Note: Descriptions are shown in the official language in which they were submitted.
:~
:~ ;
~ 104~088
.:
This invention relates to a novel polypeptide of the
formula
.:;- .. - "
- 1 2 3 4 5 6 7 8
; H-Cys-Ser-Asn-Leu-Ser-Thr~Cys-Val
9 10 11 12 13 14 15 16
~-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu
; 17 18 19 20 21 22 23 24
-His-Lys-Leu-Gln-Thr-Tyr-Pro-~rg [I]
:~ 25 26 27 28 29 30 31 32
' -Thr-Asp-Val-Gly-Ala-Gly-Thr-Pro
j ;~,.~ -NH2
. : .
and acid addition salts and complexes thereof. -
- The polypeptide of the formula CI] has serum calcium
. reducing activity and as tests upon rats have shown, it has an activity
of 3700 - 6000 MRC units for serum calcium reducing activity. It has
an activity which is fifteen times higher than that of human calcitonin
and sheep calcitonin and twenty tlmes higher than that of porcine or
cattle calcitonin. The said activity is the same or higher as compared
,
with the most active of known salmon calcitonin.
This novel compound has important therapeutic properties
: :
and may be prescribed for the purpose of reducing serum calcium content -~-~
:; 20 in diseases such as hypercalcemia, an endogenious calcitonin deficient
disease caused by dysthyroidism or hyperparathyroidism. The novel -
~;~ compound can be prescribed for osteopathy requiring calcium, such as
osteoporosis, osteomalacia, fracture, fibrous dysplasia of the bone or
rachitis caused by corticosterone therapy or inactivation after meno-
pause or external in~ury, and is especially suited to therapy in
combination with calcium or phosphorus. ~lso, the novel produce can
be used for therapy of Paget~s disease and for therapy or prevention
of peptic ulcers. The compound of the present in~entlon is more stable
: in serum, liver or kidney than the known calcitonins.
~ 30 -
;.
",,
. ~
;,
... . .
104~088
Synthesis of the compound can be made by the con-
ventional peptide synthetic method, that is to say, an amino acid and/
or peptide is reacted by condensation in the order of the amino acid
sequence of formula [I~, and the disulflde bridge is bonded by oxidation
of the mercapto group at any stage of constructlon of the peptide unit.
If desired the product may be con~erted to its acid addition salt or
complex.
The protective groups ~or the synthesis of the starting
materials or intermediates are conventional protective groups for
peptide synthesis and should be easily removable by hydrolysis, acid
decomposition, reduction, amlnolysis or hydrazinolysis.
For example, the amino group may be protected con-
ventionally by an acyl group such as formyl, trifluoroacetyl, phthaloyl,
benzensulfonyl, p-toluenesul~onyl, o-nitrophenylsulfenyl or 2,4-
.
dinitrophenylsulfenyl group; an aralkyl group such as benzyl, diphenyl-
methyl or triphenylmethyl (these groups may optionally substituted by a
lower alkoxy group such as o-methoxy or p-methoxy group); a benzyl-
- oxycarbonyl group such as benzyloxycarbonyl, o-bromobenzyloxycarbonyl,
p-bromobenzyloxycarbonyl, o-chlorobenzyloxycarbonyl, p-nitrobenzyloxy-
carbonyl, p-methoxybenzyloxycarbonyl, p-phenylazo-benzyloxycarbonyl or
~- p-(p'-methoxyphenyazo)-benzyloxycarbonyl group; an aliphatic oxycarbonyl
group such as cyclopentyloxycarbonyl, trichloroethyloxycarbonyl, t-
amyloxycarbonyl, t-butoxycarbonyl or diisopropylmethoxycarbonyl, or an
aralkyloxycarbonyl group such as 2-phenylisopropoxycarbonyl, 2-toryl~
isopropoxycarbonyl or 2-p-diphenyl-isopropoxycarbonyl, These amlno
- groups can be protected by forming enamin reacted with 1,3-diketone
such as benzoylacetone, acetylacetone or dimedone.
The carboxyl group can be protected by its amide ~ormation,
hydrazide formation or esterification. The amide group is substituted
by a 3,4-dimethoxybenzyl or bis-(p-methoxyphenyl)-methyl group.
:.
.'' ;
; - 2 - ;
1~41~8
The hydrazide group is substituted by a benzyloxycarbonyl, trichloro-
ethyloxycarbonyl, trifluoroacetyl, t-butoxycarbonyl, trityl or 2-p-
diphenyl-isopropoxycarbonyl group. The ester group is substituted by
an alkanol such as methanol, ethanol, t-butanol or cyanomethylalcohol;
an aralkanol such as benzylalcoholl p-bromobenzylalcohol, p-chloro-
: : .
benzylalcohol, p-methoxybenzylalcohol, p-nitrobenzylalcohol, 2,4,6-
trimethylbenzylalcohol, benzyhydryalcohol, benzoylmethylalcohol, p-
bromobenzoylmethylalcohol or p-chlorobenzoylmethylalcohol; a phenol
- such as 2,4,6-trichlorophenol, pentachlorophenol, p-nitrophenol, 2,4-
dinitrophenol, p-cyanophenol or p-methanesulfonylphenol; or a thiophenol
such as thiophenol, thiocresol or p-nitrothiophenol. The hydroxy group
in serine, threonine or tyrosine may optionally be protected by
.:
esterification or etherificatlon. A group protected by esterification
is, for example, a lower alkanoyl group such as an acetyl group; an
aroyl group such as a benzoyl group; or a group derived from carbonyl
such as benzyloxycarbonyl or ethoxycarbonyl. A group protected by
- etherification is, for example, a benzyl, tetrahydropyranyl or t-butyl
group. Protection of the hydroxy group can be effected by a 2,2,2-
trifluoro-l-t-butyloxycarbonylaminoethyl or 2,2,2-trifluoro-1-benzyl-
oxycarbonylaminoethyl group. However, it is not always necessary to
protect these hydroxy groups.
The mercapto group in cysteine can be protected by, for
example, acylation or alkylation. A group protected by acylation is
for example an acetyl, ethoxycarbonyl, benzoyl, ethylcarbamoyl, benzyl-
oxycarbonyl or substituted benzyloxycarbonyl group. A group protected
by alkylation is, for example, a t-butyl, acetoamidomethyl, benzylthio-
methyl or benzyl group; an arylmethyl group such as 3,4-dimethylbenzyl,
- p-methoxybenzyl, diphnylmethyl, dimethoxybenzhydryl; or a trityl group,
phenylcyclohexyl or thienyl-2-cyclohexyl group.
