Note: Descriptions are shown in the official language in which they were submitted.
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C-nucleoside derivatives and their use in the detection
of nucleic acids
The invention concerns C-nucleosides and derivatives
thereof as well as their use for labelling, detecting
and sequencing nucleic acids.
Nucleic acids are of central importance in living nature
as carriers or transmitters of genetic information.
Therefore since their discovery by F. Miescher they have
stimulated a broad scientific interest which has led to
the elucidation of their function, structure and
mechanism of action. The increasing knowledge of these
basic mechanisms in molecular biology has made it
possible in recent years to make new combinations of
genes. This technology opens for example new
opportunities in medical diagnosis and therapy and in
plant breeding.
An important tool for elucidating these relationships
and to solve problems was and is the detection of
nucleic acids with regard to their specific detection as
well as with regard to their sequence i.e. their primary
structure.
The specific detectability of nucleic acids is based on
the property of these molecules to interact or hybridize
with other nucleic acids by forming base pairs via
hydrogen bridges. Nucleic acids (probes) labelled in a
suitable manner i.e. provided with indicator groups, can
thus be used to detect complementary nucleic acids
(target).
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The determination of the primary structure (sequence),
i.e. the sequence of the heterocyclic bases of a nucleic
acid, is carried out by means of sequencing techniques.
This knowledge of the sequence is in turn a prerequisite
for a targetted and specific use of nucleic acids in
problems and methods of molecular biology. In the end
sequencing also utilizes the specific hybridization
among nucleic acids. Labelled nucleic acid fragments are
also used for this as mentioned above.
Consequently the suitable labelling of nucleic acids is
an indispensable prerequisite for any detection method.
Radioactive labelling with suitable isotopes such as 32p
or 35S was already used for this at an early stage. The
disadvantages of using radioactive reagents are,
however, obvious: such work requires specially equipped
facilities and permits as well as a controlled and
complicated disposal of the radioactive waste.
Furthermore the reagents for radioactive labelling are
expensive. It is not possible to store such labelled
samples for long periods due to the short half-lives of
the above nuclides.
In recent years there have therefore been many attempts
to circumvent these serious disadvantages i.e. to get
away from radioactive labelling. In doing so the high
sensitivity of this type of label should be retained as
far as possible.
Major advances have in fact already been achieved [see
for example Nonradioactive Labeling and Detection of
Biomolecules (Kessler, C., pub.) Springer Verlag,
Berlin, Heidelberg 1992].
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An essential prerequisite for any detection of a nucleic
acid is that it should be previously labelled and - as
explained above - if possible in a non-radioactive
manner. Whereas the radioactive labelling of nucleic
acids is usually carried out by enzymatically catalysed
incorporation of corresponding radioactive nucleoside
triphosphates, non-radioactive labelling must be
achieved by incorporating a suitable signal or reporter
group.
Non-radioactive indicator molecules that have proven to
be suitable among others are above all haptens (such as
biotin or digoxigenin), enzymes (such as alkaline
phosphatase or peroxidase) or fluorescent dyes (such as
fluorescein or rhodamine). These signal groups can be
attached to or incorporated into nucleic acids by
various methods.
A relatively simple procedure is for example to label
the 5' end of an oligonucleotide provided with a
terminal amino function by means of activated indicator
molecules of the above-mentioned type. However, this
only enables one or a few indicator molecules to be
introduced into only a low molecular oligomer whereas a
denser labelling of longer chained, high molecular
nucleic acids with the goal of achieving a higher
sensitivity must usually be achieved by incorporating
nucleoside triphosphates provided with reporter groups
by means of polymerases along the lines of a de novo
synthesis.
Corresponding methods are known to a person skilled in
the art as nick translation [Rigby, P.W. et al. (1977),
J.Mol.Biol. 113, 237] and random primed labelling
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[Feinberg, A.P. & Vogelstein. B. (1984) Anal. Biochem.
137, 266]. A further method is the so-called 3'-tailing
reaction with the aid of the enzyme terminal
transferase.
