Dimensional probes of binding and activity

WMO-W20/86 SS.OO+ .OO Fwpmoa PIen Ltd.

Tetrah&tmVol. 42.No. 7. pi. 1917to 1%1,1986 Printedin Gnat Britain.

TETRAHEDRON DItENSIOfML

NELSON

Roger

J.

Adams

REPORT NUMBER 198

PROBES

LEON&RD’

and

Laboratory,

University

of

OF BINDING

AND ACTIVITY

SHIVFIYOGI

School

of

P.

Chemical

Sciences,

Illinois

61801

Urbana,

Illinois,

HIREMCITH~

(Receiued in LISA 10 December 1985) CONTENTS

Introduction Linear

1918

............................

heterocyclic

Benzene

bases

ring

spacer.

........................

6,6,+Rlng

systems

..................

6,6,6-Ring

systems

.......................

Heterocyclic

ring

spacer

6,6,5-Ring

systems

6,5,6-Ring

system.

6,6,6-Ring

a’nd

Clngular

ring

ring

spacer

hcterocyclic

Benzene

ring

. _.

.

1919 1924 1927

.....................

6,6,6,6,6-Ring

Bentocyclobutadiene Naphthalene

1919 ._.

1927

....................... ....................... .......................

system.

A,b,b,S-

1919

......................

1928 1929 1930

systems

............... .................. ......................

1930

spacer

bases

1931 1933

.....................

spacer

1933

systems

1933

........................ ....................... Other ring systems ....................... Sent heterocyclic bases. ...................... Dihydrobenzene ring spacer .................... Ribosyl derivatives. ........................ 6,6,5-Ring systems ....................... 6,6,S-Ring

1938 1938 1939 1940

6,5,6-Ring

systems

.......................

1944

6,5,5-Ring

system.

.......................

1945

6pbr6,5-Rlng Mono-,

di-,

and

lin-Benz+QMP linnBen=-cfiW

system

derivatives

1949

.............

1952

5’-pyrophosphate.

adenosyl)-Fe-(5’-adonosyl)

ldenosyl

Px-(lin-benzo-St-

and triphosphodeoxyribosyl

finalogue

of

coensyme

Summary

and

S,m

Prospects.

tetraphosphate pentaphosphate

derivatives.

.........

......................

.......................

Department

and

....

1954 1954

1955

............ of

1954

1955

................. the

...........

)-p-(St-•denosyl)

Mono-v di-B

from

1949 1950

.......................... ..........................

Px-(~-8rn20-51-

leave

1946 1949

3’(2’1,5’-bisphosphate

Px,P_rDi-lIbenzoadenosine

Karnataka,

............

........................... ..........................

L*Benzo-NF. bLL?-Bcnzo+TP.

R*fcrcnc=g

1945

.......................

triphosphoribosyl

~w2enroadanosine

Wn

1937

Chemistry,

India 1917

Gulbarga

1957 University,

Gulbarga,

1918

N. J. LEONARD and S. P. HIRWUTEI

INTRODUCTIdN One the

of

the

discernment

substrates sites of

important of

or

on

or

enzyme

consequences

of

induction

or or

lnalogues

of

information

conformational

purines

major

of

six-membered

fused

replacement,

addition,

derivatizing

agents

f luore5cent,

or

ribofuranosyl

of

With

available

purinea,

of

specific

sets

of

nuclei: (31

ribosyl

uniti

natural

substrate

the

the

synthetic where

information the

extent

designed

Of

to

as an

the

naturally

been

subject

five-

and

else

to

by

the

addition

of

blocked, Third,

the

attached

with

other

sugar

or

departs Enzymic

has

heterocyclic

been

and of

are

and

enzyme

which

examine, these

where

the in

from and/or provided base

,

,

for

metal not

levels

have is

where

predictable

biological when

nucleoside,

binding

and

the

been

it or

enzyme

a particular nucleotide

in in

the be

compound level.

two

the of

the

to

the

binding.

this

laboratory not

procedure5 application

discussed, is

In

the

chronological

We shall

standard

represents will

on

relative

utilized.

follows

activity

or

reached

final

requisite

conformational

necessarily

being

the

the

activity

and

of

the

S,-hydroxyl

for

ion

use

also possible

dimensional

location at

but

concept

detail

the

proper

the

the

when of

NMR spectroscopy

spectroscopy shall

the

of

in

proteins.

with

characterization at

(31

of

size

probes

from

obtained

triphosphorylation

and

with

flexibility

be

the

toward

came

the

moiety

dimensional

terms,

to

from

assess

initiated

experimentation

results

cofactor;

the

was

nova

dimensional

adeninel

encouragement

levels

of

to

(e.g.,

de

differ

defined

possible

quantitative

N_ribosidation

methodology it

concept

and

that

cases,

best

synthesis

di-,

is

the

cofactors,

synthesizing

by

purine

the

valuable (11

we to

it

the

fluctuation

fluorescence

laboratories

techniques.

and

of

adenine,

In

determination or

of

dimensionally

(21

sections,

only

at

most

mono-,

(4)

and

other

of

the

replaced

that

When

five

reached:

heterocycle;

in

in

the

be

for

sites.

enzymes

heterocyclic

order,

synthesis

and

or

originals. been

the

have of

sterically

ribonucleotides

orobes,

required

essentially

and

subsequent

the has

in

corresponding

Second,

to

approach,

or

conformational

can

of

especially

probes.

answers

are

analysis

and

binding

demonstratede

probes,+e

these

periphery

a

area

take

substitution

led

group

dimensional or

fluorescent

provision

stages

relative5

a different

these

enzyme-coenzyme

There

has

or

the

been

we

are

confer

specificially

If

nitrogen5.

that

in

6teric,

examples,

by

of

may

their

have

function

the

altered

Purina5

ribonucleosides,

the

as

been

deowyribofuranosyl

the

changes.

serve

a5 First,

relocation

intact

introduced

bases,

space

to

to

lnZymR.e

an

derivatives

has or

and

analysis

inhibition

and

spatial,

analogue for

modification.

rings

bases

binding

the

moieties.

We have analogues

an

of

interest

Compound5

electronic,

cofactor

photoaffinity

or

sugar

natural

the

their

acid

is

possible

itself

regard

active

in

The

type% which

this

the

compounds

enzyme.

considerable

nucleic

for

or and

types

been

coenzymes.*

regarding

substrate,

occurring

pseudo

and

requirement5

inhibitor,

In

of

of

reaction5

requisite

employed

testing an

analogue

effect. has

is

various

product

occurring

nuclcotider,

provide

a

there

naturally

nucleosides,

include

of

physiological

chemistry,

and of

their

detail,

that

synthesis

enzymatic

and

in

strategy

substratetel

substitution

formation

studying

enzymes

definition,

One the

i5

this

indirect

the

natural

of

between

enzymes.

the

the

acid

three

i.e.,

the

mimic

aspects

interaction

cofactors,

within

to

nucleic

the

characteristics

designed

direct

fundamental

and discuss but of

whenever

described,

new such

whether

Dimensiona pro&a of biudingand activity

i.INEIlR

Ring

Benzene

as

pyrimidine

Svstema.

to

henzcr\m ring trivial the

three

to form

of ring

linearly

many X-ray

of

would

found

viva

would

routes,*

of

the

one

the

(lin-benzohypoxanthinc) -

exhibits

interest

since

stages

in

Z-hydrogen% shift

the the

the

66% D&F early best

of p&

the

is

sites*

case

clr one

(2)

from

has

with

it

a

natural

e-g.9

an8fopue

the

the

Nlr

I@‘.

the

bsaic

miqht

and

be hoped

subStancf?

while

and natural

in

but

the

Step

derivative

penultimate

in vitro

in mouse sarcoma possible

This

compound comparison enzyme

the

analysis

were of

systems

determined

a stronger analogue at

hydrolyzed

to

adenosine

in

base and

slightly

is hydroXyzed

of

sop&rate P-r

and

sol~ftion~*~ and

adcnine, substrates pti.

in

about

inoaine.*’

11.7 it

in

was would

The completed

terms

fntrntfnrl at

4-t

Stacking

to be 5.6 than

reward

to

At

the

aqueous

natural

J&-brrbPnzohypoxanthine

fatter same

‘H NMR chemical

alkaline calf

and

virus.

the

nucleotides

2 is

synthesis

the Tht

to replace

facilitated

permitted

(5;) of

generated

formation found

Separation

the

step,

and has

was

it

converging 1.

otherwise

focus

m-benzoadenine

substrata

several

2,

of

any

by

in Scheme

hydrogenation Ln-benzo

activity

m-bcnroadeninr

with

achieved

affercd an immediate -iin-benzoadmine activity. With #d*nosina desminaae from

normal

the

this

imidaroC4~5-g3quinrno~in-2~7~~-one

catalytic

. 1-e.. that out

in

and natural

stager

provided

subsequently

various

was

of

&,&-benroadenine that

of

and

The formal

system.

L_n vitro+ for

the of

known accurately

type

analopue

nitration

by deuterium.

values

recognized

synthesis

inhibits

solution,”

be carried

anrymatic

antiviral

synthesis

assignments

this

initial

disposition

aneloquc

13 Ls abbreviated

c(i-merraptopurineIS5

selectively

interactions Since

it

the

we gave

Stretches

binding

pathway (2)


synthesis

oftrr

drug

The

shown.

ring

acCivity for

metabolic

El-mercaptoimidszoC4,5-q3quinalolinazoLine, intermediate

normal

similar

after

This

antileukemia

atoms>r benzene

by a

to which linear

ring

the

s;sparrtcd

different.

was necessary

was straightforward.

as

The stretched-out

m-bcnzoadenine

a convenient

and

isomers

that

the

interesting be

is

the

the

in which

model.~

(2).

adcnine

if a compound of

would

be very

The Synthesis

of

unobstructed

to poSsess

in

the

are

system

to

answer

ta

compounds

purine

numbering

carbon

width

determinations.

be expected

substance

purine

refers

of

additional

Xn general,

transport

the

center

the

At

retaining

were

the

four by 2.4

adenine.

nucleus

the

The prefix

into

tactually

of

of

designed

with

“stretched-out”

or

in compound 2 , and

structure

advantage

af

ring

an iaxtcnded

a spacer

adenine

it

imidrzole

were

be associrted

was 8-aminoimidazoC4,5-p3quina2olinr

rings

benzene

that

the

experiments

might

name m-benzoadenine.

insertion

N7,

and

target

Our first

properties

what

ring

synthetic

BtasEs

METERacYCtxC

Spacer

h,&,S-Rin~ question

1919

crf

observed

rnuto~a~ 83% thm rate fhe

contrast

in

N. J. L~ONARU and S. P. HIREMAW

1920

the

behavior

not

converted

of

a complex and has

enzyme

broad

xanthine

that

ldenosina

laterally

of

the

synthetic

precursorsr variety.

division

and 4b should moiety

help

, catalyzrs

oxidaec

oxidation acid

can

adenine

from

buttermilk.

to uric

of

acid

a conversion

at

im

m-benrohypo-

findings

rate

indicrte

lccommedate

(2)

activity

demonstrated

prompted

to prepare

cytokinins

are

differentiation.~e cytokinins

the

2.4

that A-

and ~benzohypoxanthinr,

define

of

the

cytokinln

of

there

for the

the

were

of

which

tricyclic

about known

nucleus

larger

benroadeniner

parameters

for

the

its

the

bring

no previously

hetrracyclic

spatial

and

analogurs

hormones

a central

activity

limits

by J&-benroadeninc

plant

Since

containing

possibility

41

central

8-~3-Methyl-2-butenylamino~im~dazoL4,S~lquinazoline of

(4bb) were

prepared

(4aP

by heating

S-methylmercaptoimidazoE4,S-@quinexoline

3-methyl-2-butenylamine The substituted

oxidase

since

hypoxanthinr

These

Iln_benzoadenine

we were

in cytokinins. solution

the

to wbenzouric

and S-benzylaminoimidazoE4,3?L3quinazoline ethanolic

of

hypowanthine.se

The natural

the

noteworthy

Xanthine

air-oxidetion

xanthine

biological

synthetic

bicyclic,

and

enzyme.

on them efficiently.

and cell

or

for

eubstrates act

and

the

lsprcirlly

is

this

then

that

deaminamr

&&I-benso natural

adeninr

by

specificity

than

l xttndrd

Because

than

of

catalyzes

substrate

faster

respectively,

cell

that

to J&-bentoxanthine

apparently both

e with

to hypoxanthine

and benzylamine,

linear-bcnzoadenines

respectively,

do possess

(31 at

cytokinfn

200

an

with

lC (Scheme

activity.

II).‘*

They

are

s&e!meII

RNH,

_

h

4

3

less

active

they

permit

Moreover,

than the they

300 nin.~e.~* the

area

fluorescent

The results

the

corresponding lower

synthesized and

activity

for

or

two

proceeding

i-de

in

the

thr

cytokinin

of

the

central

nucleus

is

enalo~ues

1.7

am do their

The

lrrgrr

in

models. above

related

to

rectangle

area

1argQr

than

central

the

nucleus

no activity.%* 6-aminoimldazoC4,S-q3quinarolinazolin-S~7~l-one

routes, via

ldenine may be

nucleus.

an even

houever,

when excited

8ctivity

times

with

and bzlYld.1

380 nm range

that

section

adenine.

models,

to be obtained

, emitting

&&-bentoadenine

area

by

ldenine

suggest

by a cross

~Benzoguanine,

ring

rrspectivs

came maximum yieldu are

defined

enclosing

have

their

H

each elaboration

one

starting of

the

with terminrl

a

substituted rings

in

(Ill,

hes

central either

born benzene

sequence.-

probes of bii

DilmMioilal

The

sequences

follow

from

standard

mater-la1

ethyl

4-nitroanthranilatc

procedures,

appears

to

be

and

more

the

through

ths

at

at

332

use

of

maximum

or

sequencr

acid

standard

(456

1921

4-chloroanthranilate the

of

provided

reactions.xa

navelength

ethyl

utilizing

CI variant

reliable.

k(carboethoxy)-4-nitroanthranilic

emits

and activity

the

former

procedure

in

starting

lirr_benzoxanthine

lin_Bentoguanine

nml

starting

neutral

is

aqueous

with

(61,

also

fluorescent

buffer

upon

and

excitation

nm.sm

The

biological

interest

to

purine

activity

synthesize

derivatives.

Christ”

by

of

the

theophylline

laterally

(71

m-Senzotheophylline

the

method

shown

in

quinazoline-2,4(ly,3Y)-dione

(71

Scheme

III

(ill.

Either

NHNkC

yielded

and

caffeine

lnalogues

extended

,

was

made

starting the

(81

(9.

by

or

made of

it

of

these

Schneller

with

amino

101

and

7-chlorohydrazino

schemelll

intermediate

12

hydrogenation methylation latter, from

of and

12,

(Y

in

=

Nl&

formic

9 along

indirectly

or acid.

with of

Y = C&NH.

m-benrotheophylline

m-Senzocaffeine the

(101

3,5,7-trimethyl was

variation

in

was

obtained

isomer.*7

l&l-benzocaffeine,

CI slight

on

the

The

established

procedure

by

catalytic on

structure

of

direct

produced

the

synthesis

wbenzo-l-

methylxanthine.~e ~Benzotheophylline this

laboratory*-

Thcophylline imally t issues, but

is

activ@‘--

diseases

are

the

was

active as

including

increase

was and

and

associated

of nlth

synthesized as

by

a potential

the

same

of

arteries. both

CAMP

abnormal

cyclic

(IBMX)

nucleotide

They and

route

(Schema

phosphodiestcrase

O-irobutyl-l-methylwanthine

inhibitors

coronary levels

also

tested

produce

cQMP

metabolism

in

of

(131

is

phosphodierterases relaxation

these

of

tissues.

cyclic

III)

in

inhibitor.

smooth Since

nuclootidcs,

marin

many

muscle several the

N. J. L.&IN&D and S. P. ~TH

1922

CHICH(CHz),

14

13 possibility

exists

of

phosphodit8terase 5ufficient

to synthesize

of

ethyl

the

of

course

of

the

the

same order

that

AS

isobutyl

compound related

iodidt

to

was

found

with

methyl

Y = Cl

to

the

encourage

to 7-chloro-

treatment

The resulting

converted

was

to

by elaboration

imocyanate by

ic

to have

procmrded

followed

in dimethylformamidr.

12 with

epecif

thtophylliner-

( 141. *q The synthesis

‘*lin-benzo-IBHX”

2-Amino-4-chloro-S-nitroben2oAte

and

with

of

3-methyl-&-nitroquinazoline-2~4~lt+,3Hl-dioner hydride

disAAren

l_&8-Benxothcophylline

inhibitors.

