togetloo'r, tlo't('roni tieol toy t. 1-it's )o'tt-rtiphtOt ooouietnc met hood . 0 ..... It is too obviously curved to alloov o'milo'ulmu-. 16 tion of bimuding paratsuetens, i)ut ...
MoLt,:cUm,_io
9,
1’lIOOMOCOILOOtIY,
The
Unwinding
dinium
544-561
of Circular
Drugs:
Deoxyribonucleic
Structure-Activity
Acid
Relations
for
by
the
Phenanthri-
Intercalation
Reaction L. Department
P.
G.
of Pholrntacolof/!/,
WAKELIN
Iledical
\I.
AND
School,
1-teceiveol
J.
WAntING
Coiinbrioije
November
Universi
t!/,
Canthridge,
Englan
(I
26, 1973
SUMMARY
L.
\\AKELtN,
ao’id
by
P.
iIoi.
at’tiomo.
;.,
AX!)
itO flit’
drugs 10,
ho’(wo’o’mi
culmon 1)NA has i)t’O’tl. thnidimie o’lunomssoiphone
?oI. ,J. : Tlio’
\\AttmNG,
P/oai’inaeot.
‘Thto’ in-it o’nat’ficonu
(io)nos
(
joluemuant(hnichimiiumui
344-561
tu so’nio’s
utowinuoling
: stnomct-uro’-moo’fivit-y
o’irculmon
deoxvniboomooncleic
loon fl-ic
imsfeno’mulmifion
re-
(i974). phio’mimin-ifhnic!inoiumu)
oof
of
no’la(ioons
drugs
an-ic! htoo’fenioophuago’
P\
cm-
12
stuc!iec! ivifhi tlo’ tibjt’cf of imivestigmotitog iioow suhstit-uo’uofs cmi the phio’moamtmif’fo’t’t ifs o’mopacify too in(o’rcalmtfe hc’(wec’ni flit’ 1)NA base imuins. Ymiria-
quatenniisimog
gm’ooup
at
poositioomi
lumovo’ lift!0’
3
ton to
o’ffo’o’t.
Tue
pro’sc’mic’t’
of
a
j)li(’ti’l substituenut at ptosifioomt 6 stromogly imuflono’iu’o’s o’x(o’nnoal moffac’limuso’tut to the DNA helix tumid oofi’eo’ts (ho’ uniwimodimig amoglo’. Initnoocluctitomo oil mi p-canhoixvl grooup on-i (-he phtemtyl ring loads too vo’muko’n bindimig. l’ro’o’ 1ou’in-iimor aniiimio gnoiups mit poositiomos 3 timid S art’ root mustomic!mitory foor fl-it’ imnfo’ro’almo(ion no’mu’(ioon, lot-if fho’ir i)nco’tt’t’ midds s(al)ilitV to flit’ o’omuiplex (mipproxiunsafely I .7 ko’mol/niooio’). 1o’mitoivtul oil tho’ 3-amssimooo groomp has little o’ffo’o’( tither tluan mo smutall no’(lu(’tioomo it-i ti-it’ umowimudinig momiglo’, whio’ro’mus ao’efylmo(ioont oil the $-mtmssimlo groou) results ins uveaker loimidimog mis vo’1l mis to loowo’ro’d uniwimothimog tinigle. Ho’l)lmoo’o’mus(’nf oil both 3- mumuc!S-aoisimuo groups i)y canbo’(h-ioixvmuniimooo
amugle oofho’n
lo’atis
sul)sfiflnemu(s
too nisankecllv
oil tinily 5.i’ ± 0.6#{176}, (lit’ Ioii-it’sf vmulue c’oin-ist’tiiio’000’o’s vliiclu suggo’sf to radical oil 1)t’oluiitOe
Itltnco(Itlc’fiofl
io_(timt--i do’nivmofivo’ lotilt’
stremgtli.
fho’so’
studio’s
mitloniss
wliio’li
still
iii )ltioe
unwimids
Struo’tuno’-mocfivify tort’
vo’moko’n bim-idinig
of amssinio (ho’
DNA
with
o’monlier
ho’en amid
no’poom’fs fm’oomn this
o’omot’o’nnno’o! uvitlo oofhc’n’
sujiont’omi1s ( 1-3) . o’ovt’ro’cl c!iuuti i’lois I tooyttl
droogs
fimidimogs
oot’k
oil tint ihiot
mimic! m’o’vo’rso’
t’ino’ulan’
t!uplex
(4, vtis
y atool
5)
liv grtttot ho’ #{176}oledical 1esettrt’h
sooiol)oorto’(l t
ic’s
S
froonu
t
is
Alt
rights
--0:1:-) 107-0
Ov Ac,t’Ioii,it
of repr”dutioro
in
nress, ny
f’,rti,
tn’. r(,-erve,1.
3 am-id 5 yields
san-iso’ mingle
a loi’avy-
mus t’thidiumui
remu-ctio)nt
too tomotinisio’ro ohial
with DNA. mit low
deduced
front
activity.
1)o’o’mu moo!op(o’o! wlio’flto’u’
a
uiov(’I
clm’ugs
tos
hutic!
flit-if
till
c’ni(o’niooui by
o on nit of . ‘Flue
imuto’no’tilmitioomi
ion’
mimi itifer-
msiomc!els
nmutioimualt’ c!o’ussamtd
soon-i-it-’ tolto’nafioomiinn (lie uvimtc!imsgtol flit’helix ( 6-8),
he
whio’hu
expo’rimnc’tifmui
(oottt-icil 544
(,t,yrjgl,t
poositioom-is
cmtltifio mt-i jiro mo’t’ss ((‘i-9)
diso’thiunto-
(lie
inifercalationo
asso’ssimig
to
at
no’la(imog
boos
(Ito’
1)iN-\.s
, limos ho’o’mo slio)wmt
unwimudimg
vitlo’ to so’misifivo’ mito’mims oil niiomuifoonimg alt erafioomns ito flue WitO(litlg oil (lie 1)NA helix mussotiIitt’(! uvith c!m’ug himodimog. mo pmum’(io’tilmun’, it
have
llIut’moomuio’mloomo , 0 ot’igimoallv flit’ into’m’o’almofimog drug
hn’oimnide
Soorio’t
mobility
too m’c’mnoive
o’hoso’c!
oil
Flus with
flit’
Itolotortoftory
by
loon flit’
nN’nooI)UC’Fmd)N
Previous
groouj)s loo’lix
o’oon’ro’Ia(icomis
ito mogm’o’o’mo’tt
mind mimi mtppmunemit
ohso’nvo’d uvitlu miniy pluo’momomifltnidiuoo’, together altenmutitomi itt flue nio’chumtmiisnuu of binuding to
nutist-,
pnoovido’d
m’t’titiim’t’muioi’mits
thimut arc
cc’ntain smotisfied,
INTERCALATION
mi nueasunahle
result coilimig
Theoretical
duplc’x
practical
have
beemi
the
of
circular
and
met-hod 3,
vaniatiomi
closed
in-i
OF
aspects
fully
PHENANTHRIDINES
super-
sti(ueusts
DNA.
pho’iianfhiridime
oil
the
docunsenuted
(1,
10). Too
date
over
for
culmur
effects
DNA,
40
drugs
have
ott
flit’
supercooiling
anutong
which
tucknowledged
rels. bt’t’nu
circular
crysfmol
amid
expo’nirneuots interc’alatintg are
structuro’
the
drugs
Iromsu
divo’rsity
tested,
for
too elucidate
capacity
a
of
pheuianfhunidimies,
stamuds
of
amu
lmuctcors
we
have
nsay
appetured
calaf-imig
at
timiderto
uncoilimig
to’sf
ordc’n
to
conspare
oil
efluidiuns.
data eters
penniitfo’d which
etltidiuns, nst’mut ol the
to
(Iii’
test
flue
of (he we
genus
in
The
wt’ne
slngg(’stio)ni
(‘o)mnplex
formmition nuopluoone
no’sul(-,
were
ansgle
oil huydroogo’mu anstimio groups
be
st ondied
DNA
givo’ water
dnings,
The Slack
L.
tot-id II.
timid
All were
out
purification,
lun(hio’n
comuspoourids
lomiving
amid
tiO)Ii
lahooratoit’io’s (huo’v
dank
at
vo’ne
Lovell,
The
hvdromxvo’t
acid
; I )\ISO,
dinto’t
I)rs.
H. with-
as
i\Imuy
tossmoyo’d
& l3ako’r f or so olvot-
loomnioogemio’ity
elo’o’fro mphoono’sis
flit’
sinpplio’r.
lou
tin’
.V ‘
sioljoho
dn’y
rluemuio’to! with
f lie
at- f lie miimoximiuumuu
inso’tl -
t boo’
ito fl-ic
1 suntumsimunises toogc’fher
by in
in-i to clo’siccafoor
drug
hvl
lorom-
by sU)plio’d,
stomned
ltvlpioet’tozine
oil
Noof f imigliminu.
(ho’ loin
tobbreviations
HI)
ethic!iun
nuuolmom’ t’xt imit’tio mn-icoeffnt’io’mof sub-
timid \\‘ooollo’
(.
tiseol l)o’o’mI
o’miehu
to
L(ol.,
0-4#{176}.Tmoblo’ i(mn
nut
nii
& Bmiko’r,
timid of
state
94
494
oil Mmuy
c’lio’o’keol
chnonsnufoograpluy
de(miils
Dr.
provic!eo!
am-ic! S. S. Berg
stm’o’migtlu
Glass-tlistillo’cl
im’luding
kindly
tim-ic!
0.02,
Coonitpmtmiy,
Dago’mihumumss.
2
P. G. Wakelin, M. J. Waring, umupimblished oliservt-ttioins. I
wore
mini
EDT.-,
I lio’moidiumut Inom
rflue
.
20
ioimtie
0.1,
i’kline.s.
cifhio’n
sfram-i(Is.
hmuvo’ varvimig
1/tenallt/,
nuido’,
flue
reo1uined
ioom-ii(’ stno’utgfh 0.3). uvmos usec! tiurougluimut-.