~ 3 ~
.' ~,~ .
:
- 104~0~38
The amino group or guanidino group in arginine can be
~ protected by a nitro, tosyl or benzyloxycarbonyl group, however the
; guanidino group does not always re~uire protection. The imino group
in histidine can be protected by a benzyl, trityl, benzyloxycarbonyl,
. .
tosyl, admantyloxycarbonyl, 2,2,2-trifluoro-1-t-butyloxycarbonylamino~
ethyl or 2,2,2-trifluoro-1-benzyloxycarbonylaminoethyl group, although
the imino group does not always require to be protected.
The peptide of the starting materials or intermediates
n,
are synthesized by condensation of amino acids or peptides -preferably
- 10 of two to four amino acids- in the order o the amino acid sequence
of formula ~I], and the peptide containing two L-cysteinyl group is
preferably produced by constructing the disulfide bond in the stage
;
of its peptide construction. For example, an amino acid or peptide
. .
having a protected ~-amino group and activated terminal carboxyl group
is reacted with an amino acid or peptide having free ~-amino group and
. .
protected terminal carboxyl group. On the other hand, an amino acid
or peptide having activated ~-amino group and protected terminal
carboxyl group is reacted with amino acid or peptide having free
, terminal carboxyl group and protected ~-amino group.
; 20 The carboxyl group can be activated by, for example, an
acid azide, acid anhydride, acid imidazolide or active ester, such as
by changing to cyanomethyl ester, thiophenylester, p-nitrothiophenyl-
ester, p-methanesulfonylphenylester, thiodylester, p-nitrophenylester,
- 2,4-dinitrophenylester, 2,4,5-trichlorophenylester, 2,4,6-trichloro-
; phenylester, pentachlorophenylester, N-hydroxyphthalimidoester, 8-hydroxy-
- piperidine ester or N-hydroxypiperidine ester, carbodiimide, N,N~-
carbonyl-diimidazol or an isoxazolium salt such as Woodward reagent. -
The carboxyl group can be activated in conventional
way by~ for example, an acld azide, acid anhydride, active ester or
- 30 carbodiimide.
- 4 -
~,~",,ij
. . . . , . . , :
- , , , ~ , . . .
,, , ~ : ': ' : `' : -
`:
~;
: ~041088
"
Pre~erred condensation reactions are the Wunsch method,
azide, active ester or Geiger methods. In the condensation reaction,
` racemization should carefully be avoided.
~ The disulfide bond in the novel peptide of the present
invention may leave tuning various stages of the reaction process, and
therefore it should preferably be constructed as fully as possible in
, . .
the synthesis. Accordingly, disulfide bond formation may preferably
be effected at the reaction step of the peptide, without containing
cysteinyl group such as protected tricosa-peptide-amide (10-32) or
,.
protected docosa-peptide-amide (No. l1 - 32), with another peptide
containing cysteinyl group such as protected nonapeptide (1-9~ or
. protected deca-peptide (No. 1 - 10). The disulfide bond formation isperformed by oxidizing the protected peptide having free a mercapto
group with iodine in glacial acetic acid, diiode ethan in organic
solvent or oxygen in pH 6.5 water or hydrogen peroxide. The protected
- mercapto group, such as protected by a trityl or acetamide group, can
:
be bonded simultaneously with removing the said protective group by
treatment with iodine in methanol.
~ In the synthesis of the novel polypeptide of the present
-i 20 invention by the Meryfield solid phase peptide synthetlc method, the
hydroxy group in serine, threonine and tyrosine can be protected by,
~; for example, benzyl; the mercapto group in cysteine can be protected,
for example, by an acetoamidemethyl, benzyl, 3,4-dimethylbenzyl or
p-methoxybenzyl group; the imino group in histidine can be protected,
for example, 1-benzyloxycarbonylamino-2,2,2-trifluoroethyl group; the
guanidino group in arginine can be protected, for example, by a nitro
group; and the side chain in glutamic acid can be protected, for example,
by a benzyl ester group. The protective group for the ~-amino group is,
for example, a t-butyloxycarbonyl, o-chlorobenzyloxycarbonyl or o-bromo~
benzyloxycarbonyl group.
' ~
.
:
~0410~38
The protected peptide is re~noved ~om the carrie~ ~esin and the
protective group is removed by anhydridous hydrogen fluoride.
.:
One-step removal of all protective groups by hydrolysis
using trifluoro acetic acid can be achieved when t-butoxycarbonyl
:,. .
group is used for amino group protection; t-butyl ester for side-chain
carboxyl group protection; t-butyl ether for hydroxyl group protection
and in serine; and threonine, tyrosine and 2,2,2-tri-fluoro-1-t-butoxy-
` carbonylaminoethyl group for imino group protection in histidine.
When synthesising the protected dotriacontapeptide using the protective
group which can be removed by trifluoroacetic acid, the mercaptogroup
, , .
is preferably protected by an acetamidemethyl, benzyl, 3,4-dimethyl-
benzyl, p-methoxybenzyl or trityl group. The acetomidomethyl or
trityl group can be removed by mercuric acetate and hydrogen sulfide ;
in organic solvent from the protected peptide, and the benzyl, 3,4-
dimethylbenzyl or p-methoxybenzyl group can be selectively removed by
sodium or hydrogen fluoride in liquid ammonia.
In the present invention, it is preferable that N-
terminal fragment consisting of first to 7-12th N-terminal amino acid
sequence is condensed with residual peptide of 8-32nd, 9-32nd, 10-32nd,
11-32nd, 12-32nd or 13-32nd amino acid sequence. The aforesaid N-
terminal fragment consisting of first to 7-12th amino acid sequence is
also condensed with the fragment up to 23rd amino acid sequence, and
the thus-constructed tetracosapeptide can be condensed with nonapeptide
consisting of the 24-32nd amino acid sequence.