Activity,

us

altering

with

desired

product,

in formic

acid.-

7-ntthyl-5-~2-methylpropy1fimidazoC4~S-p3quinaroline-6~8IS~,7~~-dionc benzo-18MXl terms

of

tions

(red

which

(141,

potency

of

blood

more active

even

at

cat

heart, but

behavior,

Ibay,

Martinson,

compounds

rAlAted

of

nucleotide

to

three

did

not

1O-Vl

inhibitors

of

separate

anblogurs

of

the

from

pig

the

active

sites

moAt of

these

phosphodiesterasre

logue

of

numbering

was

Q-coupling

exhibited

In a search parasitic scope

worms,

of

described closure ll&l&

to

but

medicinal

product

compari5on

the

properties

the

protons.

expanded

to hypoxanthine, (allopurinolf that

have and

for

the

that the

har

been

led

or

without

previou5ly imidazole

against

ring

Wvmem

reported

but

known Anthelminticr. thr

8-8za-7-deaza

<181.---has

related

of

carbethoxyamino

compounds

in homsterr of

with to

was used there

body

IbenzohypoxAnthine

was similrr

activity

the

linear, of

the

2-position,

to complete

gout

The lrck

the

in ridding

of

w-wf

urea.-

benzrnoid

of

to

18HX.

ir2-dimethylbenzimidazolc-

the

on the

than

genaral

by

synthesis

of ana-

I activity 2 for

with

activity the

peak

indicrtcd

was urrd

with

inhibitors the m-brnzo

(see

effective

group

cevlrnicum

controlling

of

methyl

and Bhaduriwa

in-structure

for

forms

stretched-out

effective

closure

S-methylisothiourea

and AncvloatomA

inhibitory

of

agents

pyraZOloC3~4-Q3pyrimidin-4(S~l-one oxidase

II

includinp

was

The methodoloQy

chloroformate

related

of

product

include

7-nitrogen.

obvious

Closely

NHR signals

The lnthelmintic

in mice

without

by

the

the

a carbothoxyamino

and ethyl

artery*

however,

inhibitor

and ring

of

lnthelmintic

Xl,

of

SeriSS

I and prAk

the

les5

substitution

nitration

nature

work *7

(Scheme

substitution.

from

Kanral,

with

on thr

more potrnt

a

Kumarr

earlier

5ubstituted R-butyl

for

were

In one ca5e,

IEMX.

by reduction

angular,

a

l-imoamyl-3-isobutylxanthine.

formm tolerated

2,3-dimethyl

resulted

followed

than

enzyme

and

lnalogues

than with

syrteml

S-carboxylatr

both

*bento

7-benzyl-l8HX

m-Bentoxanthine

rather

of

the

CoronAry

in pre55,w4r

of

, 3-irobutyl-8-t-butyl-1-methylxanthine,

3-iaobutyl-lr8-dimethylxrnthinr

nanthines,

peak

kinasr

7-brnzyl-3-irobutyl-1-methylxAnthine,

3-isobutyl-8-methoxymcthyl-l-mPthyl-l-methylxanthine~ While

l xaminAtion

of

protein

In an article

14.w*

was

In terms

CAMP-stimulated

the

prepara-

mbenzo-IElIlX

IBMX (131.

report

phorphodiesteramr

benro-separated

of

the

than

affect

In

phomphodiesterAA8

brain),

Active

and Wells

14 As

different

and bovine

of

(Un_

hydrogenAted

less

it

a concentration

Schnellcr,

cyclic

with

theophylline

desirable

activity

linear

inhibition

cells,

than

Additional

WAS catalytically

sodium

1-isobutyl

its

u5e as

metabolic

lnalopue. xanthim

a mAjor

di5ordAr5.n

Dimcnaional probw of binding and activity

The

obvious

target

molecules

lin_benroallopurlnol their

substrate

or

independently

activity

from

through

esterificatlonr

two

lnalogues

liO_benxo

(171

xanthlne

laboratories

stages

and

The

schenm

of

synthesis

has

of

been

The

IUl.ee-ea

5-methyl-6-nltrolndarolc

protection, or

through

acid

(191,

hydrolysis,

6-aminoindazole-S-carboxyllc

determination

(161,

with

of

are

for

oxidase.

(Scheme

started

appropriate

reduction, to

with

synthesisee.eemee

proceeded

sequence,

of

pyrazoloC4,3-g3qulnazolln-5~6~l-one

Cuny-Llchtenthaler and

synthesis

lin_bentolsoallopurinol

inhibitor

m-benzoallopurinol, reported

for and

(16)

1923

(101

oxidation, variations which

this

of

was

cyclized

then

IV

18

16

19 t

to

u-benzopurinol

(16)

laboratoryee

started

nitroanillne

(201

nitrous

which also

then

the

this

ring

The one

of

and

thermal

was

converted

Lamination 16.e’

with

cuprous

the

compound

was

dione

was

could

be

with of

effected

(Scheme

tion

of

of the

formycin

by

starting

E.

(161 derlvatlves.e-

yield

0.19,

lmlsslon

maximum

17 The

liltbenzoadenlne 16 was

was

to

21 ,

followed to

heating

in

7-ethoxy, reported

the 300

synthesis

in

from

our

4-bromo-2-methyl-S-

by

ring

closure

with

6-aminoindazole-S-carboxamide formic

and

to

obtained

acid.

Intermediate

7(W_l-keto

yield

the

directly

from

7-amino the

19

derivatives

of

derivative

16.

of

intermediate

19 by

pyratoloC3r4-q3quinazolin-S~7~l-

with

the

following is

a

also

of

was

isomer sequence as

sequence

to

ethanol

fluorescence

nm upon

similar

17 es

found In

position

recognizable

synthetic

derivative

Lienzoallopurinol

for

bromine

m-benrolsoallopurinol,

Compound

L(SHl-keto

16 by

The

the

guanidine.-

and

IV).-

C-•glycone

to

formamide. of

hydrolysis

7-methoxy,

6-methyl-5-nitroindazole, 16

partial

cyclited

latter

with

cyanide

and

to

closure

synthesis

(171,

heating

displacement

reduction,

acid,

(221, was

by

with

excitation

was

of to

la,

namely,

that

described

the

benzologue

applied

to

of

the

for

the

construc-

well. be

fluorescent, the

solution,

lifetime at

was 315

as

4 ns r

nm.-

were

the

fluorescence and

the

quantum

fluorescence

N. J.

1924 With

regard

oxidase, uric

with

acid

to

of

lo-&

and

in

oxidase from In

was

range

esrmtially Lichtenthaler’s

their

(or

behavior

with

an

unincubated

rate

of

uric

formation are

true

properly have

sample.-

that

activity of

compound

with

interest

and

piperonylidene

7

(SH)

requirement

acid.-

active

acid

is

folate

sites

of

26

were (25f,

has of

compare inhibitors

rate

of

its

6(7H_)-keto can

reduced

by

the

a

be

derivative called

has

Jahn-

double

activity.

that

more

making

substitution

of

acid

and

lllopurinol,

the

factor

uric

Lichtenthalrrr

the

this Finallyr

at

the

originated

a

3tCHw)r

4

with

been

lumazinr

25

in

undertaken

ring

in

lxtendinQ both

latter

may of

be

of

humans

the is

of

a simpler

viewed

riboflavin.

as 80th

24

(24)

an 24

“inside-out” and

so

oriented.

pteridine

~benrolumazinr

a

more

folfc

synthesis

properly

and

the

defininp

metabolism

direCtinQ

terminal

Thus.

product

in

has purpose

the

(2%).

the

23

lies

the

the in

achieved

The

irradiation

for

involved

synthesiztd.*m

vitamin

laboratory

compounds

enzymes

on

that

to as

known,

biological and

xanthine conclusion

were

experiment

a hematopoietic

problem

success

our

described

Schneller’s

poultry.

synthetic

namely,

1.6

acid.*’ Folic

substitution

immediate

previously

in

substrate,

its

-

inhibitor.-*-

owidare

as

does

AS

been

unsymmetrical

More

lumichrome

in

true

(16)

and

nucleus has

a

m-benzorllopurinol

of

A recalcitrant the

compound

study

positions

benzo-separated

the

that

ayrtem.

further

as

Cuny,

oxidase

K,

the

derivatives)

inhibitor.oo of

two

for

while

reduce

A

found

the

derivative.-*m-

a substrate

xanthine

allopurinol

m-brnzoallopurinol

.

of

not

derivative

thiobarbituric

completely

-

as

xanthine

for

m

synthesis

7-amino

apainrt

the

(piperonyl),

alternative the

of

allopurinol,

while

as

OXyQenAted

m-benzoallopurinolr

a competitive

7(8Y)-keto

behaved

hypoxanthine

and for

identical,

LLn_benzoallopurinol

did

Thus,

nearly

hypoxanthine,it

or

~benzoallopurinol

sipnificantly.

derivative

that

their

The from

bs

of

hypoxanthine.

the

samples

of

constants

served

(151

behavior

formation

the

to

from was

to for

rate

of

u-benroallopurinol

found

oxidized

lllopurinol

inhibitors,

inhibitor

also

reported

experiment,

the

acid

reported

dietary

readily

laboratory

with

whereas

were

values

xanthine the

of consumption

Michaelis

~-bonzoallopurinol

controlled

praincubatedm*

-

,

of

of

with

compared

we

rate

the

apparent

indistinguishable

a carefully

500

that

of

The

(16)

acceptor,

disappearance

.-*

measured

the

was

conclusion

of

P. HIRBMATXI

S.

electron

indication

rate

and

~bcnroallopurinol

final

same order

lirr_Senroallopurinol Our

of

the an

the

the

similarly

M,

activity

as fas

with

be

substrates, x

the

formation

hypoxanthinet them

to oxyQen

bONAFlD

26

ring and

1925

28

29,R=H 30,R=CH,

27

could

by

be obtained

chloride

by glyoxal

29 and 30 with

annelation

urea

with

(Scheme

of

hydrogen

27a was

had served,

quanine.X5 neutral

solution

an aqueous

solution

comparison

of

the

The preferred analogue

of

the

which

of

hydrogenation

of

with

the

independent

in our as

lumichrome

synthetic

followed

exhibiting

(23)

(23)

of

all

route

is

The

l ame

route

to

131),

intermediate fluorescrnce)~*

fluorescence,

(23-26)

to m-benrofervenulin

fcrvenulin

t27a).

2,6,7-tri-

61 more quantitative,

compounds

the

a and

direct

would

be deeirable.

the

stretched-out

(32),

involved

and

*bento-

yellow-qreen

blue.

four

system from

a bluish-green

has

of

a

chAoroformamidine

tricyclic

preferred

hydro-

30 yielded

synthesis

and 2,Pbutanedione.

lumazine

fluorescencr

antibiotic

by reaction

produced

by

described of

(23)

re8pectively. on reaction

peroxide

isolation,

Compound 26 is

2,4rS-triaminobrnzoate

Catalytic

(271

without

aqueous

ethyl

confirmed

lminoquinazoline-4(%-H_)-one (28)

V).

derivative

baeic

The structure

of

and 2,3-butanedione,

tetrahydroquinoxaline oxidation

27~

conversion

of

the

32

intermediate

12 (Y e NHNHe)

orthoformate There the

has

orange

followed been

methyl

(33,

related

the

by catalytic

no report

of

the

starting imocyanate to

ll>,

hydrogenation or

give

followrd

the

reaction

derivative with

absence

The closely with

to

ethoxymethylene

preeence

~benzofervenulin.

made cimilarly, with

to

related of

methyl

with

palladium

of

triethyl

on charcoal.a7

antibiotic

ectivity

&&benzoreumycin

for

(3%

wae

2-amino-4-chlorobenzoato

7-chloro-3-methylquinazoline-2,4(lJ$3~)-dione by

nitration

and

hydrazine

displacement

to

give

1926

N. J. bONARD

and S. P. I-CRl&m??i

35

36

7-hydrazino-3-methyl-6-nitroquinazolfn-2~3~~-dione in Scheme VI.‘” through

the

(331. far

en has

Nalidixic

of

(371, that

which

is

casr

of

a% in

the

be called

as

naltdixfc

is

of

made by Yardis,

Cai,

Lin_benzo

analoguer

bacterial

activity

level

nalidixic

(381

its

and

6381 posseseed at

V = N. but groups

to prevent quinolinone methodology.

the and

in

the

Details one mafor with

exhibiting

rongeners

to

of

the

!E

L!

Y

tie

H

N

CH

He

H

N

M

H

N

M

N

cti

N

N

M

-C&i-

He

N

cti

of

to or work, of

involved

side

various

Tanaka

or

than

of

the

type position

substituted products.

ring6

and anti-

the

therapeutic

and Nagate*“’ Pa = Et,

compounds

annelation quinoline

with

blocking

synthetic

properties

greater

38,

series8

trimethylene,

angular

their

fluorescence

syntheses

strategy

left-hand

this

been

have

Eltr

that

were

ac-

dispropor-

biochemical

A’

).-

of

DNA gyrasr.

H

equrl

m-benza

a methyl ,

formation the

Prior

acid.

and shoe

-@k-

ueeful

concentrations

progenitor

H

who found

and Sauter,-

compound

nitrogen

thus

bactericidal

bacterial

-fcK,,*-

available,

es

common gram-

the

enhanced

mutants

inhibitors

acid

the

reristancw,

chromosomal

act

v**

have

-tC&

X = CH,

lS1,

-in-brnrofrrvenulin

most of

Et

a related

to

wes effected

ubenzotoxoflavin,

infections

, some of which

against

compounds of

for

tract

to plarmid-mediated

activity

These derivatives

for

related

(351

could

bactericidal

urinary

lnalogues

insusceptible

superior

resistance.4e ]iDenzo

36,

which cause

synthetic

appear

tionately

~benzoreumycfn

derivative

compound

lcid

of

(34,

to

synthesic.se

bacteria

thoueands

convuroion

ethoxymethylene

isomer

resieted

negative tivity,

The final

R’

had -

38 8re

H,

reported Re -

not

between

trimethylene The right-hand

closed

by st8ndard

H,

yet the

two

bridge %lde

Heterocyc

1 ic

Ring

6,6,5-Rina (391

were

made

relationship

spacer

however,

that

of

i.e.,

the

if

the

by

one

activities

distinct

an

presented

of

according

to

they

the

Taylor

and

pteridines.

cofactor.

time

that of

&&-benzo

in

still

over

to (411

be

Taylor

of

are

simpler

be

the

precursor

methodology

(441

in

is noteworthy

on

contraction,

reminiscent

Inbaskaren

acetic

acid

two counts.

The

of

as

in the

6x

to

a

trace

of

hydroxide.

morpholine The

second

stirrinq

point

is that

41

of

was

course

as any

on

is

and

The

that

was

on

1,3.6,0-

the

heating

obtained

compound

42

in

synthetic by

a facile

ethanol

in 5x aqueous was

from

precursor

rearrangement,

temperature

diimino

obtained

of work

1,3-dimethyl-

VII).

acid-type

at room the

probes

(431

first

(451

or on

systems

strictly,

activity

synthesized

(Scheme

(44)

a benzyiic

1,3-dimethyl-6-imino-5-phenoxyiminourscil

examples

properties

2,6_diaminopurine

ago

4,5-diimino-1,3-dimethyIimidazolidin-a(l)-one ring

as

dimensional

(421

glacial

of

ring

tetramethylimidazoC4~~-b_lpyrimidoCS.4-glpyrarin-2,5.9~1~,6~,S~-trione common

that

offer

regarded

as

5~7-diaminoimidazoE4~S~_lpyrimidoE5,4-e~-one

a

being

binding

availability

hydrogen-bonding

to

years

and

extra

cannot

Most

provided

time,

made.

related

twenty-five

recently,

been

a

a comparison

synthetic

and

has

the

the

Introduction,

at

function

but

to

a raid,

dimensional

the

activity

cannot

their

be

rings

analogue.

They

the

of

into

relationship

electronic

information are

a

kind.

as

substrate

illustrate

bear

should

parameter

feature Any

of

containing

peripheral

interest,

be of

to

one

the

basis

It

Inclusion as

pyrido-extended

some

in

only

molecule.

course

the

purineo

analoquea

extraneous

intended

analogue

Shermanno

or

the

on

recently.4T

alter

same

provided

then,

More

of

alteration

Correlations,

to

extended

systems

scant

more

an

with

are

description

represent

the

and

as

A pyrazino-separated by

the

activity

Unfortunately,

kind.

at

substrate

ring

discusred Substituted

advisable

will

section

were

stretched-out

substrate

for

biological

use

introduces

A,

of

triheterocyclic

exhibiting

well.

the

reversed)

this

to

ring,

2.4

and

synthesized

it,is

interpretation

in

ago

pyridines.-

scekinq

central

(or

relative

since

is

available

preserved

pyrazoloC3’~4’:6,53pyridoC2~3-g3pyrimidines

years

been

sites,

about

possibilities

of

have

(401

dimensions in

separated

Substituted twenty

tetrasubstitutcd

binding

nitrogen

is

about

to

pyridine

probes

spacer

System.

highly

of containinQ

sodium reactive

1923

N. J. JXONAllD and S. P. HIRIIMA?M

42

43 with

the

representative

compound, that

the

comparison

consequences 42

and

in

43

are

insolubility, cyclic

Q-phenylenediamine

1.93-dim.thylprrabrnic between

other

might

nucleotide

laterally

of

as

be

failed

and

by

rout.

heterocyclic

yellow.

the

In of

47

43,

were

being

The

has

a

for final

4.

is

of

point

tricyclic

as

out

interesting

despite

testlnp

an

relative

inhibitor

of

a purin.

ring,

the

Hofmann

interest.-

compounds its

example

a pyrazlno

from

least,

dlcarbonyl

authors

The

at

46 compounds

corresponding

synthesis.

candidate

Insertion

compound

the

react. reactivity

Compound

a reasonable

to

to

carbonyl

phosphodiesterase.

extended

rearrangement

imine

areas

described

whereas

acid,

of

ring

Various

pterldin.

