T7 RNA polymerase activation and improvement of the transcriptional sequencing by polyamines

Article abstract of DOI:10.1016/S0968-0896(00)00156-5

We examined the possibility to improve the effectiveness of the in vitro transcription system using T7 RNA polymerase by coexistence with organic bases. The effect of the additives was evaluated by measuring the amount of RNA products in comparison with t

Full text of DOI:10.1016/S0968-0896(00)00156-5

Bioorganic & Medicinal Chemistry 8 (2000) 2185±2194
T7 RNA Polymerase Activation and Improvement of the
Transcriptional Sequencing by Polyamines
Masaaki Iwata,a,* Masaki Izawa,b,c Nobuya Sasaki,b,e Yoko Nagumo,d
Hiroyuki Sasabea and Yoshihide Hayashizakib,e,y
aBiopolymer Physics Laboratory, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi,
Saitama 351-0198, Japan
bLaboratoryforGenomeExplorationResearchProject, GenomicSciencesCenter(GSC)andGenomeScienceLaboratory, RikenTsukuba
Life Science Center, the Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan
cResearch and Development, Nippon Gene Co., Ltd, 1-29, Tonya-machi, Toyama 930-0834, Japan
dDepartment of Nutrition, Tokyo Kasei University, 2-15-1 Inariyama, Sayama, Saitama 350-0013, Japan
eCREST, Japan Science and Technology Corporation (JST), 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan
Received 22 March 2000; accepted 6 June 2000
AbstractÐWe examined the possibility to improve the e€ectiveness of the in vitro transcription system using T7 RNA polymerase by
coexistence with organic bases. The e€ect of the additives was evaluated by measuring the amount of RNA products in comparison with
that of the control system (without additive). We found that four commercial bases and a series of ethylated polyamine analogues newly
designed were active enhancers in the following activation order, 1,8-bis(ethylamino)octane>1,8-octanediamine>1,5-bis(ethylamino)-
pentane>cadaverine>1,8-bis(ethylamino)-4-azaoctane>spermidine 1,18-bis(ethylamino)-5,14-diazaoctadecane>agmatine. It was
shown that RNA products were corresponding, only in the presence of active enhancers, to the full length size of the template
DNA, and that sequencing signals were enhanced by the presence of active enhancers with high ®delity so that the transcriptional
sequencing was further re®ned to be a highly sensitive sequencing method from a small amount of linear dsDNA. These results
suggest that T7 RNA polymerase was activated by the speci®c binding of the polyamine additive to produce RNA transcripts with
®delity to the template DNA. Therefore, it is expected that the transcriptional sequencing in the presence of active enhancers might
be a powerful and sensitive method, in place of the prevalent DNA ampli®cation method, for genomic science projects and clinical
and practical gene diagnoses. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
to transcribe the template DNA inserted downstream
from either SP6, T7, or T3 phage promoter.1 3
The rapid and precise production of single stranded RNA
transcripts of de®ned length from DNA templates using
bacteriophage promoters and puri®ed RNA polymerase
(RNAP) has been, and continues to be, of fundamental
importance for research on post-transcriptional RNA
modi®cations, ribozyme, and eukaryotic translation
mechanism. Moreover, this technology is now widely
used to prepare RNA templates for in vitro translation
and to generate highly sensitive hybridization probes for
Southern and Northern analyses, RNase protection
mapping, in situ hybridization, and cDNA libraries in
the genome projects. The usual method of in vitro
transcription is to use a bacteriophage RNA polymerase
Several plasmid vectors containing multiple cloning sites
located between two such promoters are commercially
available. By choosing the appropriate phage RNAP one
can produce large amounts of RNA, complementary to
either strand of the template DNA. However, T7
RNAP shows some intrinsic limitations which can lead
to low transcription levels despite the presence of a
strong promoter.2,4 7
PCR method8,9 is employed as the fundamental ampli®-
cation procedure of genomic DNA and cDNA in many
®elds of molecular biology. In the direct sequencing of
the PCR product from genomic DNA and cDNA,10
however, there have been pointed out the problems
inherent to the cycle sequencing method; intervention of
the accurate sequencing reaction by remained 20-deoxy-
ribonucleotide 50-triphosphates (20-dNTPs) and PCR
*Corresponding author. Tel.: +81-48-467-9379; fax: +81-48-462-4647;
e-mail: iwatama@postman.riken.go.jp
ySecond corresponding author. E-mail: yosihide@rtc.riken.go.jp
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00156-5

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M. Iwata et al. / Bioorg. Med. Chem. 8 (2000) 2185±2194
primers.11 These two are presently inactivated by use of
exonuclease I and shrimp alkaline phosphatase. But
these enzymes are very expensive and the enzymatic
reactions are time consuming. These disadvantages pre-
vent the highthroughput production of sequence data in
the projects of genome and cDNA sequence analysis.
reaction utilized for the screening test was carried out at
37ꢀC for 1 h in a mixture containing Tris-Cl, MgCl2,
DTT, GMP, each of rNTP, [a-32P]UTP, and wild type T7
RNAP in the presence of organic base of interest and
the linearized template dsDNA, which was derived from
pBluescript KS(+) plasmid by PvuII restriction enzyme.
Amount of T7 promoter-dependent RNA transcripts
produced on the template DNA was measured by electro-
phoresis followed by autoradiography. Relative e€ect of
the additive organic base was indicated by `activity
index', which is the quotient of the intensity magnitude
of the measured radioactivity of RNA transcripts in the
presence of the additive divided by that of the control
transcription assay (in the absence of the additive).
Organic bases, which enhanced the transcription activity
of T7 RNAP, and activity indices are summarized in
Table 1.
We have recently developed a new RNAP-based sequen-
cing method called `transcriptional sequencing',12,13
which is characterized by a rapid isothermal sequencing
reaction by use of mutated T7 RNAP with an improved
incorporation rate of 30-dNTPs as sequencing terminators.
This procedure can help to overcome serious problems
in the direct sequencing reaction of genomic DNA sim-
ply because, in the transcriptional sequencing, RNAP
recognizes and polymerizes only ribonucleotide triphos-
phates (rNTPs) along the template double stranded (ds)
DNA.13 Since the stability and amounts of RNA tran-
scripts can be a€ected by the elongation rate, we have
faced requisition for further re®nement of the transcrip-
tion reaction with respect to rapid, accurate, and long
elongation of RNA for the purpose of the establishment
of the advanced micro-sequencing technology. We have
supposed that this requirement would be achieved by
improvement of the in vitro transcription system.
Among many commercially available organic bases
examined over 40 samples, only four simple acyclic
polyamines, agmatine, cadaverine (1,5-pentanediamine),
spermidine, and 1,8-octanediamine, were found to be
e€ective enhancers of the RNAP transcription reaction.
The e€ectiveness was in the order: 1,8-octanediamine>
1,5-pentanediamine>spermidine>agmatine. In contrast,
all of newly synthesized polyamine analogues, which were
synthesized according to the pathways illustrated in
Scheme 1 and characterized by the typical feature of
their structures with ethylated terminal amino groups,
were found to be, more or less, e€ective activators.
Among them, BET-8 (5D), BET-5 (5E), BET-43 (5G), and
BET-484 (5He) were the intensive top four in this order.
