U.S. patent application number 14/579361 was filed with the patent office on 2015-04-23 for construction of cell penetrating nucleic acids.
This patent application is currently assigned to Steffen Panzner. The applicant listed for this patent is Steffen Panzner. Invention is credited to Steffen Panzner.
Application Number | 20150112051 14/579361 |
Document ID | / |
Family ID | 39939414 |
Filed Date | 2015-04-23 |
United States Patent
Application |
20150112051 |
Kind Code |
A1 |
Panzner; Steffen |
April 23, 2015 |
CONSTRUCTION OF CELL PENETRATING NUCLEIC ACIDS
Abstract
This invention provides pH-responsive zwitterionic nucleotides
and nucleic acids comprising said nucleotides, wherein said
zwitterions are constituted from one or more anionic
internucleoside linkages and one or more cationic moieties and said
zwitterionic nucleotides further comprise either one or more
hydrophobic moieties or one or more TEE's with the general
structure (I) Hydrophobic element-pH-responsive hydrophilic
elements (I).
Inventors: |
Panzner; Steffen; (Halle,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Panzner; Steffen |
Halle |
|
DE |
|
|
Assignee: |
Panzner; Steffen
|
Family ID: |
39939414 |
Appl. No.: |
14/579361 |
Filed: |
December 22, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13003410 |
Feb 10, 2011 |
8957191 |
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PCT/EP09/58921 |
Jul 13, 2009 |
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14579361 |
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Current U.S.
Class: |
536/23.1 |
Current CPC
Class: |
A61P 43/00 20180101;
C07H 21/00 20130101; A61K 47/554 20170801; C12N 2310/18 20130101;
C12N 15/11 20130101; C07H 21/02 20130101; A61K 47/545 20170801;
C07H 21/04 20130101; A61K 47/543 20170801; A61K 47/542
20170801 |
Class at
Publication: |
536/23.1 |
International
Class: |
C12N 15/11 20060101
C12N015/11 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 11, 2008 |
EP |
08104731.8 |
Claims
1. A pH-responsive zwitterionic nucleotide, comprising one or more
anionic internucleoside linkages, preferably selected from the
group consisting of phosphate diesters, phosphothioates and
phosphodithioates, one or more cationic moieties, comprising a
nitrogen base, and either one or more hydrophobic moieties or one
or more transfection enhancer elements {TEE's) having the general
formula Hydrophobic element-pH-responsive hydrophilic elements (I),
wherein said hydrophobic moieties or said hydrophobic element of
the one or more TEE's are linear, branched or cyclic chains with 40
units, wherein said chains preferably consist of carbon atoms,
preferably in form of hydrocarbon or methylene groups, and wherein
said pH-responsive hydrophilic element is selected from weak acids
having a pKa of between 2 and 6, or zwitterionic structures
comprising a combination of weak or strong acidic groups with weak
bases, having a pKa of between 3 and 8.
2. The pH-responsive zwitterionic nucleotide of claim 1, comprising
a nucleoside and wherein said cationic moieties are substituted to
the 2', 4' or 1' position of the nucleoside.
3. The pH-responsive zwitterionic nucleotide of claim 2, wherein
said nucleoside comprises a ribose, furanose, pyranose or hexitol
ring structure.
4. The pH-responsive zwitterionic nucleotide of claim 2, wherein
said nucleoside comprises a hemiacetal or acetal structure.
5. The pH-responsive zwitterionic nucleotide of claim 1, wherein
log D of the nucleotides at pH4 is greater than-1.
6. The pH-responsive zwitterionic nucleotide of claim 5, wherein
characterized in that their log D of the nucleotides at pH4 is
smaller than +3.
7. The pH-responsive zwitterionic nucleotide of claim 1, wherein
said nucleotide comprises a cationic moiety having a pK equal or
greater than 7.5 and one or more TEE's, having a hydrophilic
element which is a carboxylic acid.
8. The pH-responsive zwitterionic nucleotide of claim 1, wherein
said nucleotide comprises a cationic moiety having a pK equal or
lower than 7.5 and one or more hydrophobic moieties.
9. The pH-responsive zwitterionic nucleotide of claim 1, wherein
said nucleotide comprises a cationic moiety have a pK equal or
lower than 7.5 and one or more TEE's having a hydrophilic element
which is a carboxylic acid.
10. The pH-responsive zwitterionic nucleotide of claim 7, wherein
said nucleotide is selected from the structures in table 12, and
comprising hydrophobic moieties with the size specified
therein.
11. The pH-responsive zwitterionic nucleotide of claim 10, wherein
said nucleotide is selected from the structures IPN 1-10 through to
IPN 1-17.
12. The pH-responsive zwitterionic nucleotide of claim 8, wherein
said nucleotide is selected from the structures in table 8, wherein
the one or more hydrophobic moieties have the size specified
therein.
13. The pH-responsive zwitterionic nucleotide of claim 12, wherein
said nucleotide is selected from the structures IPN2-1 through to
IPN2-24.
14. The pH-responsive zwitterionic nucleotide of claim 9, wherein
said nucleotide is selected from the structures in table 16, and
comprising hydrophobic moieties with the size specified
therein.
15. The pH-responsive zwitterionic nucleotide of claim 14, wherein
said nucleotide is selected from the structures IPN3-10 through to
IPN3-27.
16. The pH-responsive zwitterionic nucleotide of claim 1, wherein
said nucleotide further comprises hemiacetal or acetal groups at
their 2' position, and comprising hydrophobic moieties with an
adjusted size as specified in table 20.
17. A nucleic acid comprising the pH-responsive zwitterionic
nucleotides according to claim 1.
18. The nucleic acid according to claim 17, wherein no more than
2/3 of all nucleotides are of IPN type.
19. The nucleic acid according to claim 18, wherein about 50% of
all nucleotides are of IPN type.
20. The nucleic acid according to claim 18, wherein the hydrophobic
moiety of the IPN nucleotides comprises between 4 and 12 carbon
atoms.
21. The nucleic acid according to claim 17, wherein the nucleotides
are selected from the species in tables 10, 15 or 19.
Description
FIELD OF THE INVENTION
[0001] This disclosure describes novel structural elements that
enable transport of nucleic acids across biological membranes, in
particular cell membranes. The elements are pH sensitive in terms
of charge and hydrophilicity and undergo a polar-apolar transition
when exposed to low pH.
BACKGROUND OF THE INVENTION
[0002] The field of this invention is the transport of nucleic
acids and more specifically the transport of oligonucleotides
across biological membranes. Penetration of such molecules is
hampered by their very hydrophilic and charged nature and efforts
have been made to reduce the hydrophilic nature of such molecules
by various means. Chemical modification of the internucleoside
linkage can eliminate the charged character of the phosphodiester
bond, e.g. by using methylphosphonates (Miller and Ts'o 1981, Annu
Rep Med Chem 23:295) or can reduce it through incorporation of
phosphorothioate bonds (Eckstein 1989, Trends Biochem Sci 14:97) or
phosphorodithioate bonds (Nielsen 1988, Tetrahedron Lett 29:2911).
Rudolph et al. (1996 in Nucleosides and Nucleotides 15:1725)
introduced phosphonoacetate derivatives of oligonucleotides and
Dellinger in U.S. Pat. No. 6,693,187 and its continuations U.S.
Pat. No. 7,067,641; US2004/0116687 and US200610293511 present
further data on the synthesis of such compounds. Phosphonoacetates
were profiled as derivatives of oligonucleotides with reduced
internucleoside charge that are highly nuclease resistant and, when
designed as single stranded oligodeoxynucleotides, facilitate
catalytic action of RNAseH upon binding to a complementary strand
of RNA (in Sheehan et al, Nucl Acid Res 2003, 31:4109-4118). The
thymidine dimers presented there display a decreased hydrophilicity
at low pH; however, the cellular uptake of an oligonucleotide
remained unchanged. In fact, cellular penetration was only achieved
after elimination of the carboxylate charge group by esterification
with methyl- or butyl groups.
[0003] In still other cases, lipophilic conjugation has been used
to improve the cellular uptake of oligonucleotides such as single
stranded oligodeoxynucleotides or double stranded siRNA molecules
(Letsinger et al. in U.S. Pat. No. 4,958,013 or Proc. Natl. Acad.
Sci., 86, 6553-6556, 1989 or by Manoharan et al. in U.S. Pat. No.
6,153,737 and U.S. Pat. No. 6,753,423 in combination with single
stranded oligonucleotides; Soutschek et al. (2004) Nature,
432(7014), 173-178 or Wolfrum et al. (2007) in Nat Biotech
25:1149-1157 for the delivery of siRNA.
[0004] Very recently, Panzner in PCT/EP2007/011188 described
nucleosides, nucleotides and nucleic acids derived thereof that are
designed for improved cellular uptake and comprise one or more
transfection enhancer elements, TEE's. The content of this
PCT/EP2007/011188 is included herein by reference.
[0005] In brief, pH-responsive transfection enhancer elements
(TEE's) have the general structure (I)
Hydrophobic element-pH-responsive hydrophilic elements (I)
[0006] The position of the hydrophilic element within the TEE
structure may vary and PCT/EP2007/011188 teaches that the
hydrophilic element can be located distal from the link between
molecule and TEE. PCT/EP2007/011188 also mentions that the
hydrophilic element can be located central within the TEE.
PCT/EP2007/011188 describes the pH-responsive hydrophilic element
as weak acids having a pKa of between 2 and 6, preferred of between
3 and 5. Said weak acids may be selected from carboxyl groups,
barbituric acid and derivatives thereof, xanthine and derivatives
thereof, wherein in some embodiments the xanthine derivatives are
pyrimidines.
[0007] PCT/EP2007/011188 also describes the pH-responsive
hydrophilic element as zwitterionic structures comprising a
combination of weak or strong acidic groups with weak bases, the
latter having a pka of between 3 and 8, preferred of between 4.5
and 7.
[0008] PCT/EP2007/011188 further gives guidance how to achieve the
specific pKa's of said hydrophilic elements, inter alia by
substitution hydroxymethyl-, hydroxyethyl-, methoxymethyl-,
methoxyethyl-, ethoxymethyl-, ethoxyethyl-, thiomethyl-,
thioethyl-, methylthiomethyl-, methylthioethyl-, ethylthiomethyl-,
ethylthioethyl-, chlorid-, chlormethyl-vinyl-, phenyl-, benzyl-,
methyl-, ethyl-, propyl-, isopropyl- and tert-butyl or cyclohexyl
groups.
[0009] The hydrophobic element of the TEE of PCT/EP2007/011188 can
be linear, branched or cyclic chains with a minimum chain length of
6 units, sometimes as short as 4 units. The hydrophobic element
often comprises more than 6 and up to 40 units, often between 6 and
20 units, wherein said units of said hydrophobic element often are
carbon atoms, hydrocarbons or methylene groups.
[0010] PCT/EP2007/011188 also teaches that branching of the main
chain of said hydrophobic element is possible and such branches may
comprise building blocks, such as methyl-, ethyl-, propyl-,
isopropyl-, methoxy-, ethoxy-, methoxymethyl-, ethoxymethyl-,
methoxyethyl-, ethoxyethyl- and vinyl- or halogen groups or
mixtures thereof.
[0011] In some embodiments of PCT/EP2007/011188 the hydrophobic
element may derive from sterols, said sterols may be further
substituted.
[0012] PCT/EP2007/011188 also mentions the insertion of one or more
heteroatoms or chemical groups into the hydrophobic element of the
pH-responsive transfection enhancer elements (TEE's). Such
heteroatoms or chemical groups may be selected from --O--, --S--,
--N(H)C(O)--, --C(O)O--, --OC(O)N(H)--, --C(O)--, --C(O)--N(H)--,
--N(H)--C(O)--O--, --CH.dbd.N--, --O--C(O)--, --N.dbd.CH-- and/or
--S--S--, amino acids or derivatives thereof, .alpha.-hydroxyacids
or .beta.-hydroxy acids.
[0013] One central disclosure of PCT/EP2007/011188 is the
hydrophilic-hydrophobic transition of TEE's in response to an
acidification of the environment and application of such knowledge
towards the design of nucleosides and nucleotides and detailed
information on the design of modified nucleosides, nucleotides,
internucleoside linkages or nucleic acids with enhanced membrane
permeability is given therein.
