U.S. patent application number 10/183525 was filed with the patent office on 2003-04-17 for rna polymerase transcription promoter and nucleic acid sequencing method.
Invention is credited to Hayashizaki, Yoshihide, Iwata, Masaaki.
Application Number | 20030073202 10/183525 |
Document ID | / |
Family ID | 18478985 |
Filed Date | 2003-04-17 |
United States Patent
Application |
20030073202 |
Kind Code |
A1 |
Iwata, Masaaki ; et
al. |
April 17, 2003 |
RNA polymerase transcription promoter and nucleic acid sequencing
method
Abstract
An RNA polymerase transcription accelerator comprising a
compound represented by the following Formula (I) or salts thereof.
1 A method of sequencing DNA in which nucleic acid transcripts are
obtained using an RNA polymerase and a DNA fragment as a template,
the resulted nucleic acid transcripts are separated, the nucleic
acid sequence is determined from the separated fractions wherein
the nucleic acid transcription reaction is carried out in the
presence of a compound selected from a group of compounds
represented by the above formula (I). The polyamine compounds above
have outstanding accelerating activity on transcription activity of
RNA polymerase. Therefore, use of the polyamine compounds in a DNA
sequencing method using RNA polymerase can make a length of DNA
sequence that can be determined in one sequencing longer.
Inventors: |
Iwata, Masaaki; (Wako-shi,
JP) ; Hayashizaki, Yoshihide; (Tsukuba-shi,
JP) |
Correspondence
Address: |
E. Joseph Gess
BURNS, DOANE, SWECKER & MATHIS, L.L.P.
P.O. Box 1404
Alexandria
VA
22313-1404
US
|
Family ID: |
18478985 |
Appl. No.: |
10/183525 |
Filed: |
June 28, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10183525 |
Jun 28, 2002 |
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09622644 |
Oct 23, 2000 |
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09622644 |
Oct 23, 2000 |
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PCT/JP99/07169 |
Dec 21, 1999 |
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Current U.S.
Class: |
435/91.2 ;
435/6.16; 564/512; 564/84 |
Current CPC
Class: |
C12Q 1/6869 20130101;
C12Q 1/6869 20130101; C12Q 2535/101 20130101; C12Q 2521/119
20130101; C12Q 2527/125 20130101 |
Class at
Publication: |
435/91.2 ;
564/512; 564/84; 435/6 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 21, 1998 |
JP |
10/363297 |
Claims
1. An RNA polymerase transcription accelerator comprising a
compound represented by the following Formula (I) or salts thereof.
11wherein: n is an integer of 1-8, R.sup.1 represents a hydrogen
atom or a p-toluenesulfonyl group, R.sup.2 represents an ethyl
group or a group represented by the following Formula (II), R.sup.3
represents a hydrogen atom or a p-toluenesulfonyl group, and
R.sup.4 represents an ethyl group or a group represented by the
following Formula (II): 12wherein, m is 1 or 2, R.sup.5 represents
a hydrogen atom, and R.sup.6 represents a hydrogen atom or an ethyl
group.
2. A method for sequencing DNA in which nucleic acid transcripts
are produced by using RNA polymerase and a DNA fragment as a
template, the resulting nucleic acid transcripts are separated, and
the nucleic acid sequence is determined from the separated
fractions, characterized in that said nucleic acid transcription
reaction is carried out in the presence of at least one compound
selected from the group of compounds according to claim 1.
3. The method of claim 2 wherein said DNA fragment comprises a
promoter sequence for the RNA polymerase, and the nucleic acid
transcription reaction is carried out using
ribonucleoside-5'-triphosphates comprising ATP, GTP, CTP and UTP or
derivatives thereof, and one or more kinds of
3'-deoxyribonucleoside-5'-triphosphate (hereinafter referred to as
3' dNTP derivatives) comprising 3' dATP, 3' dGTP, 3' dCTP, 3' dUTP
and derivatives thereof.
4. The method of claim 2 wherein the 3' dNTP derivatives are
labeled, and nucleic acid sequence is determined using the
label.
5. The method of claim 3 wherein the 3' dNTP derivatives are
labeled, and nucleic acid sequence is determined using the label.
Description
TECHNICAL FIELD
[0001] The present invention relates to polyamine compounds which
accelerate the transcription reaction of RNA polymerase
(hereinafter referred to as RNAP). Further, the present invention
relates to a DNA sequencing method using RNAP with the polyamine
compounds and an initiator of nucleic acid transcription reaction
by RNAP.
BACKGROUND TECHNOLOGY
[0002] A DNA sequencing method is one of the most important means
in molecular biological field. One of the most useful nucleic acid
sequencing method at present is a direct transcription sequencing
method (W096/14434) using RNAP such as T7 RNAP and a terminator of
RNA transcription reaction (for example,
3'-deoxyribonucleotide-5'-triphospha- te, 3'dNTPs). This method is
an outstanding method utilizing the RNAP transcription reaction for
sequencing nucleic acid sequences of DNA products amplified by
polymerase chain reaction without removing primers and
2'-deoxyribonucleoside-5-triphosphates (2'dNTPs). Recently, it has
been suggested that the RNAP transcription can be improved by the
addition of a compound.
[0003] Under the circumstances, it is hoped that a compound with
RNAP promoting effect will be developed. It has been reported that
as a transcription from DNA to mRNA proceeds, a concentration of
natural polyamines increases in in vivo system (C. W. Tabor H.
Tabor. Ann. Rev. Biochem. 53, 749-790(1984) and J. Marton and D. R.
Morris, "Inhibition of Polyamine Metabolism", (P. P. McCann, A. E.
Pegg, and A. Sjoerdsma, eds)). This report suggests that a natural
polyamine participates in RNAP transcription process."
[0004] M. Flugier, C. Florentz, M. W. Hosseini, J. M. Lehn and R.
Giege, Nucleic Acids Research, 22(14), 2784-2790(1994) disclosed
that the transcription activity of T7 RNAP is promoted by linear
and cyclic synthetic polyamines. However, it is pointed out that
the evaluation method of a transcription promoting ability in the
report of M. Flugier et al has some problems. In fact, by using an
evaluation method used by the present inventor, it has been found
that polyamines described in the report have only extremely low
accelerating effects. Therefore, the investigation of products
having higher accelerating activity for RNAP transcription activity
and an improvement of a sequencing method is highly desired.
[0005] An object of the present invention is to provide novel
synthetic polyamine compounds with higher and outstanding
accelerating activity for RNAP transcription activity than
synthetic polyamines described in the state of the art, and to
provide a method of DNA sequencing which can determine longer DNA
sequences at a time by using the novel polyamine compounds which
accelerate RNAP transcription activity.
DISCLOSURE OF THE INVENTION
[0006] The present invention relates to a RNA polymerase
transcription accelerator comprising a compound represented by the
general formula (I) below or a salt thereof. 2
[0007] wherein, n represents an integer from 1 to 8,
[0008] R.sup.1 represents a hydrogen atom or p-toluenesulfonyl
group,
[0009] R.sup.2 represents an ethyl group or a group represented by
the general formula (II),
[0010] R.sup.3 represents a hydrogen atom or p-toluenesulfonyl
group,
[0011] R.sup.4represents an ethyl group or a group represented by
the general formula (II). 3
[0012] wherein, m represents 1 or 2,
[0013] R.sup.5 represents a hydrogen atom,
[0014] R.sup.6 represents a hydrogen atom or an ethyl group.
[0015] Compounds 5, 10, 15a-g, and 18-23 described in the following
Examples are represented by the above general formula (I). The
relation between compounds 5, 10, 15a-g, and 18-23, and the general
formula (I) are shown in Table 1 below. In the table, No.sup.a)
represents numbers of compounds in schemes below.
1TABLE 1 Correlation between compounds 5, 10, 15a-g, and 18-23 and
the general formula (I) No.sup.a) R.sup.1 R.sup.2 R.sup.3 R.sup.4
R.sup.5 R.sup.6 N m 5 H formula (II) H formula (II) H Et 2 1 10 H
Et H formula (II) H Et 1 2 15 H formula (II) H formula (II) H ET a:
2 2 b: 3 c: 4 d: 5 e: 6 f: 7 g: 8 18 H Et H formula (II) H H 1 2 19
H formula (II) H formula (II) H H 6 2 20 Ts Et Ts Et -- -- 6 -- 21
H Et H Et -- -- 6 -- 22 Ts Et Ts Et -- -- 3 -- 23 H Et H Et -- -- 3
-- H represents a hydrogen atom, and Et represents an ethyl group
in the table.
[0016] Salts may be either inorganic or organic acid salts. The
inorganic acid salts include hydrochlorides, bromates and the like.
The organic acid salts include acetates, citrates and the like.
Among these, bromates are preferred, but not limited thereto.
[0017] A method of DNA sequencing using RNA polymerase
transcription accelerator
[0018] The present invention relates to a method of DNA sequencing
wherein nucleic acid transcripts are produced by using an RNA
polymerase and a DNA fragment as a template, the resulted nucleic
acid transcripts are separated, the nucleic acid sequence is
determined from the separated fractions, characterized in that said
nucleic acid transcription reaction is carried out in the presence
of at least one compound selected from the group consisting of
compounds represented by the above-mentioned general formula
(I).
[0019] The method of sequencing DNA of the present invention is
characterized in that the nucleic acid transcription reaction is
carried out in the presence of at least one of RNA polymerase
transcription accelerators of the present invention.
[0020] When about 0.5-5 mmol of at least one of these compounds to
1 unit of an RNA polymerase is coexisted in the nucleic acid
transcription reaction using RNA polymerase, sequencing of longer
strands can be possible even if the amount of RNA polymerase used
is not increased. Further, the amount of RNA polymerase or a
template used in the nucleic acid transcription reaction can be
reduced by the use of the compound of the present invention when
the lengths of the DNAs to be subjected are not to be
elongated.
[0021] A method of DNA sequencing is described below. Methods of
DNA sequencing in which nucleic acid transcripts are obtained by
using RNA polymerase and a DNA fragment as a template, the resulted
nucleic acid transcripts are separated, and the nucleic acid
sequence is determined from the separated fractions are already
publicly known. Moreover, a method for enzymatically synthesizing
nucleic acid transcription products by RNA polymerase using a DNA
fragment comprising a promoter sequence for the RNA polymerase, a
method for separating nucleic acid transcription products, and a
method for determination of nucleic acid sequence from separated
fractions are also publicly known. Therefore, for these purposes,
any known methods, conditions, and equipments can be suitably used
in this invention.
[0022] There is no limitation to the DNA fragment used as a
template except that it comprises a promoter sequence for RNA
polymerase. For example, the DNA fragment comprising a promoter
sequence can be a DNA product amplified by polymerase chain
reaction. Further, a nucleic acid transcription generation reaction
in the method of the present invention can be carried out without
removal of a primer and/or 2'-deoxyribonucleoside-5'-triphosphate
and/or derivatives thereof used in the polymerase chain reaction
from the amplified DNA product. The polymerase chain reaction used
for the above DNA amplification can be a method which is widely
used as PCR method. Further, the DNA fragment comprising a promoter
sequence may be a DNA fragment which has been cloned using an
adequate host after ligating the promoter sequence and a DNA
fragment to be amplified. That is, in the present invention, there
is no limitation to a DNA sequence to be amplified, a primer, and
conditions for the amplification and the like.
