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7 Improving Binding Affinity of Oligonucleotides by Conformational Restraint of the Phosphodiester Backbone – α,β-Constrained Nucleic Acids
12 Unnatural Nucleoside Analogs for Antisense Therapy
β H O
α,β- D-CNA, 45 (RC5', SP)
(α = -sc, β = ap)
β H O
O P Oε
X = O, Y = S, 49
X = S, Y = O, 50
O P O
Figure 12.13 (a) Structure of a,b-D-CNA showing conformation of the appended six-membered
dioxaphosphorinane ring system. (b) Structures of other members of the CNA family .
Naturally Occurring Backbone Modifications
Protecting the backbone phosphodiester linkages from nuclease-mediated degradation is crucial for improving the drug-like properties of ASOs. Stabilization of the
phosphodiester backbone has been realized by replacing one of the nonbridging
oxygen atoms with isosteric elements such as sulfur, nitrogen, and carbon or by
12.8 Naturally Occurring Backbone Modifications
replacing the phosphodiester linkage entirely with a non-phosphorus linking
backbone . In general, strategies that rely on isosteric replacement of the
nonbridging oxygen have been more successful because of compatibility with
methods for solid-phase oligonucleotide synthesis and because of superior properties observed in biological assays.
The Phosphorothioate Modification
Perhaps the single most successful oligonucleotide modiﬁcation to date is the
phosphorothioate 55 backbone modiﬁcation in which one of the nonbridging
oxygen atoms is replaced with sulfur (Figure 12.14) . Phosphorothioate-linked
nucleotide dimers were ﬁrst synthesized and shown to be resistant toward
nuclease-mediated degradation by Eckstein in the mid-1960s . However, it was
not until 2007 that Wang et al. found that nuclease digestion of bacterial DNA
yielded fractions that were resistant to further degradation . Closer analysis of
these fractions revealed the presence of PS-linked dinucleotide units. The authors
proposed that the PS modiﬁcation is used by bacteria to improve nuclease stability
of certain regions in their genome. However, other interesting applications related
to regulation of gene expression cannot be discounted . In a more recent study,
the PS modiﬁcation was found to protect bacterial DNA from peroxide-mediated
oxidative decomposition .
The phosphorothioate linkage presents several features that make it attractive for
oligonucleotide therapeutics. The PS backbone improves stability versus nucleasemediated degradation, does not signiﬁcantly impair the ability of the ASO to form
duplexes with RNA and DNA, and perhaps most importantly, supports RNase Hmediated cleavage of complementary RNA . This makes PS DNA ASOs
functionally competent and able to downregulate gene expression through the
RNase H mechanism. In contrast, the closely related methyl phosphonate 57 and
phosphorothioate (PS), 55
phosphodiester (PO) oligonucleotide
methyl phosphonate, 57
Figure 12.14 Structures of selected oligonucleotide backbone modifications.
12 Unnatural Nucleoside Analogs for Antisense Therapy
the phosphoramidate 56 linkages, which replace the nonbridging oxygen atom with
carbon or nitrogen, are less successful . Both these linkages are neutral,
reduce solubility, and impair hybridization with complementary nucleic acids
except when used sparingly, and also lack the ability to generate a functional
response as they do not support the RNase H mechanism. Only one neutral
phosphorodiamidate linkage used in the context of morpholino oligonucleotides
has been extensively investigated in animals and also entered human trials
[144,145]. Interestingly, a recent study showed that certain marine bacteria possess
the genetic apparatus for the biosynthesis of methylphosphonic acid .
However, it is not yet known if the bacteria incorporate the methylphosphonate
moiety into its natural nucleotide or nucleic acid pools.