47

synthesized

4H-lmidatoE4,S-23pteridines

as

have

As we shall

diuretics.

been

found

in

nature

as

se.

their

below, Q-rlbitol

derivatives.

5.9.6 the

- Ri n a Seta

benzene

rinpe

l

upon

the

re

rin9, not

only

location

.

but

The when

stretched of

the

thlophene it

is out

sulfur,

used but l .9.

ring as tilted 48.

ie an

considered

to

internal

specer,

outwards

in

Triheterocyclic

l

be

biolsosteric

the

with

perlpherrl

direction compounds

dependent of

interest

pro&e of bindingand activity

-onal

48 here

that

contain

Hitiris-

for

Structures

49 a contra1

potential

of

terminal

which

be good

inter

al ia. two

condensation

of

of

of

of

Compounds

or

The final

trimethyl

to give

the

conversion

of

of

this

type

O-Q-ethyl or

51 isolated

tures

of

this

analysis

of

propyl to have of

the

strong

of

moiety

compounds

product

(52,

of

oxidizing by heating

be

the

or

first

a trialkyl

methyl

in

the

products

which

occur8

by

displacement Et).

with

a mixture

6-amino-lr3-dimethyl-S-formyluracil

of

X-ray

also

The struc-

of

type

in

33 are

autorecyclinq

of

the

in

on El_lo,-

10-r

reported the

has

6-amino-1,3-dimethyluracil

, product

the

crystallographic

by benzylamine

Compounds

no rubstituent

of

example

orthoformate.

established

and a remarkable

Compound S3,

recently

were

9 Re -

ability

to

with

8-•thoxy

R* m 3-Pr

alcohols.-‘**

most

iw stated

a pyrimidine

family

member

the

4-alkylamino-

54

separately,

of

a central

intermediates

CR*>,

53 from

activities

0

0

O_•lkylation

They

6-alkylamino-3-methyluraciln

fR m = C&Is

Se

also

and have

basmr.

made by

companion

ere

with

corresponding

51 to

been

with

orthoformate

duality

acid

containing

have

and

IOU toxicity.

and oxidase

systems (Slf

&-chloro-S-fcrmyl-+m*thyluracil

triethyl

nucleic

deaminasm

rings

made by Clark

and P certain

purine

ring

pyrimidine

activity

represent

the

Triheterocyclic

terninrl

in dim*thylformamid* !52.-

examples

those

among those

are

system

an analysis

pyrimidines.-

reaction

ring

nervous

like for

E&stem.

and

substituted

rings

candidates

b.&.&-Rina pyridine

thiophene

central

49 and SO are

pyrimidine

would

1929

oxidation

bren

made

and

a Vilsm*i*r

reaction.

in

tetra1in.w The analoque thermolysis along the

the

of

rings of

any biological

33,

but

with

a pyrazine

6-•zido-193dimethyluracil

pathway

terminal

irradiation of

of

were

tested

facing

in

for the

ultimate opposite

i-arido-1.3-dimethyluracil activity

of

spacer

(341,

in foreamide,-

these

conversion direction isomers

has

formed

and to 34.

was

in methanol.two

was

been

the

by

the

intermediates The

product

No direct reported.

isomer

having

of comparison

N. J. L.BONARLIand S. P. HIREWTH

1930 6.6~6*5-

and 696,6rbrb-Rinq

1,2,4,5-bcnzanotctraminc formic

acid-L14

alloxan

concentrated

imidazoC4,5-

with

Svstcms.

with

klloxazine

alloxazine

-0.252

at

HCl to

has

The product (molar

yield

the

a reduction

V relative

to

the

in absolute

ethanol-dimcthylformamidc

may also

bc

as

rrgardcd

quinoxalinc

unit,

fruorescent

product

alloxan

113

was

eimilar the

pentacyclic

reduced

azodicarboxylate

ring to

(52).-

series

compounds

first

were

with

5-doazaflavins. for

of

and at

LiClO.

low

unit

in

The

maxima were

nm.

Compound 55

scparatcd

are

heated

S!!f.~*

V in comparison

and 375

by a

discussed

below.

The

1,2,4,5-benrcnetetramine to

UV maxima at related

be 56, 285,

to

a Kind 390,

of

and 420

to 5-deazaflavins

by a route

prepared

could

oxidation alcohol of

shifts

be oxidized

for

g&.

-0.590

compared

secondary occurs

under

type

58,

through

the

by dfethyl

the

the

Ag/c1gCl of

-1.13

the

findings

the

linear

of

9 with

in

58

*C in for the

were the

absence

sunlight.

SO-180

in 24 hours of

the 23

V for

compounds

presence

oxidized

at

clcctrode

~a.

temperature

conditions

for

potentials

valuee

raised

QC) in

to

and 7-protons

redox

5-dcaraflavin

being

according

the

at

these

9 cyclopentanolr

S-

V fi.

with

alcohols (lo-15

the

Their

greater.

were

temperature

representative

compounds

been

The double-headed

oxidizing

Autorecycling

and

which

10 ppm or

transfer

monomeric

the

with

rystem

-0.082

presumed

was

of

1,3~9~11,12t14-hexaazapentaccne-2~4,8~10~1~,~_,~_~11~~-

effective

of

of

more closely

end have

97,

rings

ratio

ring

UV absorption 270

naphthalcne

system,

each

system

one-electron

a base

the

condensationr

The NMR chemical

dimethylformamide

SCE.*e

molar

molecules at

tctraones

the

to

double-pteridinc

Similar

of

in width

initial

(S-deazaisoalloxazines) partially

in which the

of

at

xanthinc

formed , when the

in

benzene-separated nm.-*

a

condensation in a_ HCl was

extended

potential

observed

of

2:l)

ratio

Yonedat

mols per

of

mol

Kuroda,

the of

I buka .*e

57 Eenzocyclobutediene We have

extended mediate

been

adeninc in

width

Ring

spacer

attempting analogue (3.9

6)

to synthesize 59,

m-bcb-adeniner

between

the

benzene

because

benrocyclobutadienothe

spacer

in m-benzoadcninc

is

(2)

inter-

(2.4

one

Koga,

6)

Dimcmional probes of biding

and

the

in

naphthalene

different

widths

permit

fine-tuning

of

interactions.*-7 namely,

-lin-naphthoadrnine

spacers

of

the

When

the

syntheses,

learned

first

a cobalt-catalyzed

and

isoquinolines an

upon

examination

have

the

the

electrocyclic

with

their

of

of

two

tions.*&

while

distinct

the

been

of

the

the

that

we

unusual

The

chloro-3-nitroacetophenonct

which

methonide be

to UR

a pair

of

We have

and

we

rationalized

also

by

study

constituted

of

these

elaborated

reac-

Two

system

2-acetamido-4in

fusion.

a di-

challenge. ring

with

that

consid-

remained

the

began by

and

found

l lectrocyclic

sequential

precursor-product

was

diverted

rearranqements.

between

could and

first

in

to

were

pyrimidoC6,5-~limidaroC4,5-q3cinnoline

accomplished.L7

a

pilot

cxtrusion.w

a m-bcb-purine

of

from

4-methoxy-1-azabiphenylene

products

mechanistic

synthesis

syntheses

have

the

the

sodium

addition

nitrogen

transformations

small-molecule

methanalic

methanol

Nevertheless,

version

(62)

of

for

of

39,

two

a diethynylpyrimidine

base

monoazabiphenylenes,

rearrangement

locus

or

Of

developed

However,

facile

acid

fe8ture

we

1,3-benzodiazocines

by

with

unusual the

of

followed

behavior

equally

&*.*O

pathways

intermediacy

I-methoxy-2-azabtphenylene,

eration

reaction.

three

hopefully

enzyme-coenzyme

extrusion

utilizinq

such

will

certain

warn unknown, thermal

The

below. rings

structural

second

either

mechanistic

reactions, the

the

of

primary

(M)),

underwent

treatment

of

postulated

examined

the

co-oligomerization

4-chloro-1,3-diaxabiphenylene

discussed terminal

restrictions

involving

precursor

A)

constsnt

that ring

the

pyridazine

(4.2 the

dimensional

we

1,3-diazabiphenylene

independent condensed

between

1931

and activity

sequence,

of

the

61 63

imidasolc,

pyridarine,

lacking

peripheral

and

palladium-catalyzed chloride

and

pyridazine (633) * an

This

200

campound,

the

extrusion

the

first

of

Ring

substituted linear

cobalt-catalyzed Diels-Alder

to

the

of

followed

tetracyclic

which

system

62

a

utilized

4,6-dimethoxypyrimidin-3-yl

zinc

by

and

closure

of

the

imidatole

of

with

the

to

the

extrusion *C

and

provide a

new

ring

system

a cinnoline of

and

spacer.

HCN rather

10-e

torr)

of

of

the

imagination.

Pyrolysis than

Nm.

compound

62

of

63

contrast,

By

resulted

imidazoC4~5+_1-1,Sdiazabiphenylene class

of

linearly

extended

is

at

in (Ll),

purine

analoques.

Spacer of

m-naphthoadenine

required

of

the

the

(4!31,e7

construction

of

the

and

We doveloped

four

rings.

contiguous

intermediates

of

lcetylenic

thermally.-

lin_naphthohypo-

requisite

intermediates.

cyclotrimerization q-xylylene

(9-amino-3H_benzimidatoC5~6-q1-

m-naphthoxanthine

naphthalene array

stretch

adeninc

of

(64),&e (66)e7

a

,

provide

synthesis,

10-aminopyrimidoC6~S-~limidazoC4t3-qlcinnoline

f2tO-260

nitrogen

synthesis

quinazoline)

the

lead

pyrolysis

rcpreuentative

Naphthalane

xanthine

of

to

in

by

analogue

vacuum

The

resulted

to

second

reaction

3.4~clinitrobromobenzen@

OC appeared

flash

rings

The

cross-coupling

rings,

angular

pyrimidine

substitution.

tetra-fi-

methodologies

&long

one

route,

components &long

the

and other

that this

insured included

generation route,

of

N. J. LL?ONARband S. P. HIREMATH

1932

Diels-Alder

g-xylylene

conditions.-

In

N-hydroxyimidr, ring.

intermediates

both

or

Whereas

imatoic

extension

site, a

(64)

whereas

of

into

(2)

was

not

A

is

under

a

suitably

too

great

is

for

well

a

with

,

tolerated

deaminame,

was fit

while

anhydride,

pyrimidine

adenosine

conclusion

a satisfactory

reaction

terminal

substituted

The

substrate.

Finkelstoin

turned

warn deaminated

a

extension

generated

conversions

anhydride

4.0

a 2.4-A

were

stepwisc

mbenzoadenine

m-naphthoadenine lateral

routes,

reached

at

adenine

the

that

a

enzyme

itself

icr not

substrate.

In

the

compare

case

an

(66-69).

of

set-ice

It wae

xanthine

substrates

entire

rhown

oxidase

and

for

of

by

earlier with

xanthine

(shown

hypoxanthine

In

Krenitsky,

as

keto

form,

67)

comparison

hypoxanthine,

m-bcnzohypoxanthinc

(69),”

with

oxidace

xanthine

order and

60 5.21

< 69 x

and The

< 66.

lo--

mol

minSX

mbenzohypoxanthine (1.57

x

than

the

10-o

and

1.01 for

of and

x

10-O

oxidation is

the

imidatole to

and

m-naphthouric

site

not on

or

produce

within

extension

of

the

imidazole

ring.”

yields

(65) and

are

long

acid enzyme

naphthalene

L-‘)

and

the

V_,,,

the

(641,

about

acid

be

unable

xanthinc

the

the

a5

ability at

each

binding

necessary

for

64,

high 4 0.97;

for other

concentration

appear

at

(K_,

3.04

the

and

of at

larger

and

lo-&

then

at

xanthine

the

molrcule)

an

oxidation

lateral in

the

mnaphtho-

fluorescence T 20.5

x and

buttermilk

oxidation

(661,

exhibiting

in

carbon

end

10-w,

a competitive

pocket

the

each

Hypoxanthine

of

the

x

valuer

to

oxidase

pyrimidine

the

in

1.72 b

behavior

carbon.

to accommodate

ethanol:

a5

(66)-

10SL,

higher

in

J&-naphthohypoxanthine

purged

order

with

(oxidation that

fluorescent. in

an

function5

that

rapidly

increase

x

similar

It

at

with that

of

The

were

pyrimidine

firmt

a5

oxidation

4.15

difference5

conclude

requires

acceptor,

values

were

extension.

at

Hitching5,L*

just

of

to

hypoxanthine

and

respectively.

Two

possible

of

wnaphthohypoxanthinc

observed

derivatives

brilliantly lifetime5

that

now

electron

oxidized

rates

and

protein)-‘,

oxidized

-lin-benzouric

the

is

of

hypoxanthine

We can

~Naphthoadcnine xanthine

of

only

are

carbon.”

oxidase

of

mol

of

oxidized

lin_benrohypoxanthinc

found

valuee

Elion,

ultimate

lirr_naphthohypoxanthinc

level

the it

we

hypoxenthine.

mnaphthohypoxanthine inhibitor

(mq

and

K_.. value

ma1 L-*)

air, actual

the

is

analogues

Neil,

a5

a direct

(628).

it

related

fcrricyanide

4-hydroxypyrimidine

oxidare,

dimensionally

nsec;

quantum 66,

i

1933

Dimensional probes of binding and activity 0.70,

7 24.8

analogues as

nsac;

exhibit

activity

fluorescent

Larger

639, @ 0.88,

spacer

envisaged

units

such

between

predictable

the

water

enzymes

are

Benren.

Ring

or

will

probes

7 33.1 inhibition

as

and

to

the

Svst.

refer

to

pounds The

and

In

(711.

and

Similar

lack

of

rings

of

are

The

no

and in

of

ring.

presents

nitrogen

reepect

hydrogen

sites

dimensional

they

also

to

ambiguity

in

involve

the

amino

(Scheme

the

are

since

VIII)

removed

on

carbon

synthesis

of

m-benzohypo-

when

The

a

more

in

steps on and

pyrimidine

slowly

ten

be

but the

usual

of

the

acid

treatment

than

with

hypowanthine

in

distal com-

a common

ring

plan..

central

is

is

differences

the

original

of

catalytic

closed

the

imidazole

of

They

the

the

are

,

reflux

oxidized

in with

of

m-

6-acetamido(741,

hydrogenation ring.70

over

A more

laboratoryr7*

to in

buttermilk

the

imidazol.

and

J&-benzohypoxanthine

9 but

hydrogenation

acid,

under

reflux

in

formic

pressure.

xanthin. two

recent

which (751,

catalytic

formic

methanol

ring

actusince

3-amino-2,6-dinitrobenzonitril.

ammonia

inter-

not

Introduction

synthesis

Schneller’s

cylizations

acid

2,

imidazol.

imidazoC4,IFflquinazolin-

Nitration

from

it

among

and

nature

71.

in

and

ring

formic

was and

for

pyrimidin.

and

through

benzene,

terminal in

w

respective

within

in

70

group,

xanthine

bind-

w-benzoadenin.

the

the

the related

6-ac.tamido-!5-nitroquinazolin-4~3~l-one

acetyl

formic

and

Our

and

and

in

the

description

(721. gave

carbon

ring

the

containing

72

isomers

approached

tetrasubstituted

the

palladium

the

to

in

structural of

ring

in

changes. was

m-Benzohypowanthlne in

Al

lnalogues.

and

angulated only

benzene occur

according

palladium

acid-toluen.,

to

(731

hydrolysis

over

central

w-benzohypoxanthin.

with

with

proximal groups

relationships

orientational

quinazolin-4(3yl-on.

starts

(7.2 purines,

adenine

(701 for

“benzo”.

spatial

likely

orobel

benzoadenin.

compresses

the

(21

found

m

rings

accordingly

the

bonding

ally

acid

uses

SfXlES

w-bentoadenine

relationship

71 reside

9(8H_l-one.

their

analogu.s.**-

activity

lin_benzoadenine

binding

nomenclature,

imidazole

benzo

with

systems,

over-extended

70 r(

nal

unaphtho

lnthracen.

and

HETERIXYCLIC

to

1 ,_& N

the

nucleotides

spatial

the

of

to

abbreviated

the

term

70,

the

to

devoid

ms .

analogous

nucleosides

enzyme *bento

rings

such

the

Spacer

6,6,5-Rina sites

A)

probable of

where

the

imidazolc

synthesis

MSULAFl

ing

of

(6.3

and

c.s..

specific

those

biphenylene

insolubility

In in

complement

pyrimidine

deterrents

“sec.

orders

were

owidas. of

oxidized

and

air

magnitude under

the

1934

N. J. LEONARD and S. P. HIRIBAKTH

same

conditions.le

probable

The

consequence

imidazole

ring

or

hypoxanthine

was

erythrocytcs

or

phosphoribosyl from

of by

slow

the

shielding

to

(HGPRTI

hypoxanthine

kanthines

with

G-G-butyl

made

as

of

in

,

substitution

part

of

heating

followed

by

deaminase

for

Heroes

virus The

with

in

or

led

to

was

no

butanol

or

that

human

GNP

or

IMP

substitution

agentr.w& pentasulfide

in

compound of

to

not

wa6

pyridine

found

life

to

in

w-benroadenine

treatment did

converted

of

E-methyl

prolongation

by

intact

synthesis

without

convertible

with react

m-benzoadcnine

(701

hydrazinc with

to

adenosinc

m-benzohypo-

xanthine.*e When

7-chloroquinatolin-4O_onr

(11

product,

7-chloro-G-nitroquinazolin-4(3H_)-one

sequence

(Scheme

isomer

.

