Phagocidal action of oxidized spermine and its analogues

Vol. 28, No. 1, 1967

BIOCHEMICAL

PHAGOCIDAL

ACTION

AND BIOPHYSICAL

OF OXIDIZED

Hiroshi Fukami, Ichiro Tomida, Toshikazu Oki**, Haruhiko

Pesticide *Research

Research Institute

**Department

Received

of

(Ia),

a naturally (Hirsh,

plasma

amine

oxidase

1962).

The

oxidation

certain

bacteria

(Bachrach,

of

has

and

nucleic

been

(IIa)

ANALOGLJES.

University, Kyoto University,

Kyoto Kyoto

University,

the formyldiamine

been

provided.

action synthesis and

to

Kyoto

to

be

of

al.,

1964).

However,

of evaluations

the

of

in

their

the

because

a pure

culture

of

T-uneven

to

interpreted

between The

in

oxidized

oxidation

with with

the

pro-

phagocidal

19

of

the

difficulty

direct

toxic

alcohol

as

effects

activities

as

of its

not

the

yet

phago-

bacteriophages. homologous

is

isothat

has

of

variety well

of

evidence

re-investigations a large

(IIa)

corresponding

any

form, for

deals

formyldiamine

in

1965).

to

NaBH4

responsible

spermine

cells

been

toxic

N,N'-bis-(2-formylethyl)-butan-1,4-diwith

oxidized

be

have

beef al.,

to

complex

al.,

et

known

coliphages

findings

by

Yamada,

mammalian the

et

reduction

communication

is

inactive

the

(IIa)

and

oxidized

1954;

spermine,

These

(Bachrach,

is

al.,

inactivate

acids

is

et

1961)

a biological

(IIa)

present

al.,

of

formyldiamine

the

The

et

polyamine,

Tabor,

1963).

established after

lating

pounds

ITS

Hideaki Yamada*, Koichi Ogata**

Kyoto

oxidized

and

et&., formation

(Tabor,

The

Kyoto Science, and Chemistry,

1953;

et

19671,

the

(IIId)

tidal

al.,

(Bachrach,

spermine

amine

Morino, and

occurring

product, (Tabor,

et

series

duct

Agricultural

AND

May 22, 1967

Spermine

terms

Tetsuo Kawasaki**

Institute, Food

for

SPERMINE

RESEARCH COMMUNICATIONS

also

compresented.

Vol. 28, No. 1, 1967

BIOCHEMICAL

Oxidized reaction

spermine

mixture

crystalline and

lume

of

150

and

1964)

by

was of

of

al.,

diamine

usually

completed

theoretical

amount

prepared

19621,

buffer,

was

from

250-500

al.,

phosphate

of

pH

1964;

Israel,

(IIa)

was

et

through

780/17mm)

was

any

difficulties

of

cata-

6.0,

oxygen.

synthesis

in

(Va)

a crystalline

salt N,

12.10:

formyldismine

The

of

of aqueous

the

analogues

calcd.

for was

to

The

corresponding

or

(IVa)

was

(Va)

formyl-

in

dry

condensation

reduced

45.55;

C, its

by

oxalate

with

(Va)

procedures,

succinamide

from

8.92;

H,

with

introduced

found:

(m.p.

subsequent

prepared

was

(Anal.

diamine

20

chloride

diamine

C18H4202N4:

similar

the

The

144-l&5"),

and

of

N,N'-bis-(3,3-diethoxypropyl)-

crude

as

by

'-bis-(3,3-dietnoxypropyl)-butan-

(m.p.

was

(IIa)

3,3-Diethoxy-

succinyl

succinamide

obtained

(Vb) the

al.,

(Tabor,

synthesis

triethylamine.

give

N,N

overnight

According

via

et

formyldiamine

steps.

with

bis-diethoxypropyl

butyl)-butan-I,&-diamine

of

N,N'-bis-(2-carboxyethyl)-

following

of

The

oil. nitrate

solution

ethanol.

with

(Israel,

the

chemical

condensed

afford

its

(IIa)

The

to

a yellow as

yo-

3 hours,

(IIId)

amount

to

as

a final

(111~)

the

84-85O).

(m-p.

tetrahydrofuran

1,4-diamine

104-105~/3ti)

tne

1964).

al.,

without

in

in

300 units

within

(IIIa),

performed

(IVa)

acid

in

equivalent

succinamide

treatment

the

(Ic)

successful

an

proceeded

by

of

oxidation.

containing

8.93;

units

N,N'-bis-(3-aminopropyl)-ethan-1,2-

N,N'-bis-(2-cyanoethyl)-

(b-p.

benzene

aqueous

spermine, et

The

apparatus.

