Note: Descriptions are shown in the official language in which they were submitted.
The present invelltion relates -to desacyl-pepsidine
(hereinafter xeferred to DA-pepsidine).
The DA-pepsidine of the present invention is a pentapep- ~
tide, i.e. valyl-valyl-4-amino-3-hydroxy-6-methyl-heptanoyl ~ ;
alanyl-4-amino-3-hydroxy-6-methyl-heptanoic acid having the
formula (I):
H3C CH3 H3C CH3
\ / \ / (I)
H3C\ /H3 H3C / H3 CH fH
CH CH CH OH CH CH OH I ~ -
~ I 1 2 1 1 3 1 2 ~
H2N-CH-CO-NH-CH-CO-NH-CH---CH-CH2--CO-NH--CH-CO--NH-CH--CH-CH2-COOH
DA-pepsidine has a strong pepsin-inhibitory activity
and thus may be useful for treating a patient suffering from a
gastric ulcer. .
The present invention also provides a process for the ~ `
: :, .,. ~ .
preparation of DA-pepsidine which comprises contacting at least
one M-acyl-pentapeptide with a bacterium belonging to Bacil;lus
pumilus
The N-acyl-pentapeptide used as the starting material
-~ in the present invention is a pentapeptide, i.e. N-acyl-valyl-
valyl-4-amino-3-hydroxy-6-methyl-heptanoyl-alanyl-4-amino-3-
hydroxy-6-methyl-heptanoic acid having the formula (II):
I H3C CH3 H3C CH3 , ~ ~,
H3C ~H3 H~C /CH3 \CH ~ CH (II) ~ ;
fH ; CH eH CI~3 CH2 fH
R-NH-C~I-CO-NH-CH-CO-NH-CH--CH-CH2-CO-NH-OH-CO-NH-CH--CH-CH2-COOH .`,~
wherein R is an acyl group.
'~ As N-acylpentapeptides of formula (II), more than ~;
~.- . :: .
ten compounds are known. The compound wherein R is acetyl group, -~
~ -:
~3~67 `~-
for ex~mple, has beerl disclosed by N. Kuwana et al, and described
in U.S. Patent No. 3,819,486, 3,878,185 and Japa~ese Patent Laid-
open Publication No. 125,583/74, wherein the said compound has
been referred to "S-PI" or "Pepsidine C". In Japa~ese Patent
Laid-open Publication No. 125,583/74 compounds are disclosed
wherein R is butyryl and propionyl group, as "Pepsidine A" and
"Pepsidine B", respectively. N~acyl-pentapeptides wherein R
is isovaleryl group or straight or branched aliphatic acyl gr~up
having 5 to 16 carbon atoms have been disclose~ by Umezawa et al
in Japanese Patent Publication No. 8996/72, Japanese Patent Laid-
open No. 29582/72 and Japanese Patent I.aid-open No. 41590/74,
wherein the said compounds have been referred to "Pepstatin".
These known N-acyl-peptapeptides are known to have a strong
pepsin-inhibitory activity.
N-acyl-pentapeptides are provided ~y cultivation of
various Actinomycetes. The aforementioned pepsi~ines, for~
exampler are provided by cultivation of Streptomyces nahiwaensis
EF 44-201 strain. The pepstatins are provided, for example, by
cultivation of Streptomyces testaceus. However, N-acyl-penta-
.
;~ 20 peptides provided by the cultivation of these Actinomycetes are
.... i: : :
usually not homogeneous, but a mixture of more than ten N-acyl-
pentapeptides havlng various acyl moieties (R) and similar prop-
erties. Thus, in order to provide particular N-acyl-pentapep-
tide, there are required very complicated processes for separat-
ing them, whereby the yield of the desired product may be con-
sid~rably reduced. ~ -
DA-pepsidine having free N-terminal has the fundamental
structure in common with the aforesaid N-acyl-pen~apeptide homo~
~. :
logues. Thus, when DA-pepsidine is first prepared according to
the process of the present invention using the mixture of various ~ -
N-acyl-pentapeptides obtained by the cultivation of above
Streptomyces strain, the resulting DA-pepsidine may be acylated
. ~ ~
2 -
' ~'`'',
by a desired acylating agent to readily produce a desired
single N-acyl-pentapeptide.
The DA-pepsidine may also be used as an intermediate
for the synthesis of other novel N-acyl-pentapeptide homologues.