In total, it was shown that the activation order was BET-
8>1,8-octanediamine>BET-5>cadaverine>BET-43>
spermidine BET-484>agmatine. It is obvious that ethy-
lation at the terminal amino group increases the activation
It is well-known that the in vitro transcription system by
use of T7 RNAP is speci®cally a€ected by the presence of
such additives as inhibitory antibiotics14 and stimulatory
natural polyamines, particularly spermidine,2,3 and that
mouse and human RNA polymerases are stimulated sig-
ni®cantly by natural polyamines.15,16 In addition, it has
been recently reported that several synthetic polyamines
exert a concentration-dependent enhancement e€ect on
T7 RNAP reaction.7 These reports suggest that appro-
priate polyamines could improve the in vitro transcription
reaction to produce rapidly and precisely large amounts
of RNA transcripts. Thus, we started the wide range of
scrutiny examination to ®nd stimulatory agents for the
T7 RNAP reaction.
Table 1. E€ect of the additive on the transcription reaction of T7
RNA polymerasea
Additive
Characterization
Activity indexb
Since there has been no logical strategy, we examined
randomly the additive e€ects of amine-bases with various
structure on the in vitro transcription system. Then the
results were fed back to further systematic study by a
series of acyclic polyamine analogues. In this article we
describe the evaluation results of the e€ect of the poly-
amine additives on T7 RNAP, key structural factors
necessary for the activation of T7 RNAP reaction, the
®delity of RNA transcripts, and the remarkable
improvement of the transcriptional sequencing by use of
stimulatory polyamine analogues.
5F
5G
BET-343/HBr
BET-43/HBr
BET-444/HBr
BET-454/HBr
BET-464/HBr
BET-474/HBr
BET-484/HBr
BET-494/HBr
BET-4104/HBr
ET34/HBr
484/HBr
BET-8/Ts
BET-8/HBr
BET-5/Ts
BET-5/HBr
1.01
3.05
1.21
1.18
1.12
1.29
2.52
1.18
2.01
1.12
1.41
1.11
4.91
1.43
3.52
5Ha
5Hb
5Hc
5Hd
5He
5Hf
5Hg
8G
9He
4D
5D
4E
5E
Results and Discussion
1,8-Octanediamine
1,5-Pentanediamine
Spermidine
8
5
4.62
3.40
2.58
2.11
Increase in the transcriptional activity of T7 RNAP by
various polyamines
34
(Gua)4
Agmatine
aSee Experimental for procedure of the transcription reaction and the
evaluation method.
bThe numerical index of the magnitude of the measured radioactivity
in the presence of the additive divided by that of the control tran-
scription assay (in the absence of the additive polyamine).
Since there was no intuitive leading principle to activate
the T7 RNAP reaction at all, we tested the e€ects of many
organic bases on the in vitro transcription system. As
mentioned in Materials and Methods, the transcription

M. Iwata et al. / Bioorg. Med. Chem. 8 (2000) 2185±2194
2187
Scheme 1. Synthesis of polyamine Hbr salts.
potential more than the original amine form itself.
Macromonocyclic polyamine (10), which has been
reported to be an intensive enhancer for T7 RNAP,7
was tested by the present transcription assay system.
The result, however, indicated the activity index close to
and not more than spermidine (data not shown). These
results indicate that we found highly potent activators
of T7 RNAP other than spermidine and 10.
speci®city of the polyamine analogues as the enhancing
e€ector, optimized by penta-, octa-, and deca-methylene
chains as the most e€ective length (refer to pentandiamine,
spermidine, octanediamine, and 5Ha±g). In all cases, the
ethyl-substituent plays an auxiliary positive role for the
increment of the enhancing e€ect. The tosyl group, in
contrast, exerts the intensive suppressing e€ect for the
reaction (refer to 4D and 5D, 4E and 5E). Peripheral
tetra-methylene chains a€ord the minor suppressing
e€ect (refer to a set of structural changes for all). The
function of tri-methylene chains are skeptical; these exert
the enhancing e€ect in the case of spermidine referred to
agmatine, but the intensive suppressing e€ect in 5F
referred to 5G. In addition, ethylation at tri-methylene
chain markedly decreases the enhancing e€ectiveness
(refer to spermidine and 8G). With respect to relation-
ship between the basicity of amino group and the intensity
of the additive e€ect, substitution of the primary amino
group by the electron donating group like an ethyl sub-
stituent is likely to increase the enhancing e€ect in most
cases. In contrast, substitution of the primary amino
group by the electron withdrawing group like a tosyl
substituent results in rapid decrease in the enhancing
e€ect.
In structural viewpoints of active polyamines, enhancers
are structure-speci®c and the enhacing e€ectiveness is
highly dependent on the distance between two terminal
amino groups rather than on content of the amino
groups. Several enhancing and suppressing structural
factors are obviously demonstrated: ®rstly, the central
methylene-chain length seems to govern the structural
Figure 1 exempli®es the electrophoretic result of a typical
case of the present transcription assay using 1,8-octane-
diamine (lane 2), BET-454 (5Hb, lane 3), cadaverine

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M. Iwata et al. / Bioorg. Med. Chem. 8 (2000) 2185±2194
as the intensive transcription activator (set B). The results
are shown in Fig. 2. Panel A indicates sequencing signals
of the set A, and panel B, sequencing signals of the set B.
In the set A, it is obvious that the sequencing signals
appear in general with relatively low intensity and that
readable sequence is limited to shorter than around 150
bp, including still several unidenti®able very weak signals
in this range. Thus, the set A experiment lacks precise
sequencing signals and is impracticable for the micro-
sequencing of DNA. On the contrary, the set B experi-
ment generates analyzable sequencing signals more than
400 bp, at least, with high ®delity and, thus, is shown to
be providing a highly sensitive sequence determination
method for micro-amount of the template DNA. These
results indicate that the potency of the transcriptional
sequencing method was further re®ned by the coexistence
of an active polyamine additive and properly extended
to the reliable micro-sequencing method for DNA
without any signal drawback.
Figure 1. Electrophoretic display of the e€ect of polyamine additives
on the transcription by T7 RNA polymerase: each lane indicates RNA
products obtained in the absence of additive (lane 1), or in the presence
of 1,8-octanediamine (lane 2), 1,15-bis(ethylamino)-5,11-diazapentade-
cane (BET-454, lane 3), cadaverine (lane 4), and spermidine (lane 5).
It is pointed out that there are some intrinsic sequencing
problems, mentioned above, in the prevailed DNA
ampli®cation sequencing method, which has been used
in the world-wide scale as a key sequencing technology
in the genome science projects; in addition, for example,
the residual template DNA causes problematic electro-
phoresis in the capillary sequencer and, as a result, the
decreased accuracy of the sequencing. Since the present
®nding supports that an amount of the template DNA can
be reduced to a few nanograms without loss of sensitivity
and ®delity, the sequencing problems accompanied by
the prevailed direct sequencing method will be completely
solved by the use of the re®ned transcriptional sequen-
cing method.17 Therefore, the transcriptional sequencing
in the presence of potent polyamine additive is highly
recommended as a powerful and sensitive method for
genome science projects and clinical and practical gene
diagnoses.
(lane 4), and spermidine (lane 5) as additives in com-
parison with no additive (lane 1). It is clearly shown that
1,8-octanediamine (activity index 4.62) and cadaverine
(3.40) highly increase the amounts of RNA transcripts
and spermidine (2.58) is quite active as well. Activation
e€ect by additive on the transcription reaction was
concentration-dependent and the best activation was
observed in the wide concentration range of 0.2±2.0 mM
in most cases (data not shown), consistent with the pre-
vious result.7 It is of special importance in the case of
the presence of activator that RNA transcripts grow up
till around 300 bp, which correspond to the full length
size of the template DNA sequence, without showing
any intervention (pausing) of the transcription reaction
which is often observed in the chain-growth process
a€ected by the additive acting non-speci®cally on T7
RNAP. Thus, it is suggested that the polyamine enhancer
in the T7 RNAP-mediated transcription should bind to
the speci®c site of T7 RNAP and play a crucial role only
for the enhancement of the precise transcription and the
elongation of full length RNA transcript.