(hue
c’fiuic!iunst-
mini
momiic’ sfnc’mugtli
stnemsgthi
I)rug
imitt’rt’sted by
homiic
Pure
asso’ss-
stabilised
give
Boots
coonusplex. (he
to
0.036) coimitainied
0.1
(lie
mu ith
boonds fromsu (Ito’ o’hrotoo phuoospItmito’ oxy-
(Ito’ eoniplerno’mtarv c’oomtspoiumids
that
vio’lol too givo’
gifts
suhsfifuo’nts
pmurficulanl (7)
mssiglut
soorne
to nun
(‘0 omucluo’t ott itt 7.0, mit roomsu
sfneisgfh,
(Cmulloioocho’nio),
wo’ro’
w
cmuso’s
imutencalated
14
vo’re pH
liuffens
16101
mist-ide
Imuvourable
)novidiuug ol vanitius
HEPES
T)NA-
poositive
unuwimudimug
mu
(hois effect
stability no’gmind
DNA
a
oil imufo’n-
circular
calculatit)ms tol iiimidimtg pmuntunicould lie coomsupmiro’d with (host’
of
mi this
capable (hue
bimidimig
oif
(hat
flue
Givo’ui
.
rnt’asurenieuifs
(itO
ioonoio’
HEPES-Nmi()H
(
METHOt)S
am-ic! Tris-HC! liuffen, ioH 7.9, adc!ifiomu oil HC1 to 0.Os st Tnis
l)\’
NmiCl ()ur
ANt)
Expen’imsso’mots buffers,
(resuitmomit of
ef hi-
drug.
be
((0
mull, based
5
MATERIALS
temsipo’ra(tnre,
type-spt’cific
intercalatimig
6
prepared
series
(lit’
2
>_4
Bu.t1’ei’s. HEPES2-NaOH
I)nilusmirY imit’mi( icon-i wt-is cjuahifa( ive : to) im-ives( igat e wiuc’fho’n a pant iculmin substit utec! plsenamitlunidimue
mono’ pro’-
___
oil
suitable
ro’latec!
mis
RESIJL’I’S,
1
7
nisedical
huomusologous
mill closely
whoich
o’xminiple
which
imuto’rcalafe,
study
mire imio’ludecl
oil
nsomsf
(heir
oil a jiootemitially
to
a
formulae
imoc!io’atecl
seo’tiomis of emio’h comiipounid
10
8
these
inuptontamuce.
the
loon f lie
is
(1omisido’r-
.‘
the
(‘Itoosen
(lie
luomologue, schenue
systemn
structural
9
c!imu-
comisfitutes a likely misore uirecise tout-
flue
5-methyl
ring
oil
S
amoto!oguo’s
dyes
whmot but
cluronsoophone
ot-i
its
nuumsslx’niuug
appropniato’ dmifmi Ion
limive
as to drug,
beeni
full
supo’rco oilpnoobmubly
gno’emi
by
and are
of ro’sttlts).
gained
Itt ant effort
diunsu,
sented.
: s(o’no)idmul
hyl
hampered
bioological
takt’nu
(see
heemu
havimug
affect
ago’mu(s
has
ansoong
fho’nn
where
tniphentylmuit’fhumiute
mum-id met
vioolo’(
imtsighf
clusiomis
half
of sul)stauuco’s
mssechanismns
(13)
al)le
or
at-id TIut’
the
in-i
3, 5, 6,
m’iuug, monud all
ethidiunu
beloow;
545
poositiomus
dinsuidiuni.
titan-i
Whi(’hi muffect the DNA by unkmsown,
notiims(t’ncalativo’,
muuin(’s
mnoore
groups
c’xausuoof o’ir-
a conipilatiomu
two)
discovered o)i
l)een
iu-ifencmulatin-ig
11 amud 12 for
It-i muddifion, imig
oil
in
1)NA
pluemuamuthunidimse
imied are
INTO
tore : -
2
-
oxide.
i1lP1,
ct-hatoo’suljiho
.V-2-
tO
546
\VAKELIN
AN!)
TABLE
(o)mpoun(l
Name index
-
phetiatit
hridiniunt
lt hid iunu broomide l)inuidimnnu
chloride
o’hloride
3,8-I)iatnitooo-.’i-tnethyl-G-phenyl-
phetioont-hridimoiton 3 8-1 )iamirotm-5-mnethylphentuot
3 ,8-1
luridiniunu
)ianuitioo-.’i-nuethyl-G-(p-o’torloooxy-
loridinium
3 ,8-1)io’torbet
C21H20N3,
Br,
Molar extinclion coefficient
Mo!
formulto
vt
0.421120,
416
.3900 (480 nnu)
335.5
6200
(480 ntis)
268.5
4200
(470 tint)
434
5930
(48.5
tot-it)
458
5920
(453
nra)
0.32C2H50H
CoHigN+,
C1 C1,
M&B
2421
C14H14N,
0.54120
M&B
3492
C21H8N302#{176}, C1,
M&B
4:’94
C22H20N30,
16101
C27H2N304,
1-12()
HC1,
o’lilotitle to-
C3H303,
1.’iH2()
lao’tato’
luoxyanuino-5-et
HI)
hyl-6-
l)heoo’1phet-iu-otot1oriditiiuni 5-iIet hyl -ii- (p-amniriophenyl aniitooplio’tiatit
Molecular
chloride
1)h0’ti’l )pho’tuotot-hridiniuni 3-Aniimooi-5-iioet luyl-6-phenyl-8-tocet nui(lophetltont
1
or No).
bromide
3,8-i)itoniitoo-.T-ethyl-6-phetoyl-
WARING
Br
538 . 5 5800
(430 tim-is)
336
6170
(430 nra)
381 .5
5570
(430 nni)
3i0
4410
(365
ntu)
548
6530
(400
non)
bro otnide
) -8-
hridiniuni
Pheroidiootn
hyl -I;. (/) -tontintophenyl ) -8-tot-i-i phetoatot hridin iutii chloride 5-iNlet hyl-6-phenylphenantliridiniton 5-Et
C20H8N3,
Cl-
0 hlooride
(‘hloorido’ itio
0-
M&B
3016
C21H20N3#{176},C1,
M&B
:3427
C2OHOON,
\I&B
17(i.i
C2oH,4NBr2, 0.51120
CH3OH
Br
l)rootnioie
3,8-I )ilorot-iuoo-.5-nuethyl-6-phenylpho’tototo t loriditi intro niethyl
of
ti-ic
booing wavo’lenugfhi
buffers
uso’c!
here.
sulpl-itito’
iio’mok iii (lie
The
c!es-ansuimuo
of diniuidmumis, 1\I&B 3427, somlublo’ in-i iiurt’l mutlueous o’x(imscticons
co mo’fficiemot
muqueoius amialogue
Bacteriophage doonusomsad
is oouily sparingly svsto’nis : ifs nioolar
HEPES-NaOH
in-i
C11380,,
R. T. DNA All
PM2 BAL-31,
Espejo, were as described
samples
c’ovalently this I)NA
abstorp( i(mmu o’oic’fflciemut(‘(hntnool-buffer msuixtunes.
o(hut’r
1)XA-binuding
drug
Iiiuodimug
2
(v/
v)
eflmimiol
(‘xtiuic’tioomi
isio’mtstnro’cl in various Bo’fwc’o’mu 50 timid
o’oomucemi(ra(ioiuo
cooo’fficiemit
flue
f’ooumuc!
nicilan he
(0)
in-
Drug
so ilUtitimis
were
wlic’rio’v(’n
p(issible
a-nd
dank tot 0-4#{176}ton sto)red DVA. (‘mill thvmsuus (‘niso’(I
somohiunsu
1)rob)o’ 15o-\\’ imig
DNA
it for
30
1)
wmos
(lie
stun--isiilt’
clanifio’cI
toni
1)o)1nuoib)tmuiited
mitt o b)uffer ec,
usitig
mu nsuo’diuno
wo’nt’
l)moth.
The
ice-wmufo’r
fluo’-
filfratiomu ivero’
(Iurooumglu sfo)n’o’c!
iromzo’mi
an-id
glass at
fibre
amid
oil (
.
(ci
iOn
o’itluo’n bonnier
iino’parec!
chino’c’f lv
o’oomutmuiriinig
- 22#{176}. lumoving
0.6
(ho’ mintco-
Sedimuuemsta-
.
by
bounudminy
34,000
rpnu
were
itt
a
nuefluod
1 ton 2 oil
mi lno’shi
loon
f 0)
aus
mis! oil
mimi tit)S(mn’bmonio’o’
mis jine-
o’oomiuplt’xes
o’micli
ultrao’o’m-itniluge noun-i ; in (ho’ latter inio’ro’niients oil mi c!rug soohin(ioonu in adclo’c!
coinuoil
extinuctioti
Drug-I)NA
Ii:,-
is
onso’ itt
(ernss respect-
valuc’s
0820
1)
(1) : in-i (lie
sommutioomu
was tin
F uilf nmucens(niluge t’ouipped oiptic’s. Details oil pnoocedunt’
clc’scnihed Pro’Pmort’ol
its
msseasuned
mit 200
70-90
DNA
in
Dr. PM2 (14).
stago’ bnornidt’
Pi\12
mu nsuoilar
comsupufatiomu
\\-muning
no
before
oil 6600. li’ij’ugat
were
Bo’o’kmssmttu nssodel wifli ultravioletvitiuslv
\ISE cool-
o’oefficic’nu(s
sedimssc’mi(mutioomu
so olutiomi
tom-i mit vltile
drugs
assunuinig
pseuby
comsfaimied
c’xpo’nimo’n(s.
efficienttot 260 nina A natytieal ulti’acen
host
pno’pane et al.
circles. At to o’thidiurn
centm’at-icimis are o’xpressed niuoolmun o’onucenutrafi(on with
(ion
\-imis dnasticmullv lomwerc’d liv (lie timid flit’ no’sttl(inug so)lutionis were biv
;
itt
mu flue
22#{176}.