This

IX)

similar

sequence

benroadenine

to

W-E

nitrated,’ (761

that

proceeded

(71I.‘O

was

abbreviated

through

the

was in

minor

carried

Scheme

was

mononitro

through

I

for

the

w-benzohypoxanthine

enzohypoxanthine

(771

oxidized

with

a 6-nitro to

u-

xanthine

IX

Scheme

f! ---,

71

76 owidase

as

rapidly

monooxidized established protons, adenine For

by

(79)

the

like

and

structure

with

of

single

imidazole

did

in

pattern.7*

not

ChloroformamidIne

6-nitroquinazolin-4(3H_)-one 2,5-diamino-6-nitroquinazolin-4~%_)-one

annelation

hydrochloride, iGO1

(Scheme

of

ring

ring

react

(781,

CIfter

but

oxidized

the

which

could

w-bcnzoguanine

et

be

of

deaminase.

al.

utilized ring

with

the

2-amino-6-chlorobcnzamide

gave

fimination

the

di,t-Benzo-

ldenosine

pyrimidine of

nitration XI.

intact.xe

with

to

was

identification

Schneller

closure

only

product

dcuterium-exchange

m-benroguanine

hydrochloride

imidazoC4,5-flquinazolin-9(8X)-onp,

the

the

m-bentoadeninc,

synthesis

m-benzohypoxanthine,

of

including

as w-benroxanthine, (71),

substitution with

hypoxanthinc The

NMR spectroscopy,

chloroformamidine correct

as

stage..

a

u-Gentowith

m-Benzohypo-

phosphorus

w-Bcnzoadeninc

conditions

the

This

thione

ammonia

hydrogenolysis.

under

but

block

is adjacent

hypoxanthine-guaninc

anthelmintic

with

plaques

animals.

it

the

hydrogen. incubated and

with

search

inhibit

by

N-l

by

preparation.“’

and

a broad

w-benzohypoxanthine

of

carbonyl

when

did

enzyme

imidazoC4,5-f3quinarolin-9(G~I-thione.70

Heroes-infected

w-benzohypoxanthine

the

the

ribotidc

nor

the

yielded

either

through

its

of

of

5-phosphoribosyl-1-pyrophosphate

have

Thiation

oxidation

bonding

converted

with

or

steric

hydrogen

not

transfcrase

guaninc

been

relatively

2-amino-S-chloroof

compound

converted

(781,

in

to

the

GO produced 7-amino-

u8ual

LXmrmional

sequence

for

imidazole

u-benroguanine l-

and

3-methyl

xanthine enzyme

The

formation.

determined

isomers 9 was

(72)

of

as

a

by

locus

of

substrate

direct

independent

w-Benzopuanine

78.

neither

N3

nor

(78)) an

methylation

synthesis

of

1 ike

inhibitor

of

of

the

separate

m-bentohypo-

the

purine

salvage

HGPRT.71

CI novel

approach

cyclohexanedionc a mixture (83)

ring

was

1935

probes of binding and activity

of

was

to

(81) formic

the

in acid

isolated

as

a

distal

system

involved

reduction

formic-acetic

anhydride

and

formamide

(+

82

component

of

a complex

minor

followed

+

of by

2-nitro-1,3-

prolonged

reflux

in

Dihydro-u-benzopurine

83).

mixture.”

81 Closely

related

C4,5-flquinoline compounds

to

the

angular

of

type

84,

u-benzoadenine

tricyclic prepared

imidazolylaminocrotonates, an

amine

to

yield

by

were

substituted

system

system

with

a

cyclization

treated

of

with

compounds

of

70

is

benzene the

85

9-aminoimidrzoSubstituted

corresponding

phosphorus type

the spacer.

benz-

oxychloride

that

were

followed

classed

by

as

anthelmintics.7’-7~ 1

R*&$ _R*& 85

84 Compounds

containing

the

same

methylimidezoC4,5-flquinoline broiled

fish,--‘-

ring

(86).

heated

beef

skeleton

which

extractV7-

NH2 /

substituted broiled

compound fish,

and

quinoxaline structures

(88) of

(87)

the is

these

include

has

been

and

fried

found

related

another

to

related potent

compounds

G6 has

be

to

beef.-

NH2 /

closely

mutagen

the

been

present

The

in

cc-methyl-

NH2 /

1

also

2-amino-3-

isolated

from

2-amino-3,G-dimethylimidazoC4,5-f3mutagen

were

isolated

established

by

from

fried

beef.-%

straightforward

The

unequivocal

syntheses. Of (G9),

the

four

possible

angular

prox-benzoisoallopurinol

benzoisoallopurinol

(92),

bentologs (90)~

three

have

of

allopurinol,

w-benzoallopurinol

w-benzoallopurinol been

synthesized

(91) (89,

90, 0

and 92)

uand

one

has

N. J. LEONARD and S. P. HIREMATH

1936

been

listed

in

structural

a patent

analogy

juxtaposition led

us

of

to

name

the

purinol”

which

structural

exact

literature to

as

serve

claim be

has

in

the

to

allopurinol.

be

precise

confused

since

(89)

isatin

is,

chloral

five-step

indazole

followed

followed

by

obtained

acid

ring

with

was

by

two

clorre

heating the

in

to

steps

of

started

azoline

in

The

oxidase

tion

a

synthesis

the

lack

original do

names

will

not

and

(93)

(Scheme

XI).

of

for

by Like

at oxidase

the with

do

not

a

that

peroxide

fused

with

pyrazoloC4.Sflquinazolin-

but

of

of

ethanol its

known

in

and

from

OL has fusion

allopurinol

inhibit

been

laboratoryee

indarole proof

in of

one

sequence

azo case

the

those

(13)

caused

as

oxidase

familiar The

a

knoun

quin-

in

Schemes

IV

with

and

X1-e’

rtanthine

pyrazoloC3,4-flquinszolinthis

substrate

was

oxidare.e70n permitted enzyme;

the

(90) by

the

to and

powerful

found

Preincubathe

similar

enzyme

w-benroisoallopurinol wanthine

avoid

other

coupling. and

(90)

xanthine

untreated

to

The

structure.

(16), of

The by

pyrazoloC3,4-hlquinto

synthesis

to

acid found

involved

internal

w-benzoisoallopurinol as

our

6-aminoindatolc

formamide.ee and

and

C-aglycone

J-aminoindazole.

recently

with

w-bentoisorllopurinol

rate

the

6-aminoindazole-7-carboxylic

inhibitor-’

same

of

u-benroisoallopurinol

with

29

The

described.

w-benzo-

benrologs

started

synthesis

independent

with

not

and

w-benzoisoallopurinol,

similar

Thus,

ability. (16)

the

route

u-benzoallopurinol

oxidase

hydrogen was

laboratory3e**e*ee

unequivocal

oxidation

identified

procedure,-

94 with which

or

One

in

from

wae

alternative

allopurinol

of 95 ,

angular

diazotization,

described (92),

nanthine

catalytic

as

6-methylquinarolin-4(3H)-onr

of

xanthine

its

to the

compounds

Sandmeyer

(90)

with

competitive

of

has

S-(isonitrosoacetamido)-

yield

accompanied

provided

product

in

acetate

routes

hypoxanthine

to

that

other

of

The

90

Lichtenthaler,

mentionedeeSaL

intermediate

these

7,9(&_,W_)-dione.be

as

the

of

was

been

routes.

the

szolin-b(M_)-one The

has

reduction,

nitration,

convergence

ee

Lichtenthaler’s

formamidine

90

the

chemical

the

the

Oxidation

recognizable

ring of

decarbowylation

route

91.

in

(29).

identical

closure

with

using

furnished

reaction

also

pyrimidinone

ring

of

to

5-aminoindazole

m-benroisoallopurinol

are

synthesized

the

regard

refers

Cuny,

PyrazoloC3,4-flquinazolin-9(By)-one,

essentially

direct

by

give

closure.

(91) call

(92)

a.

by to

diaromethane

Niementowski

we

isoallopurinol

(90))

was

w-benzoallopurinol

which

formycin

names

and

“w-benzoallo-

without who

through

89

carbonyl

of

systematic

with

hydroxylamine

w-Benroallopurinol compounds

the

synthesized

94 and

by

the

or

was

starting

treatment

in

pyrimidinonr

Anyone

but

deserve are

XI

hydrate

formamide

the

abbreviated

sequence

intermediate

9(BH_)-one,

the

that (19)

1iterature33~3a*3~

designations, guides.-.ee

The

compoundm

Allopurinol

instead

analogy

our

scheme

two

to to

appeared

may

a

name

m-benzo&gsoellopurinolee

satisfactory

in

The

by

nitrogens

w-Benzoallopurinol Jahne’

of

(91).-

related

pyrazole

this

of

conform

to

owidation

preincubation lose

nearly

u-benzosecondary

all

to

1937

Dimenaiooal probes of binding and activity

mechanism

of

their

K__‘s.

tors,

it

torily

is

at

probes

Other

that

active

Rina

earlier),

only

in

unpromising linear

xanthine

concentrations as

isomers

oxidase

xanthine

can

be

hence

and

(100)

benzolumazine Christs4e

ethyl

101

(102)

with

related

substituted

possible

a201

ire,

pyrazine

compounds

synthesized

ine

is

(96).

system),

comparable oxidase

to

inhibi-

accommodated are

satisfac-

informative

system). system)

100,

of

the

have

spatial

class

of

this in The

which ring.

type,

vitro key was

two

of

against

which

Streptococcus

intermediate condensed

(Rx

in with

the

by

a research

= Re

faecalis synthesis

representative

104,

and at

was

by

and

the

w-

w-

Schneller

which

was 101

can

Werbel-o

= CHe,

of

and

3-chloropyrazine-2-

program

and

Elslaqer,

made

analogue

represented

mresulted

9 I)TOX-

(97)

103.

da-thieno

the

that

(99)

of

to

in

m-benzowanthine

analogues been

also

led

the

Johnson,

used isomers

include

Treatment

In

antagonists,

that

w-benzocaffeine

2-mercaptoacetamide furnish

to starting

Thiophene

representative.

to

used

u-benzotheophylline

m-benzopteridines.

active Pg/ml.

to

folate

compounds

The

(6,5,6-ring

which

similar

h aye

(6,6,6-ring

chloroformate

Compounds

chemistry

Christ*7.ee

(6,6,6-ring

type of

carbonitrile

2.5

Employing and

m-benzotheophyll

benzolumazine

were

inhibitors

and

of

pr0ducts.e’

(98)

as

are

relatively

angular site

Schneller

benzolumazine

with

are

Svetem~.

ser ice,

angular

(see

clear

the

and

they

.-

benzo in

allopurinol While

I?’

be

ring-closed (Scheme

XII).

regarded

investigating synthesized or

Re = CHe

concentrations

or

C.He)

below

2,4,5,6-tetraminoquin-

1,2-diketones

to

elaborate

the

1938

N. J. LEONARD and S.

P.

HIRJMATH

scheme XIII

ri

106

hllH,

B

108

Y

SO=-, Nallhc i

a,

‘Na

II,

Nae!%O+;

SO,H

c_, alloxa”

101 1.3” extended, Ishikawa,

and

angular

flavin

Manabe*’

that

analogue

shows

(105)

quinoCE,7-qlptaridine-9,ll~7ti,lOIj~-dione Scheme

XII I

activating

(106 iO5

+

as

phenylhydrazine, primary

and

107 + an

secondary

semiquinone

radical

in

be

useful

vitro

105.

only

oxidation

and

metal

of

not

as

The

model

The active bent

(out

Ring

uting

of

in

the

be

iguous.

cant

adenine,

plane)

tween

the

representatives

suggest for

planes

of give

acid

sequence

shown

capable

and

ambient

Fe=*

in

of

oxidized

temperature.

Corn’’ gave the

the

The

bent

metal

ion

complexes

metalloflavoproteins

but

also

as

BCISES

poses imidazole

where

the

the

the

rings

reactivity

of of

terminal the

bears

(111)

through

compound

113

initiation,

and

with

sodium

iodide

113

The

Diels-6lder

with war

and

two treated

di-t-butyl

cyclosddition

the

cycloaddition

and

directly

under

product

114

was

espe-

they

not

need

relation

same

a

14S0 WC

to

the

angle

have

be-

made

the

4,9-dihydro-

4,9-dihydro-u-

shown

in

Scheme

converted

N-bromosuccinimidr

and

Finkelstein

converted

contrib-

coplanar,

was

acetylenedicarboxylate

Its

the

namely.

route

of

be

that

anthracene.

(112)

equivalents

biochemically

whether

must

with

to

the

information.

shown

dihydro-m-bcnropurines. (110)

the

to

adenine 2 has

dihydroanthrecene,

rings,

bent

of

as

bear

that

of

type

question

4,9-dihydro-u-benzopurines

of

(2),

a different

1,3-Diecetyl-4,5-dimethylimidazolin-2(l~~-one dibromo

ZP*

that

the

the were

wbenzoadenine

imidazoC4,5-qlquinazoline-2,S~l~,7Ij~-dione benzouric

at and

Shinkai,

derivatives,

with

Znm*

HETEROCVCLIC

(109)

m-benropurines

first

Cdm*,

should

and

systems The

corresponding

dihydropyridine

findings

structure

pyrimidine

those

by

ions

by

2,4,7-Trimcthyl-

Spacer derivative

of

terminal

cially

made

metal

Complexes

systems

synthesized

catalysts.

4,9-dihydro analogue

was

hydroquinonc

ions

BENT Dihydrobenzene

for

acid.

alcohols

coordination

been

ability.

Complexed

agent

L-ascorbic

tetrahedral

may

(105)

108 -P 105).

oxidizing

and

has

metal-coordinative

reaction in to

XIV.=L to free

the radical

conditions

dimcthylformamidc. the

anhydride

115

by

Dimensional probes of biding

1939

and activity

Scheme XIV

116

112, R = H 113, f?= Ba,

NBS;

b,

the

simple

amount

expedient

of

refluxing

of

formamidine by

brief

c_r Tsclli,

in The

Compound in with

not

sion

will

of

be

ribosyl

synthesis

of

angular.

of

as

derivatives substrates ribosyl the

new

of

the

and

are

place

are

di-,

Second,

effected

116

was

the

on

the

occurring

ribosidation.

in

properties

classical

desired

the

along among

procedure

faces

alkylation/riboeidation straightforward offers isomers. coupling

some *-

simplicity

in

*T

is

and

when

NMR for

nitrogen

heterocycles.

reaction

with

ammonia

This heterocycle

the

analogue is

and

depend that of

nitrogen

to

determine

are

authentic

the

depends the

site

syntheses into

of

feasible. favored

the

differences

of

uncharted

method

a developing

or

that

several spin-spin

alkylation/ribosidrtion involve

locus

This

loci

are

of

*9rl-*T:

in

a

the

third

synthesis upon

of be

from

possible

models a

may

products.

many

introduced upon

scout

For

a number

always

to

of

below).

elaborated

N-ribosidatcd

there of

not

enzyme

group

is

or E-ribosyl

category (see

discus-

the

the

heterocycles.

used

the on

as

general

may be

We have

heterocyclic

introduce

of

oxidase,

linear

serve

coenzymes

strategy

syntheses

determination

to

broader

syntheses

approach

Most

and

preceded

f3-B-ribofuranosyl,

when

not

aride treatment

air

a class,

analogues

the

N-alkylated

does

the

xanthine

versions,

&ri4osidated

nitrogen

difficulties

a positive

of

concentrated

As

including

i.c.,

when

to

110 by

with

has

been

extended

their

of

of

time-consuming.

of

absence

activity.

and

unequivocal

and but

It

bases

various

115

trimethylsilyl

111

that

has

are

ribosyl,

new

with

the

section

that

ribonucleoside. on

the and

a catalytic of

precursor

110

effort

triphosphates,

the

(Cb)&iNa*

MRIVRTIVES

major

structures

with

with

in of

lengthy

precursors

First,

alkylation

of

the

toluene

was

biological

acid

and

of

open.

already

naturally

nucleic

4,

yet.

nuclei

possessing

synthetic

5’-mono-,

the

that

heterocyclic

establishment

avenues

from

derivatives

compounds

rearrangement

Activity

available

recognized

or

116

RIBffiYL It

benzene Curtius

N-methylpyrrolidone ure.s.

toluane;

&NC-I

anhydride

acetate

is

114

acid.

melting

thereof,

fv

heating

acetonitrile.

