The Unwinding of Circular Deoxyribonucleic Acid ...

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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

(lie

5-methyl

2. If- will

sumssmsumirv

givo’mi

WARING

of

Tho’

Sc’atcluard

1)holt

loin

himudimtg

oil

this

INTERC’ALATmoN t-.-

OF

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28

24

24

S20

S20

20

20

16

16 0

0.02 drug

Ft

The closed

0.04

0.06

molecules

a . 1 . EfJ’ect.

bound

ct/i iii

of

solvetit

was

o’iro’oolar

inoolecotlo’s,

0.08

per

o, no

11i’Pl’S-N

0.1

0 drug

I ) to 10(1 (lint i(liu on (B) lomoffer, iooriic strength

(.

tiOH tilloowioig

on

ioloi’ot ifictot

otntontbiguoous

too l\l2

( Fig.

T)NA

2B)

almos(

amid idenfic’mul

flue sansue

then,

muo sigt-iificamtt

ence

Introductioonu On

the

mueutral

ring

inoolo’culo’

the ducec!. spectro

‘Flit’

point

&B

shift

bimuclimug

tot

515

was mint.

lotinc!

ol o’t-huidmumss,

,

with

closed

t001(l nicked

so’dinieou

comi-

l-

bo’cmouso’

the

a clear

oil

flit’

flit’ o’sfmiblishnuent (11,

15,

Substituents

at

8

J)OSitiOl!8

Pmunticultur looguo’s poissessimug

a(to’mi(ion tvmis ntimc!ificmtfioomus

fiomus,

of

112).

l’cause

ntight(

the

this

o’fhsicliuni

chargt’d bi(lf It stmuhiliso’

oobso’nva(i

om

o’monlio’n’ c’oontc’lusitmmis abo mint oof o’Ieo’t n’oms(mu(io’ loro’o’s in oof flit’ info’rcalmufo’c!

of

of

neglected, mu am-i approoxi102 iru, sitg

ctimpbo’x

20).

flue

70%

(of

01000ffeso)lved

(hue o’ciuivmibo’mice (least miccurmufc’)

nut obo’culo’,

(7)

3B), than

booundaries

go’s(irig to- 5-fold boowo’n’ tuffimuity tlsmomi thumuf oil o’fhtichiuns. Hmovinug no’gmircl too flit’ dipomimir iooriic

mutid \Vmining

nesultimug

ito

cooonpoonetots

Os frootn

c!nug was btounicl at In fact, if (lie two

mimi istoshestic (Fig. fight

t’o’soolved

poimits a-f- low values of r’ tine bemisf-soiuares fitted bin-ic vio’bds mato’ bimudinug o’oonus(mtmt oil S X

2) 1)u( re-

frooon

t)0#{176}

comtipoooients

tio’ twom I )NA

cooetlicieti

moccoonds well with fl-it’ cit omuuimuautf nob’

(he coomumiwimidimig

too

t-atioii

chanmto’(o’n

with

c’urvo’! bo’ss tinily

of

ioon

nucleotide

D.V.-1 ctintaitted

I of II!2

l)repttrati(ot-i

o’ grouping

msiemusun’ed od

eoefflcit-io

0.08

per

I )N:

oif tiriog bindioig 0 tonI L , results

midc!ed po)inf.

0.06 bound

The

munuuphofenic

3A ; ‘1tii)bt

Altlioougli

sinsce

imu-

pres-

is sui)stmomu(iably

wmus

Scato’limincl plot is c!o’cioledby the himoding is ussmoriilc’sflv (hat

3492)

(Fig.

oif bumidimig

opltooto ousiet rio’ isuefhit

spectral

at-i

tine pnmoo’ticalI descnibo’t! : the

is mutot toffecto’o! stno’mugtlo

oil flue

in-i e(Iuic!iuuis.

yic’lds

(\1

Clearly,

boy ti-ic

mu p-cmorhoxvlat ring

sequemico’s which vo’rse oil (htt)se jtnst anigle

t’thuidium 2).

s(ahilistofioin-i

of

6-phuenuvl

oil

(Tablo’

is affoonc!ed

(i-heniyl

limic’ar

paramsieters

tliooso’

coniditions

(‘omisplc’x

oil the

smotislmuo’toorilv bimocling

with

under

(ercalmo(ed

is

yio’lcls

0.04

molecules

ta-lion

Se(liuiCn

0.02.

resolved bounottories. T[’lie tihso’isstt sloows the average level togetloo’r, tlo’t(’roni tieol toy t 1-it’ s)o’tt-rtiphtOt ooouiet nc met hood . closet! atid tiio’ko’d ci roles, respect ivt’ly ; #{149} , weightaverage boundtories. drug

0.02

nucleotide

(hat

(lie

and

3

suggestiomi

of

fhic’ 3 , 5-cliamitim-io loomis

(R1

pm-id to mit these

liyc!roogent

I”ullo’r groups

honuds

omxygt’mss of (hut’ 1)homSl)huafo’ groups P#{176} olvmsuc’beofiolo’ s( nt-muds , helping flue

conuplo’x.

Blooo’kinig

(lie

and amuapoosi-

too oil to)

5-mumisimiti

INTERCALATION

OF

28

PHENA

NTHRIDINES

INTO

t)NA

551

NH2

‘CH3

10 24 ‘ml ‘C

‘ci0-

S2.

20

5

16 B 0

0.02

0.04

drug molecules

The

circular

legend

to Fig.

together,

nnolecioles. 1. The

taker-i

group

this

O)f

shows

the (lucre

oil

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

precise

analytical

Blot.,

54, 247-279.

coonstitution.

J.

,J. ifoi.

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