Experimental
General molecular characterization methods
1
The H and 13C NMR spectra were recorded on JEOL
GSX-500S (500 MHz) and JNM-GX 400-a FT-NMR
spectrometers with Me4Si as an internal standard in
CDCl3 unless otherwise mentioned; chemical shifts (d)
and coupling constants (J) are in parts per million
(ppm) and hertz (Hz), respectively. Signal assignments
Transcriptional sequencing in the presence of polyamine
additive
Since we have recently developed the transcriptional
sequencing method which is characterized by the combi-
nation of a rapid isothermal transcriptional sequencing
reaction with the e€ective use of the chain termination
reaction,12,13 it is of our practical interest whether or not
the improvement of the transcription system by addition
of a polyamine activator will endorse the microanalysis
of DNA sequences for which the sensitive and precise
transcription must be inevitably achieved. For the
examination of this possibility, the transcriptional
sequencing was carried out by use of extremely low con-
centration (10 ng) of the template DNA in the absence of
any additive (set A) or in the presence of 1,8-octanediamine
1
were performed by measurement of H±1H COSY and
1H±13C HMBC spectra. IR spectra were obtained on a
Shimadzu FT-IR 8100M spectrophotometer. Merck
silica gel 60 (Art. 7734, 0.063±0.02) was used for column
chromatography, and fragmented Merck precoated
silica gel 60 F254 plates (Art. 5715, 20Â5 cm), for TLC.
Product spots on TLC were detected either under a UV-
light lamp or in an iodine vapor bath. The uncorrected
melting points were measured in a bilayered coverglass
(18 m/m, thickness 0.13±0.17 mm) with a micro-melting
point apparatus (Yanagimoto Seisakusho, Serial No.
2647). Elemental analyses were performed in the Micro

M. Iwata et al. / Bioorg. Med. Chem. 8 (2000) 2185±2194
2189
Figure 2. Sequencing signals of the transcriptional sequencings from a small amount of the template DNA: in the absence of any additive (panel A)
and in the presence of 1,8-octanediamine (panel B).
Analysis Laboratory of Molecular Characterization in
IPCR.
N1,N4,N9,N12-tetra(p-toluenesulfonyl)-4,9-diaza-1,12-
diaminododecane (3A) was derived. A mixture of 3A
(0.17 g), potassium carbonate (0.14 g) and bromoethane
(57 mg) in 40 mL of DMF was stirred at rt for 3 days
and ®ltered through Celite. The ®ltrate was evaporated
under reduced pressure and the residue was chromato-
graphed on a silica gel column eluted with chloroform:
acetone (95:5 v/v). The fraction indicating Rf=0.4 on
TLC (chloroform-acetone (95:5 v/v)) was collected and
evaporated to dryness to a€ord N1,N4,N9,N12-tetra(p-
toluenesulfonyl)-1,12-bis(ethylamino)-4,9-diazadodecane
(4A, 0.11 g, viscous liquid, 58% yield). Anal. calcd for
C42H58N4O8S4: C, 57.64; H, 6.68; N, 6.40. Found: C,
Materials
Agmatine, cadaverine (1,5-pentanediamine), 1,8-octa-
nediamine, and spermidine (1,8-diamino-4-azaoctane)
are commercially available (Sigma-Aldrich) and used as
additive without further puri®cation. The other poly-
amine analogues were newly synthesized through the
processes shown in detail as follows (see also Scheme 1).
Cyclic polyamine [38]N6C10 (10) was synthesized by
Wako Pure Chemical Co., Ltd.
1
57.46; H, 6.60; N, 6.39. IR(KRS): n 1350 and 1550cm
1
(SO2). H NMR data: d 1.07 (t, J=7.32 Hz, Et), 3.19
General synthetic procedure
(quar, J=7.32 Hz, Et), 1.86 (quin, J=7.32 Hz, CH2 of ±
(CH2)3±), 3.11 (t, J=7.32 Hz, CH2 of ±(CH2)3±), 3.13 (t,
J=7.32 Hz, CH2 of ±(CH2)3±), 1.58 (m, J=7.32 Hz, CH2
of ±(CH2)4±), 3.10 (quin, J=7.32 Hz, CH2 of ±(CH2)4±),
2.41 (s, CH3), 2.42 (s, CH3), 7.29 (d, J=8.30 Hz), 7.31 (d,
J=8.30 Hz), 7.66 (d, J=8.30 Hz), 7.67 (d, J=8.30). 13C
NMR data: d 13.91 (Et), 43.14 (Et), 28.71 (C of ±(CH2)3±),
45.46 (C of ±(CH2)3±), 46.51 (C of ±(CH2)3±), 25.75 (C of
±(CH2)4±), 48.39 (C of ±(CH2)4±), 21.48 (CH3), 127.07,
127.12, 129.69, 129.77, 136.20, 136.65, 143.16, 143.31. A
mixture of 4A (0.10 g) and phenol (0.21 g) in 33%-HBr
AcOH (10 mL) was heated at 75 ꢀC for 20 h and evapo-
rated to dryness under reduced pressure. Dark brown
residue was washed several times until colourless with
ether at ®rst and then with ether±methanol mixed solvent
to give 5F as colourless powder (87% yield).
Purchased 1,n-diaminoalkane was tosylated with p-tolue-
nesulfonyl chloride in pyridine in the presence of triethyla-
mine to give N1,Nn-di(p-toluenesulfonyl)-1,n-diamino-
alkane (M). The reaction of M with alkylating reagents (N)
was performed under several conditions depending on
the structure of the aimed compound. After the con-
nection of M and N, the terminal groups of the product
were treated by the conventional transformation meth-
ods18,19 to give chain-elongated and nitrogen per-tosylated
polyamines which were important intermediates for
further modi®cation.
1,12 - Bis(ethylamino) - 4,9 - diazadodecane 4HBr (5F).
Starting with N1,N4-di(p-toluenesulfonyl)-1,4-diamino-
butane as M and N-(3-bromopropyl)phthalimide as N,

2190
M. Iwata et al. / Bioorg. Med. Chem. 8 (2000) 2185±2194
1,8-Bis(ethylamino)-4-azaoctane 3HBr (5G). Starting
with N-(N3-p-toluenesulfonyl-3-aminopropyl)phthalimide
as M and N-(4-bromobutyl)phthalimide as N, N1,N4,N8-
tri(p-toluenesulfonyl)-1,8-diamino-4-azaoctane (3B) was
derived. A mixture of 3B (0.16 g), potassium carbonate
(0.18 g) and bromoethane (48 mL) in 40 mL of DMF was
stirred at rt for 3 days and ®ltered through Celite. The
®ltrate was evaporated under reduced pressure and the
residue was chromatographed on a silica gel column
eluted with chloroform:acetone (95:5 v/v). The fraction
indicating Rf=0.4 on TLC (chloroform:acetone (95:5
v/v)) was collected and evaporated to dryness to a€ord
N1,N4,N8-tri(p-toluenesulfonyl)-1,8-bis(ethylamino)-4-
azaoctane (4B, 0.11 g, viscous liquid, 65% yield). Anal.
calcd for C32H45N3O6S3: C, 57.89; H, 6.83; N, 6.33.