The nucleoside triphosphates which have been used up to
now in these methods are almost exclusively
appropriately modified derivatives of the heterocyclic
bases adenine, guanine, cytosine and thymine in the
deoxyribonucleotide series, or adenine, guanine,
cytosine and uracil in the ribonucleotide series. Such
derivatives are for example described by Langer et al.,
Proc. Natl. Acad. Sci. USA 78, 6635 (1981); Muhlegger et
al., Biol. Chem. Hoppe-Seyler 371, 953 (1990) and in EP
0 063 879. In this case the building blocks that occur
naturally in DNA and RNA are used in a labelled form
i.e. in a form provided with signal groups. The main
disadvantages of the N-nucleosides are the sensitivity
of the N-glycosidic bond towards acidic pH conditions
and degradability by nucleases.
Furthermore individual C-nucleosides (see e.g.
Suhadolnik, R.J. in Nucleoside Antibiotics, Wiley-
Interscience, New York 1970) and their therapeutic use
(antiviral or cancerostatic) has been known for a long
time.
In addition fluorescent C-nucleoside derivatives and
their incorporation into DNA and RNA oligonucleotides
has been described (WO 93/16094). The so-called inherent
fluorescence of these C-nucleosides is, however, many-
fold less with regard to quantum yield than of special
fluorophores such as fluorescein or corresponding
rhodamine derivatives. A further disadvantage of the
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autofluorescent C-nucleosides is their relatively low
excitation and emission wavelengths. As a consequence
detection systems which are based on such derivatives
only have a low detection sensitivity and on the other
hand spectrally interfering influences of the
measurement environment (such as biological material,
autofluorescence of gel matrices etc.) become very
apparent.
The known nucleosides and nucleoside derivatives thus
have a number of disadvantages which in particular have
adverse effects on the detection of nucleic acids.
Hence the object of the invention is to provide
nucleoside derivatives modified with signal groups for
the detection of nucleic acids which do not have the
said disadvantages i.e. are in particular more stable
and at the same time able to be processed enzymatically
and are suitable for the detection of nucleic acids at a
more practical wavelength.
The object is achieved by pyrrolo-[3,2-d]pyrimidine,
pyrazolo-[4,3-d]pyrimidine and pyrimidine-furanosides of
the general formulae I-V:
R2 R2 R2
N R3 N N~R3 N
N, ~ \ I
R ~
, N Ra R, N R4 Ri N R3
R9-0 O R9-O R9-Q
O p
Ra R R8 R R8 R
R7 R6 R7 Rs R7 Rs
I II III
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R2 R
1
N N-IR3 N ~R2
R1 N R3
Ry-Q R9-O--' 0
R8 0 R R$ R
R7 R6 R7 Rs
IV V
in which R1, R2, R3, R4 can be same or different and
represent hydrogen, halogen, hydroxy, thio or
substituted thio, amino or substituted amino,
carboxy, lower alkyl, lower alkenyl, lower alkinyl,
aryl, lower alkyloxy, aryloxy, aralkyl, aralkyloxy or
a reporter group,
R5 and R6 each represent hydrogen, hydroxy, thio
or substituted thio, amino or substituted amino,
lower alkyloxy, lower alkenoxy, lower alkinoxy, a
protecting group or a reporter group,
R7 represents hydrogen, hydroxy, thio or substituted
thio, amino or substituted amino, a phosphoramidite
or H-phosphonate group, an ester or amide residue
that can be cleaved in a suitable manner or a
reporter group,
R6 and R7 together form a further bond between C-2' and
C-3' or an acetal group,
R8 represents hydrogen or a hydroxy, thio or substituted
thio, amino or substituted amino group,
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R9 represents hydrogen, a mono-, di- or triphosphate
group or the alpha, beta or gamma-thiophosphate
analogues of these phosphoric acid esters or a
protecting group,
as well as possible tautomers and salts thereof.
Any detectable groups come into consideration as a
reporter group such as in particular haptens, a
fluorophore, a metal-chelating group, a lumiphore, a
protein or an intercalator.
Those compounds of the general formulae I to V are
preferred in which the acetal function of the residues
R6 and R7 is substituted by a reporter group.