The said condensation reaction can be achieved by the
azide method starting from the peptide having an azide or hydrazide
terminal. Also it can preferably be achieved by starting from the
peptide having free terminal carboxyl group, the so-called Wunsch method,
or by analogous method.
f~ '`J!~ '~i;~' ,. ~
:
` 10410~38
The N-terminal ~ragment, such as decapeptide consisting
- of 1 to 10th amino acid sequence, heptapeptide 1-7, octapeptide 1-8,
nonapeptide 1-9, undecapeptide 1-11 or dodecapeptide 1-12, can be
produced by the same process.
The decapeptide sequence can be produced by connecting
the amino acid se~uence of 1-5th with 6-lOth, sequence of 1-6th with
7-lOth or sequence of 1-4th with 5-lOth. The other fragment, consisting
` ~ of a different amino acid sequence, can naturally be used for that
synthesis. For example, the said peptide can be synthesized from
condensation of the first amino acld unit with 1-lOth amino acid
L' sequence. The fragments can be synthesized by connecting the amino
acids and/or lower peptide units in the amino acid sequence from the
C-teminal. A protective group for alpha-amino group in cysteine is
- preferably a group which can be split by acid decomposition, such as a
. t-butoxycarbonyl group. At the same time, a protective group for the
mercapto group is preferably split. Examples of the protective group
-, .
are acetamidmethyl, benzyl, 3,4-dlmethylbenæyl, p-methoxybenzyl and
; trityl groups. In the condensation reaction by azide, active este~
or acid anhydride method, it is not necessary to protect the terminal
carboxyl group in decapeptide. The carboxyl group can also be pro-
tected by esterification by alcohols such as methanol, benzyl alcohol
or other alcohols. The ester group such as methyl ester can be removed
by dilute sodium hydroxide solution or by conversion to hydrazide, and
the benzyl ester group can be removed by catalytic hydrogenation. The -
amino group of the intermediate is protected by a conventional protective
group, such as a benzyloxycarbonyl, trityl, t-butoxycarbonyl or 2-p-
diphenyl-isopropoxycarbonyl group. The carboxyl group can be protected,
if required, by conventional esterification. The hydroxyl group in
serine and threonine can be protected, if necessary, by esterification
using t-butanol, benzyl alcohol or other alcohol.
-- 7 --
""'''`~
... ,: .
.....
` 104~0~8
: ;.
:
- The benzyloxycarbonyl, p-nitr~benzylester and benzyl
ester group are split by catalytic hydrogenation in the presence of
palladium/carbon. The N-trityl group is spllt by aqueous acetic acid,
. . .
; and the t-butoxycarbonyl group can be decomposed by trifluoroacetic
; acid. The o-Ditrophenylsulfenyl group is split by hydrogen chloride,
hydrogen cyanide or sulfurous acid in organic solvent. The diphenyl-
isopropoxycarbonyl group is split by a mixture of acetic acid-~ormic ~;
acid-water (7 : 1 : 2). Methyl ester, ethyl ester or p-nitrobenzyl
- ester is changed to hydrazide by using hydrazine hydrate. The method
;; 10 ester group can be split by dilute sodium hydroxide solution, and t-
butyl ester is split by trifluoroacetic acid. The S-trityl group is
removed by mercuric acetate and hydrogen sulfide. The S-benzyl group
can be removed by sodium in liquid ammonia and, simultaneously, any
p-nitrobenzyl ester is split. Disulfide bridge formation can be
achieved by the process hereinbefore described.
A peptide having C-terminal consisting of amino acid
sequence from 8 - 13th to 32nd, which is condensed with N-terminal
peptide hereinabove, is preferably synthesized by connecting the C-
terminal fragment f- 17-32nd amino acid sequence with 8-13 to 16th amino
acid sequence such as 8-16th, 9-16th, 10-16th, 11-16th, 12-16th or
13-16th amino acid sequence. Also the said peptide can preferably be
synthesized by connecting the C-terminal amino acid (amino acid No. 32)
or C-terminal ~ragment such as peptide of amino acid sequence of 31~32nd,
29 - 32nd, 27 - 32nd, 26 - 32nd, 25-32nd, or 24th - 32nd with amino acid
or lower peptide consisting of 2 - 4 amino acid in the order of residusl
amino acid sequence. For example, C-terminal fragment 11th - 32nd can
. - be produced by condensation of amino acids such as prolineamide,
threonine, aspartic acid, arginine, glutamine or glutamic acid and lower
peptides such as dipeptide 29 - 30, dipeptide 27 - 28, tripeptide 21 - 23,
tetrapeptide 16 - 19 and tripeptide 11 - 13 in the order of amino acid
;. :
:: .
': ~ ' ' '
. , : . - ., . . , .. , :
`
~(~410~38
sequence from C-termin~l side, by active ester method or Geiger method.
Preferable protective groups for each grsup are: ~-amino group by
t-butoxycarbonyl group; side chain carboxyl group by benzyl ester group;
-amir.o group by o-chlorobenzyloxycarbonyl or diisopropylmethoxycarbonyl
group; hydroxyl group of serine, threonine and tyrosine by benzyl group;
and amino group in guanidine group of arginine and imino group of
histidine by tosyl group.
The aforesaid C-terminal fragment from 8-13th to 16th
amino acid sequence can be synthesized by condensation of amino acid
sequence of 8-13th, 9-13th or 10-13th with that of 14-16th, or by
- condensation of amino acid sequence of 8-12th, 9-12th or 10-12th with
s the amino acid sequence of 13-16th. This fragment is used for the
condensation with the fragment of the continued amino acid sequence
such as hexadecapeptide or fragment thereof hereinbelow explained.
The gamma-carboxyl group in glutamic acid can be protected by a t-butyl-
ester group. The ~-amino group in lysine can preferably be protected
by a t-butoxycarbonyl group. The hydroxyl group in serine may be
~ protected, if necessary by a t-butyl ether group. -
- The amino acid sequence of 17-32th fragment can be
synthesized by various m~thods. The said fragment can preferably be
synthesized by the condensation of nonapeptideamide consisting of
- 24-32nd amino acid sequence with the foregoing heptapeptide consisting
of 17-23rd amino acid sequence, or condensation of the said nonapeptide
; with the fragment of 20-23rd or 21-23rd amino acld sequence followed by
condensation of the residual foregoing amino acid sequence. The said
heptapcptide may preferably be synthesized by condensation of tri-
peptide 17-19th or tetrapeptide 17-20th amino acid sequence with
residual amino acid fragment up to 23rd amlno acid by the azide method.