A strain of Bacillus pumilus preferable for use in
the process of the present invention is Baclllus EF 49-210
~nov. sp.) strain, which has been separated from the soil at
Kawaguchiko machi, Yamanashi Prefecture, Japan. The microbiolog-
` ical properties of the strain are as follows:
~1] Microscopic observation (bouillon agar nutrient
medium)
1. Morphologies: Rods with rounded ends, occuring singly or ~ -
in pairs or on rare occasions in threes. ;-~
~` 2. Size: 0.4 to 0.7 by 1.0 to 4.0 microns. ~ ;
3. Motility: Motile.
-- 4. Flagella: Peritrichous
5. Spores: Formed
6. Gram stain: Positive
7. Acid fast stain: Negative
[23 Cultivation view ~ ;~
1. Bouillon agar slant medium (at 30C for one day)
,. . . .
Growth spreading flat, milky yellowish to slightly yellowish,
and weakly lustered~ No change in color of nutrient medium. :;~
2. Bouillon agar flat medium ~at 30C for 1 to 7 days)
Growth spreading flat, dendroid, milky yellowish; slightly ~ :;
yellowish~ smooth colonyj weakly lustered and translucent
to opaque. No soluble pigment. ~;~
3. Broth medium (at 30C for 2 days)
Formed pellicle on surface. Turbidity is weak and uniformO
i 30 4. Gelatin medium (at 20C, 27C and 30C for 3 days) `~
Growth weakly, but liquefies the gelatin. It is not clear -;
by stab culture.
; .
.,. -: . ... ,. .,. , - ,.. . .. . - . .... ....... , .... , .... . . . , ~
5. Litmus milk (at 30C for 6 days) ; `
Rather ~cidic, and weakly liquefied af~er 6 day cultivation. ~`
No coagulation. ;
6. Potato (at 30C for 2 days)
Growth wet and spreading slimy. Potato darkened-
[III] Physicological properties.
1. Nitrate reduction property : Negative
2. Denitrogen reaction : Negative
3. Methyl red test : Positive
4. Voges-Proskauer reaction ; Positive ;
5. Formation of indole : Negative ;
6. Formation of hydrogen sulfide : Negative
7. Hydrolysis of starch : Negative
8. Utilization of citric acid : Positive in Koser's
medium
Negative in Symmons'
medium
9. Utilization of the source of inorganic nitrogen~
Weakly positive with potassium nitrate.
~, ~eakly positive with ammonium sulfate.
10. Formation of pigment : Negative
11. Urease : Negative -
` 12. Oxidase : Positive
13. Catalase : Positive
14. Range of growth:
a) pH (brot~ nutrient medium at 30C for 2 days with ~i
shaking)
Growth in sterilized medium at pH 5 to 9.8.
~ b) Temperature (broth nutrient medium for 2 days with ~ ~-
`' shakingj ~-
Growth at 20C to 45C.
c) Concentration of salt (broth nutrient medium for ~ ;
2 days with shaking) ;~ -
.
4 ~ -~
Growth within one day, in the concentration of0-5 -~ ?
to 7.0~ of NaCl. Growth within two days, in the
concentration of 10~ of NaCl.
15. Oxygen requirement: Facultative anaerobic.
16. O-F test by Hugh-Leifson method;
Acid is formed in both aerobic and anaerobic. No
gas formed. ~-
17. Fermentation test and acid formation with carbohydrates are
as follows:
~cid formation ~ -
~ : .
No. Carbohydrates aer _ iC anaerobic .. ;~
-~ 1 arabinose - +
2 xylose + +
3 glucose + +
4 mannose + +
;-; . ...
fructose + +
. - , . . 6 galactose + +
7 maltose - +
8 sucrose + +
9 lactose - +
trehalose + +
11 sorbitol - + ;
12 mannitol - +
13 inositol + + '
14 glycerol + +
; 15 starch - +
.
;l No gas formed in any of the carbohydrate.
Acid (+), Sight acid (+), No acid (~
By comparing the data of the above mentioned properties
of the bacterium with those described in Bergey's Manual of
Determinative Bacteriology, the seventh edition, this bacterium
is considered to be a variant of Bacillu pumilus, and the said
~ 5 ~
.' .,., .. .,......... .. , ... '
bacterium was nominated as Bacillus ~ llus ~ chi.
This bacterium EF 49-210 (ATCC No. 31132) strain has been deposi-
ted to Agency of Industrial Science and Technology, Fermentation
Research Institute in Japan as FERM-P No. 2677. It was also
recognized that there is suitably used for the process of '
; the present invention the known bacterium B~clllus pumilus
IFO-12092 and IFO-12110 which were stored in Institute of ~;
Fermentation Osaka in Japan.