Furthermore those compounds have proven to be
particularly suitable in which the reporter group is
bound to the aglyconic or furanose ring via a so-called
linker group. Appropriate linker groups are known to a
person skilled in the art (see e.g. Muhlegger, K. et al.
(1990) Biol. Chem. Hoppe-Seyler 371, 953-965 or Livak,
K.J. et al. (1992) Nucl. Acids Res. 20, 4831-4837).
In addition compounds of the general formulae I to IV
are preferred in which R1 represents hydrogen, hydroxy
or an amino group, R2 represents hydroxy, an optionally
substituted amino group or a reporter group, R3 and R4
represent hydrogen, halogen or a reporter group, R5
represents hydrogen, R6 represents hydrogen or hydroxy,
R7 represents hydrogen, hydroxy, thio, an optionally
substituted amino group, a phosphoramidite or a reporter
group, R6 and R7 together represent an acetal function,
R8 represents hydrogen and R9 represents a triphosphate
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function.
In accordance with one aspect of the present invention
there is provided a compound of formula V:
R,
N ) N~ Rz
~
O R3
R9-O
R$ O R
R, R6
V
wherein R1, R2, R3, can be same or different and represent
hydrogen, halogen, hydroxy, thio or substituted thio, amino
or substituted amino, carboxy, lower alkyl, lower alkenyl,
lower alkinyl, aryl, lower alkyloxy, aryloxy, aralkyl,
aralkyloxy or a reporter group, R5 and R6 each represent
hydrogen, hydroxy, thio or substituted thio, amino or
substituted amino, lower alkyloxy, lower alkenoxy, lower
alkinoxy, or a reporter group, R7 represents hydrogen,
hydroxy, thio or substituted thio, amino or substituted
amino, a phosphoramidite or H-phosphonate group, or a
reporter group, R6 and R7 together form a further bond
between C-2' and C-3' or an acetal group, R8 represents
hydrogen or a hydroxy, thio or substituted thio, amino or
substituted amino group, R9 represents hydrogen, a mono-,
di- or triphosphate group or the alpha, beta or gamma
thiophosphate analogues of these phosphoric acid esters,
wherein the lower alkyl is C1-C6, the lower alkenyl is C2-C6,
the lower alkinyl is C2-C6, the aryl is -C6H5, the lower
alkyloxy
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is -O- (CH2)nCH3 with n=0-6, the aryloxy with --O--C6H5r
aralkyl is -(CH2) nC6H5 with n=1-6, is aralkyloxy-O- (CHz) nC6H5
with n=1-6, the lower alkenoxy is -O-alkenyl with C2-C6, and
the lower alkinoxy is --O--alkinyl with C2-C6, as well as
possible tautomers and salts thereof.
In accordance with another aspect of the present invention
there is provided a use of compound V as previously
defined: as a substrate for DNA and RNA polymerases; for
labeling nucleic acids; for detecting nucleic acids in DNA
sequencing; and for in-situ hybridization.
In accordance with yet another aspect of the present
invention there is provided oligonucleotides which contain
the compound V previously defined.
In accordance with still a further aspect of the present
invention there is provided nucleic acids which contain the
compound previously defined.
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Preferred compounds of the general formula V are those
in which R1 represents hydroxy, a thio or amino group
which optionally can be substituted or a reporter group,
R2 and R3 represent hydrogen, lower alkyl or a reporter
group, R5 represents hydrogen, R6 represents hydrogen or
hydroxy, R7 represents hydrogen, hydroxy, thio, an
optionally substituted amino group, a phosphoramidite or
a reporter group, R6 and R7 together represent an acetal
function, R8 represents hydrogen and R9 represents a
triphosphate function.
It is expedient to synthesize ttie new modified
C-nucleosides by startirig with naturally occurring
precursors. Thus for example forrnycin A as an adenosine
analogue can be deaminated to form formycin B (an
inosine analogue) . This can in tur_n be halogenated and
be further substituted nucleophilically by which means
it is possible to produce a series of interesting new
compounds such as those of the inventive type.