The PS modiﬁcation improves nonspeciﬁc binding of oligonucleotides to various
proteins . This in turn has proved to be a double-edged sword. Unmodiﬁed
nucleic acids have poor pharmacokinetic properties and are rapidly ﬁltered by the
glomerulus into the urine and excreted . Improved binding of PS-modiﬁed
ASOs to plasma proteins prevents the ASO from being ﬁltered and excreted by the
kidney and allows distribution to peripheral tissues. The PS backbone also
promotes ASO binding to cell surface proteins. This allows the ASO to be
internalized into the cell without the aid of any specialized delivery vehicles
[148,149]. In animal experiments, this property enables robust and reproducible
antisense pharmacology especially when targeting genes expressed in the liver.
Recent studies have shown that PS-modiﬁed ASOs also show robust antisense
effects in a number of other tissues such as kidney, muscle, adipose tissue, and the
CNS following direct injection . However, the promiscuous protein binding
properties of PS-modiﬁed ASOs can result in undesirable effects such as
nonspeciﬁc activation of immune receptors resulting in mild to moderate injection
site reactions and ﬂu-like symptoms in human trials . These undesirable
effects were more serious for ﬁrst-generation designs, which typically comprise 20mer PS DNA but were greatly mitigated by the introduction of second-generation
designs, which are chimeric oligonucleotides with reduced PS DNA content (8–14
Naturally Occurring Heterocycle Modifications
The heterocyclic nucleobases in oligonucleotides interact with the RNA receptor
through Watson–Crick base pairing. The ability of an ASO to form these drug–
receptor interactions is the basis for maintaining the afﬁnity and speciﬁcity of the
antisense mechanism. While a very large number of heterocycle-modiﬁed nucleosides have been described in the antiviral and anticancer arena, very few have
found utility in nucleic acid medicinal chemistry. An impediment for using
heterocycle-modiﬁed nucleosides in antisense technology is the concern that
metabolism of modiﬁed ASOs could generate nucleoside metabolites that could
get incorporated into genomic material, compete with natural nucleotide pools, or
12.9 Naturally Occurring Heterocycle Modifications
5-Methyl cytosine 58 C5-propyne T 59 C5-propyne C 60
R = H, G-clamp, 63
R = CNHNH2,
guanidino G-clamp 64
Figure 12.15 Structures of some C5-substituted pyrimidine analogs.
interfere with the functioning of polymerases. There is, however, one class of
heterocycle modiﬁcation that is found ubiquitously in nature and used
extensively in nucleic acid medicinal chemistry to improve duplex stability and
nuclease resistance, as well as mitigate the immunostimulatory properties of
certain oligonucleotide sequence motifs. This modiﬁcation is the C5 methyl
substitution on pyrimidine nucleobases, which in turn inspired the design of
new classes of heterocycle modiﬁcations for use in nucleic acid medicinal
chemistry (Figure 12.15).
5-Substituted Pyrimidine Analogs
The fundamental structural difference between DNA and RNA is the 20 -hydroxyl
group in RNA and the methyl group at the C5 position of thymine 57. The presence
of 5-methyl group on cytosine nucleobases (58) in DNA was recognized in the late
1940s while its presence in RNA was discovered much later [153,154]. The 5-methyl
group on pyrimidine nucleobases stacks between the hydrophobic nucleobases in
the major groove and improves the thermal stability of oligonucleotide duplexes. In
addition, the 5-methyl group also improves stability of DNA from nucleasemediated degradation. In the early 1980s, several studies showed that a signiﬁcant
number of the cytosine nucleobases in eukaryotic DNA were methylated at the
5-position. Interestingly almost 90% of the methylation occurred predominantly at
CpG (deoxycytidine-phosphate-deoxyguanosine) dinucleotide units. In contrast,
bacterial DNA shows only minimal levels of methylation of cytosine nucleobases. It
was later found that reversible methylation of cytosine nucleobases in eukaryotic
DNA is a strategy used by Nature to prevent transcription of certain genes and
12 Unnatural Nucleoside Analogs for Antisense Therapy
represents a marker of epigenetic (control of gene expression without a change in
sequence) regulation of gene expression .