The alpha-amino group in these amino acids can be protected with a
- 30 benzyloxycarbonyl group, and the ~-amino group is protected with a
- .
:" .~ 9 _
,~" - - ' ' .
10~1088
,
t-butoxycarbonyl group. The hydroxyl g~pu~ in threonine and tyrosine
may not be necessary to protect, but is preferably protected with a
t-butyl ether group. The hydroxyl group in tyrosine is protected
simultaneously with the alpha-amino group by a benzyloxycarbonyl group,
and the said protective group can be removed by hydrogenation.
The aforesaid nonapeptide of 24-32nd amino acid sequence
is preferably synthesized by condensation of tripeptideamine 30-32nd,
tetrapeptideamine 29-32nd, pentapeptideamide 28-32nd or heptapeptideamide
26-32nd sequence with the fragment consisting of the foregoing amino
acid sequence. The said condensation can be preferably performed by
the W~nsch method. The gamma-carboxyl group is preferably protected by
a t-butyl ester group.
The protected tricosapeptideamide 10-32nd sequence can
be obtained by condensation of amino acid sequence of 17-32nd with
heptapeptidehydrazide 10-16th sequence by the azide method.
- Another preferred method for the synthesis of amino acid
sequence 10-32nd protected tricosapeptideamide may be condensation of
, ~` C-terminal fragment of 24-32nd with the fragment of 10-23rd amino acid
. -: . sequence.
The a~ino acid sequence of 10-23rd fragment can be
synthesized by various methods. A preferable method for synthesis is
the condensation of fragment having lO-13th amino acld sequence with
the decapeptide of 14-23rd sequence, or the condensation of the said
. . .
: fragment 10-13th sequence with the fragment of 14-16th, 14-19th o~
14-20th sequence in the decapeptide followed by condensation of the
residual fragment up to 23rd amino acid sequence.
The protective group of C-terminal fragment having
protected alpha-amino group hereinabove, for example tricosapeptideamide
of 10-32nd sequence or docosapeptide amide of 11~32nd sequence, is
removed by a suitable method. ~or example, the trityl group is split by
-- 10 --
'
.. ..
'~ ' ' , ~: ' ` ': '. :
: . ~, .
....
` 104~0t~8
.
aqueous acetic acid. ~iphenyl isopropoxycarbonyl, benzyloxyca~bonyl
and t-butoxycarbonyl groups are removed by a mixture of glacial acetic
acid, formic acid and water; hydrogenation; and trifluoroacetic acid,
respectively.
The azide method or W~nsch method is pre~erable for the
condensation of N-terminal amino acid sequence having a disulfide bridge
and at least having a protected alpha-amino group, such as nonapeptide
of 1-9th sequence or decapeptide of l-lOth sequence, with C-terminal
fragment having free alpha-amino group.
-: 10 Thus the dotriacontapeptideamide having protected a
alpha-amino group, protected ~-amino group, optionally protected side
chain carboxyl group and/or hydroxyl group, is obtained. These pro-
- ::
tective groups are split by the process hereinbefore described, and
finally the product [I~ can be obtained.
`- Dotriacontapeptideamide having a free mercapto group
can be synthesized by the same method as for protected dotriaconta-
peptideamide The protective group for the said mercapto group should
be removed at the final stage of synthesis, and the disulfide bridge is
completed by the method previously described; then the product ~I] can
` 20 be synthesized.
The novel polypeptide rI] of the present invention can
- be obtained in the form of free base or a salt thereof. The free base
may conventionally be obtained from its salt. The free base can be
changed to its pharmacologically acceptable salt by reacting with an
inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric
acid, or phosphoric acid, or an organic acid such as formic acid, acetic
acid, propionic acid, glycole acid, lactic acid, pyruvic acid, oxalic
acid, succinic acid, malic acid, citric acid, tartaric acid, benzoic acid,
benzenesulfonic acid or toluenesulfonic acid.
-- 11 --
`
.
10410~8
.
The novel polypeptlde can be converted into a complex
thereof by addition of various inorganic or organic substances. The
said complex form is composed by adding a kind of inorganic or organic
substance to a long chain polypeptlde, and means the unknown construction
of the compound having a long term activity when administered. Examples
of the said complex forming substance are inorganic compounds derived
from metals such as calcium, magnesium or zinc, especially phosphate,
pyrophosphate or polyphosphate of the said metals. Further examples of
the said complex forming organic substance are non-antigenic gelatine,
- 10 CMC, polyglutamic acid or the like.
The abbreviations in this in~ention have the following
meanings.
BOC : t-butoxycarbonyl,
, AOC : t-amyloxycarbonyl,
DIP : diisopropylmethoxycarbonyl,
. . .
Cbz : benzyloxycarbonyl,
ClCbz: o-chlorobenzyloxycarbonyl,
Bzl : benzyl,
Mbzl : p-methoxybenzyl,
:, .................................................................... ..
Tos : tosyl,
-- O~t : ethyl ester,
OBzl : benzyl ester,
ONP : p-nitrophenyl ester, -
OSU : N-hydroxysuccinylimide ester,
Cys : L-cysteine, Gly : glycine,
Ser : L-serine, Lys : L-lysine
Asn : L-asparagine, Gln : L-glutamine,
Leu : L-leucine, Glu : L-glutamic acid,
Thr : L-threonine, His : L-histidine,
Val : L-valine, Tyr : L-tyrosine,
- 12 -
: -
.
.. . . . . .
", ,:' ' " ': '' ' '
104~08;~
~ro : L-proline,
Arg : L-arginene,
Asp : L-aspartic acid,
Ala : L~alanine,
....
TFA : trifluoroacetic acid,
TosOH : p~toluenesulfonic acid,
CHA : cyclohexylamine~
- DCHA : dicyclohexylamine,
~ THF : tetrahydrofuran,
.:
DMF : dimethylformamide~
AcOEt : ethyl acetate,
DCC : dicyclohexylcarbodiimide,
,.; WSC : N-ethyl-N~-dimethylaminopropyl-carbodiimide,
~ HOBT : l-hydroxybenzotriazole,
~ .. .. . .