Comsspmumiy . Soolufioimis wo’ne pm’o’jitined by
mum-it!nnt’c!mumn powo’n lo’vo’l ultrasootoic disinfegroofomn
visc’omsitv stinu’mifioimo, hlt(’ns
musmiino(muimtec!
type
iron n Signusmu Clienuicmol ctomn(moiniing 2 msug/nsul am-ic! soomticatimsg
pro’paned
lromzt’nu at DNA (highly
salt,
hionno mgo’moisimig the
irt’shly
here
cloosed c’xposed
cleofido’s,
vam’ianif.
ifs provided
used to by Espejo
used
1)uffo’rs wmos thuo’ro’looro’ do’fernitino’d liv o’x(rapolaf inng too zeroo ethamtcol o’oiuio’o’mit-rmifio ott (he
and
kimudly
c’onosplex
iuudivic!utul successivo’ bufft’r mine
tilt nmuco’iuf nifugo’ P#{176}ol2DNA mit 260
c(’lI
ins buffo’r mint
oil 0.600
INTERCALATION (i.e.,
91 .LI%mil-i muuclo’o(ides).
is more
ecconomical, DNA
same
but itt sample
As
use
(ho’
lattt’n
cleotide limited
\vlueuu
ratios soluhilify.
DNA
before which tainunc’mtt changes
in
c!ark
present-ed muot heemu
ity,
buoyancy,
by
at
H
call
on (c)
3016, 1765,
425 400
mum; i-u-i.
Equilibi’in cells
(16)
separated chamber imu the
having
itt
a water
(he
drug
uro’nsso’n(s
loin a
rmufioo,
91
3427,
bath
500
muc(’tmnacy by
ui
were at
comucentrafiout
uso’d
(drug
ii
i)NA
#{176}
attainc’oniupiefe
comssplo’xes
wo’ne flit’
lromn
by
too dett’rnuimue
the
also
hoouuud
iier
in
flue
t’quationu
mint-
su1)jc’cte(!
\V Iuc’mut’ver
.
plttft’d
Scafchuand
msicas-
(ho’ I)iridimug
t’oomuuplexes amialysis
tlio’ spo’o’-
dialysis
utuomio’cules
ito
3-n-u!
to
p ssihile
ic)rmss nequinc’ol
(17),
Jut
mis
chuosc’uu Ion lo)llows : ef lii-
mini;
M&B
coonsf
nuocfc’d
muuemuuliname drug
stirred
20#{176} fur
DNA
in-i each
it-i onue soolutioni
after chamsuben
flit’
nounusber
For
dromgs
oil 1)indimsg which
sift’s
himud
are fypio’muilv curved DNA is uso’d ; see
(to
po’n
miuo’io’oofido’.
DNA
such
(o’spc’cimoily
ivlieoi , but
Dm5CUSStON)
limiemun plot
am-id high
M &B 1765 ulfnmivioolc’t
roreSc’mut 0’ values
1)IO(S circular wlsem
-iva-s oohsenved
9B) (01
w luich was
at ioonic ahsoonp(iori
mu
himidimig
sfno’migth by the
mu exo’c’ss
jirc’c’Iuclecl mul)toVe mihout 0.1
; huo’ne a slight o’stimmite
hindinug
ultnmic’t’mifniiugmotio)mo
iviths
oil
riunis. for
those
j)loots
(18). Ion
by oil
the log
itt
flit’
in
nismido’ (ti detc’ncoomuchitioomos iclc’ni-
fl-it’ ultrmucemotnilugmu-
rio micc’ooumit
effects
ethuidiumit
dicated jut
used
pno’ssune
Such
miec’o’sstury
natlo)s
o’xpo’ninuo’mits.
o’xpeninssents, possibile
uias
mit high
\Vluile o’venv effoort wms nsuimio’ himidinug ntufioos unolo’n ticmtl
0.1) the ire(’ c!rug
deto’rmuuimimiticont oil (see I”igs. SB amid
o’xtnmipooltifioin
(ho’
negligible
to equilibrium
20 lur,
coomoc’enofnmutiomi, K coomosfmumif , mimd -ii is
ivhuerc’ (‘ is the free drug is (I-it-’ imutrimisic assoocimufio)ni
(iou-i
consparfmo’mtts
PM2
(1)
Kn-Km’
panmimuio’fo’ns wo’re o’stimuia(ed lnoomii ti 1(’mOSt-squmunt’s fittod limit’. Jut (woo (‘mises (\I &B 3427 itt
was
dialysis u-r
0’ -= C
reasomimubly
using
in-i buffer.
37S
appropriate
They
vo’nify
un amid
comistmucted
ul( nmucc’mo(nilugal by
(huc’
2 mg/mssl
Specially
91
to
o)r o’quiiibniunu
were
cleo(ido’), (lucy
pre-
pno)c’c’doire
a Visking
mit-id the other.
by SP
The
I)NA,
two
wi(lu
curves
ofomsuetnic
sam-
and M&B 3492, 470 mint; minis; jilio’nuidiumsi and M&B
in dialjsis. by
loaded
were
Bimdimsg t ro)pho
nio’asure-
emihuamuced
DNA,
M&B
50
4594, nnu).
1)MS().
i)o)imt
usuado’
onsieter.
the
dinsidiumuu, 2421, 460
flit’
nsoiar for
spo’ctro-
Umiicmimsi
fhtynius
Pi\12
o’omoo’emitra-
M&B 6390 (440
in-i 20
of
by
following
nuixtures: 16101,
oil equihl)niuniu
thc’
dimtuethyl
drug flue
po’nliormo’d
dissoocimofiomu
oil
determuuined
1W
were
nsuemusured
isosbesfic’
itt ref. 1 . W’ avc’leng(hss nuc’asunenusenuts wt’ne as
dium, \I&B
mit-
; (hey viscoos-
described
a 0
respects
(1oinufroils uiic’nf
quartz semimicroc’uvet-tes volumsies of (a) hiuffen,
3.60-nil
buffer-DMS() (450 iins);
ab-
went’
nsso’fhsod
was
souuicato’d
described l)ilidiflg
30 nun
recording
using
sioo’c( ropluof
0)fhi(’r
lc’asf
total
using
coeffic’iemu(s
loon
dissoocimuted
volumsue
flue
t’oeffi-
used;
was
equal
muid
o’xtinc(iomi
1 went’
cconuplex
o’stimusated
40to’s.
cotucemtfra(iou.
1800
omuietnic
nst’msureinemit
buffer,
(v/v) 6460
deft’nminued to connect for
mu (‘let-in
15),
40-n-ins-light-path confainsing
of and
or by re(‘o)efficients
(occurno’d, t 1uam-i(itative (mug bimidimig veno’ (1,
series
5J)
\\luero’
no 01)110itt
extinic(iomu
Occasional
oil connplexes
mu Umticam
photonueto’n.
suIpluooxidl(’,
(Ito’
Ta})lt
(ho’
, usimig
nicaily
s(’mimic’noot’uvt’f
sido’
in
oil ant
itu-
temperature
DNA
addifioomi
to drugs drug
rophto ofouno’f
fno’o’ drug
sido’
tiomu was
Ultraviolet-visible
,s1)o’(’tna
viouslv
at
dc-
547
tiuan(z
listed
oifho’r
n)NA
spect
tho’
c’iems(s
go’mienally
maiuufaimsimsg
room
as directly adjusted or
or po)imuts uuuo’uuts of
with (lie
ultracentnifugation, Sedimemuta(ioon
Speclrop/molomelo’y.
so)rptiomi
were
instanutanteous.
heicone sansples.
to
sir-icc the spo’ctral oiut bimiding appeared
performed
the
per nu-
of sedinsenotafiomi, he adequate for
equilibrium, omccurred
ant’ have
a-il
1”tor
drug
for
comntemucemiueutfassunued to
were
few’houns running
(b)
finues,
high
required imi all cases
practically
checks
of
cor five
was
in contact-
oil that
to) be
were
were the was
specf
mm-light-path
methoods
method
except
dc’to’rmimuc’d
oil the
attributable
diffo’remuf
flue
using
too four
nuo diffenemscc’s
oil
employed
pies
up
method
INTO
ro’use
pt’rnsitting
sample
fectable,
(he
lattc’r
PHENANTHRIDINES
either case the amoount of DNA is the same : 54.5 nmooles oil
clo’ootides. the
The
OF
(hue
o’ffo’c(s
lie
the
taken
ct1uilib-
tint’ kutoownu
too ho’ mit’ghigihle
That
are
drugs
lack .r
could oti
tho’v studied
likel’
oil sigmdficaiit vs.
(misc,
to lie
luo’re
wlio’nt-’
is
imi-
curvature .o’ is
the
WAKELIN
545 disfamico’
Inoomsu tii(’
sec!inssemttatioomt
axis
oil
nootmufionu,
expc’nimenots
(1,
jut
AN!) flue
15).
RESULTS
A
oil
in-i ‘Tmihle
flue
pnimic’ipmul ho’ io)umod
no’lo’n to this (able while finclimigs with each drug are “nthstit-uents
at
himidimig 0 ‘(
hidiunsu
(limit
mon-id ethyl c!iffc’reuice; supo’ncoiling who’re (‘losed m-is a dntng
nuoolo’c’ulo’s oumi1i(af-ivo’
ho’ rc’spoomise fIn’
two
hooumtdany,
cent-ned
(or 0.052 po’r
muu(’leo)fide.
oil closed are
cir’lo’s
Titus
aspc’cts (to flue
PracticallY
was
oif
a
at positioii group angle
how the
leads
which
hindimig
of
am-id poinut.
In
ooccurs, at 504
2421
wo’nc’ thero’foore sfreusgfh (Fig.
momugles loin
o’quiva-leiuce
is ro’mucIted, as nuoo(o’d pnc’viously (1, 4, 5). Ho’lix umuvinudimig
flit’
(woo
drugs
muiay
he
io)mnc
c’coniipared
Tue
insing (hue o’oiuatioomu (2)
c1t
wluo’no’
v,-1 mum-id v2
m’totioos ion
each
rc’spo’o’tive
mint’ (lit’ drug,
amid
umuwimidimig
loon o’(luic!iunss
c’o1t-iivmult’t-ice timid
mumugles
mus 12#{176} (7),
I)ilidiflg
(lie
are
(1).
