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7 Improving Binding Affinity of Oligonucleotides by Conformational Restraint of the Phosphodiester Backbone – α,β-Constrained Nucleic Acids

7 Improving Binding Affinity of Oligonucleotides by Conformational Restraint of the Phosphodiester Backbone – α,β-Constrained Nucleic Acids

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424



12 Unnatural Nucleoside Analogs for Antisense Therapy



O



(a)



O



Bx



O

O α

P

O

O

β H O



Bx



O

α,β- D-CNA, 45 (RC5', SP)

(α = -sc, β = ap)



(b)

O



O



O



O

O α

P

O

O

β H O



O

O

P

O

O

Bx



O O

O

P

O

O



O



Bx



O



O P

O

Bx



O

LNA–α,β-D-CNA, 51



O



O



Bx



H



Bx



O



O

X

P

Y

O

O



Bx

β

γ



O



O

O P Oε

ξ

O

O

Bx



O

α,β,γ-D-CNA, 52



Bx



X = O, Y = S, 49

X = S, Y = O, 50

O



O

δ



O



O



α,β-P-CNA, 48

phostone-CNA



O



Bx



H



Bx



O



O





H



O



α,β-D-CNA, 47

(SC5', RP)



α,β-D-CNA, 46

(RC5', RP)



O



O

P



O

H



O



O



Bx



O



O



O



O



O



Bx



Bx



Bx



O

δ,ε,ξ-D-CNA, 53



O

ν°



Bx



O

O P O

O

O



Bx



O

ν°,ε,ξ-D-CNA, 54



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 [129].



12.8

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 [22]. 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.

12.8.1

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) [46]. Phosphorothioate-linked

nucleotide dimers were first synthesized and shown to be resistant toward

nuclease-mediated degradation by Eckstein in the mid-1960s [138]. 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 [139]. 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 [140]. In a more recent study,

the PS modification was found to protect bacterial DNA from peroxide-mediated

oxidative decomposition [141].

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 [142]. 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

O

O P

O

S

phosphorothioate (PS), 55

O

O P

O

R3R2N

phosphoroamidate, 56



O



O



O

O P

O

O



Bx

R1

O



Bx



O

R1

O

O P

O

phosphodiester (PO) oligonucleotide

Me

methyl phosphonate, 57

Figure 12.14 Structures of selected oligonucleotide backbone modifications.



425



426



12 Unnatural Nucleoside Analogs for Antisense Therapy



the phosphoramidate 56 linkages, which replace the nonbridging oxygen atom with

carbon or nitrogen, are less successful [143]. 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 [146].

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 [147]. 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 [25]. 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 [150]. 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 [151]. 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

nucleotides) [152].



12.9

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



O



H3C



4

5



6 N



NH2



3



H3C



NH

2



O



1



S



N



NH

O



N



N



O



O



C5-Thiazole 61



O



N

N



R

N

H

H

N



O



NH

NH



N



NH2



O



5-Methyl cytosine 58 C5-propyne T 59 C5-propyne C 60



O



N



Me



O



N



5



N



Thymine 57



Me



N

O



Phenoxazine 62



O



H O

H



N

N



O



N



N

H



N H



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).

12.9.1

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



427



428



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 [153].

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 [155]. 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) [156]. 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 [161].

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].



12.10

Outlook



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



References



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encouraging results obtained from clinical trials using RNase H active gapmer

ASOs. Increasing nuclease stability and affinity for complementary RNA have been

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while improved affinity for RNA is critical, the utility of any given nucleic acid

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stability in biophysical experiments. Improved affinity is important to bind

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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

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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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