~ MeOH : methanol,
.,
EtOH : ethanol,
~- AcOH : acetic acid,
The following examples illustrate the present invention
but not construed as limiting upon the-scope thereof as defined by the
appended claims.
The assay method for serum calcium reducing activity,
the carrier and developing system for thin layer chromatography and
conditions for amino acid analysis are as follows:
[Assay metho~
Sample is diluted adequately with 0.1 N sodium acetate -
0.1% albumin solution. Male rats are subJected indi-
vidually to intramuscular injection with 0.2 ml. of the
- respective diluted solutions. After one hour, all the
rats are killed to obtain their respective blood, and
serum calcium ~alue of each blood sample is determined
- 13 -
: ~,
,
~. . ' , , , ~ :
;: : ~ : :-
::
10410E3~3 ~
, .
. ..
by atom~c abso~ption spectrophotomery, On the other
hand, Research Standard B, which ls an extract
:;. .
` obtained from the thyroid gland o~ the hog, is diluted
~` - so as to give dilutions o~ 2.5, 5~ 10 and 20 MRC mu/0.2 ml.
respectivel~. Male rats are subjected to intramuscular
. . .
i~ injection with 0.2 ml. of the respective diluted
solutions. Following the same procedure as above, the
;~ serum calcium value of the rats is determined after
~ one hour of in~ection. From the potency of the corres-
; - : -
ponding Research Standard, the potency of the calcitonin ;~
' in the sample is determined.
Thin layer chromatography (TLC)
~ . . :
'Carrier: silica gel G. -
cellulose `
. Solvent system for developer:
1. CHC13 - MeOH - AcOH 95 : 5 : 3 ~
2. CHC13 - MeOH - AcOH 85 : 10 : 5 ~;
3. CHC13 - MeOH - AcOH 80 : 15 : 2
4. CHC13 - EtOH - AcOEt 5 : 2 : 5
5. CHC13 - MeOH - AcOH - H20 10 : 10 : 1 : 1 (lo~er layer)
6. Benzene - AcOEt 2 : 1
7. n-BuOH - pyridine - AcoH - H20 15: 10 : 3 : 12 -~
8. methanol `
- 9. CHC13 - MeOH - AcOH 80 : 15 : 5
[Amino acid analysis~ -
Sample (20 gamma) is hydrolysed by 6 N HCl ~0.25 ml.)
at 105C., for 24 hours, and dried out under reduced pressure. Residue
is dissolved in 0.2 N citrate buffer solution (pH 3.25, 1.2 ml.) and
.. , :
1 ml. of the solution is subjected to amino acid analysis.
;.',
.. . .
- 14 -
. .
.',," ~ ~:
. --: ~ . , .: ~ . . : :
' ' ' ' :.. '~ '' '' ~ '
1 0 4 1 0 ~ 8
~`
EXAM~LF 1
Production of:
...
H-Cys-Ser-Asn-Leu-Ser~Thr~Cys-Va
Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-
.: .,
.:~ His-Lys~Leu-Gln-Thr~Tyr-Pro-A~g-
>' Thr-Asp-Val-Gly-Ala-Gly~-Thr-Pro-NH2
:.,
`~ 720 mg. of BOC-Lys(DIP)-Leu-Ser(Bæl)-Gln-Glu(OBzl)-Leu-His-Lys(DIP)-
. .
Leu-Gln-Thr(B~l)-Tyr(Bzl)-Pro-Arg(Tos)-Thr(Bzl)-Asp(OBzl~Val-Gly-Ala-
Gly-Thr(Bzl)-Pro-NH2 was dissolved in 5 ml. of T~A at -5C. ~fter
stirring for 35 minutes at room temperature, the solution was con-
centrated in vacuo and ethyl ether was added thereto to form a pre-
' '' ,
cipitate. The precipitate was dried over sodium hydroxide. The dried
~ precipitate was dissolved ~n 1 ml. of DM~. To that solution were added- 30 ml. of HOBT, 45 mg. of DCC and 220 mg. of BOC-Cys~Ser-~sn-Leu-Ser-Thr-~ys-
Val-Leu-Gly-OH at -5 C. and the mixture stirred for 2 hours at OC.
and overnight at room temperature. To the reaction mixture was added
water. The thus-formed precipitate was collected by filtration, washed
thoroughly with water and ethyl acetate, then dried in vacuo to obtain
740 mg. of crude product. The above 640 mg. of crude material were
treated with hydrogen fluoride at 20C. for one hour in the presence of
3 g. of phenol and 2.5 ml. of anisole. After distillation of hydrogen
- fluoride, the residue was dissolved in 200 ml. of 0.5 mole acetic acid.
The solution was washed with 150 ml. of ethyl acetate and passed
througll a column (2 X 20 cm.) of Dowex l X 2 (acetate form) and the
eluate was free~e-dried to obtain 590 mg. of the powder.
500 mg. of this powder were dissolved in 50 ml. of water
- adjusted by HCl at pH 4.37, and the said solution was charged on a column
a~4~zr~
- 15
~.,. ~
~,.
,. . , ' ' '
- ,, :. : . ~
:: :
10410~8
.
,
,. .
(2.0 cm. X 63 cm.) packed wlth carboxymethyl cellulose by 0.01 mole
am~onium acetate (pH 4.37), then washed with 4 1. of 0.01 mole acetate
buffer (pH 4.37) and 8 1. of 0.01 mole ammonium formate (pH 4.37).
Thereafter, the column was gradiently eluted with 12 1. of ammonium
formate ~0.01 mole (pH 4.37) - 0.07 mole (pH 6.0)~ . The eluate was
collected in separate 100 ml. ~ractions and the active fractions
checked by bio-assay were gathered and freeze dried to obtain the
: .
active powder.
~; 50 mg. of this active powder was dissolved in 5 ml. of
: 10 0.1 N formic acid, passed through a column (1.2 cm. X 250 cm.) of
~t
Sephadex G-50, and eluted with 0.1 N formic acld at a flow rate of
10 ml./hr. Active fractions were collected and freeze-dried to obtain
the product.
m.p. : 220 C. (decomposed).
r~D = -75 (c=1, 0.1 N-HCOOH).
~I Potency = 4000 MRC u/mg.
., : .