Found: C, 57.61; H, 6.88; N, 6.32. IR(KRS): n 1350 and
1550cm (SO2). H NMR data: d 1.08 (t, J=7.32 Hz,
Et), 1.09 (t, J=7.32 Hz, Et), 3.18 (quar, J=7.32 Hz, Et),
3.21 (quar, J=7.32 Hz, Et), 1.87 (quin, J=7.32 Hz, CH2
of ±(CH2)3±), 3.13 (t, J=7.32 Hz, CH2 of ±(CH2)3±), 3.15
(t, J=7.32 Hz, CH2 of ±(CH2)3±), 1.57 (m, J=7.32 Hz,
CH2 of ±(CH2)4±), 3.12 (t, J=7.32 Hz, CH2 of ±(CH2)4±),
3.12 (t, J=7.32 Hz, CH2 of ±(CH2)4±), 2.42 ±(s, CH3), 2.43
(s, CH3), 7.29 (d, J=8.30 Hz), 7.31 (d, J=8.30 Hz), 7.67
(d, J=8.30 Hz), 7.67 (d, J=8.30 Hz), 7.68 (d, J=8.30 Hz).
13C NMR data: d 13.91 (Et), 14.00 (Et), 42.84 (Et), 43.12
(Et), 28.69 (C of ±(CH2)3±), 46.91 (C of ±(CH2)3±), 48.40
(C of ±(CH2)3±), 25.65 (C of ±(CH2)4±), 25.75 (C of ±
(CH2)4±), 45.44 (C of ±(CH2)4±), 46.43 (C of ±(CH2)4±),
21.47 (CH3), 127.04, 127.07, 127.12, 129.65, 129.69,
129.75, 136.27, 136.66, 136.98, 143.05, 143.18, 143.31. A
mixture of 4B (0.10 g) and phenol (0.28 g) in 33%-HBr
AcOH (10 mL) was heated at 75 ꢀC for 20 h and evapo-
rated to dryness under reduced pressure. Dark brown
residue was washed several times with ether until
colourless and then with ether±methanol mixed solvent
to give 5G as colourless powder (91% yield).
25.79 (C of terminal ±(CH2)4±), 46.94 (C of terminal ±
(CH2)4±), 25.94 (C of central ±(CH2)4±), 48.02 (C of
central ±(CH2)4±), 21.48 (CH3), 127.04, 127.09, 129.64,
129.70, 136.20, 136.63, 137.01, 143.03, 143.18. A mix-
ture of 4C (0.11 g) and phenol (0.24 g) in 33%-HBr
AcOH (10 mL) was heated at 75 ꢀC for 20 h and evapo-
rated to dryness under reduced pressure. Dark brown
residue was washed several times with ether until col-
ourless and then with ether±methanol mixed solvent to
give 5Ha as colourless powder (91% yield).
1,15-Bis(ethylamino)-5,11-diazapentadecane 4HBr (5Hb).
Starting with N1,N5-di(p-toluenesulfonyl)-1,5-diamino-
pentane as M and N-(4-bromobutyl)phthalimide as N,
N1,N5,N11,N15 -tetra(p-toluenesulfonyl)-1,15-diamino-
5,11-diazapentadecane (3Cb) was derived. A mixture of
3Cb (0.19 g), potassium carbonate (0.15 g) and bromo-
ethane (40 mL) in 40 mL of DMF was stirred at rt for 3
days and ®ltered through Celite. The ®ltrate was eva-
porated under reduced pressure and the residue was
chromatographed on a silica gel column eluted with
chloroform:acetone (95:5 v:v). The fraction indicating
Rf=0.4 on TLC (chloroform:acetone (95:5 v/v)) was
collected and evaporated to dryness to a€ord
N1,N5,N11,N15-tetra(p-toluenesulfonyl)-1,15-bis(ethyla-
mino)-5,11-diazapentadecane (4Cb, 0.17 g, viscous liquid,
87% yield). Anal. calcd for C45H64N4O8S4: C, 58.92; H,
7.03; N, 6.11. Found: C, 58.75; H, 6.97; N, 6.08. IR(KRS):
1
1
1
1
n 1350 and 1550cm (SO2). H NMR data: d 1.05 (t,
J=7.32 Hz, Et), 3.17 (quar, J=7.32 Hz, Et), 1.55- 1.57
(m, CH2 of terminal ±(CH2)4±), 3.11- 3.13 (m, CH2 of
terminal ±(CH2)4±), 1.26 (quin, J=7.32 Hz, CH2 of cen-
tral ±(CH2)5±), 1.50 (quin, J=7.32 Hz, CH2 of central ±
(CH2)5±), 3.06 (t, J=7.32 Hz, CH2 of central ±(CH2)5±),
2.41 (s, CH3), 2.42 (s, CH3), 7.28 (d, J=8.30 Hz), 7.30 (d,
J=8.30 Hz), 7.67 (d, J=8.30 Hz), 7.68 (d, J=8.30 Hz).
13C NMR data: d 13.98 (Et), 42.79 (Et), 25.73 (C of term-
inal ±(CH2)4±), 25.79 (C of terminal ±(CH2)4±), 46.97 (C
of terminal ±(CH2)4±), 47.96 (C of terminal ±(CH2)4±),
23.80 (C of central ±(CH2)5±), 28.41 (C of central ±
(CH2)5±), 48.42 (C of central ±(CH2)5±), 21.47 (CH3),
127.02, 127.07, 129.64, 129.67, 136.68, 136.99, 143.03,
143.13. A mixture of 4Cb (0.16 g) and phenol (0.33 g) in
33%-HBr AcOH (13 mL) was heated at 75 ꢀC for 20 h
and evaporated to dryness under reduced pressure. Dark
brown residue was washed several times with ether until
colourless and then with ether-methanol mixed solvent
to give 5Hb as colourless powder (88% yield).
1,14-Bis(ethylamino)-5,10-diazatetradecane 4HBr (5Ha).
Starting with N1,N4-di(p-toluenesulfonyl)-1,4-diaminobu-
tane as M and N-(4-bromobutyl)phthalimide as N, N1,
N5,N10,N14 -tetra(p-toluenesulfonyl)-1,14-diamino-5,10-
diazatetradecane (3Ca) was derived. A mixture of 3Ca
(0.18 g), potassium carbonate (0.15 g) and bromoethane
(40 mL) in 40 mL of DMF was stirred at rt for 3 days
and ®ltered through Celite. The ®ltrate was evaporated
under reduced pressure and the residue was chromato-
graphed on a silica gel column eluted with chloroform-
acetone (95:5 v/v). The fraction indicating Rf=0.4 on
TLC (chloroform:acetone (95:5 v/v)) was collected and
evaporated to dryness to a€ord N1,N5,N10,N14-tetra(p-
toluenesulfonyl)-1,14-bis(ethylamino)-5,10-diazatetra-
decane (4C, 0.22 g, viscous liquid, 88% yield). Anal.
calcd for C44H62N4O8S4: C, 58.51; H, 6.92; N, 6.20.
Found: C, 58.43; H, 6.89; N, 6.16. IR(KRS): n 1350 and
1,16-Bis(ethylamino)-5,12-diazahexadecane 4HBr (5Hc).
