1.9 mmol) were dissolved in dioxane (0.5 mL). The mixture
was stirred at ambient temperature for 20 h and evaporated to dryness. The
residue was purified by silica gel chromatography (1% pyridine and 10% MeOH
in CH2Cl2) to yield 0.49 g (75%) of the product 2a as a white foam. Compound
2a: 1H NMR (500 MHz, CDCl3) d 9.50 (br s, 1H), 7.65 (s, 1H), 7.40 (m, 2H), 7.33–
7.17 (m, 7H), 6.85 (d, J = 8.8 Hz, 4H), 6.42 (dd, 1H, J = 8.8 Hz, 5.7 Hz), 5.47 (d, 1H,
J = 5.9 Hz), 4.16 (br s, 1H), 3.81 (s, 6H), 3.49 (dd, 1H J = 10.8 Hz, 2.3 Hz), 3.47
(dd, 1H, J = 10.7 Hz, 2.0 Hz), 2.84 (m, 2H), 2.77 (m, 2H), 2.66 (m, 2H), 2.61 (m,
2H), 2.49 (1H, dd, J = 13.9 Hz, 5.6 Hz), 2.43 (m, 1H), 1.36 (s, 3H); 13C NMR
(125 MHz, CDCl3) d 172.3, 164.3, 159.07, 159.05, 151.2, 144.5, 135.9, 135.6,
135.5, 130.44, 130.40, 128.46, 128.35, 127.5, 113.6, 112.3, 87.5, 84.8, 84.2, 76.0,
55.6, 38.1, 37.5, 37.2, 28.6, 11.9; HRMS(ESI): found 739.2258 [M+K]+.
Compound 2b was prepared from 1b as described for 2a. Compound 2b was
obtained as a white foam in 74% yield. Compound 2b: 1H NMR (500 MHz,
CDCl3) d 8.60 (s, 1H), 8.27 (s, 1H), 7.37 (m, 2H), 7.28–7.18 (m, 7H), 6.78 (m, 4H),
6.49 (dd, 1H J = 7.0 Hz, 6.8 Hz), 6.02 (m, 1H), 5.56 (m, 2H), 5.43 (m, 1H), 5.28
(m, 1H), 4.77 (m, 2H), 4.33 (br s, 1H), 3.77 (s, 6H), 3.47 (dd, 1H, J = 10.6 Hz,
4.3 Hz), 3.44 (1H, dd, J = 10.6 Hz, 4.0 Hz), 3.07 (m, 1H), 2.86 (m, 2H), 2.78 (m,
2H), 2.68 (m, 1H), 2.64 (m, 2H), 2.55 (m, 2H); 13C NMR (125 MHz, CDCl3) d
208.7, 173.0, 159.2, 152.8, 152.2, 151.4, 150.3, 145.1, 142.4, 136.1, 132.5, 130.6,
128.7, 128.6, 128.0, 127.3, 122.7, 118.2, 113.3, 87.3, 85.5, 85.0, 75.9, 66.9, 64.2,
55.4, 37.9, 37.5, 37.4, 28.5, 28.4; HRMS(ESI): found 832.2567 [M+K]+.
9. Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397.
Figure 2. RP HPLC chromatograms of the crude product (8 and 9) mixtures, grad-
ient elution from 15% to 35% acetonitrile in 0.1 mol Lꢀ1 Et3N+OAcꢀ buffer over
20 min.
in dichloromethane (1 h at 25 °C, N2 atm).14 The cyanoethyl groups
were removed as described above and the oligonucleotides 5 and 6
were released by treatment with a mixture of 50 mmol Lꢀ1 hydra-
zinium acetate (1 h at 25 °C).15 The excess hydrazinium acetate
was capped by acetone, and the mixtures were filtered. For solubil-
ity reasons, the resins were washed with water, the filtrates were
evaporated to dryness and the resulting oligonucleotides 8 and 9
were then subjected to RP HPLC. Crude RP HPLC chromatograms
of the product mixtures are outlined in Figure 2 (note RP and SP-
stereoisomers). Yields of the oligonucleotides 8 and 9 were 70%
and 60%, respectively. The authenticity of the products was verified
by MS(ESI); 8: found 1031.2 [(M-2)/2]ꢀ2 and 9: found 1170.2 [(M-
2)/2]ꢀ2. For the preparation of these oligonucleotides 8 and 9, the
present linker worked successfully, leaving the 2,2-bis(ethoxycar-
bonyl) 3-(pivaloyloxy)propyl phosphate protection intact. In this
preliminary study the demonstrated compatibility of the linker is
limited to the preparation of short oligonucleotides bearing ester
groups. Further studies with more biologically relevant longer oli-
gonucleotides would be advisable. It may, however, be noteworthy
that the chemistry related to the linker is well known from the lev-
ulinoyl protection, the compatibility of which has been studied
extensively with a wide range of functional groups and automated
oligonucleotide synthesis.
10. Knorr, R.; Tzeciak, A.; Bannworth, W.; Gillessen, D. Tetrahedron Lett. 1989, 30,
1927–1930.
11. Stengele, K.-P.; Pfleiderer, W. Tetrahedron Lett. 1990, 31, 2549–2552.
12. Poijärvi, P.; Heinonen, P.; Virta, P.; Lönnnberg, H. Bioconjugate Chem. 2005, 16,
1564–1571.
13. Hayakawa, Y.; Wakabayashi, S.; Kato, H.; Noyori, R. J. Am. Chem. Soc. 1990, 112,
1691–1696.
14. Virta, P.; Karskela, M.; Lönnberg, H. J. Org. Chem. 2006, 71, 1989–1999.
15. Procedure for the release of oligonucleotides: Resin (4–6, 30 mg, containing
0.5 lmol of the oligonucleotide) was suspended in a mixture of 50 mmol Lꢀ1
hydrazinium acetate (0.5 mL, NH2NH2ꢁH2O/AcOH/pyridine, 0.0124/1/4, v/v/v).
After 1 h mixing (at 25 °C), acetone (0.5 mL) was added and the suspension was
mixed for an additional 15 min. After capping of hydrazine, the resin was
filtered and washed with water (3 ꢃ 0.5 mL). The filtrate was evaporated to
dryness, the residue was dissolved in water, and then the product (7–9)
mixture was subjected to RP HPLC.
In conclusion a novel and very simple orthogonal linker and its
applicability for the synthesis of base-sensitive oligonucleotides
have been described. The procedure for the nucleoside derivatiza-
tion is exactly the same as with the succinyl linker arm, but cleav-