(heir
Tmking
uniwimodinig
4o
companisoni
with leads
that toi
5.3#{176} ±
mimigle
(ti
booumid dinuidiumsu nutolecule is o’molculmuted l)e 1 1 .s#{176} ± 2.2#{176} (Ttuhlo’ 2) , mtoot siguiificmimttly
o!iffo’reuit. As in-i tI-ic o’mise oil ethuidiuns, soorjofiommt I)(’tuI oil dimisidium shuii(o’c! DNA,
5 1 3 u-imu mind iuso’fn’it’ drugs too linsits rolloot
t ho’ro’biv
o’uualiliuig
o’stintatioomi
0)1
(Ito’
wo’m’o’ pnmio’f io’mohly oiuanit flit’
jut this
a!)amid
o loomigo’r wmivc’lemig(his oni I)imtditig vio’lo!ing to clo’mur iso)sho’stic pooimst
DNA
up
101w itotil(’
too
muss:u
insipoiso’d )
,
if mutively
o (limit
by
I3oth hcoumid
(withiims flit’
(hue
nicked
an
front
ioltiic
strength
the
2)
(hue
sfan(imug
value
comisiderahbv
lower,
nm(’ bo’lone
flue
lo’mucc’ l)tMtit. nsal
sharp
This
mit nsodenate
DNA
o’xpooso’d
.
higlt
circular
PM2
attributable supercoiliiug,
At
poimu(-
(he
sanue
a-nd lall
(into’
exhibits
the
(he usual
drugs. The cloosed circles
observed
at
I’ig. 1) itt two mu the absentee thc’ne
bow
no’specfs: of drug is
is a perceptible
toowards
behiavioour too high
t(o
this
o’quivaleuice
geno’nally (ci.
isosbesfic with M&B
in (he sansue buffer unwimudimig mumtgle of
circles
that
strength a 6-fold spectral
at
cloosed
ant
the
equiva-
is o’mifirely
nuoor-
strength
(10)
ioonic
I)o’lore
ho’Iix-uniwimudinug
with
circular drugs
(13,
16).
spectno-
noo o’stinssmtes
oil
miti(1 limos ht’o’mi oohso’nvo’d
0.06-0.07
oil
huffo’r
to mit
no opho of oi-
himudimig.
vmiluo’s
stro’migtlu
oil sensitivity tmmiso’fm’ic
ii
spo’c(
0)1 lc)fllC
amid rise oil (he
e(luidliumTl calculated
mu
without-
o’oinducted 2). of
an
occurs, there is shift
lint
decreaso’ so’o’nu with jut o’rcalmi(imig shai)e oil (hue S2 iiltot for (lit’ diffo’ns
flue visible is depressed
a
At
still ratios
accompamuied by mum. Expenimemuts
0.7#{176} (Table
oil flue
0520
pen
for
is effected,
Itypochnonuic
DNA sluows f-he fall to nemsioval amid reversal amid
is formed DNA, no
ooccurs with and a similar
vaniaf-iomo
1520
altera(M &B strength
to
miximug
of un-
drastic
buffer
shift pioint
(lit’
altered
spectrum,
sto’midv
in-i S2-i tul(o’n
1{enioval
an
is irrevensjble.
0.5 no precipifatio)ru msuoolar t’xcc’ss of drug,
c!ecnc’ase
or com-
to
precipitate is added (he
moult’. Tluo’ sam-iso’ mupplies to (ht(’ b)o’Ismuviour ol flue noicko’d circles, which sluow (he typical b)im-idim-ig rmitio wifls o’thidiunu
but
as
of the drug low ionic
canelully
absorpt-ioomu
isoshestic
imidisfinuguishu-
2),
6 (R). reflects
precipitationu
hathochnomic
flue
a
atugle
detectable
ionuic strenugfh oil 0.2 precipitation l)tmf at low- drug to nucleotide
(diniidiumuu)
pnein
(Table unwindimig
a red, gelafimious the drug stolutiomi
and
(hose iodide
1X 174 neplicative beans a quaternised
itt
(-ioomis in tho’ behaviour 2421) . mu buffers
matter
of
ethidiuns.
j)htettyl
(
added
5-suhstjtuenuf
to
0.1) whemi
be
on drug
parameters
winding
DNA
substitution
propidium
muo difft’nemuce
bimidimsg
f-he
oil methyl
the
of drug action DNA (1). This
Howuio altena-
detectable.
msiay for
Substituents
5 makes uto (ho’ charac-
miusd qummititmifive
drugs
cii
comparc’d.
tol
results
that
to
was
repointed
availablo’.
stated
do-it’
ethyl
these
are
he
hindimig
ausuinuopropyl
mia-tune
sedimenta-
are
fairly
for
To study foirm
o’cmuivalence region, circles cosedimenit
homumud
conusfami(-s
cans
viously
pared
revo’rsal
(etluidiunn)
booth
in
is
effects
ini(enclumitsge
umunesoolvo’d
0.050
tioomi
1 (lie
oin-i t he
amid
it
again
DNA
with an minid nicked
simigle
arooumid
The
at poosi(iooui plots shuoow
rt’nssoovmul
hindimug
even,
limos little or no mimigle con on (ho’
1”ig.
the
groups both
tenistic
t)i
Jut
oil P\12
o’videmut
is
gnomup tnnwinsdinug
mit-id climsidium
(jtomt ho’huaviour It
5 (R4).
/)oSitiOfl
constmuuits.
ro’sul(s c’cmnuvo’nit’utt
(he expo’ninuuenfal do’scnibed mu (unit.
(0)
oil (ho’ quafermtisimug o’flc’o’( oom flue helix
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5-methyl
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20).
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nut obo’culo’,
(7)
3B), than
booundaries
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ito
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Os frootn
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The
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spectral
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is mutot toffecto’o! stno’mugtlo
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The
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to Fig.
together,
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taker-i
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shows
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plot-, I .3
X
about
sedimutetitatioti
an
i0
1)00th
anuuiuuo
mott
bimuding
denivmi(ive
joist
(ti
level
0.1
i
secoimto!any lmuteo! tot woon(huv
gro oups
at
amtglo’ at-id
in
(his
too
(hat ploot
must)
sitc’s
o’thuidiumui
there
hut
de(ernuimoe
linear,
the
covo’ned ( he Scatmoithoughi
mo
become
popu-
It
is uiote-
iooorf-ion of f-his Scatto the c-axis, yic’ldimtg of
“stromig”
hintdimog
\Vhile biniding
mtiooro’ sites
clo’nivmotive,
moto
to uttic’lecotic!e flue existence of
which
cmonumuoot l’ tint’
(B).
dimolysis
muboottt 0.3.
muimsiber
buffo’r
DNA,
too
netosomitobly
the
luypochuromnic
to
0.33’mitic’lo’ofic!o’. Ion
plot
FA1uilibnmunu
f-hue linio’ar extnapobttft’s
dicanhe(hiooxv as
to Scatcitard
amid
muboive
85% in
coinipotietits
Over flue range (‘xl)c’ninsetits
hinic!imtg
nunuiher
l)NA
at huigiio’r drug shoiwmi) indicated valuc’s
oil
(limit
(lie
cu c’fhtidmutiu msiointoaco’t-yl drug
is
mini mojipmurent
c!rmosticalby,
this
plot
wt’re
sites
of
t’tsipltivt’d
(Fig. SB). sediniemitatiomu
Sctut-
(FlI)
fornu
twoo
tim-i I)imid!inlg
(Iuerefoorc’
bimiding l)y (lie
chard
the
ic l)ooimtt mippo’ans.
is(OSht’sf
(‘hmund
vmuluo’ oobservo’d
(limit-i (limit Like the
shift
cotitaimued as described
mi ha(lsoc’hinonsuic
spo’ctrtul \\.mis
to
the
it
DN,-1
P1112
symbols
hitditg
presented
shuoows
a,oil
Prel)aratiomi
with
of drug
data
ho’ uniwimdimig
niuemt(ioomic’d,
coefficient,
few PtOitits ra(ioos (miot
biloa’ko’d
hoxyamino
muuore
(do’.s-pheioyldinoiilioorn) 0.50. The 1)NA
0.02. The by o’quihihi-
oof o’tliidiuni.
are
3.1#{176}, (hue loowest
c’oomusfmumit falls
am-ic!
wmovelemigthu
iiimuding o’omnstanf cmulculatec!, imtdic’afinug
f-h-ic’ phuo’mamt(itnidimtt’s, bower 2).
in-i
tumid f he Although
con-i himidimsg oil mum-iisosbo’stic
tiffintity
3 mmcl 5)
a valuo’ 14-bold ( Fig. 5; Tabbo’
.
loonig
groups
carbef
to oil
average
results 4),
tu sa(isftuctoory
-i-i.mis
strength
n-itea.siorentents
imisufficiemit
approximsuate
106
poositoomis
is redtu’o’d
the the
ionic stno’migfhu o!eternsuirut’d
coonistruct
16 101 , having
for
tite
Itmili(Ito’bimidimug
\Vht’mu both
ott
mu bafhioochunoomuuic in
\Vhile
to
2421
ioiooic
4594)
drug spectrum is mio evideutce
dimulysis.
available
0.05 V
IId’B
represetits
(M&B
po)inut- it-i buffer of bimtdim-ig wmi.s therefore
chard
Effect-
abscissa
shift
0.1
buffer,
uniwimidimig amigle (Fig. coonustant falls sontewliaf
derivative
rium
between
fromuu spectrophotometric
hiypcichronuic peak DNA,
A.
acetybation
l)V
a lowered bindimsg
0.08
FtG. 2. Interaction was HEPES-Na)11
solvemit
closed
0.06
bound per nucleotide
a
precise
too 1\-I2
DNA
giveti,
it
is (‘erfaimi
muvailmublo’ pno)bmul)ly
to
2-3
the (mites
miuam-iy.