[0023] The polymerase chain reaction for the amplification of the
DNA fragment comprising a promoter sequence can be performed, for
example, in a 20 .mu.l volume of solution containing 10-50 ng of
genomic DNA or 1 pg of cloned DNA, 10 .mu.M of each primer, and 200
.mu.M of each 2'-deoxyribonucleoside-5'-triphosphate (dATP, dGTP,
dCTP, dTTP) using a DNA polymerase such as a Tag polymerase.
[0024] Provided that, either one of primers for the polymerase
chain reaction, or an insert DNA amplified is required to have a
promoter sequence for RNA polymerase, which will be described
hereinafter. In the direct transcription sequencing method, two
types of primers, one of which has a phage promoter sequence, are
used in the PCR amplification, or an amplified insert DNA having a
phage promoter sequence is used. As a result, the resulted PCR
products can be subjected to an in vitro transcription using RNA
polymerase which is activated by the above promoter sequence.
[0025] The promoter sequence for the RNA polymerase can be
appropriately selected in view of the RNA polymerase to be
used.
[0026] In the method of the present invention, a nucleic acid
transcript such as an RNA transcript is synthesized from a DNA
fragment comprising a promoter sequence. Since the DNA fragment
comprises a promoter sequence for RNA polymerase, recognition of
this promoter sequence by the RNA polymerase enables to synthesize
the nucleic acid transcript such as an RNA transcript.
[0027] Nucleic acid transcripts such as RNA transcripts can be
Synthesized by reacting, for example,
ribonucleoside-5'-triphosphates (NTPs) such as ATP, GTP, CTP and
UTP or derivatives thereof (provided that, one of the NTPs may be a
compression suppressed ribonucleotide derivative) and one or more
3'dNTP derivatives in the presence of the above-mentioned nucleic
acid transcription initiator and the RNA polymerase. The term "3'
dNTP derivative" is herein used as a generic term for indicating 3'
dATP, 3' dGTP, 3' dCTP, 3' dUTP and derivatives thereof. At least
four kinds of ribonucleoside-5'-triphosphates (NTPs) each having a
different nucleic acid are necessary for a synthesis of a
transcript even if a part of the NTP such as ATP is a
derivative.
[0028] When 3' dNTP derivative is incorporated into the 3' end of
the transcription product such as RNA or nucleic acids, the
synthesis of RNA or nucleic acid is interfered due to lack of 3'
hydroxy group. As a result, RNAs or nucleic acid fragments with
various lengths in which a 3' dNTP derivative has been incorporated
into their 3' ends are generated. With respect to each of four 3'
dNTP derivatives each having a different nucleic acid, such
ribonucleoside analogues are produced. Upon preparation of four
ribonucleoside analogues, determination of RNA or nucleic acid
sequence can be done [Vladimir D. Axelred et al. (1985)
Biochemistry Vol. 24, 5716-5723].
[0029] One or more of the 3' dNTP derivatives can be used in a
nucleic acid transcription reaction. When the nucleic acid
transcription reaction is performed using only one of 3' dNTP
derivatives, four kinds of transcription products each having at
the 3' end a 3' dNTP derivative different in nucleic acid are
obtained by carrying out nucleic acid transcription reaction four
times. Through one nucleic acid transcription reaction, a
transcription product comprising a mixture of various RNAs or
nucleic acid fragments having the same 3' dNTP derivative at the 3'
end and different molecular weight is produced. The resulted four
kinds of transcription products can be independently subjected to
separation and sequence determination described below.
Alternatively, two or more of four transcription products are mixed
and the resulted mixture can be subjected to separation and
sequence determination.
[0030] When two or more of 3' dNTP derivatives are used for one
nucleic acid transcription reaction at once, a reaction product
containing two or more transcription products each having at the 3'
end a 3' dNTP derivative different in nucleic acid can be obtained.
This product can be subjected to separation and sequence
determination described below. It is preferred to use 2 or more 3'
dNTP derivatives for a nucleic acid transcription reaction at once,
because the number of nucleic acid transcription reaction
procedures can be reduced.
[0031] Further, transcription of nucleic acid such as RNA is
carried out by using RNA polymerase in the presence of four
ribonucleoside-5'-triphos- phates each having different nucleic
acid and is terminated by the 3' dNTP derivatives. As a result,
with respect to each nucleic acid, RNA or nucleic acid ladder can
be produced for sequencing. In particular, in the present
invention, it is preferred to carry out the nucleic acid
transcription in the presence of four
ribonucleoside-5'-triphosphates each having a different nucleic
acid, separation of the resulted transcription products and
determination of four nucleic acid sequences at once.
[0032] RNA polymerase
[0033] The RNA polymerase used in the method of the present
invention can either be a wild-type RNA polymerase and a mutant RNA
polymerase. The RNA polymerase is preferably a mutant RNA
polymerase wherein at least one amino acid has been modified to
have higher 3' dNTP derivatives incorporation ability than that of
the wild-type RNA polymerase. The "wild-type RNA polymerase" herein
includes all naturally occurred RNA polymerases, and a modified
wild-type RNA polymerase which has substitution, insertion or
deletion of amino acids which are not the modification for
obtaining increased incorporation ability of 3'-deoxyribonucleotide
or its derivatives compared to that of the corresponding wild-type
RNA polymerase. That is, wild-type RNA polymerases artificially
modified with a purpose other than that described above are
included in the above "wild-type RNA polymerase". However, it is
suitable make sure that such a substitution, insertion or deletion
of amino acids to the extent that activity of RNA polymerase is
maintained.
[0034] Examples of the "wild-type RNA polymerase" include RNA
polymerase derived from T7 phage, T3 phage, SP6 phage, and K11
phage. However, it is not limited to these RNA polymerases.
[0035] The "wild type RNA polymerase" according to the present
invention includes naturally occurring thermostable RNA
polymerases, and naturally occurring RNA polymerases artificially
modified (i.e. having substitution, insertion and/or deletion of
amino acids) in order to impart thermostablity. However, it is
suitable to make the modification for imparting thermostablity to
the extent that the activity of RNA polymerase is maintained. The
mutant RNA polymerase of the present invention prepared by using a
thermostable RNA polymerase as the "wild type RNA polymerase" shall
be thermostable. As a result, for example, it can be used in PCR to
synthesize RNA fragments for sequencing in situ, i.e., during PCR,
by using the PCR product as a template.
[0036] T7 RNA polymerase has been known to be a promoter specific
RNA polymerase with an extremely high specificity. The nucleotide
sequence and production method of T7 RNA polymerase are reported in
Davanloo et al., Proc. Natl. Acad. Sci. USA., 81:2035-2039 (1984).
Its large-scale production has been already described in Zawadzki
et al., Nucl. Acids Res., 19:1948 (1991). This phage-derived RNA
polymerase can pursue the transcription reaction with a single
polypeptide, unlike RNA polymerases of E. coli and higher organisms
(Chamberlin et al., Nature, 228:227-231, 1970). Therefore, it is a
particularly excellent material for analyzing the mechanism of
transcription, and many mutants have been isolated and reported.
Further, the results of its crystallographic analysis are mentioned
in Sousa et al., Nature, 364:593-599, 1993.
[0037] As other promoter specific RNA polymerases of high
specificity, 3 kinds of RNA polymerases derived from T3 phage which
infects E. coli, SP6 phage which infects Salmonella, and K11 phage
which infects Klebsiella pneumoniae have been well known.
[0038] The four kinds of RNA polymerases mentioned above are quite
similar to one another in their primary structure of amino acids,
sequence of promoter and the like as described hereinafter.
[0039] The above-modified RNA polymerase has an increased ability
of incorporating 3'-deoxyribonucleotides and derivatives thereof in
comparison with the ability of a corresponding wild type RNA
polymerase. Wild type RNA polymerases poorly incorporate
3'-deoxyribonucleotides in comparison with ribonucleotides, which
has obstructed their use in nucleotide sequencing. In contrast, the
modified RNA polymerase has the ability of incorporating
3'-deoxyribonucleotides and derivatives thereof at least twice
higher than that of wild type. The incorporation of
3'-deoxyribonucleotides tends to be decreased especially when
3'-deoxyribonucleotide derivatives are labeled with a fluorescent
tag. The modified RNA polymerase can also improve incorporation of
such 3'-deoxyribonucleotide derivatives.
[0040] A mutated or mutant RNA polymerase is a polymerase of which
at least one of amino acids in a corresponding wild type RNA,
polymerase has been modified. Such modification of an amino acid
may be not only substitution of amino acid but also insertion or
deletion of amino acid. The mutation of amino acid is, for example,
substitution of tyrosine for at least one amino acid residue in a
naturally occurring amino acid sequence. The amino acid to be
replaced maybe, for example, phenylalanine. However, the amino acid
to be replaced is not limited to phenylalanine, and any amino acid
may be replaced so long as it can enhance the ability for
incorporating-3'-deoxyribonucleotides and other ribonucleotide
analogues relative to ability for the corresponding
ribonucleotides.
[0041] Example of the mutant RNA polymerase includes mutant T7 RNA
polymerase F644Y and L665P/F667Y. The numbers indicate an amino
acid number counting from the N terminal of the polymerase protein.
For example, F667 means that the amino acid residue No. 667 is F,
and F667Y means that the amino acid residue F No. 667 is Y
substituted by F. These sustain the RNA synthesis activity
sufficiently and have an improved ability for incorporating 3'
dNTPs. The strong bias observed in the wild-type has been
considerably decreased. Use of T7 RNA polymerase F644Y or
L665P/F667Y having such characteristics enables a nucleotide
sequence determination method utilizing transcription products,
which is of more excellent practical applicability in comparison
with a nucleotide sequence determination method utilizing a DNA
polymerase.
[0042] E. coli strains pT7RF644Y (DH5 .alpha.) and pT7RL665P/F667Y
(DH5 .alpha.), which produce the mutant T7 RNA polymerases F644Y
and L665P/F667Y respectively, were already deposited at the
National Institute of Bioscience and Human-Technology with
international deposition numbers of 5998 (FERM-BP-5998) and5999
(FERM-BP-5999) respectively on Jul. 2, 1997.
[0043] The aforementioned mutant RNA polymerases can be produced by
preparing a nucleic acid molecule encoding a RNA polymerase,
introducing a mutation into the nucleic acid molecule so that one
or more nucleotides in one or more regions should be mutated, and
collecting a modified RNA polymerase expressed by the mutated
nucleic acid molecule. Preparation of the nucleic acid molecule
encoding RNA polymerase, introduction of mutation into the nucleic
acid molecule, and collection of the modified RNA polymerase can be
performed by using conventional methods.
