Oligonucleotide mimics for antisense therapeutics: Solution phase and automated solid-support synthesis of MMI linked oligomers

Full text of DOI:10.1021/ja9533959

J. Am. Chem. Soc. 1996, 118, 255-256
Oligonucleotide Mimics for Antisense Therapeutics:
255
Solution Phase and Automated Solid-Support
Synthesis of MMI Linked Oligomers
Franc¸ois Morvan, Yogesh S. Sanghvi,* Michel Perbost,
Jean-Jacques Vasseur, and Laurent Bellon
Medicinal Chemistry Department, Isis Pharmaceuticals
2292 Faraday AVenue, Carlsbad, California 92008
ReceiVed October 6, 1995
Antisense oligonucleotides (AOs) have shown great promise
as agents for inhibiting gene expression.1 In principle, AOs
interfere in a sequence-specific manner with processes such as
translation of mRNA into protein. In recent years, significant
advances have been made in chemical modifications of AOs
that can enhance both their stability and their potency.2 One
of the main focal points of the research has been the complete
replacement of natural phosphodiester (PdO) backbone with
synthetic linkages.3 Among the various surrogates of the PdO
backbone studied in our group, we have selected methylene
(methylimino) (MMI) as a linkage of choice for advanced studies
and for incorporation into AOs.4
The MMI linkage is achiral and neutral, readily incorporated
into AOs, and stable under physiological conditions (Figure 1).
AOs containing MMI linkages hybridize to the complementary
RNA with high affinity and base-pair specificity. NMR and
modeling studies have indicated that the 3′-CH2 group of the
MMI linkage shifted the sugar conformation to a desired 3′-
endo pucker, thus helping the AOs to preorganize into a
preferred A-geometry for duplex formation.5 Biological studies
showed that incorporation of MMI linkages into a phospho-
rothioate (PS) AOs substantially improved the pharmacological
properties of the parent oligomer.6 Our prior incorporation of
the MMI linkage into AOs has been achieved by a nucleosidic
phophoramidite dimer, creating alternate PdO or PdS/MMI
linkages. This procedure, therefore, does not enable the
synthesis of oligonucleosides7a that are uniformly modified with
MMI linkages.
Figure 1. Structure and attributes of the MMI linkage.
can be further automated. As a demonstration, chimeric
oligomers have been assembled on SS utilizing a standard DNA
synthesizer. To prepare an oligonucleoside connected via MMI
linkages only, four essential nucleosidic units (1, 2, 5, 6) were
synthesized.
2′-Deoxy-5′-O-phthalimidonucleosides8 1a-d served as a
precursor for the 3′-terminal unit. The nucleosides 1a-d were
successfully anchored onto the SS (CPG) via a succinyl linker9
in good yield (∼35-40 µmol/g). To avoid the side reaction of
incoming 3′-CHO nucleosides (5 or 6) with unprotected NH2
groups left on the CPG, methylation (HCHO/NaBH3CN/AcOH)
of the SS provided fully protected CPG units 2a-d. Alterna-
tively, CPG units 2a-d can be prepared from the commercial
CPG loaded with 5′-O-DMT deoxynucleosides in three steps.
For example, CPG anchored with 5′-O-DMT thymidine was
treated with acid to remove the DMT group, followed by a
Mitsunobu reaction,10 and capping off the CPG NH2 groups
via methylation provided 2a (30 µmol/g). The bifunctional units
4a-d were prepared from 3′-C-styrene nucleosides11 3a-d.
Mitsunobu reaction12 of 3a-d provided the 5′-O-phthalimido-
3′-C-styrene nucleosides 4a-d in excellent yields. One-pot
oxidative cleavage (OsO4/NaIO4) of 4a-d gave 5a-d, generat-
ing the 3′-CHO functionality. Syntheses of the 5′-terminal units
6a,c,d have been published.11 Preparation of 2′-deoxycytosine
derivative 6b was accomplished via triazolation and amination
procedures.13 Nucleoside 7 was prepared from 3a via 5′-O-
FMOC protection14 and oxidative cleavage of the 3′-C-styrene
group. Phosphitylation14 of 1a furnished 8 in 70% yield.
SP synthesis of T4 was accomplished in the following manner.