The important 2'-deoxy-nucleosides are synthesized by
deoxygenating the above-mentioned naturally occurring
ribonucleosides such as e.g. formycin A. In this case
the deoxygenation reaction according to Barton is mainly
used nowadays (Barton, D.H.R. & Motherwell, W.B. (1981)
Pure Appl. Chem. 53, 15).
Furthermore the C-nucleosides can be synthesized
chemically as described for example in detail by K.A.
Watanabe in "Chemistry of Nukleosides and Nukleotides"
3, 421-535 (L.B. Townsend, publ.) Plenum Press, New York
and London, 1994.
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The use of the compounds according to the invention to
label nucleic acids with diverse defined signal groups
and thus to detect and sequence nucleic acids has proven
to be particularly advantageous.
The C-nucleosides according to the invention of the
general formulae I to V have a number of advantages
especially compared to the classical N-glycosidically
linked nucleosides and nucleotides such as adenosine,
guanosine, cytidine, thymidine, uridine and
corresponding phosphoric acid esters thereof.
One advantage is the chemical stability of the
C-glycosidic bond for example towards acidic pH
conditions. A further important advantage is the
stability of these compounds towards enzymatic
degradation by endonucleases and exonucleases. These
enzymes are present in biological material and can
severely interfere with the nucleic acid detection. On
the other hand it is known that DNA and RNA polymerases
are very discriminating with regard to accepting
nucleoside 5'-triphosphates that have been modified to a
greater or lesser extent i.e. with regard to recognizing
and incorporating such nucleotides as substrates in the
de novo synthesis. In particular experience has shown
that the attachment of signal groups to nucleotides
influences the incorporation and incorporation rate.
The fact that the nucleosides according to the invention
and derivatives thereof are incorporated very
efficiently into nucleic acids by suitable polymerases
such as e.g. by the methods described above of nick
translation or of random primed labelling cannot be
easily inferred from the state of the art and it must
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thus be regarded as surprising for a person skilled in
the art.
The said methods are generally used in nucleic acid
detection e.g. for the quantitative detection by
blotting techniques on membranes or also in microtitre
plates.
In the sequencing i.e. the detection of the sequence of
a nucleic acid, a complementary opposite strand is newly
synthesized on the nucleic acid to be sequenced with the
aid of a short (start) oligonucleotide (primer) and a
polymerase.
In the in situ hybridization for the detection of
certain genes or genome sections the same basically
occurs in the cell i.e. the specific incorporation of
labelled nucleotides.
The above-mentioned primers i.e. short-chained
oligonucleotides should form stable base pairs with the
template strand as well as not be attacked by endogenous
nucleases in order to ensure an optimal function.
This is fulfilled by oligonucleotides which contain the
C-nucleosides according to the as building blocks
invention instead of the classical N-nucleosides.
The same applies to long-chained polynucleotides and
nucleic acids which contain such C-nucleoside building
blocks. These are also a subject matter of the present
invention.
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Corresponding oligonucleotides as well as their
preparative precursors the nucleoside phosphoramidites
and nucleoside H-phosphonates are therefore also a
subject matter of the invention.
Nowadays oligonucleotides are usually synthesized by
known methods in automated DNA/RNA synthesizers.
Such methods of synthesis are essentially based on the
step-wise reaction of the above-mentioned
phosphoramidites or H-phosphonates and thus the
continuous linkage of these monomeric building blocks to
form oligomers (see e.g. T. Brown & D.J.S. Brown in
Oligonucleotides and Analogues-A Practical Approach
(1991) (Eckstein. F., publ.), IRL Press at Oxford
University Press, Oxford, New York, Tokyo).
Legend 5
Figure 1:
I and II denote pBR 328 DNA labelled by DIG-dUTP
incorporation and III denotes pBR 328 DNA labelled by
DIG-3-O-succinyl-s-amino-caproyl-[7-amino-3-(2'-deoxy-0-
D-erythropento-furanosyl)-1H-pyrazolo-[4,3-d]-
pyrimidine-5'-triphosphate incorporation.