[0014] As further described in the PCT/EP/2007/011188, the
nucleobases contribute to the log D of a nucleic acid; their
average log D at pH 7.4 is about -1.3 for DNA and -1.4 for RNA; the
respective values at pH4 are -1.7 and -1.8 for DNA or RNA. The
nucleobases therefore contribute a pH-dependent value of log D to
the entire structure.
[0015] PCT/EP2007/011188 is also disclosing contributions of an
average unit of the backbone, said contributions are -2.5 and -3
per abasic nucleotide in phosphodiester DNA and RNA, respectively,
and -2.0 and -2.4 for the phosphorothioate building blocks.
[0016] The table 1 below integrates these values and provides a
survey for the log D values of abasic polynucleotides and nucleic
acids with average base use. The "monomer increment" describes the
log D contribution for each additional nucleotide in a nucleic acid
structure, the offset is the extrapolated log D for 0 nucleotides
and the log D of larger structures is calculated as log
D(n-mer)=offset+n*monomer increment, wherein n represents the
number of monomer units in a nucleic acid.
TABLE-US-00001 TABLE 1 logD values for nucleic acid structures. The
table shows calculated logD values for monomers to tetramers of
abasic nucleic acids and the resulting monomer increment and offset
values from these values. For the calculation of logD values of
statistical 20mer oligonucleotides, the contribution of average
nucleobases was also taken into account. oligomer with abasic
oligomers nucleobases # of monomers 1 2 3 4 monomer increment
offset 20 deoxy pH 7.4 -5.6 -8.1 -10.6 -13.2 -2.5 -3.0 -79.2 pH 4
-4.6 -7.9 -10.5 -13.0 -2.5 -2.9 -86.8 2' OH pH 7.4 -5.8 -8.9 -11.9
-15.0 -3.0 -2.8 -83.2 pH 4 -5.0 -8.8 -11.9 -14.9 -3.0 -2.8 -90.8
PTO/ pH 7.4 -5.3 -7.2 -9.1 -11.1 -1.9 -3.3 -74.3 DNA pH 4 -3.6 -6.9
-9.1 -11.1 -2.0 -3.0 -82.6 PTO/RNA pH 7.4 -5.2 -7.7 -10.1 -12.5
-2.4 -2.8 -78.4 pH 4 -4.4 -7.6 -10.1 -12.5 -2.4 -2.8 -86.0
[0017] According to these calculations, oligonucleotides and longer
nucleic acids are highly polar structures with log D values between
-75 and -90 for average 20 mers.
[0018] Also, the nucleic acids become even more polar at lower
values of pH; this is a contribution of the nucleobases, not the
backbone.
OBJECTS OF THE INVENTION
[0019] Cellular uptake, that is penetration of nucleic acids across
biological membranes, remains challenging and alternative
approaches for this problem still represent a major technical need
for this class of substances.
[0020] It is therefore an object of this invention to provide novel
nucleic acids or their designs with improved cellular
penetration.
[0021] It is a specific object of the invention to further improve
the hydrophilic-hydrophobic transition of nucleic acids in response
to external pH stimuli.
BRIEF DESCRIPTION OF THE INVENTION
[0022] The present invention provides pH-responsive zwitterionic
nucleotides and nucleic acids comprising said nucleotides according
to claims 1 and 17. Further advantageous embodiments of the
invention are described in the dependent claims.
[0023] This invention provides pH-responsive zwitterionic
nucleotides, wherein said zwitterions are constituted from one or
more anionic internucleoside linkages and one or more cationic
moieties and said zwitterionic nucleotides further comprise either
one or more hydrophobic moieties or one or more TEE's.
[0024] The ion-pair forming cationic moieties and the
internucleoside linkages are in close spatial proximity. In many
embodiments of this feature, the cationic moieties are substituted
to the 2', 4' or 1' position, preferably to the 2' position of the
sugar ring of the nucleotides and form zwitterionic structures with
the internucleoside linkage that is connected to the 3' position of
said nucleotide. In other aspects, said cationic moieties may form
zwitterionic structures with the internucleoside linkage that is
connected to the 5' position of said nucleotide.
[0025] Structures within this definition are called Ion-Paired
Nucleotides, IPNs, throughout this disclosure.
[0026] The formation of the zwitterion is dependent on the charged
state of both participating ions. Phosphate-based internucleoside
linkages such as phosphodiesters or phosphothioates have a pK of
about 1 to 1.5 and are fully charged at all pH values greater than
2.5. Zwitterion formation is therefore guided by the availability
of the charged cation and is thus dictated by the pK of the CM. A
high value of pK results in constant zwitterion formation, a
moderate or low value of pK makes this zwitterion formation a
function of the pH of the medium. Zwitterion formation between the
CM and an internucleoside linkage is a first major contribution to
minimize the log D of a nucleic acid. An additional reduction of
log D can be achieved through use of TEE's, which also provide a
hydrophilic-hydrophobic transition in response to the pH of the
medium.
[0027] Now, in a first aspect of the invention, pH-responsive
nucleotides are provided that comprise one or more zwitterionic
structures and one or more TEE's, said zwitterions are constituted
from the anionic internucleoside linkage and a cationic moiety,
said cationic moiety having a pK equal or greater than 7.5.
[0028] In a second aspect of the invention, pH-responsive
nucleotides are provided that comprise one or more pH responsive
zwitterionic structures, said zwitterions are formed between the
anionic internucleoside linkage and a cationic moiety, said
cationic moiety having a pK equal or lower than 7.5. In some
embodiments of that aspect, additional hydrophobic elements are
present.
[0029] In a third aspect of the invention, pH responsive
nucleotides are provided that comprise one or more pH responsive
zwitterionic structures and one or more TEE's, said zwitterions are
constituted from the anionic internucleoside linkage and a cationic
moiety, said cationic moiety having a pK equal or smaller than
7.5.
[0030] The pH in physiological environments may vary and the
structures of this invention can be designed to react to such
variability. In many aspects, such variation will take place within
the interval of pH7.4, which is the physiological pH in the
circulation and in many body fluids, and a pH of about 4 to 5,
which is reached upon endocytic uptake of outside materials, in
some tumor areas or at places of ongoing inflammation. A low pH is
also found in the urine and in the intestine as well as in the
stomach. In preferred embodiments, the pH-sensitive zwitterionic
nucleotides of this invention undergo a hydrophilic-hydrophobic
transition between pH7.4 and pH4.
[0031] Explicit reference is made to PCT/EP2007/011188 for the
definition of TEE's, their hydrophilic and hydrophobic elements and
their architecture and their response to the pH of the medium.
[0032] In the second aspect of this invention, such TEE may be
absent and the pH responsive character of the IPN is mainly
contributed from the cationic moieties. In this case, one or more
hydrophobic moieties may be present, said hydrophobic elements
share the design parameters and the architecture described for
TEE's, essentially they are the hydrophobic moieties of a TEE, but
the weak acid of the TEE is absent in this aspect of the
invention.
[0033] In many aspects of the invention the anionic internucleoside
is selected from the group of phosphate diesters, phosphothioates
or phosphodithioates. It is of course possible to use different
internucleoside linkages in a nucleic acid and it is further
possible to introduce other substitutions at the internucleoside
linkage as long as the internucleoside linkage remains negatively
charged.
[0034] In some embodiments of this invention, the cationic moieties
are connected to the 2' position of a nucleoside. In other
embodiments the cationic moieties are connected to the 4' position
of a nucleoside. In yet other embodiments, the cationic moieties
are connected to the 1' position of a nucleoside, replacing the
former nucleobases at C1.
[0035] In particular aspects of this invention, the TEE or the
hydrophobic moieties are directly linked to a cationic moiety which
in turn provides a linkage to a nucleotide.
[0036] In other aspects of this invention the TEE's or the
hydrophobic moieties on the one hand and the cationic moieties on
the other hand may have individual grafting positions at C1, C2 or
C4 of the nucleosides.
[0037] In preferred aspects of the present invention the
hydrophilic-hydrophobic transition of the TEE occurs between pH4
and pH7.5 and the TEE has a guiding pK of between 3 and 6.5. It is
known from the reference PCT/EP20071011188 that the TEE may
comprise weak acids or zwitterions, the guiding pK mentioned here
is the one which determines the hydrophilic-hydrophobic transition
in either case.
[0038] Some TEE's may be alkylcarboxylic acids. Other TEE's may
comprise a sterol and still others may comprise the carboxylic acid
of a sterol.
[0039] An important element of this invention is the construction
and design of nucleic acids comprising one or more IPN's. The
nucleic acids of the present invention are oligonucleotides or
polynucleotides and in some embodiments no more than 2/3 of all
nucleotides of said nucleic acid are of IPN type.
[0040] In other embodiments only nucleotides at one or both flanks
of an oligonucleotide or polynucleotide are of IPN type.
[0041] In further aspects, the present invention comprehends
pharmaceutical compositions comprising nucleic acids further
modified with one or more IPN's and pharmaceutically acceptable
vehicles therefore.
[0042] In still other aspects, the present invention comprehends
the use of a pharmaceutical composition according to the present
invention for the treatment or prophylaxis of inflammatory, immune
or autoimmune disorders, cancers or metabolic diseases of humans or
non-human animals.
[0043] In another aspect, the present invention comprehends the use
of nucleic acids further modified with one or more IPN's for the in
vivo, in vitro or ex vivo transfection.
DEFINITIONS
[0044] For clarity, the following definitions and understandings
are used for important terms of the invention:
[0045] IPN1
[0046] . . . means Ion-Paired Nucleotides according to the first
aspect of this invention.
[0047] IPN2
[0048] . . . means ion-Paired Nucleotides according to the second
aspect of this invention.
[0049] IPN3
[0050] . . . means Ion-Paired Nucleotides according to the third
aspect of this invention.
[0051] Log P
[0052] . . . is the ratio of the respective concentrations of a
compound in the 1-octanol and water partitions of a two-phase
system at equilibrium. The octanol-water partition coefficient (log
P) is used to describe the lipophilic or hydrophobic properties of
a compound.
[0053] Log D
[0054] . . . is the ratio of the equilibrium concentrations of all
species (unionized and ionized) of a molecule in 1-octanol to same
species in the water phase.
[0055] The partition coefficient for dissociative mixtures, log D,
is defined as follows:
log D=log(.SIGMA.(c.sub.i.sup.H2O)/.SIGMA.(c.sub.i.sup.org)),
where
c.sub.i.sup.H2O is the concentration of the i-th microspecies in
water and c.sub.i.sup.org is the concentration of the i-th
microspecies in the organic phase.
[0056] Log D differs from log P in that ionized species are
considered as well as the neutral form of the molecule. Log D is
therefore the log P at a given pH of the medium.
[0057] Log P and log D values can be determined experimentally by
measuring the partition of a molecule or its ionized forms in
octanol/water systems. Experimental values have been generated for
a vast amount of individual compounds and expert systems allow
extrapolating log P and log D values for novel species. One such
expert system is ACD/Labs with the modules ACD/Log P or ACD/log D
and ACD/Labs 7.06 has been used for calculations within this
disclosure.
[0058] Nucleic acids
[0059] . . . as used herein are the polynucleotides or
oligonucleotides defined below, including, without limitation, DNA
or RNA.
[0060] Polynucleotide
[0061] . . . as used herein refers to any polyribonucleotide or
polydeoxyribonucleotide, which may be unmodified RNA or DNA or
modified RNA or DNA. Thus, for instance, polynucleotides as used
herein refers to, among others, single- and double-stranded DNA,
DNA that is a mixture of single- and double-stranded regions,
single- and double-stranded RNA, and RNA that is mixture of single-
and double-stranded regions, hybrid molecules comprising DNA and
RNA that may be single-stranded or, more typically, double-stranded
or a mixture of single- and double-stranded regions.
[0062] Oligonucleotides
[0063] . . . as used herein are defined as molecules with two or
more deoxyribonucleotides or ribonucleotides, often more than
three, and usually more than ten. The exact size of an
oligonucleotide may depend on many factors, including the ultimate
function or use of the oligonucleotide. Oligonucleotides can be
prepared by any suitable method, including, for example, cloning
and restriction of appropriate sequences and direct chemical
synthesis by a method such as the phosphotriester method of Narang
et al., Meth. Enzymol., 68, 90-99, 1979; the phosphodiester method
of Brown et al., Method Enzymol., 68, 109-151, 1979 the
diethylphosphoramidite method of Beaucage et al., Tetrahedron
Lett., 22, 1859-1862, 1981 the triester method of Matteucci et al.,
J. Am. Chem. Soc., 103, 3185-3191, 1981 or automated synthesis
methods; and the solid support method of U.S. Pat. No.