~ Amino acid composition:
,
Cys/2 2,13 (2), Ser 286 (3), Leu 5.10 (5),
~ Thr 3.48 (4), Val 2.20 (2), Gly 3.00 (3), Lys 1.98 (2), Glu 3.27 (3), -~
; 20 His 0.95 (1), Tyr 0.90 (1), Pro 1.96 (2), Arg 1.03 (1), Asp 2.07 (2),
Ala 0.94 (1) - -
.~ R value = 0.65 - 0.71,
. carrier: cellulose,
developer : n-BuOH - pyridine - AcOH - water
(15 : 10 : 3 : 12)
- The startlng material is produced as follows: Production of
partial amino acid sequence 11 - 32: BOC -Lys(DIP)-Leu-Ser(Bzl)-Gln-
Glu(OBzl)-Leu-His-Lys(DIP)-Leu-Gln-Thr(Bzl)-Tyr(Bzl)-Pro-Arg(To)-Thr-
:: (Bzl)-Asp(OBzl)-Val-Gly-Ala-Gly-Thr(Bzl)-Pro-NH2.
~ ~r~ark
16 -
'''
.
` 1041088
(1) Production of Boc-Thr(Bzl)-pro-NH2
BOC-Thr(Bzl)-OH (67.7 g.), Il-~ro-NH2-HCl (33.O g.) and HOBT (29.6 g.)
are added in TH~ (220 ml.). Thereto ls added WSC (40.0 ml.) under cooling
at -5C. stirred for one hour at -5 C. and then overnight at room
temperature. The reaction mixture i8 concentrated in vacuo and ethyl
:~ ,
acetate (800 ml.) is added to the residue, which is then washed twice
. .
with 1 N HCl (400 ml.) and twice with 5% sodium bicarbonate solution
(300 ml.) and water. After dehydration by anhydrous magnesium sulfate,
the organic layer is concentrated in vacuo. The oily material is
charged for silicagel (450 g.) column chromatography. DeveloFing by a
mixture of benzene-ethyl acetate (1 : 1), ethyl acetate and methanol
in this order and eluting fractions are checked by silica gel thin
layer chromatography. Active fractions are collected and dissolved in
~- ethyl acetate (500 ml.) then washed twice with 5% aqueous sodium bi-
carbonate (300 ml.) and water. A~ter drying with anhydrous magnesium
sulfate, concentrate in vacuo. The residue is treated with n-hexane
to obtain BOC-Thr(Bzl)-Pro-NH2 as white powder (54.5 g.;yield 61.4%).
Rfl = 0.48, ~ D = -14.0 (c~l, DMF).
Elemental analysis (C21H31NO3):
C~ H% N%
- found: 62.04 8.01 9.90
calculated:62.20 7.71 10.30
(2) Production of BOC-Ala-Gly-OBzl:
BOC-Ala-OH (94.6 g.), H-Gly-OBzl.TosOH (168.8 g.) and HOBT (67.6 g.) are
dissolved in THF (450 ml.) and WSC (91.5 ml.) is added thereto, then
stir for one hour at 0C. and overnight at room temperature. Reaction
mixture is concentrated in vacuo and residue is dissolved in ethyl
acetate (800 ml.). lN HCl (500 ml.) is added, the precipitate is
removed by flltration, and the ethyl acetate layer is washed with
: 30 1 N HCl (400 ml.), twice with 5% sodium blcarbonate solution and water
'
- 17 -
.
, ~ :
, .
;
-. ~0410B8 :-~
. .
in this order. A~ter drying anhydrous magnesium sulfate, concent~ate
in vacuo. Residue is treated with n-hexane to obtain crude material
(152.5 g.). Recrystallization i9 carried twice by ethyl acetate-n-
hexane to obtain BOC~Ala-Gly-OBzl (144 g " yield 85.7%). m.p. 87-88.5 C.
TLC: one spot, RFl ~ 0.59.
(3) Production of BOC-Ala-Gly-OH:
BOC-Ala-Gly-OBzl (101 g.) is dissolved in THF (500 ~1.) and hydrogenized
! under 5% palladium/carbon (7 g.). After 6 hours, catalyst is removed,
concentrated, dissolved in THF (800 ml.), then again hydrogenized under
5% palladium/carbon. After 4 hours, catalyst is removed and con-
-~ centrated in vacuo, and the residue is solidified by treating with
n-hexane. The solid residue is recrystallized by ethyl acetate-THF-n-
hexane to obtain BOC-Ala-Gly-OH (72 g., yield: 97.4%) with m.p. 129 -
~ 132C.
; TLC: one spot (solvent system 2). ~] D ~ 8.87 (c=l, DMP).
Elemental analysis (C]oH18NzO5):
C% H% N~
found: 48.80 7.41 11.29
calculated: 48.77 7.37 11.38
(4) preparation of BOC-Ala-Gly~Thr(Bzl)-Pro-NH2:
: BOC-Thr(Bzl)-Pro-NH2 (10.0 g.) is added to TFA (30 ml.) at -5C.,stirred for 30 minutes, and concentrated in vacuo. Residue is treated
with ether, filtered and dried over sodium to obtain
H-Thr(Bzl)-Pro-NH2.TFA.
BOC-Ala-Gly-CH obtained in (3) (6.08 g.) and HOBT (3.34 g.) are added
1II TIIF (100 ml.) aI1d WSC (4.5 ml.) is addcd tIIcreto at -5C. (pH 4.0).
After stirring at -5C. for one hour and at room temperature for 7 hours,
- the mixture is cooled at -5C., WSC (0.8 ml.) added and pH adjusted to
- 4.0, and stirred at -5C. for one hour and at room temperature for
: 30 overnight, and concentrated ir. vacuo. Residue is dissolved in ethyl
~' , .
- 18 -
~ . .
", ,t-~
1041()88
:
acetate (400 ml.), washed with saturated sodium chloride solution,
twice with 5% aqueous sodium bicarbonate (100 ml.), 1 N HCl saturated
with NaCl (100 ml.) and water (50 ml.), in this order. Ethyl acetate
layer is dried with anhydrous sodium sul~ate and the a~ueous layer is
extracted with chloroform ~hich is washed twice with water to combine
with the ethyl acetate layer. Organic layer is condensed in vacuo and
the residue is chromatographed using silica gel ~150 g.). The column
-- is flushed with ethyl acetate and methanol, and methanol eluate is
condensed in vacuo. n-hexane is added into the residue to crystallize
lo the BOC-Ala-Gly-Thr(Bzl)-pro-~l2. m.p.: 106-116C.