[0044] For example, a mutant T7 RNA polymerase can be constructed
by the following method. By using an expression vector inserted
with a T7 RNA polymerase gene as template, an expression plasmid
comprising a region between the HpaI, and NcoI restriction sites in
the C-terminus side of T7 RNA polymerase gene which is introduced
with a mutation by PCR is constructed. Subsequently, this
expression plasmid can be transformed into E. coli DH5 .alpha.,
which can then produce a large amount of a mutant T7 RNA polymerase
protein upon addition of isopropyl-.beta.-D-thio- galactopyranoside
(IPTG).
[0045] Inorganic pyrophosphatase
[0046] In the method of the present invention, a nucleic acid
transcription generation reaction is preferably performed in the
presence of inorganic pyrophosphatase. With the use of inorganic
pyrophosphatase, stable sequence data can be obtained since bias to
incorporation ability can be cancelled. The bias is such that
incorporation of 3'-deoxyribonucleotide and the derivatives thereof
into a polyribonucleotide sequence is difficult compared to
corresponding ribonucleotide and incorporation into the sequence
varies depending on the kind of nucleic acid among ribonucleotides
and 3'-deoxyribonucleotides. That is, it decreases differences in
peak altitudes (intensities of signals) corresponding to each
labeled ribonucleotide, thereby precision of sequence determination
is improved and it makes possible to obtain more accurate
sequencing data.
[0047] Pyrophosphorolysis occurs due to increase of pyrophosphate
produced by DNA synthesis, and it acts to promote the reaction so
that the resulting DNA product should be decomposed. As a
consequence, the pyrophosphorolysis inhibits the sequencing in the
dideoxy sequencing method utilizing a DNA polymerase. As for this
fact, it has been known that, when an inorganic pyrophosphatase is
used in the dideoxy sequencing method utilizing a DNA polymerase,
it inhibits the pyrophosphorolysis and thus affords stable
sequencing data [Japanese Patent Unexamined Publication (KOKAI) No.
Hei 4-506002/1992].
[0048] The pyrophosphorolysis is also effective in the sequencing
method-utilizing an RNA polymerase. More stable sequence data can
be obtained by performing the nucleic acid transcription reaction
in the presence of inorganic pyrophosphatase since differences in
peak altitudes (intensities of signals) corresponding to each
labeled ribonucleotide can be reduced.
[0049] Inorganic pyrophosphatase (EC.3.6.1.1) is commercially
available, and for example, it is sold by Sigma as INORGANIC
PYROPHOSPHATASE and by Boehringer as Pyrophosphatase. While the
amount of inorganic pyrophosphatase to be used may depends on the
degrees of activities of inorganic pyrophosphatase and RNA
polymerase, it is suitably in the range of 10.sup.-6 to 10.sup.-2
units for 1 unit of RNA polymerase.
[0050] Compression suppression ribonucleotide derivatives
[0051] In the present invention, it is preferred to use a
compression suppression ribonucleotide derivative in a nucleic acid
transcription reaction, so that compression can be suppressed and
precision of sequencing determination can be improved. Accuracy of
the sequencing determination can be improved by suppressing the
compression.
[0052] The compression suppression ribonucleotide derivatives are
ribonucleotide derivatives that can suppress compression in
sequencing analysis. The compression suppression ribonucleotide
derivatives can be selected from, for example, ribonucleotides with
base (nucleic acid) analogues instead of bases (nucleic acids). The
base analogues can be either natural compounds or synthetic
compounds. The synthetic compounds include, for example, those in
which some carbon atoms constituting a purine ring or a pyrimidine
ring are substituted by nitrogen atoms or those in which some
nitrogen atoms constituting a purine ring or a pyrimidine ring are
substituted by carbon atoms. Alternatively, it can be a compound in
which various substituents are introduced into its purine or
pyrimidine ring.
[0053] Examples of the compression suppression ribonucleotide
derivatives, ribonucleotides having base analogues, include
deazaribonucleoside-5'-tri- phosphates. Moreover, the
deazaribonucleoside-5'-trophosphates include
7-deazaribonucleoside-5'-triphosphates and
3-deazaribonucleoside-5'-triph- osphates.
7-deazaribonucleoside-5'-triphosphates include 7-deazaATP,
7-deazaGTP and derivatives thereof, and
3-deazaribonucleoside-5'-triphosp- hates include 3-deazaCTP,
3-deazaUTP and derivatives thereof.
[0054] Other Examples of the compression suppression ribonucleotide
derivatives include deaminoribonucleoside-5'-triphosphates. An
amino group is present in bases of ribonucleoside except for
uracil. Each GTP, ATP and CTP has an amino group on 2.sup.nd
position of a purine ring, 6.sup.th position of purine ring and
4.sup.th position of pyrimidine ring respectively. The
above-mentioned deaminoribonucleoside-5'-triphosphates are
ribonucleotides in which the amino groups are decomposed, for
example, N.sup.2-deaminoGTP, N.sup.6-deaminoATP and
N.sup.4-deaminoCTP, and derivatives thereof. Further,
N.sup.2-deaminoguanine is the same substance to inosine, and
N.sup.2-deaminoGTP may be abbreviated as ITP.
[0055] The other examples of the compression suppression
ribonucleotide derivatives include derivatives having substituted 1
or 2 hydrogen atoms of amino group present in bases of
ribonucleotides by organic groups except that hydrogen atoms.
Examples of such derivatives are N-alkyl substituted
ribonucleoside-5'-triphosphates (provided that an alkyl is a lower
alkyl with carbon atom number 1-6, and the substitution is mono- or
di-substitution). Provided that, the substituent can be selected
from other organic groups except an alkyl group. Examples of
ribonucleoside-5'-triphosphates substituted by N-alkyl are GTP
substituted by N.sup.2 -monomethyl, CTP substituted by
N.sup.4-monomethyl, ATP substituted by N.sup.6-monomethyl and
derivatives thereof.
[0056] In a nucleic acid transcription reaction, the compression
suppression ribonucleotide derivative can be used instead of
ribonucleoside-5'-triphosphates for bases which easily form
compression selected from four ribonucleoside-5'-triphosphates such
as ATP, GTP, CTP and UTP or derivatives thereof. The compression
suppression ribonucleotide derivatives can be used for 2 or more
ribonucleoside-5'-triphosphates, if desired.
[0057] A deazaribonucleoside-5'-triphosphate can be used as
compression suppression ribonucleotide derivative for one of four
ribonucleoside-5'-triphosphates are [7-deazaATP, GTP, CTP, UTP],
[ATP, 7-dezaGTP, CTP, UTP], [ATP, GTP, 3-deazaCTP, UTP], and [ATP,
GTP, CTP, 3-deazaUTP]. The deaza NTP can be substituted by other
compression suppression ribonucleotide derivatives. Compression
suppression ribonucleotide derivatives such as 7-deazaNTPs are
commercially available. It is also possible to use together 2 or
more compression suppression ribonucleotide derivatives, or to use
together both a compression suppression ribonucleotide derivative
and a usual ribonucleoside-5'-triphosphate for the same nucleic
acid.
[0058] When compression suppression ribonucleotide derivatives are
used in place of a part of or all ribonucleoside-5'-triphosphates
which are GTP or derivatives thereof, it is appropriate to use
together guanosine, guanosine-5'-monophosphate (GMP),
guanosine-5'-diphosphate (GDP), oligoribonucleotides represented by
the general formula N.sup.1(N).sub.n-31 1G or oligoribonucleotides
represented by the general formula N.sup.2(N).sub.n-1G together. In
this case, the above-mentioned compression suppression
ribonucleotide derivatives can be, for example, 7-deazaGTP,
N.sup.2-deaminoGTP or N.sup.2-monomethyl substituted GTP.
[0059] When the compression suppression ribonucleotide derivatives
are used in place of a part of or all
ribonucleoside-5'-triphosphates which are ATP or derivatives
thereof, it is appropriate to use together adenosine,
adenosine-5'-monophosphate (AMP), adenosine-5'-diphosphate (ADP),
oligoribonucleotides represented by the general formula
N.sup.1(N).sub.n-1A or oligoribonucleotides represented by the
general formula N.sup.2(N).sub.n-1A. In this case, the compression
suppression ribonucleotide derivatives can be, for example,
7-deazaATP, N.sup.6-deaminoATP or N.sup.6-monomethyl substituted
ATP.
[0060] When compression suppression ribonucleotide derivatives are
used in place of a part of or all ribonucleoside-5'-triphosphates
which are CTP or derivatives thereof, it is appropriate to use
together cytidine, cytidine-5'-monophosphate (CMP),
cytidine-5'-diphosphate (CDP), oligoribonucleotides represented by
the general formula N.sup.1(N).sub.n-1C or oligoribonucleotides
represented by the general formula N.sup.2(N).sub.n-1C. In this
case, the compression suppression ribonucleotide derivatives can
be, for example, 3-deazaCTP, N.sup.4-deaminoCTP or
N.sup.4-monomethyl substituted CTP.
[0061] When compression suppression ribonucleotide derivatives are
used for some or all ribonucleoside-5'-triphosphates which are UTP
or derivatives thereof, it is appropriate to use together uridine,
uridine-5'-monophosphate (UMP), uridine-5'-diphosphate (UDP),
oligoribonucleotides represented by the general formula
N.sup.1(N).sub.n-1U or oligoribonucleotides represented by the
general formula N.sup.2(N).sub.n-1U together. In this case, the
compression suppression ribonucleotide derivatives can be, for
example, 3-deazaUTP, N.sup.2-deaminoUTP or N.sup.2-monomethyl
substituted UTP.
[0062] Nucleic acid transcription initiator
[0063] In a nucleic acid transcription reaction of the method of
the present invention, it is preferred to use together the
above-mentioned compression suppression ribonucleotide derivatives
and nucleic acid transcription initiator from the viewpoint of easy
start of nucleic acid transcription reaction. The nucleic acid
transcription initiator can be selected from, for example,
ribonucleoside, ribonucleoside-5'-monophospha- te,
ribonucleoside-5'-diphosphate, oligoribonucleotides represented by
the general formula N.sup.1(N).sub.n (wherein, N.sup.1 represents
ribonucleoside, ribonucleoside-5'-monophosphate or
ribonucleoside-5'-diphosphate, N represents ribonucleoside-5'-mono
phosphate and n represents an integer of 1 or more) and
oligoribonucleotides represented by the general formula
N.sup.2(N).sub.n (wherein, N.sup.2 represents a group represented
by a formula (1) below, N show ribonucleoside-5'-monophosphate and
n represents an integer of 1 or more). 4
[0064] More specific examples of the nucleic acid transcription
initiator include guanosine, guanosine-5'-monophosphate (GMP)
guanosine-5'-diphosphate (GDP), oligoribonucleotides represented by
the general formula N.sup.1(N).sub.n-1G (provided that, N.sup.1
represents ribonucleoside, ribonucleoside-5'-monophosphate, or a
ribonucleoside-5'-diphosphate, N represents a
ribonucleoside-5'-monohosph- ate, n represents an integer of 1 or
more, G represents guanosine-5'-monophosphate) and
oligoribonucleotides represented by the general formula
N.sup.2(N).sub.n-1G (wherein, N.sup.2 represents a group
represented by the formula (1), N represents a
ribonucleoside-5'-monohpos- phate, n represents an integer of 1 or
more, G represents guanoside-5'-monophosphate).