N,N'-bis-(3-hydroxypropyl)-butan-l,k-diamine

propylsmine

The

of

a Warburg

N,N'-bis-(2-carbomethoxyetnyl)-butan-I,&-diamine

oxidation

H,

the

in

(Yamada,

reaction

enzymatic

attempt

or

LiAlH4

pmoles

potassium

were

similar

reduction

(IIIb)

30

30"

RESEARCH COMMUNICATIONS

N,N'-bis-(3-aminopropyl)-hexan-1,6-diamine

No

* et

of

spermine

(Ib)

by

The

at

oxidase

of

ml.

consumption

oxidized

of

amine

pmoles

3.0

prepared

consisted

plasma

lase

the

was

AND BIOPHYSICAL

C, N,

235-245" excess

to

45.46;

11.81%). (decomp.))

of

recrystallization

oxalic from

N,N'-bis-(b,f+diethoxy4,4-diethoxybutylamine (IW)

(m.p.

(b.p. 101-102~).

Vol. 28, No. 1, 1967

Table

BIOCHEMICAL

I.

Effect

AND BIOPHYSICAL

of oxidized spermine various bacteriophages.

of The incubation mixture suspension, 0.1 ml of oxidized lution buffer, pH 7.4. The The number of phage (plaque layer method (Adams, 1959).

on

RESEARCH COhVvWNlCATlONS

the

viability

contained 0.1 ml of the indicated bacteriophage spermine (0.6 umoles) and 0.8 ml of the diincubation was carried out at 30" for 90 minutes. forming unit, PFU) was determined by the agar-

PFU/ml Bacterio-

Host

Concentration

Phase

0.6

0 Escherichia

T2

coli

2

II

T3 T4 T5 T6

3 IO8

109

9 105


It

1 109

9 lo8

11

2

II


IO8

4 IO8

3 lo8

II

6 lo8

6 lo3

x +3oc

11

4 107


I,

2


MS2

11

4 109

+x174

11

2

T7

Salmonella

P22

15

Salmonella

I34 M2

IO8


107

1 107

typhimurium

5 IO8

3 IO7

anatum

4 IO8

2

1 107

4 105

11

IO4

5 lo8

2

SPIO

II

4 IO3

3 103

Al

II

2

105


Bacillus

F4

The

(mM)

subtilis

Brevibacterium

107

lactofermentum

2

108


P465

II

6 lo8


~~85111

11

8 lo7


1128T

Pseudomonas

glycinea

3 105


I2418

Xanthomonas

phaseoli

PK66

Streptomyces

griseus

9 IO8 6 lo8

1 IO3

bis-diethoxybutyl

phosphate

(m-p.

C20H4404N2-2H3P04:

diamine 148-150°, c,

(Vb)

Anal.

41.95;

was found:

H,

8.80%).

21

crystallized C,

41.46;

in H,


a state 9.09;

of calcd.

its for

BIOCHEMICAL

Vol. 28, No. 1, 1967

The is

phagocidal

summarized

series

in

were

only

E l5

PK66

of

dized

phages

are

summarized

almost

and

activity

same

were

not

inert

spermine, -NH-(CH2)n-hWthe

that

of

that

T5

results

and

and

(IIa)

is

that

both

are formyldiamines

on

a linear

in-.

function It

is

between

coliphages

oxi-

of

as

and

such

as

even

at

in

much

this

well

for

groups

of for with

the the the

the

and

T .

In

5

phagal

22

T

the

hydro(IIIa),

a separate

experiment,

y-aminobutyr-

direct

indoleacetaldehyde (6

mM).

evidences

phagocidal

action

-CHO

and

the

is

not

clear,

nucleic

same

acid

concentration

It

5

the

hydroxypropyl-diamine

and

give

5 at

cyanoethyl-

butyraldehyde,

terminal action.

as

the

higher

paper

T 2 andT

Essentially

However,

T2

of

(IIa)

inactivated

spermine.

(111~)

responsible

react

The

formyldiamine

imidazoleacetaldehyde

essential

(Bacillus

spermine.

(IIa)

(Vb).

phages

presented

Al

interaction

polyamines

aldehydes,

phage

coli); lactofermentum);

in

the

by

inactivated.

synthesized

oxidized

(Va)

other

also

formyldiamine

oxidized

both

reported

h (Escherichia

oxidized

to

carbomethoxyethylfor

inactivate

formyldiamine

proceeded

compounds

phenylacetaldehyde,

The

spermine

chemically

The

with

been

inhibited

acids.