- 11.5 g. (Yield: 87.3%).
TLC: one spot, Rf4 = 0.29, Rf5 = 0.52
(5) BOC-Val-Gly-OEt
BOC-Val-OH.DCHA (219.2 g.) is added to ethyl acetate (1 1.). The
solution is washed twice with 1 N HCl (600 ml.) and water (SOO ml.) in
this order. After drying with anhydrous sodium sulfate, the solution
is concentrated in vacuo. THF (100 ml.) and dichloromethan (400 ml.)
are added into the residue, and H-Gly-OEt.HCl (69.8 g.) and triethyl-
amine (70 ml.) added thereto. DCC (103.2 g.) is added thereto at -5C.
and stirred at -5C. for 1 hour and at room temperature for overnight.
: .
- Acetic acid (10 ml.) is added to the reaction mixture, sti.rred for
1 hour and a filtrate obtained. The filtrate is washed twice with
1 N ECl (500 ml.), twice with 5% sodium bicarbonate solution (500 ml.)
` and water in this order. After drying with anhydrous sodium sulfate,
concentrate in vacuo. Residue is recrystallized three times using ethyl
.,
acetate-n-hexane to obtain BOC-Val-Gly-OEt (105.4 g.), m.p.: 95 - 96 C.
Yield: 69.7%.
, (6) Preparation of BOC-Val-Gly-OH:
BOC-Val-Gly-OEt (96.8 g.) is dissolved in methanol (150 ml~) and -;
1 N NaOH solution (368 ml.) at ~5C. is added thereto. After one hour
."' ~ .
- . - 19 - '
'
; ~ ! ,
, r,
. . . ' ~ ~
::
~04~0~8
of stirring, pH is ad~usted to 8.0 by addition of 1 N HCl. Methanol is
removed by concentration in vacuo and aqueous layer is washed with ethyl
ether (100 ml.). The aqueous layer is adjusted to pH Z.O by addition
of 1 N HCl. The aqueous layer is ext~acted ~our t~mes with ethyl
acetate (200 ml.) 9 and the extract is dried by using anhydrous sodium
sulfate and thereafter concentrated in vacuo. The residue is treated
with n-hexane and recrystallized from ethyl acetate-n-hexane to obtain
BOC-Val-Gly-OH (85.5 g., yield 97.4%). m.p.: 112 - 117 C. (decomp.)
TLC : one spot, Rfl = 0.35.
(7) Preparation of BOC-Val-Gly-~la-Gly-Thr(Bzl)-Pro-NH2 :
BOC-Ala-Gly-Thr(Bzl)-Pro-NH2 (10.5 g.) is added to TFA (35 ml.) at -5 C.
After stirring for 30 minutes, concentrate in vacuo. Residue is treated
with ethyl ether, precipitated material is filtered and dried in vacuo
over sodium hydroxide to obtain H-Ala-Gly-Thr(Bzl)-Pro-NH2.TFA.
THF (100 ml.), is added thereto, and triethylamine is added at -5C.
to adjust pH to 5Ø BOC-Val-Gly-OH (5.49 g.) and HOBT (2.66 g.) are
added thereto. To this mixture WSC (3.61 ml.) is added dropwise at
-5C, and the mixture stirred at -5C. for 1 hour and at room temperature
for overnight. Reaction mixture is concentrated in vacuo, and the
. ,. ~
residue is dissolved in chloroform (300 ml.). The solution is washed
twice with NaCl saturated 1 N HCl (200 ml.), twice with NaCl saturated
; 5% sodium bicarbonate solution (200 ml.) and saturated NaCl solution
(75 ml.) in this order. After the solution is dried with anhydrous
sodium sulfate, the organic layer is concentrated in vacuo. Recrystal-
`~ lization from methanol-ethyl ether gives BOC-Val-Gly-Ala-Gly-Thr(Bzl)-
Pro-NH2 (12.0 g.) (yield: 88.3%).
m.p.: 121 - 135 C. (decomp.). TLC: one spot. (solvent system 5).
[~ 28 = -10 30 (c=l, DMF)
Elemental analysis (C33H51N709.2 H20)
': ~
~,
; ~ " '' ' ' '~, ' ' ' '` '
'`: ` ' ~'.` `' .
:
~- ~041088
-~ C% H% N%
found: 56.67 7.43 13.90
calculated: 56.72 7.50 14.03
(8) Preparation of BOC-Asp(OBzl)-ValrGly-Ala-Gly~Thr(Bzl)-Pro-NH2:
TFA (100 ml.) is added to BOC-Val-Gly-Ala-Gly-Thr(Bzl)-Pro-NH2 (33.1 g.)
at -5C.; stir for 30 min. and concentrate in vacuo. Ethyl ether is
added to the residue and the precipitated ~aterial is dried in vacuo
~- over NaOH. The DMF (80 ml.) is added and triethylamine is further
added to ad~ust the pH to 7.5. After adding HOBT (950 mg.) and
BOC-Asp(OBzl)-OSU (30.2 g.) thereto, pH is ad~usted to 7.5 by adding
N-methylmorpholine and the mixture stirred for 2 days. The pH is
adjusting during stirrîng at pH 7.5 by adding N-methylmorpholine.
; Then adding BOC-Asp(OBzl)-OSU (2.0 g.) again, adjusting pH to 7 by
.. :
addition of N-methylmorpholine and stir for overnight. A large amount
of water is added to the reaction mixture, and the precipltated sticky
j- substance is separated by decantation to crystallize by treatment with
ethyl ether. Aqueous layer is extracted with chloroform and the
extract is concentrated in vacuo. The residue is treated by addition
of water to precipitate a sticky substance. The precipitated sticky
: . .
substance is crystallized by treatment with ethyl ether. The solids
are combined and recrystallized from methanol-ethyl ether four times,
and washed with hot ethyl acetate-ethyl ether, then dried to obtain
BOC-Asp(OB~ ~Val-Gly-Ala-Gly-Thr(Bzl)-Pro-NH2 (34.2., yield 79.6%).
- m.p.: 195 - 199 C. TLC : one spot. Rf5=0.46; ~ D P-18.27 (c-~l, DMF).