4,458,066.
[0064] Transfection
[0065] . . . is used widely to specifically describe the
disappearance of a concentration gradient across a biological
membrane in vivo, in vitro or ex-vivo. It comprises transport
across, or diffusion through, penetration or permeation of
biological membranes irrespective of the actual mechanism by which
said processes occur. The agents to be transfected may comprise
nucleic acids, polynucleotides or oligonucleotides.
[0066] REF1
[0067] . . . means the PCT/EP2007/011188, all content of which is
included herein by reference.
[0068] Cationic Moieties (CM's)
[0069] The cationic moiety of this invention is a nitrogen base and
can be a primary, secondary, tertiary or quarternized nitrogen. The
chemical structures comprising the CM can be linear, cyclic or
branched and the structures may have saturated bonds. The CM may
also comprise unsaturated bonds or it may be of aromatic character.
The CM may thus be selected from the group comprising alkylamines,
alkenylamines, alkylammonium salts or alkenylammonium salts, cyclic
amines, their quarternized homologues and the like.
[0070] The CM may also comprise more than one nitrogen atom and may
thus be selected from the group comprising guanidinium salts,
imidazols, pyrazols, imidazolines, imidazolidines, pyrazolines,
pyrazolidines, pyrazines, piperazins, pyrimidines, pyridazins,
hydrazines and the like.
[0071] The CM may also comprise further heteroatoms and in some
embodiments such heteroatoms are oxygen or sulphur atoms.
Morpholino groups are a specific representative of a CM comprising
heteroatoms. Amongst the oxygen-substituted amines, moieties that
are substituted in .beta.-, .gamma.- or .delta.-position of the
nitrogen atom are used with preference. CM's may therefore comprise
nitrogen bases having one or more of hydroxyethyl-, hydroxypropyl-,
hydroxyisopropyl or hydroxybutyl groups. Of course, these CM's may
also comprise other substituents such as lower alkyl groups
comprising between 1 and 6 C-atoms and mixed substitutions are
possible within the valency of the nitrogen atom.
[0072] The CM's may also comprise acetal, hemiacetal, ester, ether,
thioether, amide or urethane functions. Preferred CM's comprise
such functions in .beta.-, .gamma.- or .delta.-position. Specific
CM's comprise the acetals, hemiacetals, esters, ethers, thioethers,
ketones, amides or urethanes of serine, homoserine, threonine,
homothreonine or 2-amino-5-hydroxycarboxylic acid.
[0073] The cationic moieties may further be selected from the
compounds listed in the table 2 below and substituted forms
thereof. Said substitutions can comprise lower alkyl groups having
between 1 and 6 carbon atoms, halogen atoms or hydroxyl or thiol
groups.
TABLE-US-00002 TABLE 2 List of some CM of the invention. (1)
##STR00001## (2) ##STR00002## (3) ##STR00003## (4) ##STR00004## (5)
##STR00005## (6) ##STR00006## (7) ##STR00007## (8) ##STR00008## (9)
##STR00009## (10) ##STR00010## (11) ##STR00011## (12) ##STR00012##
(13) ##STR00013## (14) ##STR00014## (15) ##STR00015## (16)
##STR00016## (17) ##STR00017## (18) ##STR00018## (19) ##STR00019##
(20) ##STR00020## (21) ##STR00021## (22) ##STR00022## (23)
##STR00023## (24) ##STR00024## (25) ##STR00025## (26) ##STR00026##
(27) ##STR00027## (28) ##STR00028## (29) ##STR00029## (30)
##STR00030## (31) ##STR00031## (32) ##STR00032## (33) ##STR00033##
(40) ##STR00034## (41) ##STR00035## (42) ##STR00036## (43)
##STR00037## (44) ##STR00038## (45) ##STR00039## (46) ##STR00040##
(47) ##STR00041## (48) ##STR00042## (49) ##STR00043## (50)
##STR00044## (51) ##STR00045## (52) ##STR00046## (53) ##STR00047##
(54) ##STR00048## (55) ##STR00049## (56) ##STR00050## (57)
##STR00051## (58) ##STR00052## (59) ##STR00053## (60) ##STR00054##
(61) ##STR00055## (62) ##STR00056## (63) ##STR00057## (64)
##STR00058## (65) ##STR00059## (66) ##STR00060## (67) ##STR00061##
(68)
##STR00062## (69) ##STR00063## (70) ##STR00064## (71) ##STR00065##
(72) ##STR00066## (73) ##STR00067## (74) ##STR00068## (75)
##STR00069## (76) ##STR00070## (77) ##STR00071## (78) ##STR00072##
(79) ##STR00073## (80) ##STR00074## (81) ##STR00075## (82)
##STR00076## (N1) ##STR00077## (N2) ##STR00078## (N3) ##STR00079##
(N4) ##STR00080## (N5) ##STR00081## (N6) ##STR00082## (N7)
##STR00083## (A1) H.sub.3C--NH.sub.2 (A2) ##STR00084## (A3)
##STR00085## (A4) ##STR00086## (A5) ##STR00087## (A6) ##STR00088##
(A7) ##STR00089## (A8) ##STR00090## (A9) ##STR00091## (A10)
##STR00092## (A11) ##STR00093## (A12) ##STR00094## (A13)
##STR00095## (AE1) ##STR00096## (AE2) ##STR00097## (AE3)
##STR00098## (AE4) ##STR00099## (AE5) ##STR00100## (AE6)
##STR00101## (AE7) ##STR00102## (AE8) ##STR00103## (AE9)
##STR00104## (AE10) ##STR00105## (AE11) ##STR00106## (AE12)
##STR00107## (AE13) ##STR00108## (AE14) ##STR00109## (AE15)
##STR00110## (AE16) ##STR00111## (AE17) ##STR00112## (AE18)
##STR00113## (AE21) ##STR00114## (AE22) ##STR00115## (AE23)
##STR00116## (AE24) ##STR00117## (AE25) ##STR00118## (AE26)
##STR00119## (AE27) ##STR00120## (AE28) ##STR00121## (AE29)
##STR00122## (AE30) ##STR00123## (AE31)
##STR00124## (AE32) ##STR00125## (AE33) ##STR00126## (AE34)
##STR00127## (AE35) ##STR00128## (AE36) ##STR00129## (AE37)
##STR00130## (AE38) ##STR00131## (AE39) ##STR00132## (AE40)
##STR00133## (AE41) ##STR00134## (AE42) ##STR00135## (AE43)
##STR00136## (AE44) ##STR00137## (AE45) ##STR00138## (AE46)
##STR00139## (AE47) ##STR00140## (AE48) ##STR00141## (AE49)
##STR00142## (AE50) ##STR00143## (AE51) ##STR00144## (AE52)
##STR00145## (AE53) ##STR00146## (AE54) ##STR00147## (AE55)
##STR00148## (AE56) ##STR00149## (AE57) ##STR00150## (AE58)
##STR00151## (AE59) ##STR00152## (AE60) ##STR00153## (AE61)
##STR00154## (AE62) ##STR00155## (AE63) ##STR00156## (AE64)
##STR00157## (AE65) ##STR00158## (AE66) ##STR00159## (AE67)
##STR00160## (AE68) ##STR00161## (AE69) ##STR00162## (AE70)
##STR00163## (AE71) ##STR00164## (AE72) ##STR00165##
[0074] Important physicochemical parameters of the CM's are their
pK values and their log D values, the latter being dependent on the
charged state of the CM. The following tables 3-5 therefore provide
the log D values at a pH that is substantially above the pK of the
CM (uncharged state) and substantially below the pK of the CM
(charged state). Some of the CM do comprise more than a single
charged nitrogen base and both pK values and separate log D values
and their increments are given for these structures. The numbering
of the compounds is equivalent to that in table 2.
[0075] Table 3-5: pK values for some CM of the invention and their
log D values in charged and uncharged form.
TABLE-US-00003 TABLE 3 CM pK1 logD > pK logD < pK .DELTA. log
D 1 8.28 -1.21 -4.31 3.1 2 4.34 -0.92 -2.92 2 3 5.56 -1.92 -3.92 2
4 8.97 -1.08 -4.18 3.1 5 8.08 -0.59 -3.69 3.1 6 5.08 -0.95 -4.04
3.09 7 5.5 -0.39 -3.49 3.1 8 4.39 -0.15 -2.14 1.99 9 -0.43 -0.43 0
10 5.45 0.18 -2.92 3.1 11 2.81 -1.17 #NV #NV 12 2.64 -1.17 #NV #NV
13 7 -1.49 -5.17 3.68 14 4.35 -0.33 -2.33 2 15 9.9 -0.33 -2.33 2 16
6.54 -1.86 -4.96 3.1 17 3.69 -0.33 -2.32 1.99 18 -0.27 0.75 #NV #NV
19 7.18 -0.16 -2.66 2.5 20 2.47 0.32 #NV #NV 21 11.26 0.37 -2.73
3.1 22 10.03 -0.23 -2.23 2 23 pK1 10.33 -1.23 -4.09 2.86 23 pK2 4.8
-4.09 -5.33 1.24 24 10.9 -1.08 -3.08 2 25 9.7 -0.72 -3.82 3.1 26
7.06 -0.48 -2.62 2.14 27 5.23 0.73 -1.77 2.5 28 1 -0.28 #NV #NV 29
1.29 -0.33 #NV #NV 30 2.34 -0.77 #NV #NV 31 8.97 0.93 -2.17 3.1 32
pK1 9.9 -1.17 -4.1 2.93 32 pK2 5.3 -4.1 -5.27 1.17 33 10.87 1.38
-1.72 3.1 40 6.58 -0.74 -3.83 3.09 41 6.95 -0.6 -3.7 3.1 42 6.58
0.55 -2.55 3.1 43 4.13 1.21 -1.85 3.06 44 2.82 -1.25 #NV #NV 45
2.93 -0.69 #NV #NV 46 5.94 -1.02 -4.09 3.07 47 5.31 -2.55 -5.65 3.1
48 6.78 -0.63 -3.13 2.5 49 9.79 0.49 -2.61 3.1 50 8.93 0.16 -1.84 2
51 pK1 9.57 -0.64 -3.7 3.06 51 pK2 4.21 -3.7 -4.74 1.04 52 9.74
-1.95 -3.94 1.99 53 8.47 -1.58 -4.68 3.1 54 6.13 -1.3 -3.31 2.01 55
5.01 -0.46 -2.89 2.50 56 9.04 1.06 -2.04 3.1 57 pK1 9.27 -0.45 -3.5
3.05 57 pK2 3.48 -3.5 -4.55 1.05 58 -3.08 -3.08 0 59 3.33 -1.4 -4.5
3.1 60 5.16 -1.21 -4.3 3.09 61 4.36 -1.21 -4.3 3.09 62 3.83 -2.09
-5.2 3.11 63 5.34 -1.9 -5.02 3.12 64 pK1 8.69 -1.93 -5 3.07 64 pK2
4.12 -5 -6.02 1.02 65 4.92 -0.57 -3.66 3.09 66 5.31 -0.39 -3.49 3.1
67 4.92 0.55 -2.54 3.09 68 3.28 -2.03 -5.1 3.07 69 6.21 -0.61 -3.11
2.5 70 5.53 -0.9 -3.98 3.08 71 6.6 0.39 -1.61 2 72 8.41 -1.52 -3.55
2.03 73 8.33 0.36 -2.74 3.1 74 6.44 -1.36 -4.46 3.1 75 4.1 -1.05
-3.04 1.99 76 -3.99 -3.99 0 77 7.37 0.92 -2.18 3.1 78 3.49 -1.04
-4.1 3.06 79 -2.25 -2.25 0 80 3.67 -1.73 -4.75 3.02 81 pK1 7.38
-1.62 -4.5 2.88 81 pK2 5.51 -4.5 -5.71 1.21 82 pK1 8.35 -0.79 -3.8
3.01 82 pK2 6.48 -3.8 -4.87 1.07
TABLE-US-00004 TABLE 4 CM pK1 logD > pK logD < pK .DELTA. log
D N1 3.91 -0.03 -2.52 2.49 N2 4.18 -1.71 -3.71 2 N3 3.15 -0.98
-3.41 2.43 N4 -1.61 -0.61 #NV #NV N5 -1.65 -0.71 #NV #NV N6 1 -0.87
#NV #NV N7 2.2 -1.19 #NV #NV A1 10.66 -0.68 -3.76 3.08 A2 10.73
-0.45 -3.53 3.08 A3 9.75 0.06 -3.04 3.1 A4 99 -2.89 -2.89 0 A5 9.16
-1.34 -4.41 3.07 A6 8.71 -1.51 -4.6 3.09 A7 7.77 -1.11 -4.21 3.1 A8
9.91 -1.12 -4.22 3.1 A9 10.01 -1.26 -4.35 3.09 A10 8.32 -1.59 -4.69
3.1 A11 9.36 -1.38 -4.48 3.1 A12 7.93 -1.43 -4.53 3.1 A13 8.24