The invention is elucidated in more detail by the
following examples:
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Example 1:
N1-Carboxymethyl-7-amino-3-(D-D-erythro-pentofuranosyl)-
1H-pyrazolo-[4,3-d]-pyridimine
70 mg (0.25 mmol) formycin A, 400 mg (2.2 mmol) ethyl
iodoacetate and 400 mg (2.88 mmol) K2CO3 are stirred in
ml methanol/water (1:1) for 3 h at room temperature.
The solution is subsequently evaporated to dryness,
taken up in 4 ml H20 and chromatographed on a
preparative HPLC (RP-18,25 x 1 cm). Water elutes (RE= 7
min) a main zone which is collected and concentrated by
evaporation. After lyophilization one obtains 48 mg of
the title compound (59 %).
1H-NMR (D20): 7.98 (s, H-5); 5.02 (d, J=7.4 Hz, H-1');
4.95 (s, CH2); 4.46 (t=6.0 Hz, H-2'); 4.21 (t, J=3.5 Hz,
H-3'); 4.07 (d, J=2.7 Hz, H-4'); 3.70 (m, H2-5').
13C-NMR (D20): 174.4 (C=O); 151.8 (C-7); 151.6 (C-5);
141.4 (C-3); 140.2 (C-3a); 122.9 (C-7a); 86.0 (C-4');
77.6 (C-1'); 75.2 (C-2'); 72.1 (C-3'); 62.3 (C-5'); 55.6
(CH2).
The corresponding 2'-deoxy derivative is produced in an
analogous manner starting with 2'-deoxy-formycin A.
Example 2:
7-Chloro-3-(2'-deoxy-(3-D-erythro-pentofuranosyl-)-iH-
pyrazolo-[4,3-d]-pyrimidine
The compound was synthesized (as described by L.B.
Townsend et al., in J. Chem. Soc. (C) 1971, 2443 for the
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ribofuranosyl derivative) starting from 2'-deoxy-
formycin B.
The latter was obtained from commercially available
formycin B by Barton deoxygenation (Barton, D.H.R. &
Motherwell, W.B. (1981) Pure Appl. Chem. 53, 15).
Example 3:
7-[1,6-Diaminohexyl]-3-(2'-deoxy-(3-D-erythro-
pentofuranosyl)-1H-pyrazolo-[4,3-d]pyrimidine
135 mg (0.5 mmol) Chloro-nucleoside from example 2 is
dissolved in 15 ml ethanol, admixed with 300 mg (ca.
2.5 mmol) hexamethylenediamine and refluxed for 3 h.
In the TLC (silica gel; chloroform-methanol 80:20) one
observes an almost quantitative conversion into the
title product. The reaction mixture is neutralized with
0.1 M HC1, evaporated and the concentrate is dissolved
in 10 ml ethanol. After removing the undissolved
components by filtration, it is chromatographed on a
silica gel 60 column with a mixture of chloroform
methanol (9:1). The combined fractions are evaporated
and lyophilized from dioxane (85 mg = 48.5 % of theory).
Elemental analysis for C16H26N603 (350.2); calculated
C 54.9, H 7.4, N 24.0; found C 55.3, H 7.6, N 23.7.
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Example 4:
7-[N-Trifluoroacetamidohexyl]-amino-3-(2'-deoxy-(3-D-
erythro-pentofuranosyl)-1H-pyrazolo-[4,3-d]pyrimidine
80 mg (0.23 mmol) of the nucleoside from example 3 is
dissolved in 10 ml anhydrous pyridine and admixed with
150 l (ca. 1 mmol) trifluoroacetic acid anhydride.
After standing for 5 h at room temperature the acylation
is complete according to TLC. The reaction solution is
subsequently evaporated in a vacuum and co-evaporated
three times with methanol. It is lyophilized from
dioxane and 100 mg (98 % of theory) of the desired
product is obtained.