II.

diamines

shown

were

due

were

phaseoli);

of

T-uneven

(Brevibacterium

gliseus)

is

of

as

(IIIb),

aldehyde,

how

rate

F4

the

series have

(Xanthomonas

homologous

observed

carboxyethyl-

~~85,

nucleic

Table

T-even

+~oc,

concentration

actions

bis-acetal

was

phagal

in

was

(IIId)

and

structurally

the

lyzed

oxidized

bacteriophages of

coliphages MS2,

~468,

various

coliphages the

the

phages;

inactivation

phagocidal

other

did

the

spennine

and

on

RESEARCH COMMUNICATIONS

on

that

that

I2418

time

that

The

and

; P465,

by

spermine shown

(Streptomyces

incubation

conceivable

was

glycinea);

and

activation

It

Other

anatum)

(Pseudomonas

subtilis)

oxidized

findings

(1963).

al.

of

inactivated

Similar

(Salmonella

both

I.

markedly

--et

1128T

it

Table

slightly.

Bachrach

of

action

AND BIOPHYSICAL

acids

that of

oxidized

internal however, to

form

the

the

BIOCHEMICAL

Vol. 28, No. 1, 1967

Table

II. on

except

Conditions that

0.5

Effect of the viability

of the pmoles

AND BIOPHYSICAL

the

incubation of the

formyldiamine of coliphages

were formyldiamine

RESEARCH COMMUNICATIONS

and its T andT 2

the

same as or its

homologues 5'

described homologues

in

Table I, were used.

PFU/ml Compound*

T

1.

*The

2.

IIa

3.

Va

4.

Acid-hydrolyzed

5.

2

T5

2 IO8

2 IO8

7 IO6

< lo2

1 IO8

8 10~

8 lo6


IIIa

2 IO8

2 IO8

6.

IIIb

2

IO8

2

IO8

7.

IIIC

2

IO8

2

IO8

8.

IIId

2 IO8

2

IO8

9.

Vb

1 IO8

2

10.

Acid-hydrolyzed

11.

Ib

12.

Oxidized

13.

Ia

14.

Oxidized

15.

Ic

16.

Oxidized

compounds I.

Va**

Vb**

8 lo6 2

Ib

Ia

Ic

used

IO8

2

3 IO7 2

were

as

IO8 < IO2 IO8
IO8

2

IO8

5 IO6

-c IO2

2 IO8

2 IO8

5 IO6

< IO2

follows:

H2N(CH2)3NH(CH2)nNH(CH2)3NH2; a,

n=4;

b,

n=2;

c,

n=6

R=COOH;

C,

OHC(CH2)nNH(CH2)4NH(CH2)nCHO;

II.

a,

n=2;

b,

n=3

R(CH2)2NH(CH2)4NH(CH2)2R;

III.

a, IV.

R=COOCH

; d,

R=CH20H

n=2;

b,

n=3

(C2H50)2CH(CH2)nNH(CH2)4NHkH2)nCH(oC,n5)2; a,

**The acid 3 hours.

b,

(C2H50)2CH~CH2~nNHCOCH2CH2CONH~CH2~nCH~~2H5)2i a,

V.

R&N;

hydrolysis

n=2; was

b,

n=3

carried

out

23

with

0.5

N H2S4,

at

30"

for

BIOCHEMICAL

Vol. 28, No. 1, 1967

biologically MS2,

inactive

480~

and

diamine

1 are

(IIa)

mechanisms

than of

progress

in

the our

and

complex, much

why

series

formyldiamines

RESEARCH COMMUNICATIONS

coliphages

susceptible

more

T-even

AND BIOPHYSICAL

of

to

the

and

$X174.

with

the

phagal

H.,

New Biochim.

T-uneven

action

Futher

series

of works

the on

nucleic

formylthe

acids

action are

in

laboratories.

References Adams, Bachrach, 768 Bachrach, 2, Bachrach, 2, Hirsh, Israel, 710 Tabor, 645 Tabor, 579 Tabor, 0964). Yamada,

M.

H., Bacteriophages, U., Tabor, C. (1963). U. and Leibovici, 357 (1965). U., Abzug, S. 174 (1967). J. G., J. Exptl. M., Rosenfield, (1964). C. W., Tabor, H. (1954). C. W. H., Tabor, (1961). C. W., Tabor, H. H.

and

Yasunobu,

W.

and

and

Interscience, Tabor,

J.,

Biochem.

Biophys.

Berkierkunst,

Med., J. S.

97, and

York,

A.,

345 (1953). Modest. E.

Biochim.

J.,

S.

M.,

J.

and

Rosenthal,

S.

M.,

Ann.

and

Bachrach,

K.

T.,

J-

Biol.

24

Chem.,

Med.

Biol.

Biol.

Commun., Biophys.

J.

Rosenthal,

J.

p. 443. Acta, 78,

Research

and

U.,

1959, Biophys.

Chem., Chem.,

Rev.

1, 208,

Biochem.,

Chem., 237,

Acta,

1511

zjg, (1962).

30, 2194