-1.27 -4.37 3.1
TABLE-US-00005 TABLE 5 CM pK1 logD > pK logD < pK .DELTA. log
D AE1 6.39 -1.26 -4.36 3.1 AE2 6.21 -0.38 -2.69 2.31 AE3 7.14 -0.81
-3.91 3.1 AE4 7.05 0.37 -2.7 3.07 AE5 7.55 -0.82 -3.92 3.1 AE6 7.52
0.5 -2.57 3.07 AE7 6.2 -2.07 -5.17 3.1 AE8 6.14 -0.77 -3.86 3.09
AE9 6.51 -1.18 -4.27 3.09 AE10 6.6 -0.55 -3.63 3.08 AE11 6.75 -1.61
-4.71 3.1 AE12 6.79 -0.24 -3.32 3.08 AE13 4.63 -1.11 -4.2 3.09 AE14
5.9 -0.65 -3.75 3.1 AE15 6.9 -0.541 -3.64 3.099 AE16 6.75 -0.32
-3.42 3.1 AE17 7.8 -0.23 -3.33 3.1 AE18 9.28 -0.39 -3.48 3.09 AE21
6.3 -2.1 -5.22 3.12 AE22 6.11 -0.58 -3.65 3.07 AE23 7.05 -1.9 -5.03
3.13 AE24 6.95 -0.59 -3.67 3.08 AE25 7.45 -1.78 -4.91 3.13 AE26
7.42 -0.46 -3.53 3.07 AE27 5.18 -2.75 -5.86 3.11 AE28 5.75 -1.46
-4.54 3.08 AE29 6.12 -1.86 -4.98 3.12 AE30 6.22 -1.23 -4.32 3.09
AE31 6.36 -2.3 -5.42 3.12 AE32 6.4 -0.92 -4.01 3.09 AE33 4.53 -1.43
-4.53 3.1 AE34 5.8 -1.36 -4.48 3.12 AE35 6.8 -1.46 -4.59 3.13 AE36
6.52 -1.51 -4.6 3.09 AE37 8.71 -1.52 -4.62 3.1 AE38 9.17 -1.61 -4.7
3.09 AE39 4.43 -2.39 -5.5 3.11 AE40 5.58 -2.59 -5.76 3.17 AE41 6.68
-2.68 -5.78 3.1 AE42 6.29 -2.73 -5.82 3.09 AE43 8.6 -2.75 -5.85 3.1
AE44 9.36 -2.83 -5.93 3.1 AE45 7.4 -1.34 -4.44 3.1 AE46 8.67 -1.44
-4.54 3.1 AE47 9.67 -1.3 -4.39 3.09 AE48 7.87 -0.99 -4.08 3.09 AE49
7.89 -0.45 -3.55 3.1 AE50 pK1 13.37 -2.15 -4.01 1.86 AE50 pK2 7.48
-4.01 -6.21 2.2 AE51 7.98 0.42 -2.67 3.09 AE52 7.95 0.42 -2.67 3.09
AE53 pK1 10.46 -1.35 -3.4 2.05 AE53 pK2 7.3 -3.4 -5.44 2.04 AE54
6.96 0.07 -3.03 3.1 AE55 7.92 0.61 -2.49 3.1 AE56 7.92 0.08 -3.02
3.1 AE57 pK1 10.14 -1.27 -3.8 2.53 AE57 pK2 6.87 -3.8 -5.37 1.57
AE58 7.13 0.76 -2.34 3.1 AE59 9.19 -0.6 -3.7 3.1 AE60 7.8 0.69
-2.46 3.15 AE61 7.36 -0.03 -3.08 3.05 AE62 7.95 -0.11 -3.21 3.1
AE63 6.41 -0.72 -3.82 3.1 AE64 6.41 -1.06 -4.16 3.1 AE65 pK1 9.67
-1.65 -4.1 2.45 AE65 pK2 7.6 -4.1 -5.74 1.64 AE66 pK1 9.51 -1.57
-4.1 2.53 AE66 pK2 7.14 -4.1 -5.67 1.57 AE67 5.99 0.33 -2.77 3.1
AE68 6.02 0.94 -2.16 3.1 AE69 pK1 10.46 -1.09 -3.8 2.71 AE69 pK2
6.17 -3.8 -5.17 1.37 AE70 pK1 10.14 -1.01 -3.66 2.65 AE70 pK2 5.72
-3.66 -5.1 1.44 AE71 pK1 9.51 -1.31 -4 2.69 AE71 pK2 6.09 -4 -5.4
1.4 AE72 pK1 9.67 -1.38 -3.9 2.52 AE72 pK2 6.32 -3.9 -5.48 1.58
[0076] Architecture of Zwitterionic Nucleic Acids
[0077] Nucleotides of the invention form zwitterionic structures
wherein the CM undergoes ion-pairing with the negatively charged
internucleoside linkage. In many embodiments, the CM is therefore
located in close spatial proximity to the internucleoside linkage.
Preference is given to structures wherein the CM is grafted on
position C1, C2 or C4 of the nucleoside backbone, as illustrated in
the structures (CM1) to (CM3) below. With more preference, the CM
is grafted on the C2 position of the nucleotide as in structure
(CM1). IPN's are often used to form nucleic acids and are therefore
drawn here in their chain form.
##STR00166##
[0078] The structures (CM1) to (CM3) represent nucleotides of a
nucleic acid; n>=2, B is any nucleobase selected from adenine,
guanine, thymine, cytosine or uracile; Y is oxygen or sulphur, R1
can be H, OH, F, --O--CH3, --O--CH2-CH3, --O--CH2-CH2-O--CH3, --SH,
--S--CH3, --S--CH2-CH3, --S--CH2-CH2-O--CH3 and CM is defined as
above.
[0079] As mentioned above, the formation of the zwitterion is
guided by the availability of the charged cation and depends on the
pK of the CM and the following analysis shall thus illustrate this
relationship.
[0080] For the analysis, the structure (CM)4 to (CM7) were used,
wherein (CM1) is present in its abasic form, Y is oxygen and the CM
is the ethoxy-homologue of compound (A4), the compounds (A5), (48)
or (AE34).
##STR00167##
[0081] Introduction of the zwitterionic structure leads to a
substantial change of the log D profile of these nucleic acids. As
shown in FIG. 1, the unsubstituted phosphoribose backbone of all
structures (CM4) through to (CM7) becomes gradually less
hydrophilic at low values of pH (more acidic than pH5). Insertion
of a constantly charged CM as in (CM4) leads to instant zwitterion
formation and results in a higher, less polar value for log D.
However, (CM4) and related structures do not respond to any change
in pH. CM's with gradually lower values of pK, such as
(CM5)>(CM6)>(CM7) do form zwitterionic structures, but now in
a pH-sensitive manner. The curves have the highest increment at the
pK of the respective CM (see also FIG. 1). Nucleic acids having the
structural unit (CM5), although being responsive towards pH, are
not preferred for use within a pH range between 7.4 and 4 since the
response falls outside this range. Nucleic acids comprising the
structural elements (CM4) or (CM5) therefore need additional
elements that confer the required pH response in between pH4 and
pH7.4 and can be further substituted with one or more TEE to yield
nucleotides of IPN1 type. Structure (CM6) yields a substantial and
structure (CM7) gives a complete response of log D within this
range of pH values, these subunits can be used to construct
nucleotides of IPN2 or IPN3 type.
[0082] FIG. 1 also allows the deduction of some quantitative
relationships. For pH>>pK (uncharged CM) the sum of
individual log D is fairly predictive for the log D of the complete
structure, as demonstrated in table 6.
TABLE-US-00006 TABLE 6 Quantitative analysis of logD for structural
elements of zwitterionic nucleic acids, pH >> pK. CM
Structure Backbone (uncharged) BB + CM logD (FIG. 1) CM4 -4.52 Not
defined CM5 -4.52 -1.34 -5.86 -5.42 CM6 -4.52 -0.63 -5.15 -5.19 CM7
-4.52 -1.36 -5.88 -5.46
[0083] At pH<<pK zwitterion formation occurs between the CM
and the internucleoside linkage, thereby reducing the log D of the
combined moiety by about 3.5 to 5 units, as shown in the analysis
in table 7:
TABLE-US-00007 TABLE 7 Quantitative analysis of logD for structural
elements of zwitterionic nucleic acids, pH << pK. CM
Structure Backbone (charged) BB + CM logD (FIG. 1) Delta CM4 -4.52
-3.7 -8.22 -3.24 -4.98 CM5 -4.52 -4.41 -8.93 -4.44 -4.49 CM6 -4.52
-3.13 -7.65 -4.19 -3.46 CM7 -4.52 -4.48 -9 -4.46 -4.54
[0084] As shown in FIG. 1, the net pH-dependent amplitude for the
zwitterion formation in these structures is about 1 unit of log
D.
[0085] The analysis also reveals a limitation of structures (CM4)
to (CM7): the mere addition of a CM often increases the overall
hydrophilicity, hence making the molecule even less permeable than
its parent compound DNA or RNA. Some molecules like the
2'IME-nucleotides in Prakash et al. (in Curr Top. Med. Chem. 2007
(7), 641-649) or the zwitterionic nucleic acids of Teplova et al.
(in PNAS 1999 (96), 14240-14245) are thus not functional in terms
of enhanced membrane permeability.
[0086] It is therefore important for practicing this invention to
either select CM's with a high intrinsic log D or to further add
hydrophobic moieties or TEE's to cure such defect and the following
description provides limitations for the log D values of IPN's as
well as algorithms that return a range of preferred sizes for the
hydrophobic moieties. For numerous of specific structures, specific
preferred values are presented within this disclosure. For such
calculations, the PCT/EP2007/011188 (also called REF1 herein)
teaches that any additional --CH2- moiety provides a log D of about
+0.5 thus providing guidance for the estimation of the size of such
hydrophobic additions. REF1 also provides structural impacts for
numerous other building blocks and any different chemical
representations can easily be calculated from such information.
[0087] It has been mentioned that zwitterion formation may occur
between the CM and the internucleoside linkage in 3' position as
well as in 5' position of the nucleotide, which provides some
ambiguity in the assignment of an IPN structure towards a specific
nucleotide. Most structures in this disclosure have been drawn with
the internucleoside linkage in 3' position, but this is not
limiting the teachings of this invention towards such architecture
by any means. Instead, the structures (CM1) to (CM3) can be drawn
as their isomers (CM8) to (CM10) as shown below and such isomer
configuration can be applied to the other structures throughout
this disclosure.
##STR00168##
The structures (CM8) to (CM10) represent the isomeric IPN's to
(CM1) to (CM3), respectively and monomers of such IPN in nucleic
acids are shown here; n>=2, B is any nucleobase selected from
adenine, guanine, thymine, cytosine or uracile; Y is oxygen or
sulphur, R1 can be H, OH, F, --O-CH3, --O-CH2-CH3,
--O-CH2-CH2-O-CH3, --SH, --S-CH3, --S-CH2-CH3, --S-CH2-CH2-O-CH3
and CM is defined as above.
[0088] IPN 2 Structures
[0089] The description given so far is sufficient to construct
complete nucleotides as IPN2 structures. The preferred
internucleoside linkage is of phosphodiester, phosphothioate, or
phosphodithioate type, but other linkages can be used as long as
they provide a negative charge. In preferred embodiments of such
structures, the pK of the CM is between 4 and 7.5, more preferred
is a pK between 5 and 6.5.
[0090] The core architecture of an IPN2 nucleotide is presented in
(CM1) to (CM3).