Example 5:
7-fN-Trifluoroacetamidohexyll-amino-3-(2'-deoxy-o-D-
ervthro-nentofuranosyl)-iH-pyrazolo-j4,3-d]pyrimidine-
5'-triphosphate
50 mg (0.11 mmol) of the protected nucleoside from
example 4 is converted by phosphorylation with POC13
into the 5' monophosphate according to the method of
Yoshikawa et al. [Tetrahedron Lett. 50, 5065 (1967)];
the desired triphosphate is obtained from this after ion
exchange chromatography on DEAE sephadex in a yield of
45 mg (59 %) according to the method of Hoard & Ott [J.
Am. Chem. Soc. 87, (1965)] by activation with
carbonyldiimidazole and reaction with pyrophosphoric
acid.
31P-NMR (0.1 M EDTA/D20/Eth3N): -5,4 (d, P-gamma); -10.7
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(d, P-alpha) ; -21.2 (t, P-(3) .
Example 6:
Fluorescein-5(6)-carboxamidohexyl-[7-amino-3-(2'-deoxy-R
-D-erythro-pentofuranosyl)-iH-pyrazolo-I4,3-d]-
pyrimidine-5'-triphosphatel
35 mg (0.05 mmol) of the trifluoroacetyl-protected
compound from example 5 is allowed to stand for 1 h at
room temperature in 5 ml concentrated ammonia solution
and is subsequently evaporated in a vacuum. The residue
is taken up in 5 ml 0.1 M borate buffer, pH 8.5 and
admixed with a solution of 31 mg (0.065 mmol) 5(6)-
carboxy-fluorescein-N-hydroxy-succinimide ester in 5 ml
amine-free dimethylformamide. It is allowed to stand
overnight at room temperature. The reaction mixture is
applied to a DEAE sephadex column (30 x 1 cm) and eluted
with a linear LiCl gradient (200 ml each time H20 to
0.4 M LiCl). 28 mg (59 %) of the title substance is
obtained after combining the relevant fractions,
evaporating, precipitating the concentrate in
acetone/ethanol (2:1) and drying.
Spectral data (0.1 M phosphate buffer, pH 9.0):
excitationmax[nm]: 495:
emissionmax:[nm]: 521
The digoxigenin-3-O-succinyl-E-aminocaproyl-[7-amino-3-
(2'-deoxy-o-D-erythro-pentofuranosyl)-iH-pyrazolo-
[4,3-d]pyrimidine-5'-triphosphate is produced
correspondingly by reacting the nucleoside with
digoxigenin-3-0-succinyl-aminocaproic acid-N-hydroxy-
succinimide ester.
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Example 7:
N1-{8-[N-tert.-butoxycarbonylj-amino-(3,6-dioxa)octyl-l-
amidomethyl}-7-amino-3-(2'-deoxy-(3-D-erythro-
pentofuranosyl)pyrazolo-[4,3-dlpyrimidine
125 mg (0.5 mmol) 2'-deoxy-formycin A, 369 mg (1 mmol)
(N1-bromoacetamido-N8-t-butoxycarbonyl)-1,8-diamino-3,6-
dioxaoctane and 1.4 g K2CO3 are stirred for 3 h at room
temperature in 4 ml methanol/H20 (1:1). It was processed
as described in example 1. 135 mg (50 %) of the title
compound was obtained after chromatography on a RP-18
column.
Example 8:
N1-{8-Amino-(3,6-dioxa)octyl-l-amidomethyl}-7-amino-3-
(2'-deoxy-(3-D-erythro-pentofuranosyl)pyrazolo-[4,3-d]-
pyrimidine-5'-triphosphate
135 mg (0.25 mmol) of the protected nucleoside from
example 7 is converted into the triphosphate as
described in example 5. After chromatography on RP-18
the Boc protecting group is removed by treating for 1
hour with trifluoroacetic acid. Finally 25 mg of the
title compound is obtained after a further
chromatography on QAE sephadex.
31p-NMR (0.1 M EDTA/D20/Eth3N): -6.4 (d, P-gamma); -11.1
(d, P-alpha); -21.6 (t, P-(3).