[0065] Further, more specific examples of the nucleic acid
transcription initiator include adenosine,
adenosin-5'-monophosphate (AMP), adenosine-5'-diphosphate (ADP),
oligoribonucleotides represented by the general formula
N.sup.1(N).sub.n-1A (provided that, N.sup.1 represents a
ribonucleoside, a rionucleoside-5'-monophosphate, or a
ribonucleoside-5'-diphosphate, N represents a
ribonucleoside-5'-monophosp- hate, n represents an integer of 1 or
more, and A represents an adenosine-5'-monophosphate), and
oligoribonucleotides represented by the general formula
N.sup.2(N).sub.n-1A (wherein, N.sup.2 represents a group
represented by the formula (1) below, N represents
ribonucleoside-5'-monophosphate, n represents an integer of 1 or
more, and A represents adenosine-5'-monophosphate).
[0066] More specific examples of the nucleic acid initiator include
cytidine, cytidine-5'-monophosphate (CMP), cytidine-5'-diphosphate
(CDP), oligoribonucleotides represented by the general formula
N.sup.1(N).sub.n-1A (provided that, N.sup.1 represents a
ribonucleoside, a ribonucleoside-5'-monophosphate or a
ribonucleoside-5'-diphosphate, N represents a
ribonucleoside-5'-monophosphate, n represents an integer of 1 or
more, C represents cytidine-5'-monophosphate) and
oligoribonucleotides represented by the general formula
N.sup.2(N).sub.n-1C (wherein, N.sup.2 represents a group
represented by formula (1) below, N represents a
ribonucleoside-5'-monophosphate, n represents an integer of 1 or
more, and C represents cytidine-5'-monophosphate).
[0067] Further, more specific examples of the nucleic acid
transcription initiator include uridine, uridine-5'-monophosphate
(UMP), uridine-5'-diphosphate (UDP), oligoribonucleotides
represented by the general formula N.sup.1(N).sub.n-1U (provided
that, N.sup.1 represents a ribonucleoside, a
ribonucleoside-5'-monophosphate or a ribonucleoside-5'-diphosphate,
N represents a ribonucleoside-5'-monophosp- hate, n represents an
integer of 1 or more, U represents an uridine-5'-monophosphate) and
oligoribonucleotides represented by the general formula of
N.sup.2(N).sub.n-1U (wherein, N.sup.2 represents a group
represented by formula (1) below, N represents a
ribonucleoside-5'-monophosphate, n represents an integer of 1 or
more, U represents uridine-5'-monophosphate).
[0068] The bases (nucleic acids) of the ribonucleosides,
ribonucleoside-5'-monophosphates and
ribonucleosides-5'-diphosphates represented by N.sup.1 in the
above-mentioned general formulas N.sup.1(N).sub.n,
N.sup.1(N).sub.n-1G, N.sup.1(N).sub.n-1A, N.sup.1(N).sub.n-1C,
N.sup.1(N).sub.n-1U, N.sup.2(N).sub.n, N.sup.2(N).sub.n-1G,
N.sup.2(N).sub.n-1A, N.sup.2(N).sub.n-1C, N.sup.2(N).sub.n-1U are
not specially limited, and can be suitably selected from guanine,
adenine, cytosine, and uridine. Further, bases of
ribonucleoside-5'-monophosphates represented by N and nucleic acid
sequence in which n is 2 or more are not specially limited.
Moreover, n is not limited by a function of an initiator, however,
according to a commercial availability, n is practically around 10
or less, preferably 5 or less.
[0069] Separation and determination of nucleic acid transcription
products
[0070] In the method of the present invention, a nucleic acid
transcription product is separated. The separation can be suitably
performed by any method which enables the separation of numerous
product molecules having different molecular weight, included in
the transcription products according to the molecular weight.
Examples of such methods include electrophoresis. HPLC can also be
used.
[0071] Conditions of electrophoresis and the like are not
particularly limited and it can be carried out in a conventional
manner. The sequence of RNA or nucleic acid can be determined from
bands (nucleic acid ladder) provided by subjecting the
transcription products to electrophoresis.
[0072] Detection of RNA or nucleic acid ladder can be done by, for
example, labeling terminators, ribonucleoside-5'-tirophosphates
(NTPs), used for transcript reaction. When the nucleic acid
transcription initiator is used, it can be performed by labeling
the nucleic acid transcription initiator. Detection of RNA or
nucleic acid ladder can also be performed by labeling 3' dNTP
derivatives used in the transcript reaction. The labeling can be,
for example, florescence, or radioactivity or stable isotope, and
florescence is preferred from safety and manipulation reasons.
Further, it is also possible to determine sequence of the
transcription products separated by electrophoresis -by measuring
mass of each transcription reaction products with a mass
spectrometer without using the above-mentioned labeling.
[0073] In particular, sequence of the transcription products can be
determined, for example, by using labeled 3' dNTP such as labeled
3' dATP, 3' dGTP, 3' dCTP, and 3' dUTP, subjecting the
transcription products to electrophoresis and detecting
radioactivity or stable isotope, or florescence from the resulted
bands. The detection can easily be performed by labeling 3' dNTP
derivatives as mentioned above since differences in radioactivity
or florescence intensities between the bands can be eliminated.
Further, detection of ladders which generate radioactivity or
stable isotope, or florescence can be carried out by using a usual
apparatus for DNA sequencing.
[0074] Sequence of the transcription products can also be
determined by using ATP, GTP, CTP and UTP labeled with
radioactivity or stable isotope, or florescence and detecting the
radioactivity or stable isotope, or florescence of bands of
electrophoresis.
[0075] Further, RNA or nucleic acid sequence can be determined by
using 3' dATP, 3' dGTP, 3' dCTP and 3' dUTP each labeled with a
different florescence and detecting four types of florescence from
the bands obtained by electrophoresis in which a mixture of various
transcription product fragments each having 3' dATP, 3' dGTP, 3'
dCTP or 3' dUTP at it end and each having a different florescence
are separated each other.
[0076] In this method, four kinds of 3' dNTPs are labeled with a
different florescence. Thus, by subjecting a mixture of four kinds
of transcription products each having a different 3' end to
electrophoresis, bands generating florescence corresponding to the
four different kinds of 3' dNTP at the 3' end can be obtained and
RNA or nucleic acid sequence can be determined with respect to four
nucleic acids at once by distinguishing the difference of
florescence.
[0077] 3'-deoxyribonucleotide derivatives described in WO96/14434
and Japanese Patent Laid-open Showa No.63-152364 can be used as the
florescence labeled 3' dNTPs. Further, florescence labels are
preferred to be florescence dyes which generate detectable
luminescence radiation by stimulation of energy absorption from a
suitable supplying source such as an argon laser.
[0078] A DNA sequence used as a template of transcription can be
determined from RNA or nucleic acid sequence determined by the
above-mentioned method. When ladders for each nucleic acid are
formed, the DNA sequence used as a template of the transcription
can be determined by integrating RNA or nucleic acid information
obtained from four kinds of ladders. Further, when ladders for two
or more nucleic acids are formed at once (in which two or more base
bands are present in the same ladder), the DNA sequence used as a
template of transcription can be determined by integrating RNA or
nucleic acid sequence information obtained from each ladder. In
particular, when ladders for four bases (nucleic acid) are formed
at once (in which bands for four kinds of nucleic acids are present
in the same ladder), the DNA sequence used as a template of
transcription can be determined from RNA or nucleic acid sequence
information obtained from the single ladder.
EXAMPLES
[0079] Synthesis of polyamine compounds
[0080] The followings are a list of references for a method of
polyamine compounds synthesis.
[0081] 1) Iwata, Yamamoto and Nakajima, Japanese Patent Laid-open
No. 08-027129 (Publication Date: Jan. 30, 1996), "Cyclic polyamine
and antiviral agent containing the same as active ingredient"
[0082] 2) Iwata and Kuzuhara, Japanese Patent No. 1857707, "Method
for producing N-alkylformamide"
[0083] 3) Iwata and Kuzuhara, Japanese Patent No. 1857749,
"Polyamine derivative"
[0084] 4) Iwata and Kuzuhara, Japanese Patent No. 1998558, "Method
for producing polyamine derivatives"
[0085] 5) Iwata and Kuzuhara, Japanese Patent No. 2123326
"N-phthalimide derivative and method for producing the same"
[0086] 6) M. Flugier, C. Florentz, M. W. Hosseini, J. M. Lehn, and
R. Giege, Nucleic Acids Research, 22(14), 2784 2790(1994)
Example 1
[0087] Synthesis of 1,12-di(ethylamino)-4,9-diazadodecane HBr salt
(5) 5
[0088] Diaminobutane as a raw material was reacted with tosyl
chloride at room temperature for 3 hours to obtain
N1,N4-di(p-toluenesulfonyl)-1,4-di- aminobutane. Using the obtained
N1,N4-di(p-toluenesulfonyl)-1,4-diaminobut- ane and
N-(3-bromopropyl)phthalimide as Chain A and Chain B, respectively,
N1,N4,N9,N12-tetra(p-toluenesulfonyl)-1,12-diamino-4,9-diaz
adodecane (3) was derived from the above Chain A and Chain B
according to the known methods (Patent Documents 1 to 5 and
References 6). Specifically, Compound 1 was synthesized, and then
this Compound 1 was reacted with N.sub.2H.sub.4 in DMF at
70.degree. C. for 1 day to obtain Compound 2. The obtained Compound
2 was first treated with 2N HCl at 70.degree. C. for 1 hour, and
then reacted with TsCl in pyridine in the presence of NEt.sub.3 at
room temperature for four hours to obtain Compound 3.
[0089] Subsequently, a mixture of the above Compound 3 (0.17 g)
anhydrous potassium carbonate (0.143 g) and bromoethane (57 mg) was
allowed to react in DMF (40 ml) at room temperature for 3 days with
stirring, filtered, and then concentrated. A substance exhibiting
Rf of 0.4 in TLC (Merck, Art. 5715, chloroform:acetone (95:5 v/v))
was collected by silica gel column chromatography (Merck, Art.