[0091] In most aspects, the log D(pH4) of an IPN2 nucleotide is
higher than -2, in preferred aspects this value is between -1 and
+3 and in some aspects it is greater than 3. Said log D refers to
the chain form for the IPN2 nucleotide and represents the monomer
increment per nucleotide, it is different from the IPN2 monomer
itself mainly due to the presence of the second charge at the
phosphate group.
[0092] The log D of IPN2 structures can be considered as a
composite from (i) the backbone elements internucleoside linkage
and nucleoside sugar, (ii) the nucleobases, if present as in (CM1)
and (CM2), (iii) the CM and (iv) the pH dependent zwitterion
formation between CM and internucleoside linkage.
[0093] Quantitative estimates for these elements have been
presented above, approximate values for (i) are -2 to -3; (ii) is
pH dependent and about -1.4 at pH T4 and -1.8 at pH4; values for
(iii) are listed in tables 3 to 5 for the unprotonated form at
pH>>pK and (iv) is about +1 for the zwitterion formation. In
many cases of IPN2 structures, the contribution of (iv) will be
absent at pH7.4 and present at pH4 and this pH-induced increase in
log D overcompensates the pH-induced decrease of log D from the
nucleobases.
[0094] The following table 8 presents specific composite log D's
for some of the IPN2 structures comprising the core unit of (CM1)
or (CM2) using the data presented in the tables 3 to 5 above. It is
of course possible to analyze structures of different chemical
origin in the same way; this analysis is illustrating, but not
limiting the teachings of this invention.
[0095] For comparison, one can calculate the composite log D of an
average nucleotide as the sum of its components: -2.5 as average
for the backbone and -1.8 for a statistical nucleobases at pH4 or
the respective value at pH7.4, the results are given in the first
line of table 8. The composite log D for IPN2 structures at pH7.4
includes the CM, which at pH4 further includes the interaction gain
of 1 for the zwitterion formation.
[0096] For some structures listed in table 8, the log D (pH4) of
the composite is already close to the preferred range of log D
(pH4)>-2 and these structures already have a substantially
reduced hydrophilicity.
[0097] In many cases, the additional presence of a hydrophobic
moiety is beneficial for the membrane permeability of the substance
and the last two columns of table 8 identify compound specific
ranges of such additions and return a preferred number of carbon
atoms in such hydrophobic segment.
[0098] Further preferred IPN2 structures therefore comprise a
hydrophobic moiety, said moiety essentially comprising between 2
and 20 carbon atoms. REF1 teaches that the specific chemical
configuration of such hydrophobic moieties has little impact on the
log D contribution; REF1 has also analyzed the impact of potential
substitutions to such hydrophobic moieties. In many cases the
specific position of the hydrophobic moiety on the core structures
(CM1)-(CM3) may also vary without substantial impact to the
physicochemical parameters.
TABLE-US-00008 TABLE 8 Physicochemical analysis of some specific
IPN2 structures having a pK between 5 and 7. CM means the compound
as in table 2, all other headings are described in the text. IPN2
moieties composite # C atoms # C atoms logD composite logD for for
CM pK >pK logD pH 4 pH 7.4 logD -1 logD +3 none none 0 -4.3 -3.9
3 5.56 -1.92 -5.22 -5.82 8 16 6 5.08 -0.95 -4.25 -4.85 6 14 7 5.5
-0.39 -3.69 -4.29 5 13 10 5.45 0.18 -3.12 -3.72 4 12 13 7 -1.49
-4.79 -5.39 7 15 16 6.54 -1.86 -5.16 -5.76 8 16 27 5.23 0.73 -2.57
-3.17 3 11 32 pK2 5.3 -4.1 -7.4 -8 12 20 40 6.58 -0.74 -4.04 -4.64
6 14 41 6.95 -0.6 -3.9 -4.5 5 13 42 6.58 0.55 -2.75 -3.35 3 11 46
5.94 -1.02 -4.32 -4.92 6 14 47 5.31 -2.55 -5.85 -6.45 9 17 48 6.78
-0.63 -3.93 -4.53 5 13 54 6.13 -1.3 -4.6 -5.2 7 15 55 5.01 -0.46
-3.76 -4.36 5 13 60 5.16 -1.21 -4.51 -5.11 7 15 63 5.34 -1.9 -5.2
-5.8 8 16 66 5.31 -0.39 -3.69 -4.29 5 13 69 6.21 -0.61 -3.91 -4.51
5 13 70 5.53 -0.9 -4.2 -4.8 6 14 71 6.6 0.39 -2.91 -3.51 3 11 74
6.44 -1.36 -4.66 -5.26 7 15 81 pK2 5.51 -4.5 -7.8 -8.4 13 21 82 pK2
6.48 -3.8 -7.1 -7.7 12 20 AE1 6.39 -1.26 -4.56 -5.16 7 15 AE2 6.21
-0.38 -3.68 -4.28 5 13 AE7 6.2 -2.07 -5.37 -5.97 8 16 AE8 6.14
-0.77 -4.07 -4.67 6 14 AE9 6.51 -1.18 -4.48 -5.08 6 14 AE10 6.6
-0.55 -3.85 -4.45 5 13 AE11 6.75 -1.61 -4.91 -5.51 7 15 AE12 6.79
-0.24 -3.54 -4.14 5 13 AE14 5.9 -0.65 -3.95 -4.55 5 13 AE15 6.9
-0.541 -3.841 -4.441 5 13 AE16 6.75 -0.32 -3.62 -4.22 5 13 AE21 6.3
-2.1 -5.4 -6 8 16 AE22 6.11 -0.58 -3.88 -4.48 5 13 AE24 6.95 -0.59
-3.89 -4.49 5 13 AE27 5.18 -2.75 -6.05 -6.65 10 18 AE28 5.75 -1.46
-4.76 -5.36 7 15 AE29 6.12 -1.86 -5.16 -5.76 8 16 AE30 6.22 -1.23
-4.53 -5.13 7 15 AE31 6.36 -2.3 -5.6 -6.2 9 17 AE32 6.4 -0.92 -4.22
-4.82 6 14 AE34 5.8 -1.36 -4.66 -5.26 7 15 AE35 6.8 -1.46 -4.76
-5.36 7 15 AE36 6.52 -1.51 -4.81 -5.41 7 15 AE40 5.58 -2.59 -5.89
-6.49 9 17 AE41 6.68 -2.68 -5.98 -6.58 9 17 AE42 6.29 -2.73 -6.03
-6.63 10 18 AE54 6.96 0.07 -3.23 -3.83 4 12 AE57 6.87 -3.8 -7.1
-7.7 12 20 pK2 AE63 6.41 -0.72 -4.02 -4.62 6 14 AE64 6.41 -1.06
-4.36 -4.96 6 14 AE67 5.99 0.33 -2.97 -3.57 3 11 AE68 6.02 0.94
-2.36 -2.96 2 10 AE69 6.17 -3.8 -7.1 -7.7 12 20 pK2 AE70 5.72 -3.66
-6.96 -7.56 11 19 pK2 AE71 6.09 -4 -7.3 -7.9 12 20 pK2 AE72 6.32
-3.9 -7.2 -7.8 12 20 pK2
[0099] To further illustrate, but not limit the teachings of this
invention, the following table 9 presents some more specific
chemical representations for IPN2 structures in nucleic acids,
wherein Y is oxygen or sulphur, B represents any of the nucleobases
or is absent, the index n denotes the length of the nucleic acid
and n is 2 or greater and i denotes the length of an alkyl chain
which here represents the hydrophobic moiety, (i-1) preferably
falls within the range specified in table 8 for the respective
compounds. More than one hydrophobic moiety can be used in the such
cases the preferred range for their size indices k and j shall be
selected to meet k+j=i-1.
[0100] From the IPN2 structures shown below it becomes clear that
the position of a given CM at the nucleoside sugar may vary and
that some CM's can be inserted in different orientations as in the
isomers IPN2-5/IPN2-6 or IPN2-13/IPN2-14 or IPN2-15/IPN2-16. Also,
the position of the hydrophobic element may vary, as shown in
IPN2-10 and IPN2-11 and the position of the graft may be variable,
as seen in IPN2-17 or IPN2-18. Eventually, independent
substitutions of the hydrophobic moiety and the CM are possible as
shown in IPN2-19 through to IPN2-24.
TABLE-US-00009 TABLE 9 Specific representations of IPN2 structures.
IPN2-1 ##STR00169## IPN2-2 ##STR00170## IPN2-3 ##STR00171## IPN2-4
##STR00172## IPN2-5 ##STR00173## IPN2-6 ##STR00174## IPN2-7
##STR00175## IPN2-8 ##STR00176## IPN2-9 ##STR00177## IPN2-10
##STR00178## IPN2-11 ##STR00179## IPN2-12 ##STR00180## IPN2-13
##STR00181## IPN2-14 ##STR00182## IPN2-15 ##STR00183## IPN2-16
##STR00184## IPN2-17 ##STR00185## IPN2-18 ##STR00186## IPN2-19
##STR00187## IPN2-20 ##STR00188## IPN2-21 ##STR00189## IPN2-22
##STR00190## IPN2-23 ##STR00191## IPN2-24 ##STR00192##
[0101] As disclosed above, the architecture of nucleic acids
comprising the inventive IPN structures may vary and different
degrees of substitution of native nucleic acids with IPN structures
are possible. Also, such substitutions may be concentrated on
specific sites within a nucleic acid, e.g. at one or both flanks of
the sequence, at the center part of such sequence or they might be
dispersed throughout the sequence. A known design in the art is a
"gapmer", wherein modified sequences at the flanks of an
oligonucleotide encompass an unmodified or differently modified
sequence in a center piece.
[0102] The table 10 gives calculated log D values for 20 mer
oligonucleotides having a degree of substitution of 50% and such
log D values have been calculated for pH4 and pH7.4 and for
different values of i, the length indicator for the hydrophobic
element. For comparison, the log D (pH7.4) of an unmodified
sequence of the same type is -81, that of pH4 is -89. The standard
assumptions giving above for log D of the internucleoside linkage,
nucleobases and zwitterionic interaction were used.
[0103] Introduction of IPN2 elements into an oligonucleotide, even
at a limited degree of substitution leads to substantial
improvements in log D and preferred oligonucleotides having a log
D-60 are highlighted in the table 10. Also, the IPN2 substitutions
minimize or even reverse the pH-induced decrease of log D at low
values of pH. In a third aspect, this analysis gives additional
guidance on the number of carbon atoms (i) in the hydrophobic
element that may be present in IPN2 structures when used in nucleic
acids at the given degree of substitution. While i=12 yields
preferred structures in almost any case, even the short element
with i=8 is sufficient for most species analyzed here. Even more
so, some species achieve substantial improvements in log D even
with i=4.
TABLE-US-00010 TABLE 10 logD predictions for 20mers of
oligonucleotides comprising 50% IPN2 structures. The CM means the
CM described in table 2 herein. 20mer oligonucleotides, 50%
substitution with IPN2 i = 4 i = 8 i = 12 CM pH 4 pH 7.4 pH 4 pH
7.4 pH 4 pH 7.4 3 -78 -80 -58 -60 -38 -40 6 -69 -71 -49 -51 -29 -31
7 -63 -65 -43 -45 -23 -25 10 -57 -59 -37 -39 -17 -19 13 -74 -76 -54
-56 -34 -36 16 -78 -80 -58 -60 -38 -40 27 -52 -54 -32 -34 -12 -14
32 pK2 -100 -102 -80 -82 -60 -62 40 -66 -68 -46 -48 -26 -28 41 -65
-67 -45 -47 -25 -27 42 -54 -56 -34 -36 -14 -16 46 -69 -71 -49 -51
-29 -31 47 -85 -87 -65 -67 -45 -47 48 -65 -67 -45 -47 -25 -27 54
-72 -74 -52 -54 -32 -34 55 -64 -66 -44 -46 -24 -26 60 -71 -73 -51
-53 -31 -33 63 -78 -80 -58 -60 -38 -40 66 -63 -65 -43 -45 -23 -25
69 -65 -67 -45 -47 -25 -27 70 -68 -70 -48 -50 -28 -30 71 -55 -57
-35 -37 -15 -17 74 -73 -75 -53 -55 -33 -35 81 pK2 -104 -106 -84 -86
-64 -66 82 pK2 -97 -99 -77 -79 -57 -59 AE1 -72 -74 -52 -54 -32 -34
AE2 -63 -65 -43 -45 -23 -25 AE7 -80 -82 -60 -62 -40 -42 AE8 -67 -69
-47 -49 -27 -29 AE9 -71 -73 -51 -53 -31 -33 AE10 -65 -67 -45 -47
-25 -27 AE11 -75 -77 -55 -57 -35 -37 AE12 -61 -63 -41 -43 -21 -23
AE14 -66 -68 -46 -48 -26 -28 AE15 -64 -66 -44 -46 -24 -26 AE16 -62
-64 -42 -44 -22 -24 AE21 -80 -82 -60 -62 -40 -42 AE22 -65 -67 -45
-47 -25 -27 AE24 -65 -67 -45 -47 -25 -27 AE27 -87 -89 -67 -69 -47
-49 AE28 -74 -76 -54 -56 -34 -36 AE29 -78 -80 -58 -60 -38 -40 AE30
-71 -73 -51 -53 -31 -33 AE31 -82 -84 -62 -64 -42 -44 AE32 -68 -70
-48 -50 -28 -30 AE34 -73 -75 -53 -55 -33 -35 AE35 -74 -76 -54 -56
-34 -36 AE36 -74 -76 -54 -56 -34 -36 AE40 -85 -87 -65 -67 -45 -47
AE41 -86 -88 -66 -68 -46 -48 AE42 -86 -88 -66 -68 -46 -48 AE54 -58
-60 -38 -40 -18 -20 AE57 pK2 -97 -99 -77 -79 -57 -59 AE63 -66 -68
-46 -48 -26 -28 AE64 -70 -72 -50 -52 -30 -32 AE67 -56 -58 -36 -38
-16 -18 AE68 -50 -52 -30 -32 -10 -12 AE69 pK2 -97 -99 -77 -79 -57
-59 AE70 pK2 -96 -98 -76 -78 -56 -58 AE71 pK2 -99 -101 -79 -81 -59
-61 AE72 pK2 -98 -100 -78 -80 -58 -60
[0104] IPN1 Structures.