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Example 9:
Digoxigenin-3-O-succinyl-E-aminocaproyl-{Nl-LB-amino-
(3,6-dioxa)octyl-l-amidomethyl]-7-amino-3-(2'-deoxy-(3-D-
erythro-pentofuranosyl)pyrazolo-[4,3-d]pyrimidine-5'-
triphosphate
17 mg (0.024 mmol) of the compound from example 8 is
taken up in 5 ml 0.1 M borate buffer, pH 8.5 and admixed
with a solution of 25 mg (0.036 mmol) digoxigenin-3-O-
succinyl-aminocaproic acid-N-hydroxy-succinimide ester
in 5 ml amine-free dimethylformamide. It is allowed to
stir for 5 h at room temperature and subsequently
chromatographed on RP-18 silica gel. After desalting and
lyophilizing 3 mg (ca. 10 %) of the labelled
triphosphate is obtained.
Example 10:
4-Oxo-7-(2'-deoxy-(3-D-erythro-pentofuranosyl)-3H, 5H-
pyrrolo-j3,2-d1pyrimidine
The compound was obtained by Barton deoxygenation from
the ribofuranosyl derivative described by M.-I. Lim et
al. in Tetrahedron Lett. 1980, 21, 1013.
Example 11:
4-Chloro-7-(2'-deoxy-(3-D-erythro-pentofuranosyl)-3H,5H-
pyrrolo-[3,2-d]pyrimidine
The compound was synthesized by halogenation with POC13
by the method described by Townsend et al., in J. Chem.
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Soc. (C), 1971, 2443.
Example 12:
4-[1,6-Diaminohexyl]-7-(2'-deoxy-(3-D-erythro-
pentofuranosyl)-3H,5H-pyrrolo-[3,2-d]pyrimidine
The derivative was obtained analogous to example 3
starting with the chlorinated compound from example 11
by reaction with diaminohexane.
Example 13:
Fluorescein-5(6)-carboxamidohexyl-[4-amino-7-(2'-deoxy-
j3-D-erythro-pentofuranosyl)-3H,5H-pyrrolo-j3,2-d]-
pyrimidine-5'-triphosphatel
The title compound was synthesized from the derivative
from example 12 via the steps of protecting the diamino
function with triflate, preparing the triphosphate and
reacting with fluorescein-5(6)-carboxamido-N-hydroxy-
succinimide ester. These individual process steps are
described in examples 4, 5 and 6.
Example 14:
5-(2'-Deoxy-(3-D-erythro-pentofuranosyl)pyrimidine-2,4-
dione-5'-triphosphate
650 mg (2.85 mmol) 5-(2'-deoxy-(3-D-erythro-
pentofuranosyl)-pyrimidine-2,4-dione prepared according
to J. Org. Chem. 1982, 47, 485 by deoxygenation of the
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commercially available 5-((3-D-erythro-pentofuranosyl)-
pyrimidine-2,4-dione (pseudouridine) is converted into
the 5'-triphosphate in a one-pot process according to
the method of Ludwig [Acta Biochim. et Biophys. Acad.
Sci. Hung. (1981), 16, 131]. Anion exchange
chromatography on QAE sephadex with a LiCl gradient
(water to 0.5 M) and precipitation in acetone/ethanol
(2:1) and drying yielded 850 mg (60 %) of the title
compound.
31p-NMR (0.1 M EDTA/D20/Eth3N): -6.8 (d, P-y); -11.0 (d,
P-a) ; -22. 0 (t, P-(3) .
Example 15:
N1-[Ethoxypropionyl]-5-(2'-deoxy-(3-D-erythro-
pentofuranosyl)pyrimidin-2,4-dione-5'-triphosphate
100 mg (0.2 mmol) of the 2'-deoxy-pseudouridine-5'-
triphosphate from example 14 is dissolved in 5 ml 1 M
triethylammonium bicarbonate buffer (pH 8.9) and admixed
with 5 ml ethanol. 3 ml (ca. 30 mmol) acrylic acid ethyl
ester is added and the mixture is stirred for 6.5 h at
room temperature. Afterwards it is no longer possible to
observe educt according to TLC (i-butyric acid/concentr.
ammonia/water = 66/1/33). The reaction mixture is
evaporated in a vacuum and co-evaporated once with a few
ml ethanol and water. The crude product is processed
further without further purification as described in
example 16.