7734, 70-230 mesh) using chloroform:acetone (95:5 v/v) as a
developing solvent to obtain
N1,N4,N9,N12-tetra(p-toluenesulfonyl)-1,12-di(ethylamino)-4,9-diazadodeca-
ne (4, 0.106 g, yield: 58%). The results of elementary analysis of
Compound 4 are shown in Table 2 below, and the results of
.sup.1H-NMR and .sup.13C-NMR of the same are shown Table 4 below,
respectively. The obtained Compound 4 (96 mg) was heated with
phenol (0.206 g) in 33% HBr in acetic acid (10 ml) with stirring on
an oil bath at 75.degree. C. for 20 hours, and then the reaction
mixture was concentrated under reduced pressure. The residue was
added with diethyl ether and stirred, and the supernatant was
discarded. This washing procedure was repeated by using a mixed
solution of methanol and diethyl ether until the supernatant became
colorless. The solvent was evaporated under reduced pressure to
obtain Compound 5 (see Scheme 1) as colorless powder.
Example 2
[0090] Synthesis of 1,8-di(ethylamino)-4-azaoctane HBr salt (10)
6
[0091] 3-Bromopropylamine as a raw material was reacted with tosyl
chloride at 0.degree. C. for 2 hours to synthesize
N-(p-toluenesulfonyl)-3-bromopropylamine. Further, this was reacted
with phthalimide at room temperature for 3 days to obtain
N-(N3-p-toluenesulfonyl-3-aminopropyl)phthalimide. Using the
obtained N-(N3-p-toluenesulfonyl-3-aminopropyl)phthalimide and
N-(4-bromobutyl)phthalimide as Chain A and Chain B, respectively,
N1,N4,N8-tri(p-toluenesulfonyl)-1,8-diamino-4-azaoctane (8) was
derived from the above Chain A and Chain B according to the known
methods (Patent Documents 1 to 5 and References 6). Specifically,
Compound 6 was synthesized, and then this Compound 6 was reacted
with N.sub.2H.sub.4 in DMF at 70.degree. C. for 1 day to obtain
Compound 7. The obtained Compound 7 was first treated with 2 N HCl
at 70.degree. C. for 1 hour, and then reacted with TsCl in pyridine
in the presence of NEt.sub.3 at room temperature for 4 hours to
obtain Compound 8.
[0092] Subsequently, a mixture of the above Compound 8 (0.157 g),
anhydrous potassium carbonate (0.178 g) and bromoethane (48 ml) was
allowed to react in DMF (40 ml) at room temperature for 3 days with
stirring, filtered, and then concentrated. A substance exhibiting
Rf of 0.4 in TLC (Merck, Art. 5715, chloroform: acetone (95:5 v/v))
was collected by silica gel column chromatography (Merck, Art.
7734, 70-230 mesh) using chloroform: acetone (95:5 v/v) as a
developing solvent to obtain
N1,N4,N8-tri(p-toluenesulfonyl)-1,8-di(ethylamino)-4-azaoct ane
(Compound 9, 0.111 g, yield: 65%). The results of elementary
analysis of Compound 9 are shown in Table 2 below, and the results
of .sup.1H-NMR and .sup.13C-NMR of the same are shown in Table 4
below, respectively. The obtained Compound 9 (100 mg) was heated
with phenol (0.283 g) in 33% HBr in acetic acid (10 ml) with
stirring on an oil bath at 75.degree. C. for 20 hours, and then the
reaction mixture was concentrated under reduced pressure. The
residue was added with diethyl ether and stirred, and the
supernatant was discarded. This washing procedure was repeated by
using a mixed solution of methanol and diethyl ether until the
supernatant became colorless. The solvent was evaporated under
reduced pressure to obtain Compound 10 (see Scheme 2) as colorless
powder.
Example 3.1
[0093] Synthesis of 1,14-di(ethylamino) -5,10-diazatetradecane HBr
salt (15b) 7
[0094] Diaminobutane as a raw material was reacted with tosyl
chloride at room temperature for 3 hours to obtain
N1,N4-di(p-toluenesulfonyl)-1,4-di- aminobutane. Using the obtained
N1,N4-di(p-toluenesulfonyl)-1,4-diaminobut- ane and
N-(4-bromobutyl)phthalimide as Chain A and Chain B, respectively,
N1,N5,N10,N14-tetra(p-toluenesulfonyl)-1,14-diamino-5,10-diazatetradecane
(13b) was derived from the above Chain A and Chain B according to
the known methods (Patent Documents 1 to 5 and References 6).
Specifically, Compound 11a was synthesized, and then this Compound
11a was reacted with N.sub.2H.sub.4 in DMF at 70.degree. C. for 1
day to obtain Compound 12a. The obtained Compound 12a was first
treated with 2 N HCl at 70.degree. C. for 1 hour, and then reacted
with TsCl in pyridine in the presence of NEt.sub.3 at room
temperature for 4 hours to obtain Compound 13a.
[0095] Subsequently, a mixture of the above Compound 13a (0.179 g)
, anhydrous potassium carbonate (0.146 g) and bromoethane (40 ml)
was allowed to react in DMF (40 ml) at room temperature for 3 days
with stirring, filtered, and then concentrated. A substance
exhibiting Rf of 0.4 in TLC (Merck, Art. 5715, chloroform: acetone
(95:5 v/v)) was collected by silica gel column chromatography
(Merck, Art. 7734, 70-230 mesh) using chloroform: acetone (95:5
v/v) as a developing solvent to obtain
N1,N5,N10,N14-tetra(p-toluenesulfonyl)-1,14-di(ethylamino)-5,10-di-
azatetradecane (Compound 14a, 0.215 g, yield: 88%). The results of
elementary analysis of Compound 14b are shown in Table 4 below, and
the results of .sup.1H-NMR and .sup.13C-NMR of the same are shown
in Table 8 below, respectively. The obtained Compound 14a (114 mg)
was heated with phenol (0.238 g) in 33% HBr in acetic acid (10 ml)
with stirring on an oil bath at 75.degree. C. for 20 hours, and
then the reaction mixture was concentrated under reduced pressure.
The residue was added with diethyl ether and stirred, and the
supernatant was discarded. This washing procedure was repeated by
using a mixed solution of methanol and diethyl ether until the
supernatant became colorless. The solvent was evaporated under
reduced pressure to obtain Compound 15a (see Scheme 3) as colorless
powder.
Example 3.2
[0096] Synthesis of 1,15-di(ethylamino) -5,11-diazapentadecane HBr
salt (15b) (Scheme 3)
[0097] Diaminopentane as a raw material was reacted with tosyl
chloride to obtain N1,N5-di(p-toluenesulfonyl)-1,5-diaminopentane.
Using the obtained N1,N5-di(p-toluenesulfonyl)-1,5-diaminopentane
and N-(4-bromobutyl)phthalimide as Chain A and Chain B,
respectively,
N1,N5,N11,N15-tetra(p-toluenesulfonyl)-1,15-diamino-5,11-di
azapentadecane (Compound 13b) was derived from the above Chain A
and Chain B according to the known methods (Patent Documents 1 to 5
and References 6). Specifically, Compound 11b was synthesized, and
then this Compound 11b was reacted with N.sub.2H.sub.4 in DMF at
70.degree. C. for 1 day to obtain Compound 12b. The obtained
Compound 12b was first treated with 2 N HCl at 70.degree. C. for 1
hour, and then reacted with TsCl in pyridine in the presence of
NEt.sub.3 at room temperature for 4 hours to obtain Compound
13b.
[0098] Subsequently, a mixture of the above Compound 13b (0.186 g),
anhydrous potassium carbonate (0.149 g) and bromoethane (40 ml) was
allowed to react in DMF (40 ml) at room temperature for 3 days with
stirring, filtered, and then concentrated. A substance exhibiting
Rf of 0.4 in TLC (Merck, Art. 5715, chloroform: acetone (95:5 v/v))
was collected by silica gel column chromatography (Merck, Art.
7734, 70-230 mesh) using chloroform: acetone (95:5 v/v) as a
developing solvent to obtain
N1,N5,N11,N15-tetra(p-toluenesulfonyl)-1,15-di(ethylamino)-5,11-di-
azapentadecane (Compound 14b, 0.173 g, yield: 87%). The results of
elementary analysis of Compound 14b are shown in Table 2 below, and
the results of .sup.1H-NMR and .sup.13C-NMR of the same are shown
in Table 4 below, respectively. The obtained Compound 14c (163 mg)
was heated with phenol (0.334 g) in 33% HBr in acetic acid (13 ml)
with stirring on an oil bath at 75.degree. C. for 20 hours, and
then the reaction mixture was concentrated under reduced pressure.
The residue was added with diethyl ether and stirred, and the
supernatant was discarded. This washing procedure was repeated by
using a mixed solution of methanol and diethyl ether until the
supernatant became colorless. The solvent was evaporated under
reduced pressure to obtain Compound 15b (see Scheme 3) as colorless
powder.
Example 3.3
[0099] Synthesis of 1,16-di(ethylamino)-5,12-diazahexadecane HBr
salt (15 c)
[0100] Diaminohexane as a raw material was reacted with tosyl
chloride to obtain N1,N6-di(p-toluenesulfonyl)-1,6-diaminohexane.
Using the obtained N1,N6-di(p-toluenesulfonyl)-1,6-diaminohexane
and N-(4-bromobutyl)phthali- mide as Chain A and Chain B,
respectively, N1,N5,N12,N16-tetra(p-toluenesu-
lfonyl)-1,16-diamino-5,12-diazahexadecane (Compound 13c) was
derived from the above Chain A and Chain B according to the known
methods (Patent Documents 1 to 5 and References 6). Specifically,
Compound 11c was synthesized, and then this Compound 11c was
reacted with N.sub.2H.sub.4 in DMF at 70.degree. C. for 1 day to
obtain Compound 12c. The obtained Compound 12c was first treated
with 2 N HCl at 70.degree. C. for 1 hour, and then reacted with
TsCl in pyridine in the presence of NEt.sub.3 at room temperature
for 4 hours to obtain Compound 13c.
[0101] Subsequently, a mixture of the above Compound 13c (0.208 g),
anhydrous potassium carbonate (0.164 g) and bromoethane (44 ml) was
allowed to react in DMF (40 ml) at room temperature for 3 days with
stirring, filtered, and then concentrated. A substance exhibiting
Rf of 0.4 in TLC (Merck, Art. 5715, chloroform: acetone (95:5 v/v))
was collected by silica gel column chromatography (Merck, Art.
7734, 70-230 mesh) using chloroform: acetone (95:5 v/v) as a
developing solvent to obtain
N1,N5,N12,N16-tetra(p-toluenesulfonyl)-1,16-di(ethylamino)-5,12-di-
azahexadecane (Compound 14c, 0.243 g, yield: 99%). The results of
elementary analysis of Compound 14c are shown in Table 2 below, and
the results of .sup.1H-NMR and .sup.13C-NMR of the same are shown
in Table 4 below, respectively. The obtained Compound 14c (233 mg)
was heated with phenol (0.471 g) in 33% HBr in acetic acid (13 ml)
with stirring on an oil bath at 75.degree. C. for 20 hours, and
then the reaction mixture was concentrated under reduced pressure.
The residue was added with diethyl ether and stirred, and the
supernatant was discarded. This washing procedure was repeated by
using a mixed solution of methanol and diethyl ether until the
supernatant became colorless. The solvent was evaporated under
reduced pressure to obtain Compound 15c (see Scheme 3) as colorless
powder.