[0105] The description given so far is also sufficient to construct
complete nucleic acid monomer units as IFN1 structures. The
preferred internucleoside linkage is of phosphodiester,
phosphothioate, or phosphodithioate type, but other linkages can be
used as long as they provide a negative charge. IPN1 structures
comprise a CM that is substantially charged at pH7.5, that is, the
pK of such structure is equal or greater than 7.5 and in some
embodiments the CM is a constantly charged cationic moiety
comprising an ammonium or guanidinium group without an explicit
value for pK. Besides their CM, IPN1 structures further comprise a
TEE.
[0106] The core architecture of some IPN1 nucleic acid monomers is
presented in table 11.
TABLE-US-00011 TABLE 11 Core structures of IPN1 elements (IPN1-1)
##STR00193## (IPN1-2) ##STR00194## (IPN1-3) ##STR00195## (IPN1-4)
##STR00196## (IPN1-5) ##STR00197## (IPN1-6) ##STR00198##
[0107] In many aspects of the inventive IPN1 structures, the weak
acid of the TEE is a carboxylic acid.
[0108] In preferred embodiments of IPN1 structures, the hydrophobic
element of the TEE is directly attached to the CM as in IPN1-1,
IPN1-4 or IPN1-5.
[0109] In most aspects, the log D(pH4) of an IPN1 nucleotide is
higher than -2, in preferred aspects this value is between -1 and
+3 and in some aspects it is greater than 3. Said log D refers to
the chain form for the IPN1 nucleotide and represents the monomer
increment per nucleotide, it is different from the IPN1 monomer
itself mainly due to the presence of the second charge at the
phosphate group.
[0110] The log D of IPN1 structures can be considered as a
composite from (i) the backbone elements internucleoside linkage
and nucleoside sugar, (ii) the nucleobases itself, if present,
(iii) the CM and (iv) the zwitterion formation between CM and
internucleoside linkage and (v) the TEE. Quantitative estimates for
the composite elements have been presented above for IPN2
structures and approximate values for (i) are -2 to -3; (ii) is pH
dependent and about -1.4 at pH 7.4 and -1.8 at pH4; values for
(iii) are listed in tables 3 to 5 for the unprotonated form at
pH>pK; the log D for the hypothetical uncharged form of the
quarternized ammonium groups were extrapolated as log
D(protonated)+3.1; this increment has been deduced from most of the
differences in other CM having a real pK. (iv) is about +1 for the
zwitterion formation. For (v), the contribution of the TEE can be
segmented into the contribution of the hydrophilic moiety and the
hydrophobic moiety to simplify the assignment of the number of
elements in the hydrophobic moiety. Data provided in
PCT/EP2007/011188 for the relationship between the number of
methylene groups in carboxylic acids and the resulting log D values
can be extrapolated to conclude the contributions of this charged
element. The resulting values are -0.9 for the uncharged and -3.4
for the charged state of this element. The TEE also comprises a
hydrophobic element, since without such element the net
contribution of the charged group to the log D is negative. For
practicing this invention, the global log D of the IPN structure is
of importance, it is less important whether the CM and its
hydrophobic additions or the TEE and its hydrophobic element
contribute the required hydrophobicity. In many embodiments of the
IPN1 and IPN3 structures this hydrophobic moiety can be common
structure of the CM and the TEE.
[0111] The following table 12 presents specific composite log D's
for some of the IPN1 structures comprising any of the core units
IPN1-1 through to IPN1-4 using the data presented in the tables 3
to 5 above. It is of course possible to analyze structures of
different chemical origin in the same way; this analysis is
illustrating, but not limiting the teachings of this invention. The
composite log D values do comprise the elements (i) to (v)
presented before and are given for both the neutral pH and an
acidic pH; these composite log D's are extrapolated for species
wherein the hydrophobic moiety of the TEE is absent. This allows
identifying preferred ranges for the size of said hydrophobic
elements and said ranges are given in the last two columns of this
table 12 for cases where the hydrophobic elements are methylene
groups. Preferred hydrophobic moieties comprise between 3 and 22
carbon atoms. REF1 teaches that the specific chemical configuration
of such hydrophobic moieties has little impact on the log D
contribution; REF1 has also analyzed the impact of potential
substitutions to such hydrophobic moieties. In many cases the
specific position of the hydrophobic moiety on the core structures
(IPN1-1)-(IPN1-4) may also vary without substantial impact to the
physicochemical parameters.
TABLE-US-00012 TABLE 12 Physicochemical analysis of some specific
IPN1 structures. IPN1 moieties composite # C atoms # C atoms logD
composite logD for for CM pK >pK logD pH 4 pH 7.4 logD -1 logD
+3 none none 0 -4.3 -3.9 1 8.28 -1.21 -5.41 -7.51 8 16 4 8.97 -1.08
-5.28 -7.38 8 16 5 8.08 -0.59 -4.79 -6.89 7 15 15 9.9 -0.33 -4.53
-6.63 7 15 21 11.26 0.37 -3.83 -5.93 5 13 22 10.03 -0.23 -4.43
-6.53 6 14 23 pK1 10.33 -1.23 -5.43 -7.53 8 16 24 10.9 -1.08 -5.28
-7.38 8 16 25 9.7 -0.72 -4.92 -7.02 7 15 31 8.97 0.93 -3.27 -5.37 4
12 32 pK1 9.9 -1.17 -5.37 -7.47 8 16 33 10.87 1.38 -2.82 -4.92 3 11
49 9.79 0.49 -3.71 -5.81 5 13 50 8.93 0.16 -4.04 -6.14 6 14 51 pK1
9.57 -0.64 -4.84 -6.94 7 15 52 9.74 -1.95 -6.15 -8.25 10 18 53 8.47
-1.58 -5.78 -7.88 9 17 56 9.04 1.06 -3.14 -5.24 4 12 57 pK1 9.27
-0.45 -4.65 -6.75 7 15 64 pK1 8.69 -1.93 -6.13 -8.23 10 18 72 8.41
-1.52 -5.72 -7.82 9 17 73 8.33 0.36 -3.84 -5.94 5 13 82 pK1 8.35
-0.79 -4.99 -7.09 7 15 A1 10.66 -0.68 -4.88 -6.98 7 15 A2 10.73
-0.45 -4.65 -6.75 7 15 A3 9.75 0.06 -4.14 -6.24 6 14 A4 99 0.21
-3.99 -6.09 5 13 A5 9.16 -1.34 -5.54 -7.64 9 17 A6 8.71 -1.51 -5.71
-7.81 9 17 A7 7.77 -1.11 -5.31 -7.41 8 16 A8 9.91 -1.12 -5.32 -7.42
8 16 A9 10.01 -1.26 -5.46 -7.56 8 16 A10 8.32 -1.59 -5.79 -7.89 9
17 A11 9.36 -1.38 -5.58 -7.68 9 17 A12 7.93 -1.43 -5.63 -7.73 9 17
A13 8.24 -1.27 -5.47 -7.57 8 16 AE5 7.55 -0.82 -5.02 -7.12 8 16 AE6
7.52 0.5 -3.7 -5.8 5 13 AE17 7.8 -0.23 -4.43 -6.53 6 14 AE18 9.28
-0.39 -4.59 -6.69 7 15 AE37 8.71 -1.52 -5.72 -7.82 9 17 AE38 9.17
-1.61 -5.81 -7.91 9 17 AE43 8.6 -2.75 -6.95 -9.05 11 19 AE44 9.36
-2.83 -7.03 -9.13 12 20 AE46 8.67 -1.44 -5.64 -7.74 9 17 AE47 9.67
-1.3 -5.5 -7.6 9 17 AE48 7.87 -0.99 -5.19 -7.29 8 16 AE49 7.89
-0.45 -4.65 -6.75 7 15 AE50 pK1 13.37 -2.15 -6.35 -8.45 10 18 AE51
7.98 0.42 -3.78 -5.88 5 13 AE52 7.95 0.42 -3.78 -5.88 5 13 AE53 pK1
10.46 -1.35 -5.55 -7.65 9 17 AE55 7.92 0.61 -3.59 -5.69 5 13 AE56
7.92 0.08 -4.12 -6.22 6 14 AE57 pK1 10.14 -1.27 -5.47 -7.57 8 16
AE59 9.19 -0.6 -4.8 -6.9 7 15 AE60 7.8 0.69 -3.51 -5.61 5 13 AE62
7.95 -0.11 -4.31 -6.41 6 14 AE65 pK1 9.67 -1.65 -5.85 -7.95 9 17
AE65 pK2 7.6 -4.1 -8.3 -10.4 14 22 AE66 pK1 9.51 -1.57 -5.77 -7.87
9 17 AE69 pK1 10.46 -1.09 -5.29 -7.39 8 16 AE70 pK1 10.14 -1.01
-5.21 -7.31 8 16 AE71 pK1 9.51 -1.31 -5.51 -7.61 9 17 AE72 pK1 9.67
-1.38 -5.58 -7.68 9 17
[0112] To further illustrate, but not limit the teachings of this
invention, the following table 13 presents some more specific
chemical representations for IPN1 structures in nucleic acids,
wherein Y is oxygen or sulphur, B represent any of the nucleobases
or is absent, the index n denotes the length of the nucleic acid
and n>2 and i denotes the length of an alkyl chain which here
represents the hydrophobic moiety, (i-1) preferably falls within
the range specified in table 12 for the respective compounds. More
than one hydrophobic moiety can be used in the such cases the
preferred range for their size indices k and j shall be selected to
meet k+j=i-1.
[0113] From the IPN1 structures shown below it becomes clear that
the attachment site of a given CM to the nucleoside sugar may vary
and that some CM's can be inserted in different orientations. Also,
the position of the hydrophobic element may vary and the position
of the graft may be variable, as illustrated in the structures
IPN1-10 through to IPN1-17.
TABLE-US-00013 TABLE 13 Specific representations of IPN1
structures. IPN1-10 ##STR00199## IPN1-11 ##STR00200## IPN1-12
##STR00201## IPN1-13 ##STR00202## IPN1-14 ##STR00203## IPN1-15
##STR00204## IPN1-16 ##STR00205## IPN1-17 ##STR00206##
[0114] As disclosed above, the architecture of nucleic acids
comprising IPN structures may vary and different degrees of
substitution of native nucleic acids with IPN structures are
possible. Also, such substitutions may be concentrated on specific
sites within a nucleic acid, e.g. at one or both flanks of the
sequence, at the center part of such sequence or they might be
dispersed throughout the sequence. A known design in the art is a
"gapmer", wherein modified sequences at the flanks of an
oligonucleotide encompass an unmodified or differently modified
sequence in a center piece.