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Example 16:
N1-(1,3-Diaminopropyll-5-(2'-deoxy-(3-D-erythro-
Qentofuranosyl)pyrimidin-2,4-dione-5'-triphosphate
The product from example 15 is dissolved in 5 ml water
and admixed with 5 ml ethanol and 2 ml 1,3 diamino-
propane. It is stirred overnight at room temperature and
afterwards concentrated with an oil pump in a vacuum
until the residue is viscous. It is taken up in a few ml
water and the pH is adjusted to 6 with dilute acetic
acid. Chromatography/desalting on RP 18 using a
triethylammonium acetate/acetonitrile gradient yielded
1000 A260 units (ca. 0.1 mmol) of the crude mixture of
the compound from which 140 A260 units (ca. 20 mol) of
the desired substance was isolated after ion exchange
chromatography on QAE sephadex using LiCl.
TLC (i-butyric acid/concentr. ammonia/water = 66/1/33):
Rf ca. 0.3: positive reaction with ninhydrin.
Example 17:
N1-Digoxigenin-3-o-methylcarbonyl-E-aminocaproyi-l,3-
diaminopropyl-propionyl]-5-(2'-deoxy-(3-D-erythro-
pentofuranosyl)pyrimidine-2,4-dione-5'-triphosphate
4.5 mg (5 mol) of the diaminopropyl derivative from
example 16 is dissolved in 1 ml 0.1 M sodium borate
buffer, pH 8.5 and 5 mg (7.5 mol) digoxigenin-3-O-
methylcarbonyl-s-amino-caproic acid-N-hydroxysuccinimide
ester dissolved in 1 ml amine-free dimethylformamide is
added. The clear solution is allowed to stand for ca.
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3 h at room temperature. The reaction solution is
afterwards evaporated, the residue is taken up in 1 ml
water and purified by means of RP 18 chromatography
(column: Inertsil, 250 x 8 mm, triethylammonium
acetate/acetonitrile). After removing the volatile
components in a vacuum and lyophilizing one obtains 7 mg
(90 %) of the title compound.
Example 18:
Tetramethylrhodamine-5(6)-carboxamido-{N1~fB-N-[3,6-
dioxa)octyl-l-amido-methyl]-5-(2'-deoxy-(3-D-erythro-
pentofuranosyl)pyrimidine-2,4-dione-5'-triphosphatek
The title compound was prepared and purified analogously
to example 17 by reacting 18 mg (30 mol) of the
triphosphate from example 16 and from 17 mg (32 mol)
tetramethylrhodamine-5(6)-carboxylic acid-N-hydroxy-
succinimide ester in 0.1 M sodium borate buffer,
pH 8.5/DMF. 10.5 mg of the TMR-labelled nucleotide was
obtained.
Spectral data (0.1 M Na borate buffer, pH 8.5):
excitationmaxLnm]: 551:
emissionmax:[nm]: 575
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Example 19:
Non-radioactive DNA labelling and detection by
incorporation of N1-digoxigenin-3-O-methylcarbonyl-s-
aminocaproyl-1,3-diaminORropyl-propionyl]-5-(2'-deoxy-R-
D-erythro-Qentofuranosyl)pyrimidine-2,4-dione-5'-
triphosphatel
The DNA labelling and the DNA detection was carried out
using the commercially available kit from the Boehringer
Mannheim Company (order No. 1093 657). The working
instructions describe all required working steps.
For the labelling reaction the dNTP mixture of the kit
was substituted by one with N1-[digoxigenin-3-O-
methylcarbonyl-E-aminocaproyl-1,3-diaminopropyl-
propionyl]-5-(2'-deoxy-p-D-erythro-pento-
furanosyl)pyrimidine-2,4-dione-5'-triphosphate from
example 17 (instead of DIG-dUTP).
The immunological detection reaction showed that the
incorporation of the compound according to the invention
according to example 17 had a detection sensitivity of
the labelled DNA which was similar to that when using
DIG-dUTP.
The result which demonstrates the detection and the
achieved sensitivity of the system are shown in
figure 1.