In the cultivation of the microorganisms to be used in ~ ~'
the process of the present in~ention, there may be used any comp~
osition of the nutrient medium and any conditions for the culti~
vation, provided that the microorganisms can grow, and fully
exhibit desired activities. There may be selected, a medium
containing for example, any organic or inorganic nitrogen sources
such as peptone, meat extract, corn steep liquor, soy-bean hydro- '
. ~ .
lyzate, soy-bean extract, yeast extract and inorganic ammonium
salts; any carbon sources such as molasses, dextrose, starch ~'
' and hydrolysis products thereof; and inorganic salts. The
cultivation is generally carried out with shaking or aerating
. ...
at a temperature ranging from 20~C to 45~C for a period of time
from one to se~en days.
It was found that the deacylating activity of the
Bacillus pumilus exists both intra- and extra- cellularly. Thus, '
:' :
in the process of the present invention, there may be used
cuIture broth, culture ~iltrate, ~hole cells, dry cells and/or
enzyme preparations obtained therefrom. ` `
The reactant N-acyl-pen~apeptide, is not always ' '~ '
required to use in a purified form. There may be used, for
example, a mixture of several N-~acyl-pentapeptides which may
,~. . .
be obtained in various purification stages such as culture
filtrate of the microorganism which produces N-acyl-pentapep~
tides, and products salted out from said culture filtrate.
-- 6 --
~'~, ' ' ~
i3~
Products obtained by extracting the culture fil.~rate with an ~ ~;
organic solvent and then removing the solvent may also be used.
In the process of the present invention, the reaction
conditions such as concentration, temperature and pH, are not
critical and can be varied over broad range depending upon the
kinds of the strain of microorganisms used and the N-acyl- ;
pentapeptide selected as starting material. When deacylation
of N-acyl-pentapeptide is carried out using Bacillus pumilus
Kawaguchi EF 49-210 strain, for example, it is preferable to use ~ ~
conditions of pH 4 to 10 and at temperature of about 30 - 50C. . ;
A reaction time of from 3 to 60 hours is suitable. By addition `
of a Co compound such as CoC12, and CoSO4 the yield of DA~
pepsidine may be increased.
Separation and purifica-tion of DA-pepsidine from the :
~ reaction mixture can be carried out using any con~entional proce- :
- aure such as solvent extraction, column chromatography, fraction~ `~
al crystallization and recrystalli~ation. `
To detect the formation of DA-pepsidine thin layer
chromatography can be used as follows:
Sample solution is spotted on a thi.n layer plate of -~
Silica Gel G (a trademark of Merck A. G.), and developed with a
: mixed solvent (I) containing n-butyl alcohol, acetic acid and
water in the ratios by volume of 3 : 1 : 1, or a mixed solvent
(II) containing n butyl alcohol, acetic acid, water and n-butyl-
: acetate in the ratios by vo].ume of 4 : 1 : 1 : 4. DA-pepsidine ;
is detected as a spot which is positive in the ninhydrin reaction
and also Rydon-Smith reaction, at Rf - 0~48 in the case of the
use of the mixed solvent (I). ~ ~-
Alternatively, it is detected by Casein plate method :
as follows:
After the de~eloped and dried thin layer plate mentioned
above is transferred to a flat board of Casein-containing agar, ~-
, ~i ' ;
a filter paper impreynated with pepsin solution is placed on
the board. Reaction is then effected at 30C for overnight,
to detect a non-decomposed casein.
DA-pepsidine of the present invention has the following
physicochemical properties:
(i) Appearance:
White needles
(ii) Solubility~
Readily soluble in acetic acid and methanol; soluble
-:
in ethanol, n-hu~anol and pyridine; and slightly
soluble in acetone and ethylether.
(iii) Coloring reaction:
Positive in both ninhydrin reaction and Rydon-Smith
reaction.
(i~) Ultra~iolet absorption spectrum:
0.1% methanol solution exhibits only the end
absorption due to the peptide bond, but no maximum
absorption is shown in the region from 250 m~ to
` 20 (v)~ Infrared absorption spectrum: ~ ;
; as shown in Fig. 1 of the accompanying drawings
: . :...
(vi) Composition of amino acid: ~:
After hydrolysis with 6N HCL for 72 hours at 110C, ~
the sample was analyzed by means of amino acid `
a~alyzer.~ A molar ratio of alanine and valine of
, 1 : 2 was confirmed.
(~ii) Molecular weight and structural formula: ~,~
Sample was acetylated by acid chIorids method,
;`~ followed by methyl-esterification by diazo methane
method. The resulting compound was subjected to ;~
...
mass spectrum analysis and determined to M+ = 657.