Hemssomval reduo’o’ (he tiioiugh
(Ito’
oil tho’ 3-amsuimoo group appo’ars umiwiridimig momigle somssewliat, himiohiug
ro’mntuins
(itiift’
to) mol-
stnootig.
552
WAKELIN
AND
WARING
28
10
24
‘ml ‘C
x104 20
5
20
16
B
0
0.02 drug
0.04
molecules
0.06 bound
FIG. cloosed
wto.s HFPI5-NtuOII
circular
iiuooiecioles.
A.
Tw()
such
coonuspoiumicls
(
1”ig.
6).
simnila-r
The
sl)ectrmi
shifts
havim-ig of 370
out
were
between
ot
seoiinienttotiot
the
average
oof b)ooflu drugs
in
clmo(a
precludimog
gave
flue
o’urvo’d
defo’rnsiiusmof
each
big ptunauisc’tcrs, l)tnt if stremigth oil iuu(enac(ioono if at all,
iroonus (hat
drugs
1)ean
So’:o(chumurc! iou-i
was ntis
t
out
the 6-phuo’moyl ring, lot-if in (lit’ light oil no’sults tout fl-it’ effect oil 6-sonhstifiieisfs st’m’ibt’d tih(oV(’ it cmm ho’ misstmtiio’d that of
mit 1)H 7.9) Thie geiu’nmil
.
this
anoomssafic
loon booth
o’losecl
is o’xactlv
as expected
mon-id tolthioiuglu
amid flue
ansimuom group
is no’hutivc’ly shape oil (lie tucked
o’it’o’les
cmult’ultufeo!
our de(Ito’ (tin-
imuo’oonseSo Pltofs (Fig.
loon imttero’milmtimig umowimuding
synnbcAs
binding
to the
ito the
form
turo’
limits
oov(’nlap (Ttible onsplo’t e renso)val
gncoups
ro’stnl(s
imig minigle
two
oif to
muo)f idemi(icmul,
comtaimoed
as
l)NA
in
plot
an-ic! mu 10-20-fold
(B).
estimssato’cl
enroot
2). of hot I-i 1iniussmon
mu a dc’finito’ly
the
comuil)onents
Soatchao’d
flue
80%
described
tomsuiuto
lower
un-iwinud-
neductiojmi
in
flue
binudinig o’o)uisfmiuu(-. This o’oompoiutid, do’s-muniinoc!insioliuntu (\1&B 3427), is oil spo’cimul imt crest
fhimut flue diffo’ro’nt,
substifuetot-
l)rep:um’atioolo
gles
vc’sfigafe stnhsfi(uerits
mo p-amiummioo
cloargo’d oiuo’nf-imtl
0.1
D.V-1
1)NA with
l)resetlted
because
tin-id c!insuidiuns.
P.112
The
tof drug
bimid-
o)i ethuic!mumn
pno’so’mu’e
level
plots,
of o’xao’t
o’lo’ar lift-It’
0.02.
(1t
shsooweol
I)XA,
341#{176}2 a,ool co)ellicient-,
mt’asooremetts
two isosbes(ie pout-its ito flue ro’gions tim-id 460 mmii. The spt’o’tnoophuotonuiefnic
b)indimsg
Booth
the
invo’sfiga(ed:
too
.1IB strength
ivhicli c!iffo’r S-subsfituo’mu(
bimiohimig
005 ‘ml
iootiic
1fft’o’t
arid \I &B 3016, of flue otnafennisinog
0
touffer,
legeoitl to Fig. 1. TI-ic aloso’isstiro’pteseoots together, ttoken from spect rooiohtotootnt’tric
l)hio’nndhiumls flue nature
0.1
nucleotide
3. lntoroictio,,
solvetit
The
0.08
per
6)
drugs, aiu-
oil (Ito’
cmi It-it o’d
to onssplo’x
s1)o’ctnmol shil at-i isosbo’s(ic c’n-imubling bimscliutg. hitidirig
opportun-iity
.
It
exhibit
in flue ptiimot
affoords
nc
of 1)NA, mini (l’ig.
imo a
climiiidmunss n(’spomichmug 1 .3#{176} (1’ig.
,
with ((I
Iiy
nsanmso’r
liimuding
‘fimio’cl with 7A),
msiemusuno’miio’uit-
At low ioomtic stno’ng(h to) Pi\12 DNA mtffects the cmutmsecl
mu-
to
3 , 5-c!iamsuinio oil am-i imi(o’r-
s mu to’ll-do
lino’seutce at 357
spo’o’t rophuoo(omsue(
(itomu coefficientthat
it
oline(’tly (ho’ noole oil ito (lie ioonmlsatio)ni
(0.02) so’dintenita-
idc’uufit’al oil
of its
with
‘(luidiumuu
oor
mon-i eomuivalo’uce ro’gio mmi coinmin umiwinidinig mumigle o)l 7 .7#{176} ± tilled
Synsul)ols)
Pit of is liuio’mun amuci vio’lds
.
TIut’
a himidimig
Scmoto’huard co onsfmiuit
nNTERCALATtON
OF
PHENANTHRmDINES
INTO
I)NA
limit of 4.6#{176}Ier boum-id (Irong mnolt’cule he calculated lromss the binditug ratio at (Ito’ omiset of the presumned equivalenco’ ro’gion. uppo’r cant
28
Replacement tioom-is 3
of the
and
do’nivative
24
S by (M
of
removing
oil
Pi\12
&B and
with
o’thuidiunu.
(hue
(lie
amid
a
o’apable
supt’rcoihuig
stunt’
umowinidimig
0
0.04 drug
4.
0.08
molecules
Effect
bound
‘IIOtB
of
per
4594
on
of P2112 D.’oA stilvent was HlPES-NaI)H
nucleotide
.sedinoentatioiobuffer,
difference
becomes
that o)f o’(hidiunu (Table stremtgfh 0.1 a signuificant apparemut
,
however.
The
enable
bimudimig
is eviclemitly
inutercalatiomu to
generate is largely
of accessil)le
drug
(oooo w’o’ak
oil sufficienit
drug
revc’nsed supencooils. o’xpt’nimo’msfal, (lie to
uuuclo’ttide
imipint
to
musooleTI-it’ nmonige rmotitos
limited by (lit’ ultraviolet absoorptioomu of the free drug in-i eo1uililinmumsu with flu’ comuuplex. The Sca(clsancl ploot ito Fig. SB show-s tha-t in this l)uffen flit’ hintdiuug ctonistamit has fallemu to 3.32 X 10 -ir’, almoost c’xae(lv 20 beimig
times
Iowt’n
same
eoonditiomss
demur (limit umuwinichimug
(limit-i (limit t
(Table
foor o’tliidium 2).
uuuc!er
Hoiwevo’n,
flue if
the
o’quivaleusce
flue
sanie
is
t’oonsiolo’ntoblo’ chtmmige itt flit’ hto’lixmonuglo’ nsusf htovo’ omo’cunro’d, mum-id!tin
was poiinif.
behaviour
o’ootmid not cemotnafiomis eating
At
(Fig.
a
oonly
loot-in-ic!: flit’
9A, oil
ro’nsce
(Tmubio’
flit’
3 , 5-do’-mimnino 0 morod t om go ‘m-iermi( e mo‘vo’rsecl
closo’o!
ti retoltur
nuuom!(’o’ules
0. 1 is
motif dime
to
nic’kimug
in
(Ito’
s(muiif,
neflo’cfirig
(ho’ y
tot-
o occur0
if
I
ho’se
seoliniio’mofimog no’somlvo’c!, the
muomlecules mis mu n’emuumoimoo’c! tombcln’ug-ino’e l)NA
oil fl-ic
it its o’m-iujilt ovo’cl.
()t/ie,
/)/l(1la1it/11’i(/l/1(’S
expo’u’imsiemofs diumis,
uvo’re
dc’nivat
ivo’ Imirge
subsfittit’nfs
st’t’ 0
ieiv
:\_
j)0m’lt0m’!fl(’d!
isoomsso’tmomsii(!munn
do’scniptioomus,
hemun
fl-it-if
tue
pno’scmuct’
mull natioms uvhio’re cml cloiseci o’iro’bo’s
pn(opomrtioono oil tho’so’ inotmot’f fraction oil fh( toofttl l)NA
prepantit
7.2#{176}.Thw ti-it’ lim-io’mir
2).
oil
muntuioigtnes
oil
momugle
in-i F’ig. 9B (ompo’ni synsb)iols), (hmuno fbotit foot (ho’ cic’s-amutinto
failonn’e
drugs. At 1)ooumudmurv
imoc!i-
umowimic!imig
plot btowo’r
in
supo’r-
oopo’m synitbools), (hio’
Scatchard was still
stno’uig(h
0.1
loon (hut’ clo’s-monuino
valte nuoot gu’o’mofo’n (hiamo o’omsstanuf, c!o’nivo’d fronsi
superc’oils
mit
sfro’nigflu
to somuue himidimig
iomtit’
70
I)NA
cirt’lo’s wmus remuuomvo’t! lot-it mit- mio’(’essihle t!m’ug to mu-
lowenim-ig
chb)nomssom
imiterac(lit-in-i (limit-
to (ho’
iouuit’
usoted
closed be reverso’cl
the
becaust’ boumud
9B.
o’milo’ulmu-
weako’n
of dimssidiuuti
0)1 the
(‘ooilitig
1”ig.
alloov
i)ut
7B),
flc’ himidin
to
of
o’lear
ntmuu (Fig.
presented
on diiuuidiumss, drug
(limit
from
of bimuding paratsuetens, must be substamititully
The
the drug is added, the So of the closed circles falls to a nuinimunut, reflectim-ig renuoval oil the right-hanuded supercoiils oniginually lireseust, but fail to rise agmuin (Fig. SA, open sym-
amid 415
curved
added
uvitli
sediwith
r(’veals
derived
is
comispoumuc!