[0115] The following table 14 gives calculated log D values for 20
mer oligonucleotides having a degree of substitution of 50% and
such log D values have been calculated for pH4 and pH7.4 and for
different values of i, the length indicator for the hydrophobic
element. For comparison, the log D (pH7.4) of an unmodified
sequence of the same type is -81, that of pH4 is -89. The standard
assumptions giving above for log D of the internucleoside linkage,
nucleobases and zwitterionic interaction were used.
[0116] Introduction of IPN1 elements into an oligonucleotide, even
at a limited degree of substitution leads to substantial
improvements in log D and preferred oligonucleotides having a log
D>-60 are highlighted in the table 14. Also, the IPN1
substitutions minimize or even reverse the pH-induced decrease of
log D at low values of pH. In a third aspect, this analysis gives
additional guidance on the number of carbon atoms i in the
hydrophobic element that may be present in IPN1 structures. While
i=12 yields preferred structures in almost any case, even the
shorter element with i=8 is sufficient for most species analyzed
here. Even more so, some species achieve substantial improvements
in log D even with i=4.
TABLE-US-00014 TABLE 14 logD predictions for 20mers of
oligonucleotides comprising 50% IPN1 structures. CM denotes
structures as in table 2 herein. 20mer oligonucleotides, 50%
substitution with IPN1 i = 4 i = 8 i = 12 CM pH 4 pH 7.4 pH 4 pH
7.4 pH 4 pH 7.4 1 -80 -97 -60 -77 -40 -57 4 -79 -96 -59 -76 -39 -56
5 -74 -91 -54 -71 -34 -51 15 -71 -88 -51 -68 -31 -48 21 -64 -81 -44
-61 -24 -41 22 -70 -87 -50 -67 -30 -47 23 pK1 -80 -97 -60 -77 -40
-57 24 -79 -96 -59 -76 -39 -56 25 -75 -92 -55 -72 -35 -52 31 -59
-76 -39 -56 -19 -36 32 pK1 -80 -97 -60 -77 -40 -57 33 -54 -71 -34
-51 -14 -31 49 -63 -80 -43 -60 -23 -40 50 -66 -83 -46 -63 -26 -43
51 pK1 -74 -91 -54 -71 -34 -51 52 -88 -105 -68 -85 -48 -65 53 -84
-101 -64 -81 -44 -61 56 -57 -74 -37 -54 -17 -34 57 pK1 -73 -90 -53
-70 -33 -50 64 pK1 -87 -104 -67 -84 -47 -64 72 -83 -100 -63 -80 -43
-60 73 -64 -81 -44 -61 -24 -41 82 pK1 -76 -93 -56 -73 -36 -53 A1
-75 -92 -55 -72 -35 -52 A2 -73 -90 -53 -70 -33 -50 A3 -67 -84 -47
-64 -27 -44 A4 -66 -83 -46 -63 -26 -43 A5 -81 -98 -61 -78 -41 -58
A6 -83 -100 -63 -80 -43 -60 A7 -79 -96 -59 -76 -39 -56 A8 -79 -96
-59 -76 -39 -56 A9 -81 -98 -61 -78 -41 -58 A10 -84 -101 -64 -81 -44
-61 A11 -82 -99 -62 -79 -42 -59 A12 -82 -99 -62 -79 -42 -59 A13 -81
-98 -61 -78 -41 -58 AE5 -76 -93 -56 -73 -36 -53 AE6 -63 -80 -43 -60
-23 -40 AE17 -70 -87 -50 -67 -30 -47 AE18 -72 -89 -52 -69 -32 -49
AE37 -83 -100 -63 -80 -43 -60 AE38 -84 -101 -64 -81 -44 -61 AE43
-96 -113 -76 -93 -56 -73 AE44 -96 -113 -76 -93 -56 -73 AE46 -82 -99
-62 -79 -42 -59 AE47 -81 -98 -61 -78 -41 -58 AE48 -78 -95 -58 -75
-38 -55 AE49 -73 -90 -53 -70 -33 -50 AE50 pK1 -90 -107 -70 -87 -50
-67 AE51 -64 -81 -44 -61 -24 -41 AE52 -64 -81 -44 -61 -24 -41 AE53
pK1 -82 -99 -62 -79 -42 -59 AE55 -62 -79 -42 -59 -22 -39 AE56 -67
-84 -47 -64 -27 -44 AE57 pK1 -81 -98 -61 -78 -41 -58 AE59 -74 -91
-54 -71 -34 -51 AE60 -61 -78 -41 -58 -21 -38 AE62 -69 -86 -49 -66
-29 -46 AE65 pK1 -85 -102 -65 -82 -45 -62 AE65 pK2 -109 -126 -89
-106 -69 -86 AE66 pK1 -84 -101 -64 -81 -44 -61 AE69 pK1 -79 -96 -59
-76 -39 -56 AE70 pK1 -78 -95 -58 -75 -38 -55 AE71 pK1 -81 -98 -61
-78 -41 -58 AE72 pK1 -82 -99 -62 -79 -42 -59
[0117] IPN3 Structures.
[0118] The description given so far is also sufficient to construct
complete nucleic acid monomer units as IPN3 structures. The
preferred internucleoside linkage is of phosphodiester,
phosphothioate, or phosphodithioate type, but other linkages can be
used as long as they provide a negative charge. IPN3 structures
comprise a CM that responds to pH between pH4 and pH7.4, and in
preferred embodiments of IPN3, the pK of the CM is between 4 and
7.5, more preferred is a pK between 5 and 6.5. Besides their CM,
IPN3 structures further comprise a TEE.
[0119] The core architecture of some IPN3 nucleic acid monomers is
presented in table 15.
TABLE-US-00015 TABLE 15 Core structures of IPN3 elements (IPN3-1)
##STR00207## (IPN3-2) ##STR00208## (IPN3-3) ##STR00209## (IPN3-4)
##STR00210## (IPN3-5) ##STR00211## (IPN3-6) ##STR00212##
[0120] In many aspects of the inventive IPN3 structures, the weak
acid of the TEE is a carboxylic acid.
[0121] In preferred embodiments of IPN3 structures, the hydrophobic
element of the TEE is directly attached to the CM as in IPN3-1,
IPN3-4 or IPN3-5.
[0122] In most aspects, the log D(pH4) of an IPN3 nucleotide is
higher than -2, in preferred aspects this value is between -1 and
+3 and in some aspects it is greater than 3. Said log D refers to
the chain form for the IPN3 nucleotide and represents the monomer
increment per nucleotide, it is different from the IPN3 monomer
itself mainly due to the presence of the second charge at the
phosphate group.
[0123] The log D of IPN3 structures can be considered as a
composite from (i) the backbone elements internucleoside linkage
and nucleoside sugar, (ii) the nucleobases itself, if present,
(iii) the CM and (iv) the pH dependent zwitterion formation between
CM and internucleoside linkage and (v) the TEE. Quantitative
estimates for the composite elements have been presented above and
approximate values for (i) are -2 to -3; (ii) is pH dependent and
about -1.4 at pH 7.4 and -1.8 at p1-14; values for (iii) are listed
in tables 3 to 5 for the unprotonated form at pH>pK and (iv) is
about +1 for the zwitterion formation. For (v), the contribution of
the TEE can be segmented into the contribution of the hydrophilic
moiety and the hydrophobic moiety to simplify the assignment of the
number of elements in the hydrophobic moiety. Data provided in
PCT/EP2007/011188 for the relationship between the number of
methylene groups in carboxylic acids and the resulting log D values
can be extrapolated to conclude the contributions of this charged
element. The resulting values are -0.9 for the uncharged and -3.4
for the charged state of this element. The TEE also comprises a
hydrophobic element, since without such element the net
contribution of the charged group to the log D is negative. For
practicing this invention, the global log D of the IPN structure is
of importance, it is less important whether the CM and its
hydrophobic additions or the TEE and its hydrophobic element
contribute the required hydrophobicity. In many embodiments of the
IPN3 and IPN3 structures this hydrophobic moiety can be common
structure of the CM and the TEE.
[0124] The following table 16 presents specific composite log D for
some of the IPN3 structures comprising any of the core unit of
IPN3-1 through to IPN3-4 using the data presented in the tables 3
to 5 above. It is of course possible to analyze structures of
different chemical origin in the same way; this analysis is
illustrating, but not limiting the teachings of this invention. The
composite log D values do comprise the elements (i) to (v)
presented before for IPN3 structures and are given for both the
neutral pH and an acidic pH; these composite log D's are
extrapolated for species wherein the hydrophobic moiety of the TEE
is absent. This allows identifying preferred ranges for the size of
said hydrophobic elements and said ranges are given in the last two
columns of this table 16 for cases where the hydrophobic elements
are methylene groups. Preferred hydrophobic moieties comprise
between 4 and 22 carbon atoms. REF1 teaches that the specific
chemical configuration of such hydrophobic moieties has little
impact on the log D contribution; REF1 has also analyzed the impact
of potential substitutions to such hydrophobic moieties. In many
cases the specific position of the hydrophobic moiety on the core
structures (IPN3-1)-(IPN3-4) may also vary without substantial
impact to the physicochemical parameters.
TABLE-US-00016 TABLE 16 Physicochemical analysis of some specific
IPN3 structures. CM denotes the structures listed in table 2
herein. IPN3 moieties composite # C atoms # C atoms logD composite
logD for for CM pK >pK logD pH 4 pH 7.4 logD -1 logD +3 none
none 0 -4.3 -3.9 3 5.56 -1.92 -6.12 -9.22 10 18 6 5.08 -0.95 -5.15
-8.25 8 16 7 5.5 -0.39 -4.59 -7.69 7 15 10 5.45 0.18 -4.02 -7.12 6
14 13 7 -1.49 -5.69 -8.79 9 17 16 6.54 -1.86 -6.06 -9.16 10 18 27
5.23 0.73 -3.47 -6.57 4 12 32 pK2 5.3 -4.1 -8.3 -11.4 14 22 40 6.58
-0.74 -4.94 -8.04 7 15 41 6.95 -0.6 -4.8 -7.9 7 15 42 6.58 0.55
-3.65 -6.75 5 13 46 5.94 -1.02 -5.22 -8.32 8 16 47 5.31 -2.55 -6.75
-9.85 11 19 48 6.78 -0.63 -4.83 -7.93 7 15 54 6.13 -1.3 -5.5 -8.6 9
17 55 5.01 -0.46 -4.66 -7.76 7 15 60 5.16 -1.21 -5.41 -8.51 8 16 63
5.34 -1.9 -6.1 -9.2 10 18 66 5.31 -0.39 -4.59 -7.69 7 15 69 6.21
-0.61 -4.81 -7.91 7 15 70 5.53 -0.9 -5.1 -8.2 8 16 71 6.6 0.39
-3.81 -6.91 5 13 74 6.44 -1.36 -5.56 -8.66 9 17 81 pK2 5.51 -4.5
-8.7 -11.8 15 23 82 pK2 6.48 -3.8 -8 -11.1 14 22 AE1 6.39 -1.26
-5.46 -8.56 8 16 AE2 6.21 -0.38 -4.58 -7.68 7 15 AE7 6.2 -2.07
-6.27 -9.37 10 18 AE8 6.14 -0.77 -4.97 -8.07 7 15 AE9 6.51 -1.18
-5.38 -8.48 8 16 AE10 6.6 -0.55 -4.75 -7.85 7 15 AE11 6.75 -1.61
-5.81 -8.91 9 17 AE12 6.79 -0.24 -4.44 -7.54 6 14 AE14 5.9 -0.65
-4.85 -7.95 7 15 AE15 6.9 -0.541 -4.741 -7.841 7 15 AE16 6.75 -0.32
-4.52 -7.62 7 15 AE21 6.3 -2.1 -6.3 -9.4 10 18 AE22 6.11 -0.58
-4.78 -7.88 7 15 AE24 6.95 -0.59 -4.79 -7.89 7 15 AE27 5.18 -2.75
-6.95 -10.05 11 19 AE28 5.75 -1.46 -5.66 -8.76 9 17 AE29 6.12 -1.86
-6.06 -9.16 10 18 AE30 6.22 -1.23 -5.43 -8.53 8 16 AE31 6.36 -2.3
-6.5 -9.6 11 19 AE32 6.4 -0.92 -5.12 -8.22 8 16 AE34 5.8 -1.36
-5.56 -8.66 9 17 AE35 6.8 -1.46 -5.66 -8.76 9 17 AE36 6.52 -1.51
-5.71 -8.81 9 17 AE40 5.58 -2.59 -6.79 -9.89 11 19 AE41 6.68 -2.68
-6.88 -9.98 11 19 AE42 6.29 -2.73 -6.93 -10.03 11 19 AE54 6.96 0.07
-4.13 -7.23 6 14 AE57 6.87 -3.8 -8 -11.1 14 22 pK2 AE63 6.41 -0.72
-4.92 -8.02 7 15 AE64 6.41 -1.06 -5.26 -8.36 8 16 AE67 5.99 0.33
-3.87 -6.97 5 13 AE68 6.02 0.94 -3.26 -6.36 4 12 AE69 6.17 -3.8 -8
-11.1 14 22 pK2 AE70 5.72 -3.66 -7.86 -10.96 13 21 pK2 AE71 6.09 -4
-8.2 -11.3 14 22 pK2 AE72 6.32 -3.9 -8.1 -11.2 14 22 pK2
[0125] To further illustrate, but not limit the teachings of this
invention, the following table 17 presents some more specific
chemical representations for IPN3 structures in nucleic acids,
wherein Y is oxygen or sulphur, B represent any of the nucleobases
or is absent, the index n denotes the length of the nucleic acid
and n>2 and i denotes the length of an alkyl chain which here
represents the hydrophobic moiety, (i-1) preferably falls within
the range specified in table 16 for the respective compounds. More
than one hydrophobic moiety can be used in the such cases the
preferred range for their size indices k and j shall be selected to
meet k+j=i-1.