Thus, the molecular weight of the sample was
. ~
i i, . : ~
- 8
^l:
~3~7
identical with the calculation value 601 of DA- ~ -
pepsidine. The peak of fragment was also identical
with the calculation value derived from the struc~
; tural formula (I) of DA-pepsidine. In addition,
aforementioned acetylated product of the sample ~-
was identical with pepsidine C on thin layer chrom~
atography, and the esterified product was also
identical with methyl ester of pepsidine C.
(viii) Pepsin-inhibitory activity: I
Pepsin-inhibitory activity oE DA-pepsidine was
determined using the method~reported by S. Murao
and S. Satoi in Agr. Biol. Chem. ~apan 34 (8)~1265 1
- 7 (1970).
. .
, It was shown that the amount of DA-pepsidine which
gives 50% inhibition against 100 ~g of pepsin in
0.82 ~g. While, that of pepsidine C is 0.86 ~g.
The present invention~will be further il~lustrated by
way of~the following Examples.
Example l
Into 0.5 litre Sakaguchi flask, was charged 0.1 litre
1 . . . .
of liquid medium (pH 7) containing I% of meat extract, 1~ of pep~
`; tone and 0.5% of NaCl, and the medium was sterilized at 120C for 10 minutes.
Each 20 flask prepared as above was inoculated respect~
lvely by l ml~of~the broth: of~3aaillus pumilus ~ ~EF 49
210 strain which was cultivated previously on the similax nutrient ~;~
medium at the temperature~;of 30C~for 24 hours , and~cultivated .
~ith shaking at~30C for~ 72~hours.
After completion of the~cultivation, the cells ~ere
remo~ed from the broth~by;centrifugatlon, and to be combined
supernatant 4 litres of~cold acetone~were added dropwise with ;~
cooling. The resulting preclpitate was suspended in di tilled
water to provide 50 ml o~ suspension.
10 g of N-acyl-pentapeptide mlxture (pepsidine C,
pepsidine B and pepsidine A in the weight ratio of 94 : 4 : 2)
were dissolved into water and 6N NaOH was added to neutralize,
thereby pro~iding 500 ml of aqueous solution. Into the aqueous
solution, 10 ml of the suspension of aforementioned acetone
precipitate were added, and the mixture was stirred in pH 8.5
at 37C for 5 hours.
The resulting reaction solution was extracted three
times with each 500 ml of n-butyl alcohol. These n-butyl alcohol
layers w~re combined, evaporated to dryness under reduced pressure
to obtain 9.8 g of the residue. The residue was dissolved in 90
ml of solvent mixture containing n-butyl alcohol, acetic acid,
water and n-butyl acetate in the volume ratio of 4 ~ 4.
The resulting solution was charyed in a column filled with 500 g
of silica gel, and t-hen subjected to column chromatography by
developing with the solvent mixture as above mentioned.
About 0.75 Iitres of the main fraction were e~apor- `
ated to dryness under reduced pressure to obtain 0.7 g of
- ~ .
residue. The residue was crystallized from ethyl alcohol to ;-
obtain 0.3 g of crude crystals. Said crude crystals were further
., ~
recrystallized from ethyl alcohol to obtain 0.17 g of DA-pepsidine
in a form of white n edles.
Melting point: Decomposed at 193C to 199C, and clear
melting point was not observed.
[a¦20 = -S7 to -60 (C = l; methanol)
Elementary analysis:for C29H55N5O8 H2O
C% ~% N% _
Calculated: 56.19 9.26 11.29
Found: 56.39 8.99 11.27
Example 2
: Into 30 1itre jar-fermentor, were charged 15 litres of ;-
liquid nutrient medium (pH 7~ containing 1% of meat extract, ~;
1% of peptone and 0.5% of NaCl, and then the medium was
,
sterilized at 120C ~or 10 minutes.
The broth of EF 49-210 strain was prepared by cultivat-
ing the bacterium at 30C for 24 hours with shaking on the
similar nutrient medium as afore-mentioned. :~
Into said jar-fermentor, 0.2 litres of the broth of
said EF 49-210 strain were inoculated and the cultivation was
carried out under the following conditions:
Time: 24 hours
Tamperature: 30C
Aeration: 15 litres per mlnute
Stirring: 350 r.p.m.