Coupling4 of 9 with 5a gave an oxime dimer (12, R ) Phth, R′
) TPS, n ) 1, B ) T), which on hydrazinolysis (H3CNHNH2)
furnished 5′-O-NH2-oxime dimer 14 (R ) NH2, R′ ) TPS, n
) 1, B ) T). Another round of coupling of 5a with 14,
followed by hydrazinolysis, provided an oxime trimer (14, R
) NH2, R′ ) TPS, n ) 2, B ) T), which coupled with 6a to
give an oxime tetramer 15 (n ) 3, B ) T). Reduction of 15
gave 16 (R ) R′ ) TPS, R′′ ) H, n ) 3), which on methylation
followed by TBAF treatment gave MMI tetramer 17 (n ) 3, B
) T) in 79% overall yield. Coupling of 3′-CHO nucleosides
with the 5′-O-NH2 nucleosides was quick and almost quantita-
tive, thus allowing the manual synthesis of 17 in <8 h.
Tetramers T3C and T2CT were assembled in a similar manner
in high yields utilizing appropriate building blocks. The general
This communication reveals a flexible synthetic strategy for
constructing AOs containing the MMI backbone in any desirable
configuration with the PdO and/or PdS backbone. We have
accomplished the synthesis of essential nucleosidic building
blocks (1-8), thus enabling us to construct chimeric7b AOs as
potential drugs. The solution phase (SP) methodology described
herein is simple to manipulate. The couplings are efficient, and
the process is transferable to solid-support (SS) synthesis, which
(1) Selected books and reviews published in 1995: (a) Therapeutic
Applications of Oligonucleotides; Crooke, S. T., Ed.; R. G. Landes Co.:,
Austin, TX, 1995. (b) Carbohydrate Modifications in Antisense Research;
Sanghvi, Y. S., Cook, P. D., Eds.; ACS Symposium Series 580; American
Chemical Society: Washington, DC, 1995. (c) Crooke, S. T. Hematol.
Pathol. 1995, 9, 59. (d) Crooke, S. T. In Burger’s Medicinal Chemistry
and Drug DiscoVery, Vol 1; Wolff, M. E., Ed.; John Wiley: New York,
1995: p 863. (e) Mesmaeker, A. D.; Ha¨ner, R.; Martin, P.; Moser, H. E.;
Acc. Chem. Res. 1995, 28, 366.
(2) Sanghvi, Y. S.; Cook, P. D. In ref 1b; Chapter 1, p 1. Mesmaeker,
A. D.; Altman, K.-H.; Waldner, A.; Wendeborn, S. Curr. Opin. Struct. Biol.
1995, 5, 343.
(3) Sanghvi, Y. S.; Cook, P. D. In Nucleosides and Nucleotides as
Antitumor and AntiViral Agents; Chu, C. K., Baker, D. C., Eds.; Plenum
Press: New York, 1993; p 311.
(4) Vasseur, J.-J.; Debart, F.; Sanghvi, Y. S.; Cook, P. D. J. Am. Chem.
Soc. 1992, 114, 4006.
(8) Perbost, M.; Hoshiko, T.; Morvan, F.; Swayze, E.; Griffey, R. H.;
Sanghvi, Y.S. J. Org. Chem. 1995, 60, 5150.
(9) Pon, R. T. In Protocols for Oligonucleotides and Analogs; Agraval,
S., Ed.; Humana Press: Totowa, NJ, 1993; p 465.
(5) (a) Mohan, V.; Griffey, R. H.; Davis, D. R. Tetrahedron 1995, 51,
8655. (b) In addition, substitution of the O3′ by a CH2 group reduces the
ring gauche effects and may enhance conformational stability. See:
Roughton, A. L.; Portmann, S.; Benner, S. A.; Egli, M. J. Am. Chem. Soc.
1995, 117, 7249.
(6) Sanghvi, Y. S.; Bellon, L.; Morvan, F.; Hoshiko, T.; Swayze, E.;
Cummins, L.; Freier, S.; Dean, N.; Monia, B.; Griffey, R.; Cook, P. D.
Nucleosides Nucleotides 1995, 14, 1087.
(7) (a) We refer to modified oligonucleotides that lack the phosphorus
atom in the backbone linkage as oligonucleosides. (b) Chimeric AOs are
oligomers that contain more than one type of modifications to create a gap
for RNase H activity.
(10) (a) Hughes, D. L. In Organic Reactions; Paquette, L. A., Ed.; John
Wiley: New York, 1992; Vol. 42, p 335. (b) Rano, T.A.; Chapman, K. T.
Tetrahedron Lett. 1995, 36, 3789.
(11) (a) Sanghvi, Y. S.; Ross, B.; Bharadwaj, R.; Vasseur, J.-J.
Tetrahedron Lett. 1994, 35, 4697. (b) Sanghvi, Y. S.; Bharadwaj, R.; Debart,
F.; Mesmaeker, A. D. Synthesis 1994, 1163.