Starting with N1,N6-di(p-toluenesulfonyl)-1,6-diamino-
hexane as M and N-(4-bromobutyl)phthalimide as N,
N1,N5,N12,N16-tetra(p-toluenesulfonyl)-1,16-diamino-5,12-
diazahexadecane (3Cc) was derived. A mixture of 3Cc
(0.21 g), potassium carbonate (0.16 g) and bromo-ethane
(44 mL) in 40mL of DMF was stirred at rt for 3 days and
®ltered through Celite. The ®ltrate was evaporated under
reduced pressure and the residue was chromatographed
on a silica gel column eluted with chloroform:acetone
(95:5 v/v). The fraction indicating Rf=0.4 on TLC
(chloroform:acetone (95:5 v/v)) was collected and evapo-
rated to dryness to a€ord N1,N5,N12,N16-tetra(p-toluene-
sulfonyl)- 1,16 - bis(ethylamino) - 5,12 - diazahexa-decane
1
1
1550 cm (SO2). H NMR data: d 1.05 (t, J=7.32 Hz,
Et), 3.17 (quar, J=7.32 Hz, Et), 1.55±1.57 (m, CH2 of
terminal ±(CH2)4±), 3.11±3.13 (m, CH2 of terminal ±
(CH2)4±), 1.56 (m, CH2 of central±(CH2)4±), 3.10±3.13
(m, CH2 of central ±(CH2)4±), 2.41 (s, CH3), 2.42 (s,
CH3), 7.28 (d, J=8.30 Hz), 7.30 (d, J=8.30 Hz), 7.67
(d, J=8.30 Hz), 7.68 (d, J=8.30 Hz). 13C NMR data: d
13.96 (Et), 42.78 (Et), 25.73 (C of terminal ±(CH2)4±),

M. Iwata et al. / Bioorg. Med. Chem. 8 (2000) 2185±2194
2191
(4Cc, 0.24 g, viscous liquid, 99% yield). Anal. calcd for
C46H66N4O8S4: C, 59.33; H, 7.14; N, 6.02. Found: C,
1,18-Bis(ethylamino)-5,14-diazaoctadecane 4HBr (5He).
Starting with N1,N8-di(p-toluenesulfonyl)-1,8-diamino-
octane as M and N-(4-bromobutyl)phthalimide as N,
N1,N5,N14,N18 -tetra(p-toluenesulfonyl)-1,18-diamino-
5,14-diazaoctadecane (3Ce) was derived. A mixture of
3Ce (0.18 g), potassium carbonate (0.14 g) and bromo-
ethane (37 mL) in 40 mL of DMF was stirred at rt for 3
days and ®ltered through Celite. The ®ltrate was evapo-
rated under reduced pressure and the residue was chro-
matographed on a silica gel column eluted with
chloroform-acetone (95:5 v/v). The fraction indicating
Rf=0.4 on TLC (chloroform:acetone (95:5 v/v)) was
collected and evaporated to dryness to a€ord
N1,N5,N14,N18-tetra(p-toluenesulfonyl)-1,18-bis(ethyla-
mino)-5,14-diazaoctadecane (4Ce, 0.17 g, viscous liquid,
92% yield). Anal. calcd for C48H70N4O8S4: C, 60.09; H,
7.35; N, 5.84. Found: C, 59.98; H, 7.30; N, 5.85.
IR(KRS): n 1350 and 1550 cm 1 (SO2). 1H NMR data: d
1.07 (t, J=7.32 Hz, Et), 3.18 (quar, J=7.32 Hz, Et), 1.58
(m, CH2 of terminal ±(CH2)4±), 3.12 (t, J=7.32 Hz, CH2
of terminal ±(CH2)4±), 3.13 (t, J=7.32 Hz, CH2 of term-
inal ±(CH2)4±), 1.24 (m, CH2 of central ±(CH2)8±), 1.50
(quin, J=7.32, CH2 of central ±(CH2)8±), 3.06 (t, J=7.32
Hz, CH2 of central ±(CH2)8±), 2.42 (s, CH3), 7.29 (d,
J=8.30 Hz), 7.29 (d, J=8.30 Hz), 7.67 (d, J=8.30 Hz).
13C NMR data: d 14.01 (Et), 42.78 (Et), 25.75 (C of
terminal ±(CH2)4±), 25.78 (C of terminal ±(CH2)4±),
46.99 (C of terminal ±(CH2)4±), 47.74 (C of terminal ±
(CH2)4±), 26.63 (C of central ±(CH2)8±), 28.72 (C of
central ±(CH2)8±), 29.10 (C of central ±(CH2)8±), 48.50
(C of central ±(CH2)8±), 21.47 (CH3), 127.04, 127.07,
129.62, 136.84, 137.01, 143.03. A mixture of 4Ce (0.16 g)
and phenol (0.32 g) in 33%-HBr AcOH (10 mL) was
heated at 75 ꢀC for 20 h and evaporated to dryness under
reduced pressure. Dark brown residue was washed sev-
eral times with ether until colourless and then with ether-
methanol mixed solvent to give 5He as colourless powder
(85% yield).
1
59.24; H, 7.07; N, 5.98. IR(KRS): n 1350 and 1550cm
1
(SO2). H NMR data: d 1.06 (t, J=7.32 Hz, Et), 3.17
(quar, J=7.32 Hz, Et), 1.57±1.58 (m, CH2 of terminal ±
(CH2)4±), 3.11 (t, J=7.32 Hz, CH2 of terminal ±(CH2)4±),
3.12 (t, J=7.32 Hz, CH2 of terminal ±(CH2)4±), 1.26
(quin, J=7.32 Hz, CH2 of central±(CH2)6±), 1.50 (quin,
J=7.32 Hz, CH2 of central ±(CH2)6±), 3.06 (t, J=7.32 Hz,
CH2 of central ±(CH2)6±), 2.41 (s, CH3), 2.42 (s, CH3),
7.28 (d, J=8.30 Hz), 7.30 (d, J=8.30 Hz), 7.67 (d, J=8.30
Hz), 7.68 (d, J=8.30 Hz). 13C NMR data: d 14.00 (Et),
42.79 (Et), 25.75 (C of terminal ±(CH2)4±), 25.79 (C of
terminal ±(CH2)4±), 46.99 (C of terminal ±(CH2)4±), 47.83
(C of terminal ±(CH2)4±), 26.30 (C of central±(CH2)6±),
28.71 (C of central±(CH2)6±), 48.42 (C of central ±
(CH2)6±), 21.47 (CH3), 127.04, 127.07, 129.65, 136.76,
137.01, 143.03, 143.10. A mixture of 4Cc (0.16 g) and
phenol (0.33 g) in 33%-HBr AcOH (13 mL) was heated
at 75 ꢀC for 20 h and evaporated to dryness under
reduced pressure. Dark brown residue was washed sev-
eral times with ether until colourless and then with ether-
methanol mixed solvent to give 5Hc as colourless powder
(89% yield).
1,17-Bis(ethylamino)-5,13-diazaheptadecane 4HBr (5Hd).
Starting with N1,N7-di(p-toluenesulfonyl)-1,7-diamino-
heptane as M and N-(4-bromobutyl)phthalimide as N,
N1,N5,N13,N17 -tetra(p-toluenesulfonyl)-1,17-diamino-
5,13-diazaheptadecane (3Cd) was derived. A mixture of
3Cd (0.16 g), potassium carbonate (0.18 g) and bromo-
ethane (38 mL) in 40 mL of DMF was stirred at rt for 3
days and ®ltered through Celite. The ®ltrate was evapo-
rated under reduced pressure and the residue was chro-
matographed on a silica gel column eluted with
chloroform:acetone (95:5 v/v). The fraction indicating
Rf=0.4 on TLC (chloroform:acetone (95:5 v/v)) was col-
lected and evaporated to dryness to a€ord N1,N5,N13,N17-
tetra(p-toluenesulfonyl)-1,17-bis(ethylamino)-5,13-diaza-
heptadecane (4Cd, 0.15 g, viscous liquid, 89% yield).