Example 3.4
[0102] Synthesis of 1,17-di(ethylamino) -5,13-diazaheptadecane HBr
salt (15 d) (Scheme 3)
[0103] Diaminoheptane as a raw material was reacted with tosyl
chloride at room temperature for 3 hours to obtain
N1,N7-di(p-toluenesulfonyl)-1,7-di- aminoheptane. Using the
obtained N1,N7-di(p-toluenesulfonyl)-1,7-diaminohe- ptane and
N-(4-bromobutyl)phthalimide as Chain A and Chain B, respectively,
N1,N5,N13,N17-tetra(p-toluenesulfonyl)-1,17-diamino-5,13-di-
azaheptadecane (13 d) was derived from the above Chain A and Chain
B according to the known methods (Patent Documents 1 to 5 and
References 6). Specifically, Compound 11d was synthesized, and then
this Compound 11d was reacted with N.sub.2H.sub.4 in DMF at
70.degree. C. for 1 day to obtain Compound 12d. The obtained
Compound 12d was first treated with 2 N HCl at 70.degree. C. for 1
hour, and then reacted with TsCl in pyridine in the presence of
NEt.sub.3 at room temperature for 4 hours to obtain Compound
13d.
[0104] Subsequently, a mixture of the above Compound 13d (0.160 g)
, anhydrous potassium carbonate (0.180 g) and bromoethane (38 ml)
was allowed to react in DMF (40 ml) at room temperature for 3 days
with stirring, filtered, and then concentrated. A substance
exhibiting Rf of 0.4 in TLC (Merck, Art. 5715, chloroform: acetone
(95:5 v/v)) was collected by silica gel column chromatography
(Merck, Art. 7734, 70-230 mesh) using chloroform: acetone (95:5
v/v) as a developing solvent to obtain
N1,N5,N13,N17-tetra(p-toluenesulfonyl)-1,17-di(ethylamino)-5,13-di-
azaheptadecane (Compound 14d, 0.152 g, yield: 89%). The results of
elementary analysis of Compound 14e are shown in Table 2 below, and
the results of .sup.1H-NMR and .sup.13C-NMR of the same are shown
in Table 4 below, respectively. The obtained Compound 14d (142 mg)
was heated with phenol (0.283 g) in 33% HBr in acetic acid (13 ml)
with stirring on an oil bath at 75.degree. C. for 20 hours, and
then the reaction mixture was concentrated under reduced pressure.
The residue was added with diethyl ether and stirred, and the
supernatant was discarded. This washing procedure was repeated by
using a mixed solution of methanol and diethyl ether until the
supernatant became colorless. The solvent was evaporated under
reduced pressure to obtain Compound 15d (see Scheme 3) as colorless
powder.
Example 3.5
[0105] Synthesis of 1,18-di(ethylamino)-5,14-diazaoctadecane HBr
salt (15 e) (Scheme 3)
[0106] Diaminooctane as a raw material was reacted with tosyl
chloride at room temperature for 3 hours to obtain
N1,N8-di(p-toluenesulfonyl)-1,8-di- aminooctane. Using the obtained
N1,N8-di(p-toluenesulfonyl)-1,8-diaminooct- ane and
N-(4-bromobutyl)phthalimide as Chain A and Chain B, respectively,
N1,N5,N14,N18-tetra(p-toluenesulfonyl)-1,18-diamino-5,14-di
azaoctadecane (Compound 13e) was derived from the above Chain A and
Chain B according to the known methods (Patent Documents 1 to 5 and
References 6). Specifically, Compound 11e was synthesized, and then
this Compound 11e was reacted with N.sub.2H.sub.4 in DMF at
70.degree. C. for 1 day to obtain Compound 12e. The obtained
Compound 12e was first treated with 2 N HCl at 70.degree. C. for 1
hour, and then reacted with TsCl in pyridine in the presence of
NEt.sub.3 at room temperature for 4 hours to obtain Compound
13e.
[0107] Subsequently, a mixture of the above Compound 13e (0.177 g)
, anhydrous potassium carbonate (0.135 g) and bromoethane (37 ml)
was allowed to react in DMF (40 ml) at room temperature for 3 days
with stirring, filtered, and then concentrated. A substance
exhibiting Rf of 0.4 in TLC (Merck, Art. 5715, chloroform:acetone
(95:5 v/v)) was collected by silica gel column chromatography
(Merck, Art. 7734, 70-230 mesh) using chloroform:acetone (95:5 v/v)
as a developing solvent to obtain
N1,N5,N14,N18-tetra(p-toluenesulfonyl)-1,18-di(ethylamino)-5,14-di-
azaoctadecane (Compound 14e, 0.173 g, yield: 92%). The results of
elementary analysis of Compound 14e are shown in Table 2 below, and
the results of .sup.1H-NMR and .sup.13C-NMR of the same are shown
in Table 5 below, respectively. The obtained Compound 14e (163 mg)
was heated with phenol (0.320 g) in 33% HBr in acetic acid (10 ml)
with stirring on an oil bath at 75.degree. C. for 20 hours, and
then the reaction mixture was concentrated under reduced pressure.
The residue was added with diethyl ether and stirred, and the
supernatant was discarded. This washing procedure was repeated by
using a mixed solution of methanol and diethyl ether until the
supernatant became colorless. The solvent was evaporated under
reduced pressure to obtain Compound 15e (see Scheme 3) as colorless
powder.
Example 3.6
[0108] Synthesis of 1,19-di(ethylamino)-5,15-diazanonadecane HBr
salt (15 f) (Scheme 3)
[0109] Diaminononane as a raw material was reacted with tosyl
chloride at room temperature for 3 hours to obtain
N1,N9-di(p-toluenesulfonyl)-1,9-di- aminononane. Using the obtained
N1,N9-di(p-toluenesulfonyl)-1,9-diaminonon- ane and
N-(4-bromobutyl)phthalimide as Chain A and Chain B, respectively,
N1,N5,N15,N19-tetra(p-toluenesulfonyl)-1,19-diamino-5,15-di
azanonadecane (Compound 13f) was derived from the above Chain A and
Chain B according to the known methods (Patent Documents 1 to 5 and
References 6). Specifically, Compound 11f was synthesized, and then
this Compound 11f was reacted with N.sub.2H.sub.4 in DMF at
70.degree. C. for 1 day to obtain Compound 12f. The obtained
Compound 12f was first treated with 2 N HCl at 70.degree. C. for 1
hour, and then reacted with TsCl in pyridine in the presence of
NEt.sub.3 at room temperature for 4 hours to obtain Compound
13f.
[0110] Subsequently, a mixture of the above Compound 13f (0.168 g),
anhydrous potassium carbonate (0.126 g) and bromoethane (34 ml) was
allowed to react in DMF (40 ml) at room temperature for 3 days with
stirring, filtered, and then concentrated. A substance exhibiting
Rf of 0.4 in TLC (Merck, Art. 5715, chloroform: acetone (95:5 v/v))
was collected by silica gel column chromatography (Merck, Art.
7734, 70-230 mesh) using chloroform: acetone (95:5 v/v) as a
developing solvent to obtain
N1,N5,N15,N19-tetra(p-toluenesulfonyl)-1,19-di(ethylamino)-5,15-di-
azanonadecane (Compound 14f, 0.174 g, yield: 98%). The results of
elementary analysis of Compound 14f are shown in Table 2 below, and
the results of .sup.1H-NMR and .sup.13C-NMR of the same are shown
in Table 5 below, respectively. The obtained Compound 14f (164 mg)
was heated with phenol (0.320 g) in 33% HBr in acetic acid (10 ml)
with stirring on an oil bath at 75.degree. C. for 20 hours, and
then the reaction mixture was concentrated under reduced pressure.
The residue was added with diethyl ether and stirred, and the
supernatant was discarded. This washing procedure was repeated by
using a mixed solution of methanol and diethyl ether until the
supernatant became colorless. The solvent was evaporated under
reduced pressure to obtain Compound 15f (see Scheme 3) as colorless
powder.
Example 3.7
[0111] Synthesis of 1,20-di(ethylamino)-5,16-diazaeicosane HBr salt
(Compound 15g) (Scheme 3)
[0112] Diaminodecane as a raw material was reacted with tosyl
chloride at room temperature for 3 hours to obtain
N1,N10-di(p-toluenesulfonyl)-1,10-- diaminodecane. Using the
obtained N1,N10-di(p-toluenesulfonyl)-1,10-diamin- odecane and
N-(4-bromobutyl)phthalimide as Chain A and Chain B, respectively,
N1,N5,N16,N20-tetra(p-toluenesulfonyl)-1,20-diamino-5,16-di
azaeicosane (Compound 13g) was derived from the above Chain A and
Chain B according to the known methods (Patent Documents 1 to 5 and
References 6). Specifically, Compound 11g was synthesized, and then
this Compound 11g was reacted with N.sub.2H.sub.4 in DMF at
70.degree. C. for 1 day to obtain Compound 12g. The obtained
Compound 12g was first treated with 2 N HCl at 70.degree. C. for 1
hour, and then reacted with TsCl in pyridine in the presence of
NEt.sub.3 at room temperature for 4 hours to obtain Compound
13g.
[0113] Subsequently, a mixture of the above Compound 13g (0.170 g),
anhydrous potassium carbonate (0.126 g) and bromoethane (34 ml) was
allowed to react in DMF (40 ml) at room temperature for 3 days with
stirring, filtered, and then concentrated. A substance exhibiting
Rf of 0.4 in TLC (Merck, Art. 5715, chloroform:acetone (95:5 v/v))
was collected by silica gel column chromatography (Merck, Art.
7734, 70-230 mesh) using chloroform: acetone (95:5 v/v) as a
developing solvent to obtain
N1,N5,N16,N20-tetra(p-toluenesulfonyl)-1,20-di(ethylamino)-5,16-di-
azaeicosane (Compound 14g, 0.177 g, yield: 98%). The results of
elementary analysis of Compound 14g are shown in Table 2 below, and
the results of .sup.1H-NMR and .sup.13C-NMR of the same are shown
in Table 5 below, respectively. The obtained Compound 14g (167 mg)
was heated with phenol (0.318 g) in 33% HBr in acetic acid (10 ml)
with stirring on an oil bath at 75.degree. C. for 20 hours, and
then the reaction mixture was concentrated under reduced pressure.
The residue was added with diethyl ether and stirred, and the
supernatant was discarded. This washing procedure was repeated by
using a mixed solution of methanol and diethyl ether until the
supernatant became colorless. The solvent was evaporated under
reduced pressure to obtain Compound 15g (see Scheme 3) as colorless
powder.