In the context of oligonucleotide therapeutics, early ASO designs did not
incorporate the 5-methyl cytosine nucleobases and instead mostly deoxycytidine
nucleotides were used. This preference was most likely driven by the commercial
availability of DNA phosphoramidites for solid-phase oligonucleotide synthesis at
the time. Researchers working with early ﬁrst-generation designs of PS-modiﬁed
DNA noticed that certain oligonucleotides produced increased beta-cell proliferation and other immunostimulatory effects . Krieg later showed that many of
these effects were the result of increased immune stimulation produced by PS
DNA ASOs containing unmethylated CpG motifs. He rationalized that since the
majority of CpG motifs in eukaryotic DNA were methylated, DNA ASOs containing unmethylated CpG motifs were most likely being recognized as bacterial DNA
in animals resulting in stimulation of the immune system via activation of Toll-like
receptor 9 (TLR 9) . Subsequent SAR studies showed that introducing a 5methyl group on the cytosine nucleobase in ASOs containing CpG motifs largely
mitigated the TLR 9 response. Interestingly, PS DNA ASOs containing unmethylated CpG motifs have been extensively investigated as vaccine adjuvants and as
cancer therapeutics for their immunostimulatory properties, although this does not
constitute an antisense mechanism, which requires Watson–Crick base pairing
with complementary RNA.
The improved duplex stabilizing properties of the 5-methyl group on pyrimidine
nucleobases resulted in a number of analogs being prepared and evaluated for
improving ASO afﬁnity for complementary RNA (Figure 12.15). Matteucci and
coworkers showed that introducing a propynyl group at the C5 position of
pyrimidine nucleobases (59 and 60) resulted in an improvement in duplex stabilizing
properties and nuclease stability relative to C5 methyl substitution [157,158]. The
improved duplex stabilizing property was attributed to efﬁcient stacking of the pi
system of the acetylinic group between the hydrophobic nucleobases in the major
groove. Further expansion of this concept resulted in the identiﬁcation of C5 thiazole
pyrimidines (61), which also improved the thermal stability of the modiﬁed duplexes
[159,160]. Replacement of the cytosine nucleobase with a phenoxazine ring (62),
which retains the ability to form Watson–Crick base pairing but extends the planar
surface area for improved stacking, further increased duplex thermal stability .
Further structural modiﬁcation led to the addition of an aminopropyloxy side chain
on the phenoxazine scaffold, which provided unparalleled increases in duplex
thermal stability (63 and 64). This analog was termed G-clamp because of its ability
to form more than three hydrogen bonds with guanosine [162–164].
The synthesis of modiﬁed nucleic acid analogs for antisense applications has seen a
resurgence over the past few years. Much of this interest has been driven by the
identiﬁcation of the RNAi pathways for regulation of gene expression and by the
encouraging results obtained from clinical trials using RNase H active gapmer
ASOs. Increasing nuclease stability and afﬁnity for complementary RNA have been
the primary metrics guiding the synthesis of new nucleic acid analogs. However,
while improved afﬁnity for RNA is critical, the utility of any given nucleic acid
modiﬁcation should not be based simply on its ability to increase duplex thermal
stability in biophysical experiments. Improved afﬁnity is important to bind
complementary RNA in a cell but not enough to produce a functional response or
modulate other ASO drug-like properties.
The design of the next generation of nucleic acid modiﬁcations and oligonucleotide designs for therapeutic applications will most likely be guided by a more
sophisticated understanding of the structural biology of nucleic acid–protein
interactions. Crystal structures of important proteins, which mediate antisense
effects, such as human RNase H1 and Ago2, are now available. It is up to the
medicinal chemist to use this resource to design new analogs that facilitate the
important nucleic acid–protein interactions to produce an optimal functional
response. Moreover, it is now becoming evident that chemically modiﬁed ASOs
interact with a host of cell surface and other proteins involved in the endocytotic
pathways. Chemical modiﬁcations that enhance productive interaction of the ASO
with these cell surface proteins can help modulate ASO uptake into the relevant
functional compartment of the cell (nucleus for ASOs and cytoplasm for siRNAs)
and improve ASO activity and therapeutic proﬁle in animal experiments.
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