Anal. calcd for C47H68N4O8S4: C, 59.72; H, 7.25; N,
5.93. Found: C, 59.66; H, 7.27; N, 6.01. IR(KRS): n 1350
1,19-Bis(ethylamino)-5,15-diazanonadecane 4HBr (5Hf).
Starting with N1,N9-di(p-toluenesulfonyl)-1,9-diamino-
nonane as M and N-(4-bromobutyl)phthalimide as N,
N1,N5,N15,N19 -tetra(p-toluenesulfonyl)-1,19-diamino-
5,15-diazanonadecane (3Cf) was derived. A mixture of
3Cf (0.17 g), potassium carbonate (0.13 g) and bromo-
ethane (34 mL) in 40 mL of DMF was stirred at rt for 3
days and ®ltered through Celite. The ®ltrate was eva-
porated under reduced pressure and the residue was
chromatographed on a silica gel column eluted with
chloroform:acetone (95:5 v/v). The fraction indicating
Rf=0.4 on TLC (chloroform:acetone (95:5 v/v) was
collected and evaporated to dryness to a€ord
N1,N5,N15,N19-tetra(p-toluenesulfonyl)-1,19-bis(ethyla-
mino)-5,15-diazanonadecane (4Cf, 0.17 g, viscous liquid,
98% yield). Anal. calcd for C49H72N4O8S4: C, 60.46; H,
7.46; N, 5.76. Found: C, 60.28; H, 7.42; N, 5.74.
IR(KRS): n 1350 and 1550 cm 1(SO2). 1H NMR data: d
1.07 (t, J=7.32 Hz, Et), 3.18 (quar, J=7.32 Hz, Et), 1.57
(m, CH2 of terminal ±(CH2)4±), 3.12 (t, J=7.32 Hz, CH2
of terminal ±(CH2)4±), 3.13 (t, J=7.32 Hz, CH2 of term-
inal ±(CH2)4±), 1.23 (m, CH2 of central ±(CH2)9±), 1.49
(quin, J=7.32 Hz, CH2 of central ±(CH2)9±), 3.06 (t,
J=7.32 Hz, CH2 of central ±(CH2)9±), 2.42 (s, CH3), 7.29
1
1
and 1550cm (SO2). H NMR data: d 1.07 (t, J=7.32
Hz, Et), 3.17 (quar, J=7.32 Hz, Et), 1.57±1.58 (m, CH2 of
terminal ±(CH2)4±), 3.11 (t, J=7.32 Hz, CH2 of terminal ±
(CH2)4±), 3.13 (t, J=7.32 Hz, CH2 of terminal ±(CH2)4±),
1.23 (quin, J=7.32 Hz, CH2 of central ±(CH2)7±), 1.25
(quin, J=7.32 Hz, CH2 of central ±(CH2)7±), 1.50 (quin,
J=7.32 Hz, CH2 of central ±(CH2)7±), 3.06 (t, J=7.32 Hz,
CH2 of central ±(CH2)7±), 2.42 (s, CH3), 7.29 (d, J=8.30
Hz), 7.30 (d, J=8.30 Hz), 7.67 (d, J=8.30 Hz). 13C NMR
data: d 14.00 (Et), 42.78 (Et), 25.78 (C of terminal ±
(CH2)4±), 46.99 (C of terminal ±(CH2)4±), 47.80 (C of
terminal ±(CH2)4±), 26.63 (C of central ±(CH2)7±), 28.69
(C of central ±(CH2)7±), 28.76 (C of central ±(CH2)7±),
48.50 (C of central ±(CH2)7±), 21.47 (CH3), 127.04,
127.07, 129.64, 136.80, 137.01, 143.06. A mixture of 4Cd
(0.14 g) and phenol (0.28 g) in 33%-HBr AcOH (13 mL)
was heated at 75 ꢀC for 20 h and evaporated to dryness
under reduced pressure. Dark brown residue was
washed several times with ether until colourless and
then with ether-methanol mixed solvent to give 5Hd as
colourless powder (91% yield).

2192
M. Iwata et al. / Bioorg. Med. Chem. 8 (2000) 2185±2194
(d, J=8.30 Hz), 7.67 (d, J=8.30 Hz). 13C NMR data: d
14.01 (Et), 42.78 (Et), 25.75 (C of terminal ±(CH2)4±),
25.78 (C of terminal ±(CH2)4±), 46.99 (C of terminal ±
(CH2)4±), 47.71 (C of terminal ±(CH2)4±), 26.72 (C of
central ±(CH2)9±), 28.74 (C of central ±(CH2)9±), 29.10 (C
of central ±(CH2)9±), 29.43 (C of central ±(CH2)9±), 48.50
(C of central ±(CH2)9±), 21.47 (CH3), 127.04, 127.07,
129.62, 136.84, 137.01, 143.03. A mixture of 4Cf (0.16 g)
and phenol (0.32 g) in 33%-HBr AcOH (10 mL) was
heated at 75 ꢀC for 20 h and evaporated to dryness under
reduced pressure. Dark brown residue was washed sev-
eral times with ether until colourless and then with ether-
methanol mixed solvent to give 5Hf as colourless powder
(89% yield).
(7:3 v/v). The fraction indicating Rf=0.3 on TLC (chlor-
oform:acetone (9:1 v/v)) was collected and evaporated to
dryness to a€ord N1-ethyl-N1,N4-di(p-toluenesulfonyl)-
N8-formyl-1,8-diamino-4-azaoctane (7B, 0.22g, viscous
liquid, 88% yield). Anal. calcd for C24H35 N3O5S2: C,
57.56; H, 6.92; N, 8.24. Found: C, 57.57; H, 6.97; N,
7.99. IR(KRS): n 1680 (CHO), 1350 and 1550 (SO2)
1
cm 1. H NMR data: d 1.07 (t, J=7.32 Hz, Et), 1.09 (t,
J=7.32 Hz, Et), 3.19 (quar, J=7.32 Hz, Et), 1.86 (quin,
J=7.32 Hz, CH2 of ±(CH2)3±), 3.14 (t, J=7.32 Hz, CH2
of ±(CH2)3±), 3.18 (t, J=7.32 Hz, CH2 of ±(CH2)3±), 1.60
(quin, J=7.32 Hz, CH2 of ±(CH2)4±), 1.67 (quin, J=7.32
Hz, CH2 of ±(CH2)4±), 3.10 (t, J=7.32 Hz, CH2 of ±
(CH2)4±), 3.34 (quar, J=7.32 Hz, CH2 of ±(CH2)4±),
2.43 (s, CH3), 6.05 (bs, NH), 7.31 (d, J=8.30 Hz), 7.66
(d, J=8.30 Hz), 7.67 (d, J=8.30 Hz), 8.16 (bs, CHO).
13C NMR data: d 13.91 (Et), 43.40 (Et), 28.23 (C of ±
(CH2)3±), 45.54 (C of ±(CH2)3±), 46.92 (C of ±(CH2)3±),
26.39 (C of ±(CH2)4±), 37.36 (C of ±(CH2)4±), 49.08 (C of
±(CH2)4±), 21.48 (CH3), 127.04, 127.12, 129.77, 135.91,
136.37, 143.39, 143.43, 161.44. A mixture of 7B (0.21 g)
and phenol (0.93 g) in 33%-HBr AcOH (10 mL) was
heated at 75 ꢀC for 20 h and evaporated to dryness
under reduced pressure. Dark brown residue was
washed several times with ether until colourless and
then with ether-methanol mixed solvent to give 8G as
colourless powder (91% yield).
1,20-Bis(ethylamino)-5,16-diazaeicosane 4HBr (5Hg).