When
plot-
obviously
too
clc’nivmufive
iO)fllC
at 365
It
of flue
strength 0.02. The I)NA prepn-irmutioon cot-itt-med 8.5% cloised circular niuooleo’imles. Symbools are do’scribed it-i the legend to Fig. 1. The abscissa represent-s the average level of (1mg binditog to ti-ic two DNA cotnponents together , determined by equi libriunsu dialysis.
10-fold lower than-i 2). In buffer of ionic
points
ol ethidium
012
ic!ems(icmul
imig nseasuremenits is
tout flue t’iro’les,
momaulysis
a Scatchard
tion tiout
16
j)oimit Spectral
isoisbestic
cules problem
yio’lcls
is still
all the usutol effects of closed tumid msicked
mom-ieoiuivalemuce
bols).
which
reversing
DNA,
ionooduces usio’uo(atiout 20
FnG.
1765)
mit poSi-
atoms-is
amigle as ethidiunt tumid (!imiuidiuun (Fig. 9A). Iii (lie low ioomiic s(remugth huffo’m’ (0.()2) it
20
coefficient The
mouusimio groups
liromsuime
n’(’f.
ml istaisef
2 1)
,
tin-id
o’lianged too flit’
Ii
1)10
loom’nsulmoo’
tinui(!iumti
, P0 isit ivo’ly mof(tic’ito’!
(for
miohuitio omitl tit
.
All
of liitimid
to do’s-mititimo t
t’c’o’throngs
lic’t t’u’t oo’vo’Iic’ 8-tunsinom
gromup
WAKELIN
:054
ANt)
WARING
4
28
3 ‘C
ici0-
24
2 S20 0
20
I 0 B
16 0’l
drug
Tie closed
solvent circular
legend
to Fig.
together,
0.2
molecules
1. The
abscissa
front
(of flit,
jiheuuamutlnidine
fotnntc! (‘oilimg
to of
remove closed
were
drugs pursued
niot
fluo’ir
of
agno’o’s
with
helix
our
(hue moitro
of
groups
POSi(itmtis ( nc’vo’rsible)
5).
Au
an
efluidmum
limoso’ mit pH
and S Icormutafion vmiltcs
all
u-i-etc
the
binding
ohiservationus the fact that to
oimi (lie repoorted
iomuic perfectly
stremigfhi.
tw-o
0.02.
It-i Imuct-, (he
analogtme
beaninug
miuetical
at
In’ its I)scuolo)-
dnmowno
iromn
drastic
St nimo’f nrc
irnporttumtt
may
the
preset-if-
ntiodificatioius
of
ef-hidiunsu
work c’an
be witliotit
is
to that-
mitt-ide
he
mit(Oni
do’nivative
quite
to
abolisltimig
(-he
stability nature
these at
14
iihtenuamt-
cant
flue
imnpoirtamut where
is ac!vamit
rO)lC
of (lie
best
shown
be
(0
fiy
in future a lueavv-
chtanactc’r
group
irac(icmully flue
geoccitt-
ageous.
omu flue
6-piietivl
a-mt jitter-
st-in-ic
coouuo’erti (lit’ o’ffect
of (h-ic iutercmula(ecl oof the oumo(ennisimig
S muppea-u’s
a
(hue o!ibncomsuo
as (1-ic’ o’fhuiclitnmn
miext co)nu(’ltisiouss subst-ifuemtts
gavo’ s(rc’uigtht
foinmsi
hta-vimig
amiahigh
drugs iotuic
(hat
1765)
well proove iuutencalafiomu
oil
The
t’v(’ni nesponise
chmiracf’nistics
plex studies
3.
comiclusioni
01
amid 3 , S-dibromuuo tot au unfavourably
discovery
(i\1 &B
ca-lmuf-iomu o’oomuuplex
I)ISCUSSION
mtuoost
Yet
noormtial
various T1’Iuc
1)o)(emo(imil.
the des-anssimo oil dimiditimsu
umu-
aussimio grotps
mub)oove a-l)Oiut
imutercalafive
and
too imuvestigate
was frustrated of am inso)luble
its
thriditic’s sttnchiec!, n-iomie was foumid which failed to nemsuoove and reverse flue supercoilimtg t)l isI2 1)NA, (he omily ri(ar failures heimug logues
above
mumualogue
of the
between RD iotoic stretigth sedintentation
oil ntoife
mottemtipt
0.4
0.3
‘ml
the superDNA. Be-
Io’ads
ool)senvtofiomts
in place 3
DNA
drugs
4 and effect
the hut
is worthy
anuuinuo-subst-ituted
(Figs.
systemsu;
DNA, further, with
the
tiialysis
it-i nuo’asunimig
I
0.1
16101 aoooi P312 DNA 0.02. The DNA preparatioot-i contaitied 70% coefficieoit, with synubols as described mi the the average level of drtmg binding to the two J)NA ct)iutpotie010S measunensietits presented itu the form of a Scatchard plot (B).
repro’setits
ring
0
nucleotide
mit-ic! reverse circulmun duplex to
imtteracfit)mu
uviuic!imsg
per
eqmnililoritom
0)1 difficulties
ol (Iu’se
bound
FIG. 5. I,oleraction was IIEPES-NaOH buffer, nsolecules. A. Effect on the
taken-i
(‘iitiS(’
I
0’3
substjtuenit pne(’ij)ita(iom
oil amid
ccomusplt’x. The at poisif ion
imnsuafenial.
The
is proohably 1)hemiomsso’nmt
INTERCALATION
OF
PHENANTHRIDINES
INTO
DNA
555
28 28
24
24
S20 S20
20
20
16
16 0
0’04 drug
FIG.
6.
molecules
Effects
of
The solvent was 85% closed circular average
level method.
which
ooccur
bound
per
pheioidiurn
0.05
ii
0.12
to ti-ic
are
if it is nuissing.
The
coomusist oil mu which
o’lectrically nuch of
is in f-he fornt to the surface
of stacked of the
22).
(he
Lackimig
(M&B plamsar
form
stacked
aggregates
nuoolecule
than
ethuidium
or djnidium.
tiomi of seconudarv kntowns to he highly ionic phenomena fact
that
are
quantitatively
conditions 6-pheiuyl
readily
ethjdmum
will unider
(15). nimsg
Thus seems
Since
22),
ring, can
(he
as
practically
flue maini too he
effect
imilornuatioini
is available
with
conicenning
i)c)tli
amu
by
are
blocked
with
(hat
flue
too DNA
(Fig.
(
16).
derivative
The
(-he
3).
Thuo’
still
miot
ro’vensmil l)ehimovio)ur
intenestimig
iii (hat
or
coitseoltien-ices
oil a- dj-tert-hutvl
conso!o’u’ivtutive
beem sl-ioo-ivii consupoounid,
it seemsus
a nonintencala(ive
to) bind
msucc’humunuism
carhethoxy
suhsf-i(uo’nts
(RD
are
16101)
4)
lmurger grootipirig insteractiomi. (Judy
are
muot least by
monigle S-amusimuo,
2) , although remtoval amid nuakes
be than
uniwimsdimug tot-hen,
ion
can oo(luen
of pnoflavimie, whuichu humus rec’cuufly to) differ markedly irons its parer-it proflavinie,
Itt by
in-itencalaf
effect
(Fig.
derivmutive
the
are
group,
acetvlation
(Tmohle prevent
to
(lucy
(-he
the
Tho’
supercoiling
parison
of the
(he distnibufiomu of bound drug hefuveen inutercalated complex and an externally stmucked comic. Moire
whemu
this
ahscissto siu(ows the spectrtophoto-
3-aniimuo in
2).
I).V-1 contto.ined
suh)s(jtuenofs,
with mu usuch nuajo)n effect ciii the
conisiderable sufficient
salt-free
concernoed
reduction-i
I’.12
that
mio apparemut
(Fig. 6; Table can l)e blocked
the
by
loin
flue
a- small
(if
1. The
nuamudatory with
of
of
0.1
nucleotide
preparati000
determined
removed
is
is the
to Fig.
Ouse,
ii
precipitates
DNA
of the 3 S-diamsuiuio it is nsouv clear
substitution without
lorma-
The
0.08
per
,
reaction.
precipitation
form
role
attached (see ref.
explicable, ontly
legend
particular, no nseans
is virtually
.
together,
the
c’cimsiplexes to inicreases the
the
in
0.06 bound
coefficient
0.036)
mueutral the drug
msuone readily
(stacked) semusitive
(15,
s(renugth
precipitates
0.04
molecules
on. sedimentation
strength
components
tumid hence
nuclu
(B)
(ionic
described
phenyl 2421)
3016 7.9
1)NA
aggregates DNA luelix
out-of-plamte
a completely
pH
two
undoubtedly 1 : 1 complexes
des-pluenyldimidium
0.02 drug
ot,o(1 M&B
buffer,
Symbols
bitiding
0
nucleotide
(A)
Tris-HC1
molecules.
of drug
metric
0’08
alnstost
ol as
our
bulky
556
WAKELIN
AND
WARING
O4
0 U C 10
02
-e 0 UI 0
10
0 wavelength,
nm
40
U
C 10
0 U) .0 10
400 wavelength, 7.
FIG.