(12) (a) Mitsunobu, O. Synthesis 1981, 1. (b) Debart, F.; Vasseur, J.-J.;
Sanghvi, Y. S.; Cook, P. D. Tetrahedron Lett. 1992, 33, 2645.
(13) (a) Divakar, R. J.; Reese, C. B. J. Chem. Soc., Perkin Trans. 1 1982,
1171. (b) Perbost, M.; Sanghvi, Y. S. J. Chem. Soc., Perkin Trans. 1 1994,
2051.
(14) Beaucage, S. L.; Iyer, R. P. Tetrahedron 1992, 48, 2223.
0002-7863/96/1518-0255$12.00/0 © 1996 American Chemical Society

256 J. Am. Chem. Soc., Vol. 118, No. 1, 1996
Communications to the Editor
Scheme 1a
Table 1. MMI Analogs of Isis 3521 as Inhibitors of PKC-R
Protein Expression18
Isis
No.
sequence,a 5′ f 3′
Tm IC50
b
c
3521 GSTSTS CSTSCS GSCSTS GSGSTS GSASGS TSTSTS CSA 52.1 100
9500 GSTSTS CSTSCS GSCSTS GSGSTS GSASGS T*T*T*C 51.5 100
10403 T*T*C′*T CSGSCS TSGSGS TSGSAS GSTSTS TS CSA 52.4 125
10404 T*T*C′*T CSGSCS TSGSGS TSGSAS GS T*T*T*C
52.5d 100d
a An asterisk indicates MMI linkage; S, phosphorothioate linkage;
C′, 5-methylcytosine. b Tm values are in °C measured with RNA
complements. c See ref 18 for experimental details. d Tm of parent 18-
mer PS oligomer was 51.3 °C, with an IC50 of 120 nM.
The water solubility of oligonucleosides can be restored by
incorporating nucleoside 7, which provides a negative charge
at the 5′-end of the molecule. For example, oxime dimer 14 (n
) 1, R ) NH2, R′ ) O-SL-CPG) was prepared and coupled
with 7 to provide a trimer (14, R ) FMOC, R′ ) O-SL-CPG,
B ) T, n ) 2). Deprotection of the latter trimer with piperidine
in CH3CN gave 14 (B ) T, n ) 2, R ) H, R′ ) O-SL-CPG),
which on standard phosphoramidite coupling14 with T gave a
tetramer, which on cleavage from the CPG, followed by
reduction/methylation, furnished 18 (n ) 2) with a negative
charge. Tetramer 18 (n ) 2), with a PdS linkage, was also
prepared in an analogous way. Three chimeric AOs were
synthesized as analogs of Isis 3521, a PS oligomer which inhibits
PKC-R protein expression at ∼100 nM concentration.18 Ma-
nipulation of the chemistries described above in an appropriate
manner19 allowed us to synthesize Isis 9500 (3′-capped), 10403
(5′-capped), and 10404 (3′- and 5′-capped) as the first examples
of chimeric AOs containing multiple MMI linkages in a row.
The 5′ f 3′ unidirectional uninterrupted conjugation of MMI
and DNA pieces has proven very useful due to the fact that the
entire synthesis was performed on a single instrument by simply
replacing the reagent bottles. The novel chimeric AOs were
found to hybridize to their complementary RNA with better
affinity and specificity compared to the unmodified PS oligomer
(Table 1) and were found to inhibit the PKC-R expression.
Tetramer 17 (n ) 3) was found to be completely resistant to
cleavage by purified exo- (SVPD) and endo- (S1) nucleases.20
Therefore, capping of an AOs at the 3′- and 5′-ends with MMI
blocks should provide significant resistance to degradation by
nucleases.
In conclusion, this work helps define chemical strategies for
SP and SS synthesis of MMI-type oligonucleosides which may
lead to a new class of chimeric AOs with improved pharma-
cological properties. The methodology also allows the MMI
portion to be further elongated with additional PO or PS linkages
while the oligomer is still attached to the SS, thus indicating
the compatibility of MMI and phosphoramidite chemistry.
These advances in the R-O-NH2 and R-CHO coupling have also
shown applications in combinatorial chemistry.21 An appropri-
ate mix of such studies should eventually lead to the discovery
of novel drugs.
Acknowledgment. We thank P. Dan Cook for his interest, support,
and encouragement throughout this project. We are also grateful to
Elena Lesnik for the Tm measurements, Lendell Cummins for nuclease
studies, Nicholas Dean for PKC-R data, Patrick Wheeler for NMR
studies, and Ramesh Bharadwaj for preparing the starting materials.