Example 4
[0114] Synthesis of 1-ethylamino-8-amino-4-azaoctane HBr salt
(Compound 18) 8
[0115] Using 4-bromobutylamine as a raw material,
N-(p-toluenesulfonyl)-4-- bromobutylamine was prepared by reacting
it with tosylchloride at 0.degree. C. for 1 hour. Subsequently,
N-(p-toluenesulfonyl)-4-bromobutyl- amine was allowed to react with
phthalimide at room temperature for 24 hours to obtain
N-(N4-p-toluenesulfonyl-4-aminobutyl)phthalimide. Using the
obtained N-(N4-p-toluenesulfonyl-4-aminobutyl)phthalimide and
N-(p-toluenesulfonyl-3-bromopuropilamine as Chain A and Chain B,
respectively,
N1,N4-di(p-toluenesulfonyl)-N8-formyl-1,8-diamino-4-azaocta ne
(Compound 18) was derived from the above Cain A and Chain B
according to the publicly known method (Patent Documents 1 to 5 and
Reference 6). Specifically, Compound 11f was synthesized, and then
this Compound 11f was reacted with N.sub.2H.sub.4 in DMF at
70.degree. C. for one day to obtain Compound 12f. The obtained
Compound 12f was first treated with 2N HCl at 70.degree. C. for one
hour, and then reacted with TsCl in pyridine in the presence of
NEt.sub.3 at room temperature for four hours to obtain Compound
13f.
[0116] Subsequently, a mixture of the above Compound 13f (0.236 g),
anhydrous potassium carbonate (0.339 g) and bromoethane (91 ml) was
allowed to react in DMF (50 ml) at room temperature for 3 days with
stirring, filtered, and then concentrated. A substance exhibiting
Rf of 0.3 in TLC (Merck, Art. 5715, chloroform: acetone (9:1 v/v))
was collected by silica gel column chromatography (Merck, Art.
7734, 70-230 mesh) using chloroform:acetone (7:3 v/v) as a
developing solvent to obtain
N1-ethyl-N1,N4-di(p-toluenesulfonyl)-N8-formyl-1,8-diamino-4-azaoc-
tane (Compound 17, 0.221 g, yield: 88%). The results of elementary
analysis of Compound 17 are shown in Table 2 below, and the results
of .sup.1H-NMR and .sup.13C-NMR of the same are shown in Table 6
below, respectively. The obtained Compound 23 (210 mg) was heated
with phenol (0.929 g) in 33% HBr in acetic acid (10 ml), with
stirring on an oil bath at 75.degree. C. for 20 hours, and then the
reaction mixture was concentrated under reduced pressure. The
residue was added with diethyl ether and stirred, and the
supernatant was discarded. This washing procedure was repeated by
using a mixed solution of methanol and diethyl ether until the
supernatant became colorless. The solvent was evaporated under
reduced pressure to obtain Compound 18 (see Scheme 4) as colorless
powder
Example 5
[0117] Synthesis of 1,18-diamino-5,14-diazaoctadecane HBr salt
(Compound 19) 9
[0118] Compound 13e synthesized in Example 3.6 (152 mg) was heated
with phenol (0.317 g) in 33% HBr in acetic acid (10 ml) with
stirring on an oil bath at 75.degree. C. for 20 hours, and then the
reaction mixture was concentrated under reduced pressure. The
residue, was added with diethyl ether and stirred, and the
supernatant was discarded. This washing procedure was repeated by
using a mixed solution of methanol and diethyl ether until the
supernatant became colorless. The solvent was evaporated under
reduced pressure to obtain Compound 13e (see Scheme 5) as colorless
powder.
Example 6.1
[0119] Synthesis of
N1,N8-di(p-toluenesulfonyl)-di(ethylamino)-octane (Compound 20) and
N1,N8-di(ethylamino)-octane (Compound 21) HBr salt 10
[0120] First, using 1,8-diaminooctane as a raw material,
N1,N8-di(p-toluenesulfonyl)-1,8-diaminooctane was prepared
according to the method described in M. Iwata and H. Kuzuhara,
Bull. Chem. Soc. Jpn., 55, pp.2153-2157 (1982).
[0121] Subsequently, a mixture of the above
N1,N8-di(p-toluenesulfonyl)-1,- 8-diaminooctane (1.0 g), anhydrous
potassium carbonate (1.54 g) and bromoethane (2.5 molar
equivalents) was allowed to react in DMF (60 ml) at room
temperature for 3 days with stirring, filtered, and then
concentrated. A substance exhibiting Rf of 0.7 in TLC (Merck, Art.
5715, chloroform: acetone (98:2 v/v)) was collected by silica gel
column chromatography (Merck, Art. 7734, 70-230 mesh) using
chloroform: acetone (98:2 v/v) as a developing solvent to obtain
N1,N8-di(p-toluenesulfonyl)-- 1,8-di(ethylamino)octane (Compound20,
0.901 g, mp: 112-3.degree. C. (recrystallized from a mixture of
acetone and methanol)). The results of elementary analysis of
Compound 20 are shown in Table 2 below, and the results of
.sup.1H-NMR and .sup.13C-NMR of the same are shown in Table 5
below, respectively. The obtained Compound 20 (876 mg) was heated
with phenol (20 molar equivalents) in 33% HBr in acetic acid (10
ml) with stirring on an oil bath at 75.degree. C. for 20 hours, and
then the reaction mixture was concentrated under reduced pressure.
The residue was added with diethyl ether and stirred, and the
supernatant was discarded. This washing procedure was repeated by
using a mixed solution of methanol and diethyl ether until the
supernatant became colorless. The solvent was evaporated under
reduced pressure to obtain Compound 21 (see Scheme 6) as colorless
powder.
Example 6.2
[0122] Synthesis of
N1,N5-di(p-toluenesulfonyl)-di(ethylamino)pentane (Compound 22) and
N1,N5-di(ethylamino)pentane HBr salt (Compound 23)
[0123] First, using 1,5-diaminopentane as a raw material,
N1,N5-di(p-toluenesulfonyl)-1,5-diaminopentane was prepared
according to the method described in M. Iwata and H. Kuzuhara,
Bull. Chem. Soc. Jpn., 55, pp.2153-2157 (1982).
[0124] Subsequently, a mixture of the above
N1,N5-di(p-toluenesulfonyl)-1,- 5-diaminopentane (1.0 g), anhydrous
potassium carbonate (1.683 g) and bromoethane (0.454 ml) was
allowed to react in DMF (40 ml) at room temperature for 3 days with
stirring, filtered, and then concentrated. A substance exhibiting
Rf of 0.7 in TLC (Merck, Art. 5715, chloroform: acetone (98:2 v/v))
was collected by silica gel column chromatography (Merck, Art.
7734, 70-230 mesh) using chloroform: acetone (98:2 v/v) as a
developing solvent to obtain
N1,N5-di(p-toluenesulfonyl)-1,5-di(ethylamin- o)pentane (Compound
22, 0.901 g, mp:112-3.degree. C. (recrystallized from a mixture of
acetone and methanol)). The results of elementary analysis of
Compound 22 are shown in Table 2 below, and the results of
.sup.1H-NMR and .sup.13C-NMR of the same are shown in Table 5
below, respectively. The obtained Compound 22 (744 mg) was heated
with phenol (20 molar equivalents) in 33% HBr in acetic acid (10
ml) with stirring on an oil bath at 75.degree. C. for 20 hours, and
then the reaction mixture was concentrated under reduced pressure.
The residue was added with diethyl ether and stirred, and the
supernatant was discarded. This washing procedure was repeated by
using a mixed solution of methanol and diethyl ether until the
supernatant became colorless. The solvent was evaporated under
reduced pressure to obtain Compound 23 (see Scheme 6) as colorless
powder.
[0125] Physicochemical analysis
[0126] To characterize the compounds, physicochemical analyses
using elementary analysis and .sup.1H-NMR and .sup.13C-NMR spectrum
analyses were performed for precursors subjected to detosylation
reaction. The results are shown in Tables 2 to 6. The yields of
detosylation reaction in which detosylated compounds were obtained
from the precursors are shown in Table 2.
[0127] The yields of detosylation reactions for deriving target
compounds from the precursors are summarized in Table 3. The
results of .sup.1H-NMR and .sup.13C-NMR spectrum analyses of the
precursors which were subjected to detosylation reaction, are
summarized in Tables 4 to 6.
2TABLE 2 Values of Elementary analysis of Ethylpolyamine
N-pertosylates (yield 4 (58) 9 (65) 14a (65) 14b (87) /
C.sub.42H.sub.58N.sub.4S.sub.4O.sub.8
C.sub.32H.sub.45N.sub.3S.sub.3O.sub.6
C.sub.44H.sub.62N.sub.4S.sub.4O.sub- .8
C.sub.45H.sub.64N.sub.4S.sub.4O.sub.8 %) Found Calcd Found Calcd
Found Calcd Found Calcd C/% 57.46 57.64 58.61 57.89 58.43 58.51
58.75 58.92 H/% 6.60 6.68 6.88 6.83 6.89 6.92 6.97 7.03 N/% 6.39
6.40 6.32 6.33 6.18 6.20 6.08 6.11 14c (99) 14d (89) 14e (92) 14f
(98) C.sub.46H.sub.66N.sub.4S.sub.4O.sub.8
C.sub.47H.sub.68N.sub.4S.sub.4O.sub.8
C.sub.48H.sub.70N.sub.4S.sub.4O.sub- .8
C.sub.49H.sub.72N.sub.4S.sub.4O.sub.8 C/% 59.24 59.33 59.66 59.72
59.98 60.09 60.28 60.46 H/% 7.07 7.14 7.27 7.25 7.30 7.35 7.42 7.46
N/% 5.98 6.02 6.01 5.93 5.85 5.84 5.74 5.76 14g (98) 17 (88) 20
(80) 22 (72) C.sub.50H.sub.74N.sub.4S- .sub.4O.sub.8
C.sub.24H.sub.35N.sub.3S.sub.2O.sub.5
C.sub.26H.sub.40N.sub.2S.sub.2O.sub.4
C.sub.23H.sub.34N.sub.2S.sub.2O.sub- .4 C/% 60.94 60.82 56.57 56.56
61.35 61.38 59.12 59.20 H/% 7.59 7.55 6.97 6.92 7.95 7.93 7.38 7.34
N/% 5.60 5.67 7.99 8.24 5.45 5.51 5.93 6.00
[0128]
3TABLE 3 Yields of detosylation compounds Compnd Yield/% Compnd
Yield/% Compnd Yield/% Compnd Yield/% 5 87 15c 89 15g 90 23 95 10
91 15d 91 18 91 15a 91 15e 85 19 94 15b 88 15f 89 21 96
[0129]
4TABLE 4 .sup.1H-- and .sup.13C--NMR Spectral Data of
Ethylpolyamine pertosylates (.delta./ppm from TMS in CDCL.sub.3,
J/Hz) .sup.1H--NMR .sup.13C--NMR Compnd Et --(CH.sub.2).sub.3--
--(CH.sub.2).sub.2-- aromatic aromatic Et --(CH.sub.2).sub.3--
--(CH.sub.2).sub.2-- aromatic aromatic 4 1.07, t 1.86, quin 1.58, m
2.41, s 7.31, d 13.91 28.71 25.75 21.48 136.20 3.19, quar 3.11, t
3.10, quin 2.42, s 7.66, d 43.14 45.46 48.39 127.07 136.65 J = 7.32
3.13, t J = 7.32 7.29, d 7.67, d 46.51 127.12 143.16 J = 7.32 J =
8.30 J = 8.30 129.69 143.31 129.77 Compnd Et --(CH.sub.2).sub.4--
--(CH.sub.2).sub.3-- aromatic aromatic Et --(CH.sub.2).sub.4--
--(CH.sub.2).sub.3-- aromatic aromatic 9 1.08, t 1.57, m 1.87, quin
2.42, s 7.31, d 13.91 25.65 28.69 21.47 129.75 1.09, t 3.12,t 3.13,
t 2.43, s 7.67, d 14.00 25.75 46.91 127.04 136.27 3.18, quar 3.12,
t 3.15, t 7.29, d 7.67, d 42.84 45.44 48.40 127.07 136.66 3.21,
quar J = 7.32 J = 7.32 J = 8.30 7.68, d 43.12 46.43 127.12 136.98 J
= 7.32 J = 8.30 129.65 143.05 129.69 143.18 143.31 Compnd Et
--(CH.sub.2).sub.4-- --(CH.sub.2).sub.n-- aromatic aromatic Et
--(CH.sub.2).sub.4-- --(CH.sub.2).sub.n-- aromatic aromatic .sup.