After -the cultivation, the microorganisms were removed
from the broth by centrifugation. Into 12 litres of the super-
natant, ammonium sulfate was added to 0.8 saturation, and the
mixture was placed in refrigerator for three days. The resulting
precipitates were suspended in distilled water to obtain 750 ml
of suspension. ~"
A mixture of 100 ml of the suspension, 400 ml of the
neutraliæed aqueous solution containîng 8 g of pepsidine C~ 292 ml
of 1/15 M phosphate buffer (pH 5.5) and 8 ml of the solution of
CoC12 ~5 x 10 2M) was incubated for 40 hours at 37C. The reaction ~`
mixture was extracted 3 times with 800 ml of n-butyl alcohol, ~`
respectively. The combined extracts were evaporated to dryness. `
The residue was dissolved in a minimum volume of the solvent
mixture containing n-butyl alcohol, acetic acid, water and n~
butyl acetate in the volume ratios of 4 : 1 : 1 4, and was ~ ;
subjected to silica gelcolumn chromatography with the similar
solvent mixture as developing solvent.
Two litres of main fractions were combined and evapor- -
ated to dryness to give 6 g of white powder. The powder was
crystallized twice from ethanol to give 2 g of DA-pepsidine in a
form of white needles.
3~
Example 3
100 Mg of DA-pepsidine obtained by the process of
Example 1 were dissolved in 10 ml of pyridine. 1.2 Ml of the 20-
fold diluted acetylchloride solution in acetone were dropped into
the above pyridine solution with ice-cooling. The mixture was
allowed to stand over-night. An aliquot of this reaction mixture ;~
~ was subjected to silica gel thin layer chromatography by using
; the developing solvent conta~ning n-butanol, acetic acid, water ;
and n-butyl acetate in the volume ratios of 4 ~ 4, follow~
ed by the dete~tion of ninhydrin reaction, Rydon-Smith reaction,
and casein plate method, whexeby the resulting product exhibited
Rf = 0.49, and it was identified with authentic pepsidine C.
The reaction solution was evaporated to dryness. The
residue was dissolved in 10 ml of methanol. Into the resulting
solution, 17 ml of ether solution of diazomethane were added.
The solution was allowed to stand at room temperature for three
hours, and then evaporated to dryness. The residue was dissolved
in methanol. By the addition of ether, 3~ mg of methylester of ` ~;
; pepsidine C. were crystallized. -
Example 4
The procedure in Example 3 was repeated except that ~
isovaleryl-chloride was used in place of acetyl chloride. The - ;;
product exhibits Rf = 0.64 on the thin layer chromatography -~
identified with authentic pepstatin A~
Example 5 ;~
One milliliter of the culture broth of EF 49-210 strain
cultivated by the similar procedure as Example 1 was mixed with `
one milliliter of aqueous solution which was prepared by~`
dissolving 10 mg of pepsidine C into water and neutralized by ~,;;
6N NaOH solution. The resulting solution was shaked at 37C for
5 hours `~
The reaction solution was subjected to the silica gel
'i - 12 -
thin layer chromatography by developing with the solvent contain-
ing n-butanol, acetic acid and water in the ratlos by volume of
3 : 1 : 1. The product exhibits Rf = 0.48, ~nd is identified with
authentic DA-pepsidine.
Example 6
The procedure in Example 5 was repeated except that , ~'~
pepsidine C was replaced with pepsidine B or pepsidine A.
' Formation of DA-pepsidine was detected with thin layer chromato- '~;
graphy in the respective cases.
Example 7 ~'
~` The procedure in Example 5 was repeated except that N-
,, isovaleryl pentapeptide ~pepstatin A) was substituted for
pepsidine C. There was also detected the formation of DA-pepsidine ~,
with thin layer chxomatography.
,~ Example 8 '~
~,~ The procedure in Example 5 was repeated, except that ~ '
';~ N-n-hexanoyl-pentapeptide or N-n-decanoyl-pentapeptide was
replaced with pepsidine C. The production of DA-pepsidine was '~
also confirmed with thin layer chromatography, in the respective ~ ',
20 ~ cases. ~'
s~ Example 9
~ The procedure in Example 5 was repeated except that ;, "~
", Bacil'lus'pumi'lus' Kawaguchi EF 49-210 was replaced with B'ac'illus ~;;`~'
. . _ . :
'~i, pumilus IFO 12092 or Bacillus''pumi'lus IFO 12110. Formation
~ of DA-pepsidine was detected with thin layer chromatography, '
'; in each case.
'?
Example 10
:~i The procedure in Example 5 was repeated except that , ~' '
one milliliter of the culture broth of EF 49-210 strain was
"~i 30 replaced 10 mg of acetone-dried cells of the bacterium. The ~',
" formation of DA-pepsidine was detected with thin layer chromato- '~ '
r~ graphy.
, . .~ ',
~,',' ~ ~ 13 ~