Tie 80.5
um
3, 88.7 DNA
lbsorption-
solvent
l)NA
curve
(curve
ao-id 1 uiig/n-ii as flue flavimse
(curve
tei’t-btifvl anialogue
1o’nsists. This furiclamisemtal nisnis oil e(hic!iunst.
of M&B
31,27 buffer,
(A ) amid ]!I&B ionic
1765
strength
(B)
0.02.
time preseioce
iii
The
of
calf
concetotu’atiot-i
of
DNA
thymus M&B
3427
added to the following final concentrations : curve 1 , zero ; omorve 2, 44.4 g/ml ; curve 4, 133 .tg/ral; curve 5, 1 mg/nul. M&B 1765 was used at 52.8 ,ut, it-i the absence of 1) or with I)NA added to 50 jig/nil (curve 2), 100 g/tnl (coorve 3), 200 mg/ml (cturve 4),
wit-h
tog/i-i-il;
,
spectra IIEPES-NaOH
was
nm
5).
stnbstituenufs of ; yet the unuwinding
oobservationi diffcncmu’e
intto’ncalm(-iomu Ito
momiv
evemit
nuay betweemu by
the
proeffect
poiuut to the media-
proflavinte there are
and several
peculiarities of RD a
in 16101
(ion
with
from
intercalatiomu
unwinding of the dip
the
DNA-hindimug
whuich
suggest
DNA atigle in (lie
msuav
differ
by
ethuidiumiu
is oS2u
behaviour (-hat
nsuuch curve
its
int-erac-
substantially : (he
helix-
lower; the shape for closed circular
INTERCALATION
OF
I’HENANTHRIDINES
INTO
28
1)XA
.).)
.
3 #{149}ml
.
‘C
x10-’
24 2 20
20 I
B
16
drug
005
01
molecules
bound
015 per
0
0.05
0.15
0.1
nucleotide
V
Fmc. 8. Interaction,between .1f(B 3427 (de.o-(umnj100 dimidium) aood P312 D.VA The DNA preparatiots ctoootaitoed 90% closed circular molecules. A. Effect- on the sedinnentatioti coefficiemot- at iomtic stret-igth 0.02 (A, #{149}) anti 0.10 (, 0) ; the S20 of closed ciro’molar moilecules is sioowt by circles, at-id that- of nicked circular moilecooles by triatogles. No special syml)(ol is used to it(licate the 820 of unresolved boutidtories; they mire shoowt a.s and 0. The abscissa repro’semts the average level of binding to the two l)NA cotuiponetots together, taken frcm spectrophotonwtrie measurements presented ill the fornu of Scatciuard ploot-s (B), where #{149} t’orresponds to ionic stretogth 0.02 and 0 corresporods to iomtic strength 0.10. PM2 in
aI)Pt’ans
DNA
high
bimudimug 820
of
shift
nicked
in the
bestic
(Fig.
SA)
o’specially at reduction
; (he
cinclesisutuiusinual;
pneseuuce
point;
sites
amioousualous,
ratios
and
flue
(deteruutined
is surprisingly
fhiespectral
of DNA by
high.
lacks
muuusuber
an
of
isos-
dialysis)
models
Expenimnents
with
mitolec-
manmuer,
‘multi
mophore
amid
posed for Accordingly, intercalates
nsaxinal
the
DNA
overlap
of
base
pairs,
the
chro-
as
pro(6, 7).
proflavine amid cthidmunu ti-ic possibility that RD 16101 “sidewise” (23) dir o)therwise
ntodifies
flue
seriously
enuf(’rtaiuied.
larly
that
suggest
iustcncala(iomu It
site woould
msmst be
be
particu-
interestinug if f-he high niumsuher of b)indavailal)le to) (his drug betokened freedom front tlue iieighbour-exclusiomu lwining
sites
too
3 , S-dimumisiiso
M&B
the bulky nature of the dicarbethoxyantino sul)S( ituents would indeed prevent inutercalatjomi in flue nionmusal ular
believed
nt’stnict
biutdimsg
by
tablisl-ied imutercaimutimog migo’nits (11). The rosost direct misso’ssnssemit- oil (lit’ groups
chronophore
binding
eciuilihniunt
o’iplc
As of
3427,
a result
cot-i (lie
sttidies
comes
front
cour
which
lao’ks
tlsenu
hintdiuog
is weaker
by
a factor
of
strength 0.02 omslv uncertainty
role
of
pht’ntan(iunidimue
the
ethidiunu
os-
with
altogether. titan
10 or
20
at
(hat ilomsic
amu! 0.10, respectively. Thie lies im (hue exact mtiagmii(ude
of (hue himudiutg constants for diussiclmumis, (he strict 3 , S-diamimio amialogue of M &B 3427.
the l)inding jut Table 2 are If
there
is
then
the
formitation
comistam-its applicable
every
indication
inicreased
free
attributable
3 , S-diamiuso groups at ionic strength
Thus,
if hydrogen
for et-luidiumsi given to oliniuidmunsu-monul thatthis is so oenergy
oil
complex
flue preseno’o’ oil in-i (he o’xtreme case to
is, 0.10,
oomily
bomuds
mire
1.7
kc’al
it ormo’cI
nnomlt’. mom
\VAKELtN
AN!)
wARmNG
V
28
4
24
V ‘C
xI0-4 S20
2
20
16 B
0
0.04 drug
0.08
molecules
bound
FnG. Fhe
1 )NA
prel):ortot
i(ltiS
:oo’
those
momoummiom gu’ooups,
So’tot chai’d
fluc’ loonveven,
imlfluo’!-ico’
0 ii
the
0011
uvitluimi
1-ito do’o’ule
t o’rmotitively,
too
Fig.
between cloosed
S. The
1)itodimog
l
Fuller
imt(em’ctulmo(ed to omiuplex. littvo’ mon imsspc)rfamit oil
(hit’
imitt’m’t’molmitioimi
(ho’ emuhumouoo’o’c!Into’
drug
site.
o’mo’ngy
Al-
o’liange
n-i-ia:’. be in((’npreto’d it-i oothio’r -i-i-toys. (to) Cho’nsiit’ttl o’vid!c’mice imio!io’tcto’s that the 3-annimuo oil o’fiuidiunsu
gn’ooup
neln’moo’to
oI’y
goofs
(24),
‘tihistifion
too
0)1
to
5-moitm’omgeii
mom-ic!climioidiumuu
is tmmiiquely
miftmio’k l) elet’tn’ophuilic whiit’h is o’vido’mce loin the
jotositivo’
o’huminge
rca-
delo-
irooni
(he
by
con-
(hue ;-mums1ini(o moifnogen
j toga 1-00
ing
I iomu muroiound f hit’ piuemum-imofhnic!iuiiumiu clinoophoore. This wooulol htovo’ (hit’ effect oil plmictlto’
1)OisitiVe
‘io:ongo’ol (‘to ‘i5t
tito’
pi-iosPluato’
‘o! o’It’o’frcostaf
moo’mono’u
ooxvgemns,
(lie
mio’gmotively
leac!imug
jo imito’m’tio’fiomo timid
oil flue systo’ns. tlomot nc’nsomvtul oil
emio’m’gy
shnoowno
o’loargc
Mt’B ci rcular
Synbols
mitt’ ro’lmofivo’lv weak, ko’mol, misoolo’ o’tuchu (ci
toniemofafioimo flit’
0#{149}05
0.15
0.1 V
plots.
mis suggo’sfo’cl
(7), thuo’’ oomuly 0.SS
tlit’ stal)ilifv ‘Tluc’v o’oould,
0
nucleotide
9. Interaction-i 90
.
Iitn(l \Vmuning eoonfnibufinig
per
t(ot1 I toi toed
B. Correspot-iolitmg olo’so’ril)ecl ito the 1O’getm(l
t’oo’liioietit
012
to
jut-
lowc’niuig
Hoowever, uve have (lie 3-aussino) group
1765 tose(1
was
(10(1 Pi12
ntolecmoles. for
ro’smolts
deterinineti
I11ect tot
toy the
sedititeti
strength
0.02
notot moofico’mubolv
moc!ded
muffect
sfmubilitv
niet
(ho’
nsightt
t mit ion
totool 0.10
spectropitotomnetric
clioo’s
stmit’kiuig
con the
tonic
TIo’ hanc’o’d
1
I).V.
A.
hod.
bimidimog.
he
ini(o’nacfionu
do-ic
(b)
too cmi-
hio’fweo’mu
the
churomtuophomro’ mom-id the I)NA i)ttse pmumns. Partimul doimimotioomu 0)1 flue lou-ic pmuins oil (lie amisino ni(ncigc’mis
would
imofti
imicrt’toso’
(hoe ( he
f-hue o’h-ironisoopluoorc domuat umider
contjugmoted ir-elec(romt
system y monoit-it-id
tumid imucno’mtse its
o’lectrooutst mi-ted
in ig (‘ttpmu’it V . Tue (mu) tip)hiO’5. (c)
auusimoo 0 gro 0-i-ills
nimg c!emisit
san-iso’ cnif ldlS!ii The pnc’seuio’o’
in-i o’thidiumsi
mood
oil
ltuo’ilitmut 0’s liydnoogo’uu boonid imtto’nac’t iott wmuto’r nsstmleo’ulo’s in-i scolutionu ; wiftoess muuuch
highier
somlul)ih(y
(hat-i
(lie
the
dinnuiditini with their
c!o’s-mumtuiuio
derivative. ( )mi c’oomssplo’x loonussmufioomi with 1)NA, (hue iiuc’nt’mose in-i o’nstnoopy cii flue s(olvo’mif sysnsighut
(cmii
o’ommoso’oitmo’miflv lie
gro’mofo’r
loon flue
dimumiuim-io o’oomoujiomtiroc!s; luo’muce (hut’ langem’ emoergy (0
(‘lititigo’.
be iniupoorfamof-
cmulatioouo do’failo’c!
lmt(roii)jc
ternuss
mo ti-ic’ o’uierge(io’s
mine
free
kuuomwmi
oil ti-it’ info’r-
netio’tioout loon ethuidiunu (19). \Ioone studio’s vitli (lie des-montiuooo cio’nivtitivt’
INTERCALATION
and
oof luo’r
3 , S-subst
imivt’stigmitiono h)indirig
too DNA,
between \Vhio’re are
wotild
these various unibracket
Sctitchuard
plots (Figs.
seeni
could probmobly
SB,
due
in nuoos(
nature
of the
ovo’r
by the
amid 9B).
o’aso’s
(of.