[0126] From the IPN3 structures shown below it becomes clear that
the attachment site of a given CM to the nucleoside sugar may vary
and that some CM's can be inserted in different orientations. Also,
the position of the hydrophobic element may vary and the position
of the graft may be variable, as illustrated in the structures
IPN3-10 through to IPN3-21.
TABLE-US-00017 TABLE 17 Specific representations of IPN3
structures. IPN3-10 ##STR00213## IPN3-11 ##STR00214## IPN3-12
##STR00215## IPN3-13 ##STR00216## IPN3-14 ##STR00217## IPN3-15
##STR00218## IPN3-16 ##STR00219## IPN3-17 ##STR00220## IPN3-18
##STR00221## IPN3-19 ##STR00222## IPN3-20 ##STR00223## IPN3-21
##STR00224## IPN3-22 ##STR00225## IPN3-23 ##STR00226## IPN3-24
##STR00227## IPN3-25 ##STR00228## IPN3-26 ##STR00229## IPN3-27
##STR00230##
[0127] As disclosed above, the architecture of nucleic acids
comprising IPN structures may vary and different degrees of
substitution of native nucleic acids with IPN structures are
possible. Also, such substitutions may be concentrated on specific
sites within a nucleic acid, e.g. at one or both flanks of the
sequence, at the center part of such sequence or they might be
dispersed throughout the sequence. A known design in the art is a
"gapmer", wherein modified sequences at the flanks of an
oligonucleotide encompass an unmodified or differently modified
sequence in a center piece.
[0128] The following table 18 gives calculated log D values for 20
mer oligonucleotides having a degree of substitution of 50% and
such log D values have been calculated for pH4 and pH7.4 and for
different values of i, the length indicator for the hydrophobic
element. For comparison, the log D (pH7.4) of an unmodified
sequence of the same type is -81, that of pH4 is -89. The standard
assumptions giving above for log D of the internucleoside linkage,
nucleobases and zwitterionic interaction were used.
[0129] Introduction of IPN3 elements into an oligonucleotide, even
at a limited degree of substitution leads to substantial
improvements in log D and preferred oligonucleotides having a log
D>-60 are highlighted in the table 18. Also, the IPN3
substitutions minimize or even reverse the pH-induced decrease of
log D at low values of pH. In a third aspect, this analysis gives
additional guidance on the number of carbon atoms (i) in the
hydrophobic element that may be present in IPN3 structures. While
i=12 yields preferred structures in almost any case, even the short
element with i=8 is sufficient for most species analyzed here. Even
more so, some species achieve substantial improvements in log D
even with i=4.
TABLE-US-00018 TABLE 18 logD predictions for 20mers of
oligonucleotides comprising 50% IPN3 structures. CM denotes the
structures from table 2 herein. 20mer oligonucleotides, 50%
substitution with IPN3 CM i = 4 i = 8 i = 12 none pH 4 pH 7.4 pH 4
pH 7.4 pH 4 pH 7.4 3 -87 -114 -67 -94 -47 -74 6 -78 -105 -58 -85
-38 -65 7 -72 -99 -52 -79 -32 -59 10 -66 -93 -46 -73 -26 -53 13 -83
-110 -63 -90 -43 -70 16 -87 -114 -67 -94 -47 -74 27 -61 -88 -41 -68
-21 -48 32 pK2 -109 -136 -89 -116 -69 -96 40 -75 -102 -55 -82 -35
-62 41 -74 -101 -54 -81 -34 -61 42 -63 -90 -43 -70 -23 -50 46 -78
-105 -58 -85 -38 -65 47 -94 -121 -74 -101 -54 -81 48 -74 -101 -54
-81 -34 -61 54 -81 -108 -61 -88 -41 -68 55 -73 -100 -53 -80 -33 -60
60 -80 -107 -60 -87 -40 -67 63 -87 -114 -67 -94 -47 -74 66 -72 -99
-52 -79 -32 -59 69 -74 -101 -54 -81 -34 -61 70 -77 -104 -57 -84 -37
-64 71 -64 -91 -44 -71 -24 -51 74 -82 -109 -62 -89 -42 -69 81 pK2
-113 -140 -93 -120 -73 -100 82 pK2 -106 -133 -86 -113 -66 -93 AE1
-81 -108 -61 -88 -41 -68 AE2 -72 -99 -52 -79 -32 -59 AE7 -89 -116
-69 -96 -49 -76 AE8 -76 -103 -56 -83 -36 -63 AE9 -80 -107 -60 -87
-40 -67 AE10 -74 -101 -54 -81 -34 -61 AE11 -84 -111 -64 -91 -44 -71
AE12 -70 -97 -50 -77 -30 -57 AE14 -75 -102 -55 -82 -35 -62 AE15 -73
-100 -53 -80 -33 -60 AE16 -71 -98 -51 -78 -31 -58 AE21 -89 -116 -69
-96 -49 -76 AE22 -74 -101 -54 -81 -34 -61 AE24 -74 -101 -54 -81 -34
-61 AE27 -96 -123 -76 -103 -56 -83 AE28 -83 -110 -63 -90 -43 -70
AE29 -87 -114 -67 -94 -47 -74 AE30 -80 -107 -60 -87 -40 -67 AE31
-91 -118 -71 -98 -51 -78 AE32 -77 -104 -57 -84 -37 -64 AE34 -82
-109 -62 -89 -42 -69 AE35 -83 -110 -63 -90 -43 -70 AE36 -83 -110
-63 -90 -43 -70 AE40 -94 -121 -74 -101 -54 -81 AE41 -95 -122 -75
-102 -55 -82 AE42 -95 -122 -75 -102 -55 -82 AE54 -67 -94 -47 -74
-27 -54 AE57 pK2 -106 -133 -86 -113 -66 -93 AE63 -75 -102 -55 -82
-35 -62 AE64 -79 -106 -59 -86 -39 -66 AE67 -65 -92 -45 -72 -25 -52
AE68 -59 -86 -39 -66 -19 -46 AE69 pK2 -106 -133 -86 -113 -66 -93
AE70 pK2 -105 -132 -85 -112 -65 -92 AE71 pK2 -108 -135 -88 -115 -68
-95 AE72 pK2 -107 -134 -87 -114 -67 -94
[0130] The teachings of this invention have been demonstrated using
phosphoribose as a backbone structure of the nucleic acids;
however, the biophysical principles used herein can of course be
applied to other and related structures. It is possible to replace
the ribose ring in the nucleosides by other furanoses, by pyranoses
or by hexitol ring structures. Also, the use of locked nucleic
acids, that is nucleic acids comprising a 2', 4' methoxy or ethoxy
bridge or the use of open structures that are devoid of the oxygen
in the furanose ring (so called unlocked nucleic acids) is well
within the teachings of this invention.
[0131] It is also possible to further substitute the backbone
structure with hydroxyl or alkoxy groups so that hemiacetals or
acetals are formed. According to REF1, any additional hydroxyl
group is expected to further decrease the log D of a given
structure by about 2 units. In contrast, only very small changes of
log D or even an increase of its value were observed when
calculating this parameter for hemiacetals or acetals of furanose
or pyranose ring structures. In consequence, shorter hydrophobic
elements are needed and the number of C atoms in these can be
reduced. The tables 19 and 20 illustrate this part of the invention
without limiting its scope.
TABLE-US-00019 TABLE 19 Alternative nucleoside structures having
hemiacetal or acetal structures. N1 ##STR00231## N2 ##STR00232## N3
##STR00233## N4 ##STR00234## N5 ##STR00235## N6 ##STR00236## N7
##STR00237## N8 ##STR00238## n >= 2, B is any nucleobase
selected from adenine, guanine, thymine, cytosine or uracile; Y is
oxygen or sulphur, the CM of this example is AE1 and can be
substituted by the other CM's mentioned throughout this
invention.
TABLE-US-00020 TABLE 20 Analysis of logD contribution for the
structures listed in table 19. The relative logD listed herein
compares the actual logD to that of the ribose form. Actual logD
values for a specific hemiacetals or acetals can be calculated by
adding this value to the logD value of the respective ribose form
from the tables shown earlier in this disclosure, in particular
tables 8, 12 and 16. The "relative number of C atoms" follow the
same logic. Structure Relative logD Relative number of C atoms N1 0
(unsubstitued ribose, 0 (unsubstituted ribose, reference structure)
reference structure) N2 +0.67 -1 N3 +0.89 -2 N4 +1.42 -3 N5 +1.18
-2 N6 +1.76 -3 N7 +2.57 -5 N8 +3.1 -6
[0132] It is therefore possible to use the hemiacetals or acetals
of arabinoside nucleosides as shown in the prototype structures
N1-N4 or those of the unlocked nucleic acids as illustrated with
the prototype structures N5-N8.
[0133] From the description made throughout this disclosure it
becomes clear that the beneficial effects of the cationic moiety
forming the zwitterionic structure, the TEE undergoing a pH driven
hydrophobic transition and the general increase of hydrophobicity
contributed by the hydrophobic moieties are by and large additive.
This allows mesoscale predictions of log D values for larger
structures such as oligonucleotides that cannot easily be predicted
on their atomistic level. It also facilitates an analysis of
oligonucleotides carrying mixed nucleotides, such as native and IPN
modified nucleotides. The additive effect of said substitutions can
also be used to design nucleic acids carrying different backbone
modifications. At the end, much more detailed predictions for
specific sequences and modification patterns can be achieved using
the mesoscale approach presented herein and such predictions are
well within the teachings of this invention.
[0134] The additive effect of the modifications to the nucleic
acids also supports the construction of nucleic acids wherein mixed
modifications of IPN's and the TEE-modified nucleotides of the REF1
are used.
[0135] The IPN of this invention or the TEE modified
oligonucleotides of REF1 both represent nucleotide structures that
undergo a hydrophilic-hydrophobic transition when exposed to a
slightly acidic environment. Depending on the type of modification,
the pH induced amplitude in log D may vary and typical values for
such amplitude are about +1 per abasic IPN2 monomer, +2.5 per
abasic IPN1 monomer or per TEE modified nucleotide of REF1 or +3.5
per abasic IPN3 monomer. As such, quite substantial changes in the
hydrophobic character of a nucleotide can be induced by the
inventive structures. As one consequence, the additional need for
hydrophobic elements has been shown to be rather moderate, with i=4
to 12 methylene groups yielding significant improvements in the
cellular permeability of larger nucleic acids even at a limited
degree of substitution.
DESCRIPTION OF THE FIGURES
[0136] FIG. 1: pH dependent log D values for IPN structures. Log D
values for compounds (CM4), (CM5), (CM6) and (CM7) have been
analyzed over a range of different pH values. Calculations were
done using the log D module of the ACD/Labs 7.00 software package.
For comparison with structures not comprising a CM, the respective
values for a methylated 3' phosphoribose are presented.
* * * * *