111,

lack

acetab!;

dihydroisatoic

with

or

t-~~-cEC-CDot-Bu;

e-toluenesulfonic

substituted in

ofq=,

NaI.

e_r foraamidine

CH&N;

nitration ring

of or

system.

Once

N. J. LEONARD and

1940 the

heterocyclic

*CNHm at ribosidated carbon

the

compound the

with

t3

in

methylthio

deactivates

the

pyrimidine

of

two

3

ribosyl

(see

2 for

establishment

of

the

and

between

differentiated

triacetates)

obtained

quinazoline fact

and

that

at

spectrum

of

‘Ii

N-l

the

of was

3.

Synthesis

by

bentyl

in of

of

ammonia

groups

and

at

150

displacement

the

of

and

other two

ben%oadenosine

(118),

or

and

on

with

matched

2,3,5-tri-Q-

of

benzylation

derivatives

products

coupling

of

by

the

well

(as

general

verified

as

8.6 -H

‘wN-l&

compound

the

the

in

Hz

and

verified

(Scheme

the

ST

NMR

of

1.2

Hz

in

definitive. as

deblocking

amino

the

3 position.Tw

coupling

8-methylthio-3-

(117)

by

the 3-benzyl

and

product

benzylated

concomitant

methylthio

1-

nitromethana.*m

ribosyl

as

the

ammonolysisr

treated

1 position

are

of

because

by

distinctive-

anhydrous

ri-

Cl-xSU3-6-methylthioimidazoC4,5-ST-

the

the

SC effected of

and

3-benzyl-8-methylthioimidazo-

the

of

ribosidation

~13-~-tri-~-acetylribofuranosyl~imidazoE4~5-q3quinazoline ethanolic

the

This

ribosidation

3 was

cyanide

relationships

Treatment

Svstems.

for

illustrated

benzylated

separated

compound

*sN-imC

the

two

were

of at

isomer

Such

NMR spectrum.‘m

b,&,S-Rinq

studies

shown

second

the

replaceable and

the

structure

structures

3 occurs

of

system)

structure

the

NMR spectral

benzylation

attachment

the

from

We

or

or

coupling

position.

for

be

of

when

and

the

HaINOI

alkylated

structures

benzylation

mercuric

identity

confirmed

substitute

*w-*F

that

selected

numbering

and

C4,5-qlquinazoline

the

spectra

formed

bromide

reveal

on

was

favors

the

derivatives

acctylribofuranosyl The

and

will

subsequently

ultraviolet

merely

8-methylthioimidazot4r5-g3quinazoline.

earlier)

would

can

‘W-label!edr

only

of

from

see

II, which

one final

but

confirmation

group,

The

the

NMR spectrum

originating

ring.

derivatives

*eC

out,

In

N position,

Scheme

the

imidazole

the

led

products

worked

stage. the

label

procedure

bosidated

is

appropriate product,

on

this

synthesis

the

S. P. HIREKMH

with

of

XV)

to

the

sugar

afford

3-4-~-ribofuranosyl-~-benroadenine.x*

&-

Assignment

of

Scheme XV

the

fl configuration

for

the

methyl

application

was groups

of

the

ison

of

with

was the

of

possible

fluorescence quantum

yield

For as

well nized of

the

as

in

to of

(0.44)

were

and

S.b

in

for

adenosine

for

water

both

they

be

water, to

study

accepted

~benzoadenosine

be

difference

at

*C

20

of

3.5

and and

with

should are

of

observed 110

be

a run

5.3

nsecf

and

useful

as

of

biological

as

substrates tll8l

and

in

on

compar-

the

the

alkaline

The

observed

side

fluorescence

dioxane.,,

activity, or

DBF,

*benso-

fluorescent

is

p&m for

6bX

Thus,

3.0.

unprotonated.

ethanol

and

The

coworkers.*m-*4

(3.7

for

the

his

systems

that

derivatives

that

shift derivative

substrate

enzyme

ensure

constant

probes that

Imbach

m-bentoadenosine

bentoadeninc

We found

aH NtlR chemical

adcnosine-related

lifetime

important

be

figures

an

order

dimensional

enzymes.

to

substrate in

the

of

found

parallel

activities

adenosine-related where

on

2& ,3’d_-isopropylidene

the

correlations

_QD-benzoadenosine compared

based

in

probes

as

it

recog-

cofactors

converted

was by

to

lin_

a

variety

Dimensional probes of binding and activity

benzoinosine of

the

(1191 of

V’,,,

order

as

size,

i.e.,

that

of

of

edcnosine,

edenine,

edenosinc.

which

a

by

an

independent was

et

behavior

This Since

u-benzoinosine

of

u-benzouric

least

es

when

the

group

in

is

fast

as

that

apparently is

that

the

deowy-

by

any Both

chemical

separation

of

nitrogen Scheme

of

the

benzoinosine

imidetole Intermediate

(1191

mentioned Cln acyclic

or

to

(1201

of

wenthine

2.4

of

normal

reported with

of

the

ring, 124

that

be

m-benzoguanoslne could

be

M

The

was

et

We concluded

the

hydrophilic

spacer

the

oxidation

that

ribosyl

in

hydrophobic

pocket

of

the

phosphorylesc,

bond

in

moiety,

an

ribofuranosyl end

guaninc,

did

not

bring

u-benzolnosinc.”

stages for (1291

prepared

10-e

finding-l-

hypoxanthinc,

ribosyl

used

x

oxidese

m-benzoinoslne

et

8.9

human

riboside

unusual

ribosyl

and

also

m-

25%

enzymatic the

nucleoslde

kanthines

is,

could

m-

dceminesc. was

gave

by

to

glycosidic

departing

for

being

ADP-induced

substrate.

in

contrast,

protected

N-7

When

of

xanthine

of

By

of the

of

M end

the

benzene-ring

binding

cleavage

of

of

inhibition.

oxidase.

interaction

restored.

case

N-l

(1191.

aggregation

of

with

the

the

that

adcnoslne

lo-’

(1201

the

A by

of

x

a particularly

steric

in

leboretory.‘e

1.42

order

m-bentoedenosinc

syntheses

lin-Benzoxenthosinc

by

derivatives

rcgioisomers

XVI.‘-

low

es

larger con-

7-ll-g-ribofurenosylwith

activity

at

m-benzoinosine

promotes

with

this

m-bcnzoxenthosinc

phosphoryletion

reaction

in

induction

hypoxanthine,

are

of

inhibitory

a

anti

u-benzoinosine

effect

constituted of

ribofurenosyl

accompanied

llS,

of The

substrate.

condition

05%

same

l-II-pribofuranosyl-u-

reaction

route

possibilities

normally

119,

the

requirement

enelogue

of

to

displaced

oxidation

of

possible

unfavorable

lnosinc

enzyme

to

of

substrate

detectable

without

oxidized

(1211

reaction

m-benzoinosine,

about

not

acid

of

isomer

M against

(1191

of

end

was

lo-*

rate

enzyme

the

corresponds

inosine

of

relative

x

matches

no

for

it

2.84

The

of

e substrete

normal

favored

at

mucosa

value

accept

the

probably

synthetic

tested

can

than

L

l

unaltered.

deeminetion

aggregation,

inhibition

is

intestinal

with

enzyme

A,

“stretched-out” gave

enzymatic

C\DP .

as

the

of

benzoadenosine

end

is

calf

arc

The

non-substrate,

product

the 2.4

features

unsubstituted

in

benzoadenine,

by

attachment

other

is

unsubstitutcd

platelet

rlbosyl

from

edenosine,

Thus,

extended

that

bcnzoedenoslne

dcrminase

substrate

adenosine.*=

the

so

obtained

edenosina

natural

laterally

formation

The

by

the

1941

groups

(1191

required

attached

to

123 -c 124 conversion with

entymatlcally

the

and to

127

-c 128

in

m-

reagents from

the

either

indicated.

m-bcnzolnosinc

above. nucleoside

related

to

m-bentoguanosine

(1251

has

been

made

by

1942

N. 1. LBONAROand S. P. HIREMATH

Scheme XVI

123

l&b

riib(Ac),

125

124

126

Burroughs

127

scientists

We,llcome

,79-

128

namely,

imidazoC4,5-glquinazolin-8o_onc

-

6-amino-3-C(2-hydrowyethokylmethyll-

(12Bal.

Their

synthesis

involved

the

12th silylation

of

benzoate,

u-benzoguanine

chromatographic

liberation

of

122a

methylamine. herpes

drug

(HSV-11 to

the In

in

the

(51, separation

by

short

Compound

which

but

did

the

3-

and the

2-(chloromethoxylethyl 7-substituted benzoate

m-benzo

no

exhibit

analogue

inhibition

of

competition

products,

ester

with

of

the

herpes

with

and

aqueous potent

simplex

acyclovir

for

anti-

virus-l binding

enzyme. order

to

obtain

deoxyribosida

2’-deoxy-lin_benzoadenosine, series,

we

used

fI-~-ribofuranosyllimidazoC4r5-g1 tri-Q-benzoyl acetate

nucleosides

in

chloride thiobenzoyl

as

afforded ca.

4:l

diacetylthiobenroyl

hydride

the

of

This

ratio.

and

and

by

then

subjected

with

FIIBN

with to

as

the

was

rlbonucleosides

was

(1171-

an

first

3’,5’-

converted treatment

H&/pyridine. free-radical initiator.

or

the

Treatment

precursor.

a mixture

PhClC=~Me,Clisomer

the

extended

analogue

B-(methylthiol-3-(2~,3~r5~-tri-Q-acatyl-

quinazoline

derivativeTL

ammonium

butyltin

is

with

the

of

showed

(Zoviraxl, kinase

of

heat-treatment

129ar

acyclovir

thymidine

rlkylation

of

and to first

corresponding 117

with

hydrowyl-

2’,5’-dip-acetyl a

separable with

The

the

mixture

of

chloroiminium

3’,5’-diacrtyl-2’-

deowygcnation Replacement

with of

the

tri-n_mrthylthio

Dimcmional probes of big group

by

means

of

ethanolic

u-benzoadenosine ribonucleoside study

was

Synthesis

with of

at

the

N-l,

ribosidation

of

was

ically

more

dat ion.

and

N-l

sites

rotational

values

respective

N-methyl

Naturally

(RP-yellow

been

and

comparison

structure

of of

the

been

by 132

may

be

the

to

result

‘-C

known

(23)

were

the

the

J&-

129-131

functions

and

therefrom

the

of

The

the

via

basis

N-2

thermodynam-

transribosi-

of

%H NMR spectra

derivatives

that

Five

Itent

as

with the

optical

with

those

of

the

structure. have

yellow

6,7-diamino

monomeric

tlaerki-Danzig,

chromatography taken

In isomers

temperature.

on

for

(15),

preceded

control

and

time. by

lllopurinol

laboratory.**

including

stage

later).

obtained

kinetic

deory-

S I-triphosphate (ire

ribosidated

of

2’-deowy-

extended

of

assigned

the

first

isolated

repeated

the I

same

of

were

lumazine

have

the

isomers,

OC afforded

Lichtenthaler-.-

conditions,

N-6

for

and

product

derivatives

mushrooms

these,

and

to

triacetylribosyl)

the

ribosidation

found

I-V)

Russula

be

of

occurring

have

Cuny in

reaction

to

stable

The

tution

the

considered

150

ribonucleosides

by

1943

linearly

DNA polymerase

proportions or

at

This

sequentially

N-S,

the

pressure

N-3).

possible

and

tribenzoyl-

protection

isomer

at

enzyme

various

N-2,

series,

indicates

acyl

the

under

dRib

m-benzoallopurinol

benzoellopurinol (Rib’

with

phosphorylatcd

particularly

attachment

ammonia

(119,

and activity

on

and

cellulose

representative

and

of

the

substi-

russpteridines

Euqsterxoo sephadex

from gels.

Of

rtl+imidazoC4,5-q3-

pteridines.

0

b-t,

“-LO” H-LO” H-i-OH

AN

kib

CH/=N

134

133

haOH

132 Finally,

amonq

generating

a

guanosine

with

methanol

.*ol-*O-

the

product

bles,

in

synthetic extended

methyl

a

6,6,S

ring

adenosine

systems, analoque

g-cyonomethanimidate

Since

is the

the

laterally

the

tricyclic

periphery.

starting

(133)

material

ribonucleoside that

of

and

is

whose

adenosine,

we

another is

by

method

the

sodium

a bicyclic structure

have

of

methowide

in

ribonucleoside more

applied

to

closely

the

name

metamorphosis

adenosine-like consistent

This

“IA’-metemorphosine.” of

an

tricyclic with

the

inosine

proposed

(134)

name

is

(disconnection

molecule. structure

The

9

intended

of

analytical 134

and resem-

S-amino-9,10-

dihydro-lO-oxo-3-(3-~--ribofuranosyl-3~-l,3~5-triazinoCl,2-q3purine trivial

of

reaction

but

the and

since

to

terminal

indicate, ring)

spectroscopic the

reactants

the formally,

to

an

data offered

were the

N. J. LLONARD and S. P. HIRBWATH

1944

opportunity

of

guanosine

different

and

C = N vs.

distinguishing of

involving

134

133

and

analysis the

of

linear

spacer

and

its

the

widrr

In

the

*H NMR spectrum,

the

clearly

1.31

nm. from

(by

to

the

release

deaminase.

It

guanosine

the

on

top

higher

probably

due

w-carbonyl

in

via

vibrational

can

also

serve

as

a

9-substituted

guanines

with

methyl

have

obtained

similar

from

9-(2-hydronyethoxylmethylguanine

ribonucleosides naturally For

acetate,

Representatives

membered cytotoxicity

of

by ring

of

a

in

vitro

not

some

against

media

with

T 444

proton

transfer in

adenoeine

since

it

reverts

The

5 min.

from

of

longer

a-a a decrease

is

to

reaction

of

general.

We

deoxyguanosine

by

of

have by

and

tricycle

and

tricyclic

bean

136 second

L-1210

pyrimidine

ring

136a-141.Lo***ow mouse

his

leukemia.

from coworkerm.

(1361

acetic

has

a central

Some

have the

been

with

acid.x9’ that

onto

For

category

sequence

ribonucleosides

135 a

and

nucleoside

50% aqueous

the

constructed

three-stage

kib

formulas

to

tricyclic

Townsend

a simple

ammonia,

conform

bib

of

In

glycerol,

of

interesting

systems

(1351

number

in

in

(1331

actually

very

ribonucleosides

ethanolic

shown

*C

shown

hydrogen

@ = 0.9SS,~Q&

well

guanosine

as of

exhibits

facility

as

&*Oi*‘-

N-H.

-38

adenosine

OC within

diadenosine-like

large

annelation are

of

products

do

6,5,6-ring

6-aminotoyocamycin

diethoxymethyl

indicative

the

state

tricyclic

here,

derived

a double-headed

from

synthesized

or

nonidentical,

ewocyclic

central

2.2

9.39,

&cyanomethanimidate

they

defined

conuisting

occurring

example,

prepared

as

in

20

vs.

were

and

established

the

(acyclovirl.x03

Although

probes

at

2.4

e.g.,at

M,

X-ray which

that

bottom,

modulation)

“protected”

N_ NaOH

f1uorQscQnt.r

Svstrmr.

yield,

excited

9-benzylguanine

metamorphosine

a change

the

matches

four,

showed

and

ICI’-

(by

of

of

proximate

processes.

0.1

6,5,6-Rinq

the

10.2

the

to

,

N-H’s

of

nsec

group

series at

quantum

1.38

with

dimensional

than

and

treatment

of

the

tcmperaturer

7 =

product

in of

We obtained

products.

Single-crystal

the

DQO-exchanqeablc

lower

and

is

of

carbonyl

at

of

6 values

peri

phase),

behavior

NH.

the

lifetime

nsec

This

energy

the

viscosity

fluorescence =

at

the

N-3 means

spectroscopic

derivative.

compound

vs.

definitive

isomeric

parallel

with

2-bromo

diffQrentiated

between

increasing

of

1-p

sought

possible

a pair

structure

we

reagent,’

other

derivative

last-mentioned

somewhat

the

by

2-bromo

by

bonding

134

ring

condensation-cyclization,

three

corresponding

tricyclic

was

the

structure

its

of

C Z N in

134 from

confirmation

with

modes

have

five-

shown

synthesis

lilb

bQQn

H

moderate of

the

Dimcn2ional probe3 of binding and

tricyclic

ribonucieoside

2-azapurine served

143,

nucleoside

as

the

which

analogue

starting

resembles

at

mat@rial.

the

edenosine

other

Selective

activity

at

diazotization

of

142 group of

at

the

the

& position

6,5,&-linear

6,5.5-Rinq heating

a

hydrazine

of

at

of

142

led

was

ring

representative

found

to

closure

of (144)

possess

this

in

“good”

in

XV) .*-

a manner

similar

to

precursor

for

methane/dimethylformamide protected by

3-

(148)

chromatography

effect

that

removal

methylthio

of

group.

and

in as

the

the

treated

scheme

for

the

acetyl

Structure

and

(147).