Supporting Information Available: Details of the experimental
procedures, synthesis, and characterization data for nucleosides, oli-
gonucleosides, and oligonucleotides (14 pages). This material is
contained in many libraries on microfiche, immediately follows this
article in the microfilm version of the journal, can be ordered from the
ACS, and can be downloaded from the Internet; see any current
masthead page for ordering information and Internet access instructions.
a Abbreviations: in 1-6, B ) thymine (a); N-4-benzoyl-5-methyl-
cytosine (b) adenine (c); N-2-isobutyrylguanine (d); Phth ) N-
phthalimido group; TPS ) tert-butyldiphenylsilyl group; FMOC )
9-fluorenylmethoxycarbonyl group; amidite ) â-cyanoethoxy-N-di-
isopropylaminophosphityl group; P ) controlled pore glass (CPG);
T-3′-PO ) 3′-phosphorylthymidine; T-3′-PS ) 3′-thiophosphoryl
thymidine; SL ) succinyl linker.
SP strategy was extended toward the synthesis of T8 (17, n )
7) and T12 (17, n ) 11) on SS in the manner described below.
Thymidine-derivatized CPG 2a was packed into a 1 µmol
column and connected to an automated DNA synthesizer
(Scheme 1). Hydrazinolysis of 2a resulted in the formation of
1,2-dihydro-4-hydroxy-2-methyl-1-oxophthalazine (10) instan-
taneously. Attempts to estimate the release of 10 utilizing the
standard UV technique was found to be inefficient (low ꢀmax).
We now report15 an efficient and highly sensitive method of
detection of 10 using the emission spectra, based on the
luminescence property of the phthalazines. Treatment of 2a
with a solution of H3CNHNH2 (3%, CH2Cl2-MeOH, 9:1 v/v)
for 2 min plus a 5 min wait step generated 5′-O-NH2 derivative
11 (B ) T) quantitatively. In the next step, coupling of 5a
with 11 was found to be most efficient when a solution of 5a
(0.1 M, CH2Cl2-AcOH, 97:3 v/v) was passed through the
column for 15 s with a 10 min wait step. Unreacted 5a was
fully recovered on evaporation of solvent and recycled for
subsequent reactions. Subsequent iterations of deprotection and
coupling steps resulted in the formation of oxime oligomer 12
(R ) TPS, R′ ) O-SL-CPG, B ) T, n ) 7, 11). A wash
cycle (CH2Cl2) was essential between each step. An average
coupling efficiency of 97-99% was obtained for the two
oligonucleosides (12, B ) T, n ) 7, 11) prepared.16 Treatment
of the CPG with ammonia released the free oxime oligomer 13
(n ) 7, 11), which was then reduced/methylated and desilylated
to furnish crude 17 (n ) 7, 11). HPLC purification of the crude
products gave analytically pure oligonucleosides. Purified 17
(n ) 7, 11) exhibited poor water solubility, which was not
altogether unexpected, as other completely neutral oligonucleo-
sides were also found to be sparingly soluble.17
JA9533959
(15) Cook, P. D.; Sanghvi, Y. S.; Morvan, F. PCT Int. Appl. WO 95/
18136, July 6, 1995.
(16) In addition to the pyrimidine oligonuclesides, we have also
synthesized several purine-containing dimers (A*T, T*A, G*A, G*T)
according to the SS methodology.
(17) For a sulfonate backbone, see: Huang, J.; McElroy, E. B.; Widlanski,
T. S. J. Org. Chem. 1994, 59, 3520. For a silyl backbone, see: Cormier, J.
F.; Ogilvie, K. K. Nucleic Acids Res. 1988, 16, 4583. For an extensive list,
see ref 2.
(18) Dean, N. M.; McKay, R.; Condon, T. P.; Bennett, C. F. J. Biol.
Chem. 1994, 269, 16416.
(19) White, M. A. In Solid Supports Catalysts in Organic Synthesis;
Smith, K., Ed.; Horwood: Chichester, U.K., 1992; p 225.
(20) For experimental conditions, see: Cummins, L.; Owens, S. R.; Risen,
L. M.; Lesnik, E. A.; Freier, S. M.; McGee, D.; Guinosso, C. J.; Cook, P.
D. Nucleic Acids Res. 1995, 23, 2019.
(21) Cook, P. D.; Sanghvi, Y. S.; Kung, P.-P. PCT Int. Appl. WO 95/
18623, July 13, 1995.