-ivitli
the
The
stacking
imuto’rcalmifo’c!
nusost
This
3B
amid OB)
cat-iso’s
but
too the
super-
is
is
1)3’
bojutes,
Scatchard
Alnc’ac!v
plots
drugs
which
comusistemit
were bomunud
that
each
to
with
the
DNA,
helix
by
12#{176}. This
ens
mumugles fromsu
atigle
was
mumgle
stntmctooral ioo’nttnrhafioons oother f lsmtnu nso’nt’ tmiwimidinug,
ool fl-ic sta’hi
ro o(mi(joomtal t l-i(
mind
demnanc!
o’omifornutmof it omit-il too bo’ t’onsid(.’ro’(L
with
oonue oobso’rvmotiomi o’xpltimnotioon
vanimutioomu of
in-i umiwimuchimig
ioimiic
s(no’nug(h
c!iffo’net-it-
whose
iron-i
et1toivmulemso’c’
varies
mu the
little
that
iouiic
if it represcuits
a- change
a-nd
stich
the
comuclusiomo
error
at
evidentee
mt
sense
son-ic
future
coirdingly. (ho’
work
woork
winding
will
(1,
nuolecules
in
1 1 , 25)
is revised,
If
.
the
25),
lx’
muffeo’fed,
in
explantaanuglo’s oil
Table
2.
suggested that l)OUflOi drug molecule
3) it was
In un-
cules
went’
miof
equihibniunis, calmted
nisight
ho’
o/12#{176}. Acc’o)rdimig
presenict’
ununtodified
it-i am
timid
imtfcrcalated
flint
(lie
simisplv too this
3 , S-diansintoo
pro)porfiouual liute
of a 6-pho’nyl
stmute
irmtc(ioonu oil
too
:im CsCl
hut’re
l)imidimog
(Fig.
10;
(29)]
amid
to
Table
witlu
(huamu (hose
0.06
to
largo’.
values o’omit-
ito (26-
ro’porf#{128}sil foir 2.83
iii
CsCI
0.05 (30)]. The high’r values lounuc! ito CsCl no doubtreflect the different nomittine o)f (Ito’ salt- at-id (hue much htight’n itonsic s(remugf Ii. The
slight
stremsgth
decreasc’
imisplied
limue in I’ig.
and,
31).
amid Kuhn plex
ins e with inucreasimg tour least-squares
data
oven
couccnifnat-iomus,
conutradict (30,
by
10 is miof statis(icmilly
simuce (lie
of salt
PM2
bimid-
oof (!rug
mis ooppooso’d
agreemcuit
louvc’n [v
tot m huit
[v
minid
favours
are in ,
i:oy other woonko’ns under coimidit-iomss oil stilt o’oiuico’nt rn-it
althomugh
1.5
rc’mosoomuiuig
sulistifuent
gncoups
at
iui(o’r-
c!ctenmisiried
luI2 rtumige
in-i (1w ratioi
extenmimullv
in satisfactoory
toot
than
plaimued
2) arc
we
tue shift is co’rtainuly valuc’s of v for (‘fhidium
DNA
defernsined parablo’
tiriglo’s /)e1 () 12#{176} mnigluf moist o’asily h)c o’xif soinuso’ frmuctioomi of (lie boumud usuole-
lower
The P\12
be adjusted acflue coniclusionis
onu relative amsgles for to that of c(hidiuns.
shuouvut
l)ooumtd
inutercalated,
seriously 10,
)ressimg proh)lem comscerns (hue lowt’r umuwimudiutg
do’nivatives
earlier
(
ac9-13),
supports
not-
angle
2 cant ev(’mut,
depo’uid tinily as counparcd
A mmmc (ions for
(1-5,
available
this
arty
presemit
several
is
that it is magnitude
date
lnu
siutce tho’v deniva(ivt’s
the
as
it-i Table
estimates frcntu
or
it is widely
estimate
Icor
stremugth
been
However,
oil o’thii-.
poimif
10). At liresemut loin (isis vaniaticin
as a provisio)nal
floe
for
mumud o!ibntomo amimuloguo’s cml o!insid3427 am-id 1765; Table 2). This bo-’-
0.01-0.5 (Fig. loss too account
forthcoming.
o‘ 1omock-
Isavo’
to noivel
a- funcfioom
to
oor imito’r-
stigmor-phosphtif
1)roposo’d in 1964 ott flue basis oif nsscdectilar model-buildimug studies (7), amid as yo’t n-ito direct experimental pnoooof oil ifs validity has
cepted
nt’spo’o’f
lomuso’ pmoin tilting,
is o1uite
diunis,
oonly
oil lomuso’ homlins with
apparo’mof-
haviour
sinoplo’st lout
tic’ mire laco’d
as
DNA
avtoilmihle
tiltinsimito’ly
clo’s-mimino junt (M&B
inovoourablo’.
omfle.
msuay
the
i.t’.,
(hit’ gc’oomuo’tny
ptossible
0 ii
mnay
uvhsio’lu
with ethuidiuutu, mussur-i-io’thuidium niolecule tiuu-
bounud
(he
wiusds
observed
this imifc’rprc’fa(ioin. ed lic’lix-umuwimudiusg 2 were all calculated
witit
Tic’ cstimat given-i in-i Table Eq. 2 bs’ com-npanison ing
molways tightly
uvitii
panmomsset
omi ox-
o’videtice,
mtxis,
fero’mto’e
the
lacilitmutimog
ro’mtdening
n-ito ussemumus (hut’
ho’hix
the shumope of bindimig iso)thd’rmits becmiuse oil the chanuges im superhto’lix free cnuo’ngy as the l(’vel oil drug loiuudimg rises (10). Curved
by
o’iommuplo’x less flue
muovensucu-it
(he
oil
prc)jioortitomi
ro’msumuimss fl-it’
w-ith
Drtg-indtnct’d D N-\. helix as
uvhuichu affects
ton by
inttc’npretafiomo
ett-isistent
of
inisfamuco’s
o’itioo’r
oof flue
l’igs.
1#{176}s12 DNA,
bituclimug,
u’o’-
inifo’rto’ro’mno’e
mmmuiim-ioogrcotmps
tenuomil
jf
whuen’o’mos or
lc’toc!s too to higher
o’rs
c’urva-
state, gromup
jniosuau’
iroomsi
rno’asune-
559
iuu(ero’almofo’cl
fl-ic’ 1)hit’m’l
chiroomsuophione
pantunsiet
liuuean
Inoontu mu variety
(hut’
msscovmul of
oontside
u-it’ro’ derived
wt’ne
SB,
it-i other
n(’stilt
h(’lictil
2 flu’s’
nanuge c(ovo’red
2B,
distiuguishu
!ONA
INTO
imig iii
hnidimies,
huo’lp to
which
if utoif mill of (he mc’uits
pltetian(
poossibilities. ed binidimug
in-i Tmtblt’
listo’d
tune
it t( cc!
n’HENANTHRIIInNEs
dihinoniso o compounud , espc’c’imuI ly oil ( ho’ t lienniuoodynmonssics oil tluo’ir
mis (ho’
such
OF
(he
sigmoitio’tomnt,
o)uily mulimtii(cc!
tho’y
conclusions
Smitit (26)
ioo!de
lif(o’cl mto!-igo-
dci nicit uiecc’ssturilv oil t’arlien
uvorko’rs
timid Boorst (27) amid Bottgo’r milso foot-in-ic! musomutewluat contu-
dc’pen-idcmice
of
e.
s(neuig(h
loor
too t’comtparo’
o our’
tom-i iomuoic
DNA.
Finally,
it
is
insfruc(ivt’
560
WAKELIN
ANt)
WARING
‘l?c
-2.00
-1.50
-1.00
log cation
FnG. -ionic
10. Equivalence
f or uomoiiimdi
tot
ptii
concentration
c-if .soipercoiliimg
)If/
-0.50
Of I1I2
Error
liars
i--i--ietit as
toti
ecpmt-itiotm
p
itidio’tite
the
utiro’soilved
k (logo
=
ttottd
ctutioti
with
mom-ic! thermopo’utio’
lmunge years
nttusihio’r mugco (32,
misemusurenstemit
well
as
each
fmr
eottmivalence deternuination.
ctoticetmtratiooti)
of
ftixicit-v
51)-i-it-i tof tloe
i)oulltltory,
do imot’l IisiomLs
t host’
t!o’rivo’c!
ironsi
0)1 I)lio’uimunifhiniohimuo’s 33). Iii (hoist’ (o’sts,
tests omi
where
The
straight
I)Ositio)ti gromups
(‘o as well mit poosifioons
wc’ usioulci ho’sifa(e luavo’ o’lticida(o’d
a
as (he
stih)sfittmo’mit
at
tioit-i
tof
art’
no’ventho’Ic’ss
phio’m-imum-itiuniclimio’s
bloo’kimig st’qtmo’ntc’e
miut’Ic’ic oil their
muss lo)r
goovo’rni
ti-it’
fhit’sc
o’ito,
too tunic!
dmo(mo onu
dntigs
imihsil)i(
(hue
l)mcfo’nia
by
stnuctunc’tictivity
(h-ic’ in-ift’rcala(iooni
mine ito goiod
cot-in studies bmisis 0)1 ac-
acid synithesis mis a conbinu!ing to) DNA ( 1 1 , 34,
So fan mis otin
reltutit
iii
bo’lio’ved
0)1 fnypmuuooosooniio’s
growfit
35).
as lro’e pnimsiary moniuinuo 3 mimic! S wmus niomtt’c!. While
too t’ltoinsi thatfii(’ nsoolo’ctnlan
agrec’nuien-it
muctivifv
oil
no’actioimu with
tiseso’
plies
tof
dtota
on
as
fuioctioii
(0
of
(hitose drugs
go,
which Iii
o’ivo.
\%‘e tittotok i\Ir. N. F. Tottv foor skilled teo’itnical tito’e , tttid tutttiv to ollc’agues for helpful t-idvicc antI nt I(Stfl. \Vo:, tore esioo’o’imollv grtoteful Ito I )rs. H. for
nicked
circles
cosedi-
is a least-squares
the drings, toogether their cheiiical
1. Waring, 2. Waritig, 3.
4.
Waritg,
5.
go’tioi’roois
sup-
M. J. (1970) \--I. J. (1970) M.
fit
to
the
with
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