(64)~~~

was of

solvent

groups

treated

formation

made

by

excess

activity.

system.

and

assignments

at

The

two

of

of

ethanolic

150 the

OC to

Scheme

was

effect

also

a

2,3,5-tri-Qusing

major 147,

nitro-

products, were

ammonia

isomeric (or

Scheme

cyanide

derivatives with

(14&, (113, which

with

mercuric

9-amino-l-~-_D-ribofuranosylbenzimidazoCS,6-qlquinatoline

XVII

the

(145),

m-benzoadenosine

presence

separately

(J-g-ribofuranosylbrnzimidazoC5~6-qlquinaroline (146)

used

l-R-pribofuranosyl

and

amino

145

m-naphththoadenine bromide

ewocyclic

containing

wnaphthoadenosine

9-(Methylthio)benzimidazoC!3,&-glquinazoline

acetylribofuranosyl

series

anti-tumor

144 XVII)

a (142)

Ibib

We synthesized

Svstem.

the

with

ethanol

Fiib

&,&.6.5-Rina

and

143.*-**07

6-bromotoyocamycin

refluxrloe

to

ribonucleoside

fin early

System.

suspension

end

143

compound

tricyclic

one

6-amfnarangivamycin

end,

at

separated 20

OC to

dirplecemcnt

of

products,

9-amino-3-

m-naphthoadenonine) were

made

the

N. J. h3NARIl and S. P. HDUMATH

1946 the

on

basis

erties

in

of

chromatoqraphic

parallel

series.Lw

with

The

related

to

long

those

naphthacene

the

its

spectrum

protonat from

second did

to

146

was of

was

Whereas

intestinal the

to

no

conversion

to

of

5.61.

as

of

lonqer

to

inhibitor.-

Whereas

for

dsaminase,

of

4.0

A from

the

satisfactory

fit

at

enzyme

adenosine,

binding

the to

the

extra

adenosinc

almost

normal

site

and

on

while

was an

is

2.4-A

of

the

it

no an

like that

too

its

a

great

lateral

for

extension substrate

calf

for

active

inactive,

established

at

that

underwent

was

deamination

going

from

to

nor

adenosine,

in

pyrimidine

deaminase

(uniquely)

146

in

upon

pK,

the

comparable

conditions,

substrate,

active

enzyme

upon

u-naphthoadenosine

for

nm,

absorbance

of

by

activity

By

340-370

ring

at

was

longer of

plj.

greater

m-naphthoadenina of

changing

converted

reaction

are of

three

protonation

m-benzoadenine

absence

of

predictably

inosine,”

substrate

The

the

first

a rate

u-benzo

bands

wavelength, and

of

of

prop-

the

spectrum

is

identical

effective

extension

site

group

by

wavelength

was

under

derivative.

affected

m-benroadenosine,

adenosine

adenosine

The

absorption

effect

The

in

wavelength

intermediate

m-benzoinosine

deamination

ribosyl

at

spectroscopic

pairs

~naphthoadenosine

lonq

electronic

pronounced

%

of

the

greatly

b-benzoadenosine

mucosa

detectable

not

UV

isomeric

anthracene.

the

bands

shifts

(both

of

in

NMR and

band6 as

those nm,

(146)

m-naphthoadenosine, rinq.

and

similar

absorption

group

show

There

ion.

118

to

392-438

lin_naphthoadenosine contrast,

the

m-benzoadenosine

related

bands,

of

wavelength

of

are

wavelength

behavior

those

a

(1181

of

were

efficient.

TIDPUI-, In

terms

substrates

of or

of

the

experimentation

stages, of

cofactors

at

phorylation

binding,

follow

phosphorylated

level

each

level.xoq

phosphate

phosphorylated one-sixth

of

to

derivatives all

known

the

the

synthesis.

next

and

(149, cnzvmes

x

may

to

been

establish

of

reauire

The

with In

used the

fact,

to

a related

natural

enzyme

di-,

and

the

third

fourth

triphosstage and

spectroscopic they

convert

integrity

especially

b-banzoadenosine

FITP or

the

mono-,

constitutes

and

be

interested

150)

from

introduction.

activity

methodology

We have

unit

the

and

upon

enzyme

differ changes,

ribosyl in

binding

immediately since

the

mentioned

enzyme

that

dimensional

of

was

DERIVATIVES

analoques

defined

St-hydroxyl

with

stage

TAX WIBDSYL

purine by

that

dealing

synthesis

DI-,

providing

merge

from of

structure

since

adenine-containina

B

I;-OH

0

0

0

OH

OH

OH

6I- o-c O-b-0” I- I &&r-ienroadenosine 5’-diphosphoryl Adenosine

5’-tetra-

phosphoryl Adenosine phosphoryl

5’-penta-

with

the

one

in (lie)

fifth

analysis

159

at

Dizncosiooal probes of binding and activity

cofactor

such

me have

as

AMP,

synthesized

*benso-ATP similar

(149~1,

triphosphates

of

benzoguanostne

for

the

to

the

by

, and

HPLC,

electrophoresisr to

(149b)

and

Moffatt

on

characterized

by

discovered muscle

that

our

various Other

was

kinase supply (and

ATP-

synthesized

benzoadenosinr

general

by

phosphate in

displacement purity

and

=*P

0

heart

with

pure

solvent*o’**z7 appeared

in in

an

a

salt

acid

in

of

symmetrical by

the

oliqophosphates the

other

and of

ATPa

St-tri

ribotides

and

completion pyruvate

of

the and

formed

the in

use the

low

of

(149d)

was

equivalence s suggested

reaction.

at

m-benro-

the

oxidation

first

by

reaction

trifluaroacetic

purified

the by

one

that

two

the

by

linear

phosphates

synthesis,

and were

ribonucleoside

terminus

and

Enzymatic

case

general

dehydrogenase

was without

adenosine

at

5’ -monophosphoromorpholidat~

(125)

The

electrophosphate

to

unsymmetrical

of

was

interconversion5

m-benzo-C\DP

to

m-benzo-

f119),~benzo-

m--benzoinosine

The

cyclic

synthesized

and

tetraphosphates.m-7.*~~***~

lactate

the

with

of

u-benzoadonosine

m-benzoguanosine

The HPLC,

Ifn-benzo-AMP,

v was

isolated

The

of

potassium

by

method of

J&-

dichloride

addition/

temperature

anhydride

and

in

Honjo.“” from

with

for

established

The

As

and

generated

achieved

FOl-

(1SOfr

acid

anion

a bisphosphorylation

product

diphorphatea

of

Nishimura, was

in

methodology.

3’(2’),5’-bisphosphate

)in-benroadenosine

possible.

produced

to

below).L*o

-phosphodiesterase

at

NMR signal.

phosphoenolpyruvate.Sx* by

the

from

and were

(120)~

xanthosine

with

synthesized

conversion

kinase

e*P

149e.f

were

adenosine some

singlet

of

(see

method

J&-benzc-CAMP

5’ -phosphoromorpholidate

nature

covenient

standard

ring-opening

3’,9’

symmetrical

dimethylformamide

thus

confirmed

by

S1-pyrophosphate

chromatography.**-“P**s=

diphosphate

we rabbit

phosphorus.“O**lr*“c

were

chloride of

The

gradient the

and

-lin-benzoadenasine

anhydrcus

from

nucleotide

application

c’,Ppdi-J&in-benroadenosina

from

3’-0

&,r-Benroadenosine

1963.****‘*m

Once

this

was

thr

of were

with

used

Marumotos

cyclfzation

group

pyrophosphoryl

method

S’-triphosphate

systems

by

enzyme

trichloromethylphorphonic

analogue

shown

l__&-benzo-ADP

-lin-benzo-ATP

applying

generated

cAMP

Sl-monophosphate.

prepared

by

of

Then,

which

this

beef

made

with

NMR spectroscopy,

with

recycle enzyme

procedure

*C.

*C,

of

we

to

specific

NMR spectra.

to

trichloromethylphosphonate

trichloromethyl

structure

incubation

adenosine

the

25

**P

to

complete

to

the

3*,fjt-monophosphates

treatment

at

DMF at of

and

phoresis,

of

were cyclic

5’-

(118)

triethyl

t-butoxide

on

the

and

and

the

as

phosphoromorpholidat~

converted

requiring

converted

Furukawa,

uniquely, and

J&protec-

m-cresol

fakanohashi,

and

and

of

was in

m-benxo-AMP

phosphoenolpyruvate,

derivatives

by

be

triphosphate

J&-bentoadenoeine

the

elcctrophoresis,

&i_D-bentoadcnosine

First,

in

of

used

could

and

(118)

with

of

used

di-r

nece8Sity

chloride

NMR spectrumt

incubation

(149b)l

we

1.120) 9 and

the

formed

5’-diphosphate

m-benzo-C\TP-1

phosphate

example*

HPLCI

m-benzo-ADP

pyruvate

generate

TLC,

we

the

and

S’-mono-c

Fujii,

was

conversion ,

( 149c) Both

Khorana.***

Imai ,

by

=‘P

m-benzoadenosine

&J_D-bcnzo-ATP

and

fluorescent

pytophosphotyl

microanalysisr

For

aliiL~*‘~

avoided

A.*--

J,_&-benzo-ADP

m-benzoadenosine

with

coenzyme

or

lin_bentoxanthorine

described

5’-nucleotidase.LO’*1~og~**

FAD, (149a)l

)nter

route

5 ‘-monophosphate

The

Honjo."'

reconversion

reaction

conditions

Masuda

the

preferred

Unprotected

steps. (149a)

of

(119),

The

NADPH,

(150)~

synthesis

J&-benzoinosine

tion-deprotectfon

NAD*,

&_&-benzo-RMP

lin_benzo-CAMP

(12!3).***

J&-benzo-AMP according

fluorrscrnt

and

methodology

AMP ,

cyclic

ADP,

thr

1947

with

accomplished

reaction

was

t with

NADN

of

f.ibenro-IMP

driven

present

pyruvatc to

to to

consume &-benzo-

the

N. J. LEONARD and S. P. HIRBIATH

1948 XUP with

xanthine

represents

the

phosphate

level.

oxidase first

the

of

phosphate

in

further

While

group.

ribotides,

the

analoques.*”

Thus,

reactions fail

-lin-benzo-GDP,

and

to

lin_benzo-GTP

under

their

course

the

of

3’,5’-

QXP NMR spectra

the

(-1.7 (3.9

When

estimation

value

readily,

the

in the

among

5’-,

3’-,

and

= 22

Hz)

and

L-r

= 20

tively, of

)p,(5’

for

the

and

‘H

the

the

are

(a,

-11.1,

chemical

for

the

the

6-H

becomes

particularly

and

lin-benzoadenine

is

0.20 in

T,

exP

d;

chemical

that

of

m-bento-

at to

‘,&.,,,.

respec-

like

and

and

-6.1,

RTP,

is

ppm from general

shifts

single

centered

of

the

(the

is When

are least

the

nucleotides.x* in

relaxation

aqueous times

syn

base

about

-11.3,

those

of

dd,

the

to

of the

of

magnitude

Head-to-tail solution (DESERT

were method).

takes

ionization

that

course,

anti

in

dilutions

phosphate

and

predominates

possess

no

conformation

unprotected,

stacked

order

at

(protonation

conformation

to is

150,

charge

ldenylates,

vertically one

and

of

sensitive

responsive

adenine

on

4-H

% 4.0)

at

are

doublets,

t;

( 149~)

149a-d

2-H

of

an

upfield

u.

QIP

CIDP and

similar

monitor

(pD

nucleotides effect

a

the

nucleotides

corresponding

analogues

the

15o.“.“e

constants

are

positions

in

at

A(S’)p,(5’)4.

for

indicative

149a-c

shifts 0-P

encountered

and

~3, -5.ET

The

)A

the

the

differentiated

by

t-11.0)

)p.(S’

in

consistently

13, -22.4, of

allowed,

constant

be

The

those

is and

readily

characterization.

ring),

and

d;

to

chemical

m-bento-AMP

those

downfield

-10.31

is

2’,3’-bisphosphates

ribonucleosides. (a,

as

are

distinquish

similar

pyrimidine

conditions

and

can

(149b)

the

and

nearly can

signals

(149d)

QlP

of

signals

show

similar

signals

such

3’,5’2’-P

lin-bento-G(S’

dd,

NMR spectra

proton),

association

substitution

the

-23.2~

~benzoadenine

the

on

for

For

infinite,

acidic

of

2 I-P

and in

constants

under

NIP

P signal

149a-f

corresponding

assignment

signals

one

(149~)

phosphates

the

equivalent 8.5)

3’-P

l-42) arc

useful

)FI (15GQ).

therefore

under

the Thus,

20

(-11.1).

a-P

on

The

m-bento-RDP

therefore

approaching place

and

convert

correlations

from

both

mixtures 5’-

to

m-benzo-CAMP

distinct in

ribonucleoside

2 ‘-phosphates

-

in

3’-P

the

observed

structure

the

of

convert

coupling

NMR phosphorus in

~-bQnzo-A(S’)pQ(5’)~-benzo-A

corresponding In

and

of

L-y

4(!3’)p*(5’)4 A(5’

the

m-bento-ATP

Hz;

and

shift

the

position.

constants

the

With

proportions.

resonance

coupling

of useful

to

levels

qeneraliza-

P-O-P

CAMP and

5’-phosphate

proportions

ppm, when

average

quite

are

of

3.88

different

5’-P

St-P

85% HQPO..,

reaction.

especially

present from

of

both

added with

phosphate

some

and

when

example,

occurs

the

failed

further

and

for

the

shifts

bisphosphorylation

average

from for

of

nucleotides

resonance

relaxation

chemical

quantitative

is

ppm) ppm)

complete

adenine

which of

analogues

observed

shifts

of

conditions. phosphate

we

case

effective

synthetase

capability

For

magnetic

upfield

relative

the

esP

downfield

(149a). e*P

the

ring,

failed

it

51-mono-

the

presence

the

kinase

various

chemical

nucleotides.

conditions, shifted

the

the

between

m-benzoadenine

shift

have

since

similarity

the

in

surprisingly

assay

2’ ,5’-bisphosphatesr that

tionXP~~X~0~~X~~~S~ striking

normal

characterizing

and

the

since

the

as

interconvert

succinyl-Co4

at

imidazole

by are

to

surprising,

ring,

the

monophosphate

to

the

in

prevented

enzyme

guanosine

and occurring

pyrimidine

others

lin_benzo-GNP

In

useful

oxidation

u-benzo-GDP

and

the

is

some

stretched-out

both

a conversion

u-bentoxanthosine,

the

150

was

such

occurs

whereas

case

oxygen of

Oxidation

&-benzoinosine, in

and

example

(pD

%

these

aqueous greater

solution, than

orientations indicated

of by

Head-to-tail

the

with those

of

stacked deuterium alternate

Dimensional probes of binding and activity

stacking

was

found

p,Nr_dimcthyl

pK_, values

The -CAMP,

side

between

the

149a-c

(7.6,

7.1,

that

reason

already in

the

to

be

synthesis

of

with

recognized

contaminated

of

accumulated

model is

AMP

for

(149a)

the

in

enzyme

CIMP in

to

the

function,

in

149asLm1

for

in

this

AMP

probably stretched-out binding

model

its helps

at

least

lin-Benzo-CAMP. kinasc

from

cAMP.~‘~ of

brain

and

activation

by

slightly

the

enzyme

can

earlier

oligonucleotide CITP.