14a 1.05, t 1.55 .about. 1.57, m 1.56, m 2.41, s 7.30, d 13.96
25.73 25.94 21.48 136.63 3.17, quar 3.11 .about. 3.13, m 3.10
.about. 3.13, m 2.42, s 7.67, d 42.78 25.79 48.02 127.04 137.01 J =
7.32 J = 7.32 J = 7.32 7.28, d 7.68, d 46.94 127.09 143.03 J = 8.30
J = 8.30 129.64 143.18 129.70 14b 1.05, t 1.55 .about. 1.57, m
1.26, quin 2.41, s 7.30, d 13.98 25.73 23.80 21.47 136.68 3.17,
quar 3.11 .about. 3.13, m 1.53, quin 2.42, s 7.67, d 42.79 25.79
28.41 127.02 136.99 J = 7.32 J = 7.32 3.06, t 7.28, d 7.68, d 46.97
48.42 127.07 143.03 J = 7.32 J = 8.30 J = 8.30 47.96 129.64 143.13
129.67 .sup. 14c 1.06, t 1.57 .about. 1.58, m 1.26, quin 2.41, s
7.30, d 14.00 25.75 26.30 21.47 136.76 3.17, quar 3.11, t 1.50,
quin 2,42, s 7.67, d 42.79 25.79 28.71 127.04 137.01 J = 7.32 3.12,
t 3.06, t 7,28, d J = 8.30 46.99 48.42 127.07 143.03 J = 7.32 J =
7.32 J = 8.30 47.83 129.65 143.10 14d 1.07, t 1.57 .about. 1.58, m
1.23, quin 2.42, s 7.30, d 14.00 25.78 26.63 21.47 136.80 3.17,
quar 3.11, t 1.25, quin 7.29, d 7.67, d 42.78 46.99 28.69 127.04
137.01 J = 7.32 3.13, t 1.50, quin J = 8.30 J = 8.30 47.80 28.76
127.07 143.06
[0130]
5TABLE 5 .sup.1H--NMR .sup.13C--NMR Compnd Et --(CH.sub.2).sub.4--
--(CH.sub.2).sub.n-- aromatic aromatic Et --(CH.sub.2).sub.4--
--(CH.sub.2).sub.n-- aromatic aromatic J = 7.32 3.06, t 48.50
129.64 J = 7.32 .sup. 14e 1.07, t 1.58, m 1.24, m 2.42, s 7.29, d
14.01 25.75 26.63 21.47 136.83 3.18, quar 3.12, t 1.50, quin 7.29,
d 7.67, d 42.78 25.78 28.72 127.04 137.01 J = 7.32 3.13, t 3.06, t
J = 8.30 J = 8.30 46.99 29.10 127.07 143.05 J = 7.32 J = 7.32 47.74
48.50 129.64 .sup. 14f 1.07, t 1.57, m 1.23, m 2.42, s 14.01 25.75
26.72 21.47 136.84 3.18, quar 3.12, t 1.49, quin 7.29, d 42.78
25.78 28.74 127.04 137.01 J = 7.32 3.13, t 3.06, t 7.67, d 46.99
29.10 127.07 143.03 J = 7.32 J = 7.32 J = 8.30 47.71 29.43 129.62
48.50 14g 1.07, t 1.57, m 1.23, m 2.42, s 14.01 25.76 26.75 21.47
136.86 3.18, quar 3.12, t 1.49, quin 7.29, d 42.78 46.99 28.72
127.04 137.03 J = 7.32 3.13, t 3.06, t 7.68, d 47.70 29.17 127.07
143.03 J = 7.32 J = 7.32 J = 8.30 29.43 129.64 48.50 20 1.10, t
1.26, m 2.42, s 14.06 26.55 21.45 142.88 3.20, quar 1.52, quin
7.29, d 42.59 28.67 127.04 J = 7.32 3.10, t 7.68, d 29.09 129.55 J
= 7.32 J = 8.30 47.51 137.24 22 1.09, t 1.32, quin 2.42, s 14.01
23.67 21.47 142.98 3.20, quar 1.57, quin 7.29, d 42.76 28.39 127.04
J = 7.32 3.10, t 7.68, d 47.37 129.60 J = 7.32 J = 8.30 137.09
[0131]
6TABLE 6 .sup.1H--NMR .sup.13 C--NMR Compnd Et --(CH.sub.2).sub.4--
--(CH.sub.2).sub.3-- NH--CHO/aromatic Et --(CH.sub.2).sub.4--
--(CH.sub.2).sub.3-- NH--CHO/aromatic 17 1.07, t 1.60, quin 1.86,
quin 2.43, s 7.66, d 13.91 26.39 28.23 21.48 136.37 3.19, quar
1.67, quin 3.14, t 6.05, bs 7.67, d 43.40 37.36 45.54 127.04 143.39
J = 7.32 3.10, t 3.18, t 7.31, d 8.16, bs 49.08 46.92 127.12 143.43
3.34, quar J = 7.32 J = 8.30 J = 8.30 129.77 161.44 J = 7.32
135.91
Example 7
[0132] Evaluation method for accelerating effect on RNA polymerase
transcription activity
[0133] An activation level was evaluated by observing an effect of
a polyamine on in vitro transcription activation using
bacteriophage T7 RNA polymerase in the reaction system below.
[0134] Each polyamine was added to 10 .mu.l of reaction solution
(40 mM Tris-Cl, pH 8.0, 8 mM MgCl.sub.2, 5mM DTT, 200 M GMP,
wild-type T7 RNA, polymerase 5U, 0.2 .mu.Ci[.alpha.-32P]UTP, dsDNA
produced by cleavage of pBluescript [Stratagene] by a restriction
enzyme Pvull 500 .mu.M as rNTP template DNA) to yield a final
concentration of 2 mM, then reacted at 37.degree. C. for one hour.
After the reaction, 10 .mu.l of formamide loading dye (98%
formamide, 10 mM EDTA) was added to the products, heated at
90.degree. C. for modification, subsequently analyzed by
electrophoresis on 4% acrylamide gel. The gel was dried after the
electrophoresis, observed for radioactivity contained in the
transcription products using the BAS2000 image analysis system
(Fujiphotofilm Inc.), and evaluated for activation level of each
polyamine.
[0135] The results are shown in Table 7. Data is given in
amplification of radioactivity compared to a blank in which a
reaction is carried out in parallel in the same condition excepting
no polyamine was added.
7TABLE 7 Accelerating effect of synthetic polyamines on
transcription activity of T7 RNA polymerase Com- Molecular
Molecular Activity No. pound Characteristics Formula Weight Level
.sup.a) 1. 5 BET-343/HBr C.sub.14H.sub.34N.sub.44HBr 582.101 1.01
2. 10 BET-43/HBr C.sub.11H.sub.27N.sub.33HBr 444.093 3.05 3. 15a
BET-444/HBr C.sub.16H.sub.38N.sub.44HBr 610.155 1.21 4. 15b
BET-454/HBr C.sub.17H.sub.40N.sub.44HBr 624.182 1.18 5. 15c
BET-464/HBr C.sub.18H.sub.42N.sub.44HBr 638.209 1.12 6. 15d
BET-474/HBr C.sub.19H.sub.44N.sub.44HBr 652.236 1.29 7. 15e
BET-484/HBr C.sub.20H.sub.46N.sub.44HBr 666.263 2.52 8. 15f
BET-494/HBr C.sub.21H.sub.48N.sub.44HBr 680.290 1.18 9. 15g
BET-4104/HBr C.sub.22H.sub.50N.sub.44HBr 694.316 2.01 10. 18
ET34/HBr C.sub.9H.sub.3N.sub.33HBr 416.039 1.12 11. 19 484/HBr
C.sub.16H.sub.38N.sub.44HBr 610.155 1.41 12. 20 BET-8/Ts
C.sub.26H.sub.40N.sub.2O.sub.4S.sub.2 508.751 1.11 13. 21 BET-8/HBr
C.sub.12H.sub.28N.sub.22HBr 362.193 4.91 14. 22 BET-5/Ts
C.sub.23H.sub.34N.sub.2O.sub.4S.sub.2 466.670 1.43 15. 23 BET-5/HBr
C.sub.9H.sub.22N.sub.22HBr 320.112 3.52 Activity level .sup.a) is
shown in a rate against transcription activity when enzyme is not
added.
[0136] It has been revealed that ethylated amine compounds have
much stronger accelerating effect than the original amine compound
from comparison between Compounds 10 and 18, and Compounds 15e and
19.
[0137] It has been also revealed that a tosyl group which is one of
substituents suppressing transcription activity of RNAP from
comparison between Compounds 20 and 21, and Compounds 22 and
23.
[0138] A difference of activity levels of tetramine 5 and Compound
15a is thought to reflect a difference in the length of methylene
chain on the both ends. Taking the length of methylene chains at
the both ends into an account, tetramethylene shows better
transcription activity accelerating effect than trimethylene.
[0139] Remarkable chain length specificity can be observed in an
influencing on the transcription activity accelerating effect which
results from a difference in methylene chain length in a center of
a tetramine. A compound with octamethylene or decamethylene shows
about twofold increase in transcription activity accelerating
effect compared to that of the other compounds.
[0140] Sequencing of DNA with a longer strand becomes possible by
addition of the transcription promoter of the present invention
even if the amount of RNA polymerase used for DNA sequencing method
is not increased. Further, the amount of RNA polymerase or a
template used in the nucleic acid transcription reaction can be
reduced by the use of the compound of the present invention when
the lengths of the DNAs to be subjected are not to be
elongated.
* * * * *