Starting with N1,N10-di(p-toluenesulfonyl)-1,10-diami-
nodecane as M and N-(4-bromobutyl)phthalimide as N,
N1,N5,N16,N20 -tetra(p-toluenesulfonyl)-1,20-diamino-
5,16-diazaeicosane (3Cg) was derived. A mixture of 3Cg
(0.17 g), potassium carbonate (0.13 g) and bromoethane
(34 mL) in 40 mL of DMF was stirred at rt for 3 days
and ®ltered through Celite. The ®ltrate was evaporated
under reduced pressure and the residue was chromato-
graphed on a silica gel column eluted with chloroform:
acetone (95:5 v/v). The fraction indicating Rf=0.4 on
TLC (chloroform:acetone (95:5 v/v)) was collected and
evaporated to dryness to a€ord N1,N5,N16,N20-tetra(p-
toluenesulfonyl)-1,20-bis(ethylamino)-5,16-diazaeicosane
(4Cg, 0.18 g, viscous liquid, 98% yield). Anal. calcd for
C50H74N4O8S4: C, 60.82; H, 7.55; N, 5.67. Found: C,
1,18-Diamino-5,14-diazaoctadecane 4HBr (9He). A mix-
ture of 3Ce (0.15 g) and phenol (0.32 g) in 33%-HBr
AcOH (10 mL) was heated at 75 ꢀC for 20 h and evapo-
rated to dryness under reduced pressure. Dark brown
residue was washed several times with ether until col-
ourless and then with ether±methanol mixed solvent to
give 9He as colourless powder (94% yield).
1
60.94; H, 7.59; N, 5.60. IR(KRS): n 1350 and 1550 cm
1
(SO2). H NMR data: d 1.07 (t, J=7.32 Hz, Et), 3.18
(quar, J=7.32 Hz, Et), 1.57 (m, CH2 of terminal ±
(CH2)4±), 3.12 (t, J=7.32 Hz, CH2 of terminal ±(CH2)4±),
3.13 (t, J=7.32 Hz, CH2 of terminal ±(CH2)4±), 1.23
(m, CH2 of central ±(CH2)10±), 1.49 (quin, J=7.32 Hz,
CH2 of central ±(CH2)10±), 3.06 (t, J=7.32 Hz, CH2 of
central ±(CH2)10±), 2.42 (s, CH3), 7.29 (d, J=8.30 Hz),
7.68 (d, J=8.30 Hz). 13C NMR data: d 14.01 (Et), 42.78
(Et), 25.76 (C of terminal ±(CH2)4±), 46.99 (C of term-
inal ±(CH2)4±), 47.70 (C of terminal ±(CH2)4±), 26.75 (C
of central ±(CH2)10±), 28.72 (C of central-(CH2)10-),
29.17 (C of central ±(CH2)10±), 29.43 (C of central ±
(CH2)10±), 48.50 (C of central ±(CH2)10±), 21.47 (CH3),
127.04, 127.07, 129.64, 136.86, 137.03, 143.03. A mix-
ture of 4Cg (0.17 g) and phenol (0.32 g) in 33%-HBr
AcOH (10 mL) was heated at 75 ꢀC for 20 h and evapo-
rated to dryness under reduced pressure. Dark brown
residue was washed several times with ether until col-
ourless and then with ether±methanol mixed solvent to
give 5Hg as colourless powder (90% yield).
1,8-Bis(ethylamino)-N1,N8-di(p-toluenesulufonyl)octane
(4D) and 1,8-bBis(ethylamino)octane 2HBr (5D). A mix-
ture of N1,N8-di(p-toluenesulufonyl)-1,8-diaminooctane20
(1.00 g), potassium carbonate (1.54 g) and bromoethane
(2.5 mol equiv) in 60 mL of DMF was stirred at rt for 3
days and ®ltered through Celite. The ®ltrate was evapo-
rated under reduced pressure and the residue was chro-
matographed on a silica gel column eluted with
chloroform:acetone (98:2 v/v). The fraction indicating
Rf=0.7 on TLC (chloroform:acetone (98:2 v/v) was
collected and evaporated to dryness to a€ord 4D (0.90 g,
mp 112±113ꢀC recryst from acetone±methanol, 80%
yield). Anal. calcd for C26H40N2O4S2: C, 61.38; H, 7.93; N,
5.51. Found: C, 61.35; H, 7.95; N, 5.45. IR(KRS): n 1350
1
1
and 1550 cm (SO2). H NMR data: d 1.10 (t, J=7.32
Hz, Et), 3.20 (quar, J=7.32 Hz, Et), 1.26 (m, CH2 of ±
(CH2)8±), 1.52 (quin, J=7.32 Hz, CH2 of ±(CH2)8±), 3.10
(t, J=7.32 Hz, CH2 of ±(CH2)8±), 2.42 (s, CH3), 7.29 (d,
J=8.30 Hz), 7.68 (d, J=8.30). 13C NMR data: d 14.06
(Et), 42.59 (Et), 26.55, 28.67, 29.09, 47.51, 21.45 (CH3),
127.04, 129.55, 135.91, 137.24, 142.88. A mixture of 4D
(0.88 g) and phenol (20 mol equiv)) in 33%-HBr AcOH
(10 mL) was heated at 75 ꢀC for 20 h and evaporated to
dryness under reduced pressure. Dark brown residue
was washed several times with ether until colourless and
then with ether±methanol mixed solvent to give 5D as
colourless powder (96% yield).
1-Ethylamino-8-amino-4-azaoctane 3HBr (8G). Starting
with N-(N4-p-toluenesulfonyl-4-aminobutyl)phthalimide
as M and N-(p-toluenesulfonyl)-3-bromopropylamine as
N, N1,N4-di(p-toluenesulfonyl)-N8-formyl-1,8-diamino-4-
azaoctane (6B) was derived. A mixture of 6B (0.24 g),
potassium carbonate (0.34 g) and bromoethane (90 mL)
in 50 mL of DMF was stirred at rt for 3 days and ®l-
tered through Celite. The ®ltrate was evaporated under
reduced pressure and the residue was chromatographed
on a silica gel column eluted with chloroform:acetone

M. Iwata et al. / Bioorg. Med. Chem. 8 (2000) 2185±2194
2193
1,5-Bis(ethylamino)-N1,N5-di(p-toluenesulufonyl)pentane
(4E) and 1,5-bis(ethylamino)pentane 2HBr (5E). A mix-
ture of N1,N5-di(p-toluenesulufonyl)-1,5-diaminopen-
tane20 (1.00 g), potassium carbonate (1.68 g) and bromo-
ethane (0.45 mL) in 60 mL of DMF was stirred at rt for
3 days and ®ltered through Celite. The ®ltrate was
evaporated under reduced pressure and the residue was
chromatographed on a silica gel column eluted with
chloroform:acetone (98:2 v/v). The fraction indicating
Rf=0.7 on TLC (chloroform:acetone (98:2 v/v) was
collected and evaporated to dryness to a€ord 4E (0.90 g,
mp 90±91 ꢀC (spontaneous cryst. upon dryness and left
standing), 72% yield). Anal. calcd for C23H34N2O4S2: C,
59.20; H, 7.34; N, 6.00. Found: C, 59.12; H, 7.38; N, 5.93.