2’ ,5’-

incorporated

at

2’,5’-bisphosphate

than

accommodate

of

CAMP

from

such

as

extension

the

3’ and

end a

of

variety

l

.

of This

not

the

in

the

utilized,

presence and

tetranucleotidr of

other

analogurs

solution. protein

concentration

not

inhibit

such

kinase, purinc

of only

be

excess th-

ring

by

to T4

nearly

as

2.4

good

A.

as

RNCI ligase

This

its

that

lengthen

3’,5’-bisphosphate

lcceptor.ss7 are

and showing

prepared used

than

activation

inhibits

mixture,

9 can

is

Our

. u-bcnzoadcnosine the

NH.

system

kinase.

in

higher

s

results

satisfactorily

activates

protein

m-bmsoadrnosine (Flp)rC

is

bisphosphatr

Thus

Our

FIMP hydrophobic

(1lE)

CAMP-dependent

we

specific.

ring

kinase

does

As

requisite

adcnylate

maximally a

less

C\TP permits

highly the

the

a

other, lJD-brnzo-

substitute

of

at

the

formed. is

The

supports

for

heteroaromatic

adenylate

adenosine.

and

anti,

will

for

but

enzyme

are

site

-lin-benzo-CAMP

lateral

It

the

CIDP.

produced.n.xxo

ADP

size

of

plus

(149~)

is

the

limiting

muscle

does

of

rtretched-

substituting

u-benzoadanosine

with

all

catalyzes

ADP

were

substrate

3’(2’),5’-bisoho~ohat directly

tried

and

muscle

of

the

we

3-iso-F\MP

a

we

cases.

synthetic

the

AMP or

AMP binding

(150)

levels

of

mbenzo-AMP

rabbit

interact

chain

The

the

skeletal

low

I&n-Benroadenosine described

define

CAMP 9 while less

3’,5’-monophosphate

as

were

natural

all

Mg**-CIDP

QD-benzo-fATP

extension

analoguc

from

of

kinasc

the

was

general,

activity,

muscle)

diphosphatcs

the

failure

that

from

totally

pig

for

m-benro-ADP

of

there

In

zero

the or

substrates.

practically

AMP and

When

of

which

in

functioning

comparison,

for

This

Addition

protein

in

,*mw

for

pocket,

both

conformation

accounts

specific

no

in

and,

system

is

system,

anticipation.

activity

our

or

plus

the

ADP.

substrates

pK,

to

nuclcotide.

to

C\TP or

and

of

(rabbit

substitution

model

sugar/base

present

one

natural

site

this

is

Mgm*-FITP

one

the

two-site

a

as

to

drawn

the

nucleotides

activity be

brings germane

activities

of

direct

is

unnatural

of

addition

and

these

for

these

the

derivatives.

range

to

kinaee

regard

for

below,

the

Since

which

specific

discuss

support

with

a

bases

values

in

information

enzyme

phosphate

of

case

with

between

evidence

demanding,

no

CIdenylate

phosphate

various

the

The

hopeful

with

intermediate

in

was

fortuitous

conclusions

that

lin-Benro-fAMP.

the

observations through

there

the

occur

lin_benzo-CARP

diminishes and

This

activity

of

the

and

phosphates

micrllas

phosphate(s)

While

enzyme

some

positive

analogues

shall

expect

the

of

can

5 mfl Mgm* t whereas

m-benzoadeninc

enzymes.

equivalence

permitted

the

the

1benzo-Ado,

interaction

that

of

conformation

(118)

in

ammonium

the

mentioned

rewarded

substrate

of

of

and

intramolecular

u-banzoadenosine.

activity

various

presence

protonation-deprotonation.

between

of

crystals

ana1ysis.w

spectroscopy

the

indicate

base

single

X-ray

m-bcnzoadenosine

the

the

to

respectively)

interaction

in

by

unchanged

quaternary

with

two-site

in

in

to

pattern

ultraviolet

no

5.6,

or

of

transfer

is

cations

been

out

as

base,

involved

a priori

should

When

are

cofactors

which

by

responded

metal

discussion

have

.-CITP

and

7.3,

closer

utilized

determined

pK_, value

intramolecular

We have

intermolecular lin_benzoadenine

chain.xxw.xwo

N+-H

molecules

values

the of

and

phosphate

(150).

divalent

be

as

-ADP,

-AMP,

phosphate

no

to

derivative

1949

an and is

N. J. JJONARD and S. P. HIREMKIH

1950

substrates the

as

lack

of

their

unmodified

specificity

of We have

lin-Benzo-ADP. synthesis kinase the

of and

use

a coupled

of

the

but

no

specific

the

and

Although beef

heart

phosphorylate

or

rat

mitochondrial

phosphate

excitation

at

properties

332

are

binding.

In

quenched

The

FM CIDP.

the

the

with

inner

membrane

substrate

for

The (149d) The

the

folding

of

greater

of

cleavage

that

of

149d

approximately

are

the

predictive is

in

close

aromatic

strong

Micrococcus

fl”orescence.‘~~ a probably

with in

The

nucleotide

units.

hydrolysis

with

venom

(149bl luteus

helical

The

phosphodiesterase,

aid

its

venom

an

intact

fragments Purified by

FIDP C&

strong

enzyme

is

reversed

is

inactivity be

does

These

of

binding

to it

upon

nsec.xxo*xeo

quenching

is,

(1121

0.40-O-44

problems

The

strongly by

of

the

of

the

ADP

transported not

across

serve

as

a

S’-pyrophosphate

by

a of

number

of

The

in

was

changes

(149d)

when

was by

presence was

the

including

it

not gel

of

alkaline

of

of enzymes,

phosphatase,

acid to

and

OC,

to

stacking

polymer

bases

find

that

from

the

showed

and the reaction

almost

was

some

00

shown

by

nuclease, alkali

no

interactions of

micrococcal by

are

system

phosphorylase

37

or

of

WV and

u-benzoadenylatc

acid) the

be

interactions

polynucleotide at

to

hydrolytic intensity

in

chromatography

Mne*

by

found

u-bento-AMP

nucleic

attributable

integrity

of

surprising

poly(m-benzoadenylic

a mixture

The

stacking

anhydride

properties.

determined

fluorescence

dramatic

intramolecular the

its

as

149d,

phosphodiestrase,

primer-independent

and

or

is

3.7

failure

increase

isolated

the

by

than

5’-pyrophosphate.SSm

structural of

is

That

moieties,

the

in

the

enzyme.

to

quenching

observation

array

the

snake

of

other

was

nor

m-bentoadenosine

is

there

WV spectrum

Accordingly, which

material,

m-benzo-&DP

from

to

m-benzo-

u-benzo-ADP

solution

and

the

strong

fluorescence

acids.

mixture

highly

1 PM lin_benzo-C\DP

indicated

by

rings

proximity

amino

polymeric of

of

is

a

remains

and

m-benzo-ADP for

was in

of

tricyclic

,

the

due

magnitude.

indicative

two

sites

2ADP

carrier.

with

of

alone

P_x,~-di-~-benzoadenosine

accompanied

orders

of

highly

using

CITP,

that

a

the

m-benzo-ATP

the

of

that

p * ,_P-diadenosine

was

two

fluorescence between

of

by

particles

by

aqueous

rate.

solution

cleavage

to

m-benzo-ATP.xe4

lifetime

to

nucleotide

hypochromism

hydrolytic than

is

is

of

to

yield

ATPase

system

stacking

done

binding

addressing

appreciable

aqueous

percentage

means

an

adenine

or

dilute

in

in

for

mitochondria at

relate

Neither

potently

fluorescence

reached ring

return

to

AMP,

hydrolyze

quantum

S pM mitochondrial

intact

ADP

converted

fluorescence

the

nitrogen-heteroaromatic analogue

l_&2-benzo-CITPl we

~iento-CIDP

site.

~111

more

valuable

conclusion

have

generates

the

not

(149a-cl

example,

by

of

permits

disproportionation

submitochondrial

fluorescence

especially this

(85%)

is

inhibited

nm and

(from

pyruvate

also

site.

and

derivatives

with

conversion

If

we

kinase.

mitochondria,

The

PM).

enzymatic

achieved

possible

This

CIMP binding

wbenzo-ADP is

with

satisfactory

this

the

be

mixture

one

(14%)

liver

ATPare 27

the

and

should

sites.

the

that

u-benzo-ADP

will

its

to

consistent

was

formation

adenylate

while

at

react

of

kinase it

ADP

Thus,

corresponds can

16 PM z.

the

a

(149bl

dehydrogenase.ixo

that

CIDP with

enzyme,

that

wbenzo-ADP

adenylate

of

observation

an

efficiency

lactate

note

m-benzo-AMP.

a-benzo-AMP

and

one

and

with

for and

we

to

u-benzo-ADP

ATP,

mentioned

*benso-ADP The

enzyme

I?TP,~.*~~

AMP site

unchanged

50

kinaae

of

&MP and

specific

from

assay

pyruvate

consideration into

already

&D-bento-fiTP

Iem

ligase.

phoaphoenolpyruvate.

of

involves

counterparts,

T4 RNA

to

and 2 5 its

snake

1951 m-bentoadenosi Uu

band

of

species

and

structure IDP,

( 1 1s) ,

ne the

polymer

was

of

of

effect

with and

loss

of

of

fluoresCWnC~~

characteristic

return

fine

enzymatic UV band

long-wavelength

hge*

and

the the

Upon

broadened.

the

polymerization

presence

with

lacked

polynucleotide

a GpU

primer

UV fine

or

Thr

alkaline

was

The

the

was by

accompanied

structure.*”

of

hydrolysisr In

reappeared. phosphorylase

and

long-wavelength

structure

extent

of

case

the

monomeric

the

fine

of

possible

m-benzo-

in

a strong

the

hypochromic

polymerization

use

not

determined. m-Bento-ADP human be

was

platelet-rich

active,

but

The

has

Roskoski’s

least

cyclic

with

kinetics to

2.0

PM,

2.4

A wider

similar

The catalytic

subunit

as

observed

well

in

of

catalytic

in

polarization.

with

m-bento-ADP

and

with

the

and

2 moles than

the

free

Perrin

and or

values

type

no

titration

catalytic

20*

the Thus,

was

amino

acid

excited

for

the

bind

also

in angle

if

not

all,

mbenzo-ADP

at

this

in

determining near

bound

affinity

of

bound

from

the

a-

holoenzymr,

140

calculated

observed

rotation

both so

or

the

both

higher

the

that

is

catalytic

there

is

Fiuorescence-

site.

or

for

agrees

skeletal

and

that

9.0

method

and

the

subunit

of

from

and

rigidly,

binding

I&

rotation

indicates

with

catalytic

with

260

of

increase by

out

The

subunit of

an

presence

concentration

of

is

was

the

by

calculated

values

which

a 5% of

catalytic

in

constant

fact,

state,

9 which

useful

change

respectively,

catalytic

adenine

residues

No

holoentymes

PM,

average

the

most,

the

carried

by

steady-

which

of

accompanied

at

II

3.4

holoenzyme,

itself

the

ADP.

was

(b)

Type

theoretical

protein

within

confirming

nucleotide

chemical

binding

site

of

subunit.‘e***-

measurement

of

fluorescence

displacement

skeletal

muscle

selected

competing

from

decrease

the

and to

with

determined

and

The of

holoenzyme

rotation

of

of

25O

the

II

of

subunit.

close

of

mole

lifetime

as

modification

The

per

3.5

inhibitor

site

and

by subunit

concentration.

K_..

of

to

adenosine

investigated

u-benzo-ADP

was

and

of

catalytic

J&l-benzo-ADP

subunit

determined &

the

polariZ*tiOn

degree

catalytic

equation.

rotation

subunit little

free

of

enzyme

to

from di-

m-benzo-ADP,

active

product.

constant

m-bcnto-ADP

catalytic

are

at

increasing the

data

the

the

titration

concentration

gave

during

respectively,

to

the

subunit

kinetically

brain

polarization

from

(a)

the

Thus,

to

spectrum

to

was

For

reaction

emission binding

of m-benro-ADP

benzo-ADP

kinase

P(M).“* binds

natural

polarization

ion

muscle

the

but

catalytic

concentrst

,

added

meaWAre-

emerged

holoenzymcs

II

a competitive

is

has

m-benzoadenosine

type

Fluorescence-oolarization

the

increasing

(9.0

fluorescence

subunit,

measuring

well

as does

the

ADP moiety

pOlarizetiOn thrt

of

protein

when

J&l-benzo-ADP

ADP.*ee

fluorescence

spectroscopy.

for

inducer showed

methodology

and

(149bl

adenine

than

interaction

dependent

that

the

the

subunit

fluorescence

to

in

active of

in

aggregation

mild

determinations

facilitation

m-benro-ADP

ATPI

very

less

realized

the

and

a

times

leboratory.Se*-**o

responds

its

the

amply

be

Comparative

3’,5’-monophosphate

state

the

to

200

of

been

triphosphates

due

at

prediction

ments”9

found

plasma.

type

displacement

titration

technique,

tenacious

binding

of II

protein

nucleotides.‘mT in

fluorescence

from

the it

of

the

polarization

m-benzo-ADP

catalytic

is

possible

nucleotide

was

from

kinase The

the

with &

increasing of

polarization

t0

m8p

moiety,

each

2y the in

used

to

subunit

this

this

of

was

tin-benzo-4DP

exact

determine

concentrations nucleotide

of

subunit.

also

catalytic

fluorescence requirements exrmple

by

the

bovine of

calculated

that

accompanied

disDl#cemen\ for variants

the

most

N. J.

1952 including

modification

multiplicity,

and

is

possible

of

lirr_bcnzo-CIDP

to

ldenine

the

St-terminal

determine

which

protein can

the

the

the

of

CIDP)

indicated

role

cCIMP at

to

the

5ame

binding,

We have of

group,

using

promote

presumably

amsume

ribo5yl

By

ions

metal

kina5e.Sm7

m-benzo-cNlP

ring,

polarity.

therefore,

(and,

CAMP-dependent that

of

the

and S. P. HIRE~UTH

LEONARD

and the

to

what

catalytic

earlier the

pho5phate

methodologyt

in

rubunit

thir

regulatory

it

extent, of

mection

mite

of

thl5

enzyme. 65 kinase by

in

the

ca5e

in

the

presence

mbenzo-ADP

KM as

protein ADP;

ca5e

that

of

the

kinase

kiname,

the

the

limit5 binding

divalent

cation

and

presence

of

Thic by

of

effect

wa5

other

with

monomeric

1 in-Benzo-&TP. degrees

of

acetate

kinase, kinare,

benzo-ATP

represent5

ability

for

bringlnq

the

wider

also

the

are

behavior to

in

kinsse

ha5

substrate,

the

which

i5

gluco5e values torilY9

the

of

for ADP

which

and

in

the

indicating

and

CIDP sites, by

yeast

same that

range the

ATP is

to

does

mite

together lowered

r PO

that ha5

with

throughout. u-benro-ATP

the at

can

an

extra

paired

the

The by

accommodate It with

their In

is

as

not

with

direction

long The

can

stretched-out

least

as

kinamc.

together

lower

Adenylate

in

CITP and,

at

efficiency.

which

unless

only

an

required

triphomphate

m-benzo-AMP,

function

is

catalyzed

conformation

with

to

for

hexokinase,

acid

enzyme

it

analogues.

CT-VI-m-bento-ATP.

not

con5imtent

activity

among

ATP

adenylate

site5,

site

for

thenucleoside

respect

rub5titute with

those

m-

natural

structural

but

dinproportionatem

can

NIP

the

in

leading with

u-benzo-ADP

the

catalyzed are

PGK,

u-benzo-ADP

to

and

of

the

analoguc, The

unique

the

m-bcnzo-&TP

Y phosphate

above

b-Benzo-ATP

converted

specificity separate

the

that

(PGK),

utilization from

ATP.

is

varying

including

kiname

case5,

than

thm of

with

enzymes,

Sphorphoglyceric

mite

u-benzo-ADP,

CC5C

benzo-CITP.

of

etringent

am

substitutes

deviation

is

observable

dimsociation

stretched-out

u-benzo+TP

indicates

by

dimcumsed

and

the as

phosphorylate

represented

premence

been

more of

kinase,

exchange

of

the

conditions.‘e”

all

known

a

the

by

a5

of

phosphoglycerate

phosphofructokinese,

in

further

a-benzo+DP

transfer

In

upon

pho5phorylation.wDSmP

(149~)

presently

dependent

increaee

rai5ed

using

phosphoryl

(PFK)

phosphoglycerete

nucleus

-PCLe-

of

protein

binding.

small

of

in

titration

was

extent

variety

than indicates

m-benzo+DP,

the

hexokinamc.m~x~o

inhibition the

a

concentrations

of

m-benzo-ATP

Permits

emtimating

20

affinity

cGMP-dependent

glucose

of

u-benzo-ATP

largc5t

G-phosphoglycerate

for

it

yeast

higher

so

of

a

the

At

concentration,

in

number

inhibitor

requirements

yeast

for

of

binding

under

that

a

was

polarization

in

hexokinase

and

CITP.

allosteric

useful

the

of

affinity

polarization

kinase

which

& cGMP-

tighter

binding

supported

about

,

enhanced

phosphofructokinase

adenylate

cofactor

brought

a

the

polarization

this

ions

inhibitor

We found

efficiency

to

m-benzo-ADP

h-fold

in

to

to

For

over

bound

competitively

similar

fluorercence

metal

Fluorescence

be

PM,

Fluorescence

hekokina5e

ADP. may

The

which

of

with

difference

m-benzo+DP

of

22

protein

inhibited

titration.*-

rigidly

a competitive

ligand into

is

cGMP-dependent woo

KS of

binds

detection.

of

yeast

a

a greater enzyme.

of

indicative

means

fluorescent dimeric

is

delineated

B-lyxose,

with

m-benzo-aDP

polarization

addition

ATP

moiety

the

kinasc, peptide

polarization

CAMP-dependent

that

fluorescence

to

there

u-benzoadcninc

within

protein

pho5phorylatable

fluorescence

i.e.,

revealed’

The

of

respect

by

than

doe5

c&MP-dependent

with

determined

dependent

the

of

AMP

of i5

difference

m-

present, in

complementary the

a

ADP,

and

pho5phorylation

of

replace

CITP.

The

analogue

bind5

5ati5fac-

dimencional

toler-

2.4-A

I(n

Dimensional probes of binding and activity

ancet of

but

the

bound

and

as

ATP.

transfer When

Substantial

gluCOse with

and

mitochondriai

ATPase,

displayed

negative

apparent

Michaelis

presence

of

the

conversion

phosphate, and

of

and

fluorescence which

with

tyrosyl

binding

are

in

and to

with

the

has

shown

n

the

the

to

London-Schmidt

tloreover,

displayed

aspattate

that

it

when the

does

and

the

for The

bound

ATCase,

to

and

plays

a

extended

ATCase

for

ATP,

with

constant

activator

group

The

catalyzes

polarization.

of

hypothesis

the

carrier.

association

amino

activity

compounds the

in

which

carbamyl

extent

quenched

the

and

by

Both

nucleotide

between

Instead,

hydrolysis

(ATCase),

The

not

phosphate

_J&fibenzo-ATP

fluorescence

is

the

determined

adenina

same

by

binding

for

HCDe-. ATP

aspartate the

juxtaposition for

similar.“*

J_&benro-ADP,

interactions

protein.

and

by

similar.*“1

u-brnzo-ATP

the

be

and

for

determined

of

to

transcarbamylase

are

was

of

process,

accord

a substrate

absence

in

L i ke

the

favorable

substrates

found

activation

phosphate

ATCase

the

residues

the

and

that

as

lia-benzo-ATP

constants

emission

indicates

as

were

&benzo-ATP

association

lin-benzo-ATP

compared

they

aspartate

of carbamyl

occurs

their

were

equal.

as

suggests not

for

were

activity

activation

alloSteriC

ATP

constants

any,

rate is

cooperativity

10 mfi HCO,-

if

in

m-benzo-ATP

lin-benzo-ATP

purified

little,

reduction

bound

1953

key

role

ATP

in

analogue

activation

vs.

inhibition.*== Uhlenbeck substrate

under

a catalyst

for

synthesis it

in

inhibit

vitro,

m-benzo-ATP

is

into

a

j&-benzo-ATP

This

enzyme

catalysis

bioluminescence induces

of

excited

titration

and

light

with suggests

change

sites

in

CAMP-and

regulatory

11.3 of

PM,

that

than

as

close with

ATP,

to ATP,

that

[Y-m=P]ATP

was

parameters

of

case

of

has

nor

does

reported

that

rifampicin-resistant of

which

is

Watson-Crick the

the

role

rx of

ATP, to

luciferase

color

of

a of

extension

in

template pairing

protein,

and

the

RNA

base

firefly

in

lateral

that

of

is

according

be

to

an -

luclferln

reduced

rate

of

the the

1uCiferaSe

Purina

in

has@

the

vicinity

ATPI

as

11.9

from

protein

used

the

for

the

m-benzo-ADP

subunit kinase

with with

for

kinase

with

the

K_m for with

v,,,

best

catalytic for

CAlae*Ihistone

in as

of

&i-D-

phospho-

paptides

water,

Leu-Arg-Arg-

&&benzo-ATP

nucleotide

m-benro-ATP

the

means

lin-_benzo-ATP

subunit.“. use

the

by

Ui th

aCCepkOrS.

observed

The

defining

kinases

substrate

phosphoryl

PM.

for

protein

a good

-lin-benzo-ATP

reported

catalytic

fluOresCenCe-pOlari2atiOn

protein

acceptor,

makes

been

of

titration

cGMP-dependent

subunit

which

cGMP-dependent

methods

CAMP-dependent

phosphoryl

synthesized the

a

have

binding

conformation

displacement

of

activities II

the

Roskoski’s

transferase type

the

Variation that

in

WElenzo-ATP

the

in

Kornberg

a as

RNA synthesis

in

u-benzo-ATP

enzyme

as serve

RNA

DNA-directed

With in

shown

it

emitter.

earlier

Ala-Ger-Leu-Gly

normal

separation

ATP.

not

a

synthesis

strong

bento-ADP.,-L.“..‘=.

or

primase,

the

production

compared

fluorescence

ATP-Catalytic

and

that

We have

displays

light

We mentioned

strand

analogue

reaction

by

chain,

(Ap1.C

does

ATP

polymerization.

duplex

for

replace Rowen

indicate

8ertsch.x3m

incremental

an

the

in

and

substrate *=a

incorporated

experiments

works

acceptable

get

to

but

T7 phage

In not

RNA.**O

into

template-directed

Scott,

system.

does

phosphate

kinase,

pCp.*a=

polynucleotide

These in

Kornberg,

and

incorporation

*a4

exacting

(Apl&

not

transfers

T4 polynucleotide

m-bento-ATP

does

directed.

&&-benzo-ATP

by

linking

ATP

polymerase,

that

catalysis

was was

20%

substrate*

other

m-Benzo-

determining

the

In

substrate.“’ H2B-(I??-3%)

kinetic

as

the

the

N. J. LFDNARD and S. P. HIREMATH

1954 phoephoryl that

acceptor,

of

#TP

as

ATP, the

binding the

32

the

PM,

the

subatrate.*ee

region

of

to

the

k&, for

&,_,‘

with

Thus,

arrangement

does

bis-5O

and

fluorescence

interaction

has

also

I’-protons base

of

and

monomer,

his-5’

association

that

A(5’1p,(SilA

and

oligophosphates

as

chain

for

the

A from

and

A

when

quantum

yield

adenylate

to

short is

by

pyruvate.*e which

of

to

,

DI-

in

reaction

of

true

As

of

,

9-, water.”

related that

the

enough

P*-(lin-

dinucleosidr

is or

with

the

are

an

it

low

the valuer

is the is

phosphate widened

fluorescence

ubcnzo-AMP,

increased.

stacking in

the

base

with

are

intramolecular

Since

intramolecularly

their

lifetime

constants

whether

terminal

compared

enzyme

indeed

penta-

inhibitor,

by

as

end1

are

between

produced

analoguer

made

association

for

one

judged

one

AMP and

intermediate

p-1

and

were kinase

experiments.

has

“open”

TRI~~OX~Ri~n

or

when

The been

reversal

broken

“extended”

and form

of

than

dADP

when

d-m-benzo-ATP

of the

the

monophosphate

showed

polyCdlATl3

with

binds ,

lower

was 1, was

made

as

the follows:

the

activity with

to

tested

in low

observed..w

the

and

pyruvate

kinase

better

better

in

morpholidatelee

kinase

the than

pyruvate

a very

via

the

pyruvate

reflecting

moiety

m-benzo-ADP

were

(149a-cl,

2’-deoxy-m-benzoadeno%ine;ew-e&

diphoephate

-lin-benro-ADP

DNA polymerase

DERIVATIVES phosphates

phosphates of

m-benroadenine

site.

into

6-, in

the

(at

effective

these

the

This

4-,

close

mixed

and

1Oe M-*

is

m-benzoadenosine

phosphorylation

activity

Ca-eePITTTP

ATP

fluorescence

to

yield the

bound

x

lifetime

both that

the

lower

~01i

of

ldenylate

of

kinase

2

9 as

u-benzodeoxyadenosine

also

to

most

p+

the

indicates

stretched-out

effect

solution

present

active

E.

A from

fluorescence

are

case

aromatic

level,

the

the

m-benzo-A(S’lpefS’lA

constant

2’-Deoxy-Lirr_benzo-ADP

is

extended

with

and

While

phosphorylation

the

ATP,

chain.

chemical

chemical

and

by

and

rdenylate

similar

aqueous

noNo-,

by

of

efficient

dilution

those

rings

respect

kinetics

2.7

signifies

corresponding

rdcnine-

earlier.“* of

which

inhibitors The

with

Af5’fpe(5’tAr

in

and

the

the that

intramolecular

infinite

The

known

muscle

by

inhibitors

in

to with

experienced.

and

m-benzo-Al.

oligophosphate

tri,

ir

strong

than

dilution,

tetraphosphate

(u.

free

fluorescence

As

by

field

nitrogen-tricyclic

with

a qualitatively

kinase

these

close that

for

indicated

shifts

at

higher

infinite

inhibition

lengthened

stacked

to

mentioned

NMR chemical

anisotropy

porcine

determined

that

is

mono,

at

two

competitive

of

A(.5’lp+(51)A

suggestive

the

at

the

compare

QD-benzo-A(5’1p4(5’lA

that

the

A(5’)p,(5’)A.e.~“I*L1. are

M-z

phosphate

of

are

magnetic

to

inhibitors

(2.4

by

evidence

J&-benzo-A(5’1p4(5’lA probes

lp

of

to

I-adenosyll-P-(5’-adenosylltetraphosphate

oliqophosphatee

for

The

-adenosvll-Pe-(5’-adenosvlloentaphosphate.

x

also

Y-phosphate

was

,51-E~-benzo-AMP-2-@,l

also

the

dimensional

2

shown

holds

P’(m-Senzo-5

of

hM, 0.06%

equivalent

the

,S’-Cm-benzo-AMP1

protons

lin_benzo-AMP, tether

potent

29

only

&-benzo-ATP is

position

not

spectroscopic

been

anomeric

diphoephatc

benzo-5’

of

kinasa

5’-pyrophosphate. of

ultraviolet

The

was was

acceptor.

interaction

and

binding

protein

P*,Pe-Di-~benzoadenosine stacking

u-benzo-ATP

J&-benzo-ATP

while

cGMP-dependent

conformational

transfer

observed

but

ADP 9 so kinane.ee

a standard level

of

than of

region*e7

the

incorporation

Nearest-neighbor

of

the showed

triphosphate

nick-translation v

dADP, the

d-m-benro-ADP At

the

phosphoenol-

accommodrtion

hydrophobic

di,

and

analysis

experiment from

Dimcssionaiprobes of bindingand act@ indicated

that

positions

within

analogue the

the

caused

incoming

template

analogue

the chain is

termination

erswntirl

deoxyribonucleotider

and

benroadenosine

can

However,

would

there

mispositioninq site

form

the

And thus

basis

of

next

our

to

by DNA polymerase

Watson-Crick

of

linkage

the

A-T

the

the

with

that of

in

that

d-m-

thymidinw in

fl51).

of

the

chain,

the

active

incoming

next

cannot

be ruled

helix

interferes

the

of

the

thw phosphodieeter

m-3’-phosphate

distortion

the

pairing

base

d-mbcnzo-A

explanation

that

I among naturrl show

pair in

terminating

with

that

base

distortion

An alternative is

corresponding

The experiments

ansloguos.

be resulting

experiments

residues. the

internal

showed

blatson-Crick

to selection

their

preventing

requisite

ldenine

into

reactions

triphotphate

3*-hydroxyl

deoxyribonucleotide.

incorporated

DNA-sequencing at

a widaned

then

significantly

while

dwoxynucleoside

strand

not

was

polymer,

1955

out

on the

with

the

pairing.ma7

$Rib

The

ring

corrin

represented

system

by

the

is _

co 27

rhombaid

152 AIWUYRUE UP COENZYWE B ,I The

analogue

synthesized

of

in our

coenxyme

benzoadenosine.**e special

152 is

hetwrolytic

the

products. of

is

in

pair,

also

detectable

the

prerequisite will

reduction

first for

the

is

moiwty

intermediate but

is

case

Campound

not

152 was

ribonuclwotidc

of

on binding

observed

indicate%

that

or

forms

detectable the

offered

Thus, (light)

bond.

dissociated.

in the does

analogue.7-Sn

s and

partially

was

moiety

on homolytic

carbon-cobalt

reductasc

catalysis

of

but,

by EPR in the

be completed.

inhibitor

J_&-benzoadcnosino

fluorescence

ribonucleotide

(l%Il~

and !5’-chloro-S’-deoxy-lin-

coenzymw

of

thw m-benzoadwnosyl

to be

the

solution

In addition,

to

the cocnzymr

competitive

of

cleavage

when

considered

cobCI3alamin

“profluorescent”

enzyme

radical and

the

cyanide)

analogue

polarization to

in

nonfluorescent

(acid*

fluorescent coenzyme

&&--benzoadenosylcobalamin

from

The fluorescwncw

advantage

compound

B,m,

laboratory

of

it

is

bound

loosely

The stabilized

adenosylcobalamin,

is

ribonucleotide

reductase

guarAnt@w

ribonuctwotidw

found

reductase

the

fluorescence

that

reaction

to be an effwctivw

from

Lactobacillm

leichmanit.

SunncIRY At43 PROSPECTS The

concept

synthesis and

of

of

dimensional

analogues

ribonucleotides

hwterocyclic formally

probes differ

by defined

basses hAvw

inserted

interesting

that

between

activity

been

of

from

binding natural

dimmAional made

terminal

in biological

with

and

activity

bicyclic

chrngws.

has

bases,

LinwArly-extended

carbocyclic

ar

heterocyclic

and

af

these

rings, systems.

some The

etimulated

compounds

hrvw with

the

ribonuclwosfdw8~

spacers shown heterocyclic

N. J. LEONARDand S. P. HIREMATH ring

mpacerm

introduce

hydrogen-bonding the

dimenmional

peripheral is

change

ribonucleotide

levels,

help

or

for

ample,

intact

with

phore

of

the

including the

of

binding defines

the

enzyme

titratfon

and

requirements probes challenge

offer

-

analogue of

of

and

the

Health,

of

Dr.

the

natural

work

U.

Probem.‘ee

Grants

S.

Public

M.

Stevenmon,

literature

which

for

at

an

the

be

The

Service.

University we have

of

this

Illinois

combined

from

authors

under

to

map

site.

or

the

requirements -

yield,

the

natural with in

any

laboratory the

for

his

the

title

exact

CIdditional a synthetic

initial of

for

lifetime, riborempect

to

total

ham been

National

acknowledge ,

of

independently

incorporated

Gtl 34125

indicative

constitute

of

from

herein

fluoro-

fluorescence

information

described

and

The

spatial

behavior.

ex-

The

example,

enzyme-•ubmtrate

of

conditions,

active

anrlopues

must

for

are

behavior

provide

to

valuable.

fluorescence-polarization

dimenmionm

that

GM 05829

Health

,

moiety

term

factors

rotate?, of

the

lxpremmion

mymtemm,

inhibitor

titration

active

the

lifetimes

techniquem

fluorescence

continue

for

particles.

enrymem.

fine-tuning

The

substrate

The

Research

of

extended, will

or

Thomas

diffuse

further-extended

enzyme-coenzyme

by

of

or

potential

to or

the

most

environmental and

of

if

the

be

the

biological

to

activator for

activity.

delineate

inhibitor

a nucleotide

laterally

Acknowledgment. supported

the

They

yield,

of

coenzymem

and

or

basis

a substrate

and which

and

that

to

in

change probes

series,

of

sensitive

made

ribonucleomide

mubmitochondrial

displacement

binding

biological

nucleotidem

picture

the

is

fluorescence

and

polarization

both

activator

intermediate

and

is

electronic

been

orientational

continue

complex

versus

but

almo

dimensional

m-benzo

mymtemm.

fluorescence

whether

and

for

with

binding

of

the

the

in

mitochondrio

an am

probably

limitations

inactivity/activity

and

by

linearly-extended

will

enzyme

changes

bamem have

qualify

in

and

lin_benzoadenylatem

polarization with

not

the

lpatial in

stacking,

mode

been

the

inhibition

responsible

at

do

particularly

have

establish

activity

is

dimenmional

tricyclic

lupplemented

Theme It

probem

definable

ham been

applied.

dimensional

only

Clngular

lubmtituentm.

strictly

They

not

changem.

the

Institutes

lmmimtance survey

Dimensional

of

Dimensionalprobesofbinding and activity

1957

-0

%ee

Buraor's

I

8. R. Baker,

!

Jnhibitorr. -G.

Chmmistrv,

WiImy-Intmrscimncm,

Wolff). see

tlmdicinal

Qmsia n New

Wiley,

Part

York

Iii

ld.

4th

(Edited

by H. E.

(1979).

Irrmvmrsiblm Enzvm

hctivm-Site-Qirmcm

of

York

Biochmmistrv

Wmbmr,

Now

(1967).

11, 864

(197231

*mm

-0,

also

e361

326

(1972). 4N.

vmtmrpcvclms

J. Leonard,

12,

9rl. J. Lmonard,

clcc. Chmm.

+J.

in B)oIoaicallv

Lmonard,

J.

by W. Vomltmr

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Rmq.

129 15,

(1979). 128

Activm

D. 0. Davms),

(1982). Princiolms

237-249.

pp.

Product*

of Nmturml

Thimmm-Stratton

Inc.,

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New

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Pure

frl. J. Leonard,

Biooolvmmrs

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R. G. llorricm,

J. Lmonard,

b RDDllmd

Chmm.

24,

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9-28

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(1985).

M. CI. Sprmckmr,

?. Ora.

. 40,

Cm

356

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Svmpomium

Aced. SSJ.

Sci.. 60,

R. Barrio,

J, Am. SW.

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Liu,

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0. E. Kmysmr,

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N.

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P. VanDmrLijn,

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H. c\. Sprmckmr,

J. Leonard,

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l mm

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