IR(KRS): n 1350 and 1550cm 1 (SO2). 1H NMR data: d
1.09 (t, J=7.32 Hz, Et), 3.20 (quar, J=7.32 Hz, Et), 1.32
(m, CH2 of ±(CH2)5±), 1.57 (quin, J=7.32 Hz, CH2 of ±
(CH2)5±), 3.10 (t, J=7.32 Hz, CH2 of ±(CH2)5±), 2.42 (s,
CH3), 7.29 (d, J=8.30 Hz), 7.68 (d, J=8.30 Hz). 13C
NMR data: d 14.01 (Et), 42.76 (Et), 23.67, 28.39, 47.37,
21.47 (CH3), 127.04, 129.60, 137.09, 142.98. A mixture of
4E (0.77 g) and phenol (20 mol equiv)) in 33%-HBr
AcOH (10 mL) was heated at 75ꢀC for 20h and evapo-
rated to dryness under reduced pressure. Dark brown
residue was washed several times with ether until colour-
less and then with ether±methanol mixed solvent to give
5E as colourless powder (95% yield).
the presence of the additive divided by that of the control
transcription assay (in the absence of the additive poly-
amine). The results are summarized in Table 1.
Transcriptional sequencing
The transcriptional sequencing was performed by the
methods reported12,13 which included recent modi®cation
as follows; a sequencing reaction mixture contained PCR
product (10 ng) from lambda phage genomic DNA
shotgun clone as the sequencing template, the ¯uorescent
30-dNTPs, inosine triphosphate (ITP, 2500 mM),21 UTP
(500 mM), ATP (250 mM), CTP (250 mM), PPase (10
units), and T7 RNA polymerases F644Y (25 units).13
The sequencing reaction was carried out at 37 ꢀC for 1 h.
The excess of ¯uorescent 30-dNTPs in the labeled product
was eliminated by Sephadex G-50 column (Amersham
Pharmacia Biotech Co., Inc.). The puri®ed product was
analyzed by ABI PRISM 377 XL DNA Sequencer
(Perkin Elmer Corp.), as shown in Figure 2.
Acknowledgements
This research was supported by Special Coordination
Funds and a Research Grant for the Genome Exploration
Research Project from the Science and Technology
Agency of the Japanese Government, and a Grant-in-
Aid for Scienti®c Research on Priority Areas and Human
Genome Program from the Ministry of Education and
Culture, Japan to Y. H. This study was supported in part
by Core Research for Evolutional Science and Tech-
nology (CREST) from Japan Science and Technology
Corporation.
Enzymes and nucleic acids
The wild type T7 RNA polymerase was purchased from
GIBCO-BRL. The mutant polymerase F644Y was puri®ed
from mutant protein highly expressed Escherichi coli
clone by Nippon Gene Co., Ltd. The rNTPs and radio-
active nucleotide were from Amersham Pharmacia Bio-
tech. The ¯uorescent 30-dNTPs were synthesized by Wako
Pure Chemical Co., Ltd.
References
1. Chamberlin, M.; Ring, J. J. Biol. Chem. 1973, 248, 2235.
2. Milligan, J. F.; Groebe, D. R.; Witherell, G. W.; Uhlen-
beck, O. C. Nucleic Acids Res. 1987, 15, 8783.
3. Sampson, J. R.; Uhlenbeck, O. C. Proc. Natl. Acad. Sci.
USA 1988, 85, 1033.
4. Dunn, J. J.; Studier, F. W. J. Mol. Biol. 1983, 166, 477.
5. Jorgensen, E. D.; Durbin, R. K.; Risman, S. S.; McAllister,
W. T. J. Biol. Chem. 1991, 266, 645.
6. Ikeda, R. A.; Ligman, C. M.; Warshamana, S. Nucleic
Acids Res. 1992, 20, 2517.
7. Frugier, M.; Florentz, C.; Hosseini, M. W.; Lehn, J. M.;
Giege, R. Nucleic Acids Res. 1994, 22, 2784.
8. Saiki, R. K.; Scharf, S.; Faloona, F.; Mullis, K. B.; Horn,
G. T.; Erlich, H. A.; Arnheim, N. Science 1985, 230, 1350.
9. Saiki, R. K.; Gelfand, D. H.; Sto€el, S.; Scharf, S. J.;
Higuchi, R.; Horn, G. T.; Mullis, K. B.; Erlich, H. A. Science
1988, 239, 487.
10. Wong, C.; Dowling, C. E.; Saiki, R. K.; Higuchi, R. G.;
Erlich, H. A.; Kazazian, H. H. Jr. Nature 1988, 330, 384.
11. Bachmann, B.; Lueke, W.; Hunsmann, G. Nucleic Acids
Res. 1990, 18, 1309.
12. Sasaki, N.; Izawa, M.; Watahiki, M.; Ozawa, K.; Tanaka,
T.; Yoneda, Y.; Matsuura, S.; Carninci, P.; Muramatsu, M.;
Okazaki, Y.; Hayashizaki, Y. Proc. Natl. Acad. Sci. USA
1998, 95, 3455.
13. Izawa, M.; Sasaki, N.; Watahiki, M.; Ohara, E.; Yoneda,
Y.; Muramatsu, M.; Okazaki, Y.; Hayashizaki, Y. J. Biol.
Chem. 1998, 273, 14242.
Transcription reaction:13
The transcription reaction (total volume 10 mL) was
done at 37 ꢀC for 1 h in a mixture (pH 8.0) containing
Tris±Cl (40 mM), MgCl2 (8 mM), dithiothreitol (DTT,
5 mM), GMP (200 mM), each of rNTP (250 mM),
[a-32P]UTP (0.2 mCi, 3000 Ci/mM), and wild type T7
RNA polymerase (5 units) in the presence of polyamine
of interest (2 mM) and the template linear dsDNA
(10 mM), which was derived from pBluescript KS(+)
plasmid (Stratagene) by PvuII restriction endonuclease.
After addition of 10 mL of formamide loading dye, the
reaction mixture was denaturated at 90 ꢀC for 2 min,
and then subjected to electrophoresis on 8% polyacryl
amide gel containing 6 M urea. The radioactivity of
RNA products in the dried gel was measured with BAS
2000 image analyzing system (Fuji Photo Film Co., Ltd).
The activation e€ect by polyamine was assayed by mea-
suring the incorporation rate of the radioactive sub-
strates analyzed with electrophoresis on polyacrylamide
gel containing 6 M urea. Electrophoresis for the selected
samples is shown in Figure 1. Relative e€ect of the addi-
tive was indicated by `activity index', which is the quotient
of the intensity magnitude of the measured radioactivity in

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M. Iwata et al. / Bioorg. Med. Chem. 8 (2000) 2185±2194
14. Chamberlin, M.; Ring, J. J. Biol. Chem. 1973, 248, 2245.
15. Blair, D. G. Int. J. Biochem. 1984, 16, 747.
16. Blair, D. G. Int. J. Biochem. 1985, 17, 23.
17. Itoh, M.; Kitsunai, T.; Akiyama, J.; Shibata, K.; Izawa,
M.; Kawai, J.; Tomaru, Y.; Carninci, P.; Shibata, Y.; Ozawa,
Y.; Muramatsu, M.; Okazaki, Y.; Hayashizaki, Y. Genome
Res. 1999, 9, 463.
19. Iwata, M.; Kuzuhara, H. Bull. Chem. Soc. Jpn. 1989, 62,
1102.
20. Iwata, M.; Kuzuhara, H. Bull. Chem. Soc. Jpn. 1982, 55,
2153.
21. Sasaki, N.; Izawa, M.; Sugahara, Y.; Tanaka, T.; Wata-
hiki, M.; Ozawa, K.; Ohara, E.; Funaki, H.; Yoneda, Y.;
Matsuura, S.; Muramatsu, M.; Okazaki, Y.; Hayashizaki, Y.
Gene 1998, 222, 17.
18. Iwata, M.; Kuzuhara, H. Bull. Chem. Soc. Jpn. 1989, 62, 198.