Elemental analysis (C44H62N6012H20):
- C% H% N~
found: 58.08 6.90 12.47
calculated: 57.88 7.07 12.27
,::
1 ~
.; . .
-., ,:,*
,: : . ::
-: ~ 104~088 ~
(9) Boc-Thr(Bzl)-AspcoBzl~al-Gly~Ala~Gly~Thr(Bzl)-pro~NH2
TFA (30 ml.) i9 added to BOC-Asp(OBzl)-Val-Gly-Ala-Gly-Thr(Bzl)~Pxo-N~12
(7.2 g.) at -5C.; stir for 30 minutes and concentrate in vacuo.
' Residue is treated by adding ethyl ether, and precipltated material is
dried in vacuo over NaOH. DMF (15 ml.) is added thereto and the pH
adjusted to 7.5 by add~tion of triethylamine. A~ter adding HOBT (100
- mg.) and BOC-Thr(Bzl)-OSU (4.0 g.), the pH is adjusted by N-methyl-
morpholine and the mixture stirred for 2 days at room temperature.
The pH is adjusted further by DMF (3 ml.) and N-methylmorpholine to
7.5, and the mixture stirred overnight.
After reaction is completed, large amount of water is ...
added thereto and precipitated material is filtered and recrystallized
from methanol-ethyl ether three times to obtain BOC-Thr(Bzl)-Asp(OBzl)-
Val-Gly-Ala-Gly-Thr(Bzl)-Pro-NH2 (6.7 g.; yield: 77.1%). m.p.: 172 -
188C. TLC: one spot. (solvent system 2 and 5)- L~] D =-13.65
- (c=l, DMF).
` Element~l analysis (C55H75N9014.2H20)
":. C% H% N%
~: .
found: 60.31 6.92 11.58
~- 20 calculated: 60.31 7.00 11.51
. . . .
(10) Preparation of AOC-Arg(TOS)-Thr(Bzl)-Asp(OBzl~-Val-Gly-Ala-Gly-
- Thr(Bzl)-Pro-NH2:
TFA (20 ml.) is added at -5 C. to BOC-Thr(Bzl)-Asp(OBzl)-~al-Gly-Ala-
` Gly-Thr(Bzl)-Pro-NH2 (6.52 g.); stir for 30 minutes and concentrate in
vacuo. Residue is treated by adding ethyl ether and the precipitated
substance is dried in vacuo over NaOH. DMF (25 ml.) is added thereto
~ and the pH is adjusted to 5.0 by adding triethylamine at -5 C. HOBT
::
~` (972 mg.) and AOC-Arg(Tos)-OH (3.2 g.) are added thereto, and WSC
(1.3 ml.) is added dropwise under cooling at -5C., then the mlxture
stirred for one hour at -5C. and for overnight at room temperature.
- 22 -
,`''1'~
'
10410~8
:.
Ethyl acetate is added to the reaction mixture, and the
- precipitate is treated with hot methanol (400 ~ ethyl ether (50 ml.),
:,
Thereafter, the mixture is treated with hot methanol (300 ml.)~ethyl-
ether (100 ml.) to obtain ~OC-Arg(Tos)-Thr(Bzl)-~sp(OBzl)-Val-Gly-Ala-
Gly-Thr(Bzl)-Pro-NH2 (7.0 g.; yield 82.7%). m.p.: 187 - 193 C. ~decomp.).
- TLC: one spot (solvent system 2 and 5) Rf2~0.42.
Elemental analysis (C69H95N13017S.H2O):
C% H% N% S%
': ."
found: 57.97 6.88 12.76 2.26
calculated: 58.01 6.84 12.75 2.24
Amino acid composition: NU3 1.28, ~rg 0.95 (1), ~sp 1.04 (1), Thr 1.50(2),
Pro 0.98 (1), Gly 1.84 (2), Ala 1.00 (l), Val 1.01 (1).
(11) Preparation o~ BOC-Tyr(Bzl)-Pro-OBzl:
BOC-Tyr(Bzl).OH.CH~ (37.6 g.) is mixed with ethyl acetate (300 ml.) and
~ l N HCl (120 ml.), separated and the ethyl acetate layer washed three
- ; times with water. After drying with anhydrous sodium sulfate, the
organic layer is concentrated in vacuo and the oily residue is dissolved
in dichloromethane (80 ml.). H-Pro-OBzl.HCl (19.3 g.) is then added
thereto and WSC (14.6 ml.) is added to the mixture at -5C.; then the
mixture was stirred for one hour at -5 C. and for overnight at room
temperature.
Reaction mixture is concentrated in vacuo, and the
residue is shaken with ethyl acetate (800 ml.) and 1 N HCl (400 ml.)
to separate the ethyl acetate layer. The organic layer is washed
twice with 1 N HCl (300 ml.), twice with water (300 ml.), three times
with 5% aqueous sodium bicarbonate (300 ml.) and twice with water
(300 ml.), in this order. The organlc layer is dried with sodium
carbonate and concentrated in vacuo. Crude product is sub~ect to column
chromatography using silica gel (400 g.). Chloroform~ethyl acetate
(4 : 1) mixture is passed through the column and the eluates are checked
. ~ ..
_ 23 -
- -,;,` '
.,;, ,`~ , .
~ 1041088
.... :
; by thin layer chromatography using solYent system 1 to obt:ain oily
BOC-Tyr(Bzl)-~ro-OBzl (43 g ) ~ 0.88.
(12) Preparation of BOC-Thr~Bzl)-Tyr(Bzl)-~ro-OBzl:
T~ (50 ml.) is added under cooling at -5C. in BOC-Tyr(Bzl)-Pro,OBzl
` (20 g.), stirred for 10 minutes at -5C. and 50 minutes at room
,-. :-:
temperature, and concentrated in vacuo. Oily residue is dried over
NaOH ~or one night, DM~ (30 ml.) is added and the pH adjusted to 7.5
by addition of triethylamine to -5 C. HOBT ~470 mg.) and BOC~Thr(Bzl)-
,.
OSU (14.2 g.) are added and the mixture stirred at -5 C. for 1 hour
and at room temperature for overnight. During stirring, the stir
reaction mixture is maintained at pH 7 by adding N-methylmorpholine.
After 2 days, dimethyl amino propylamine (1 ml.) is added and the
` mixture stirred for a further 2 hours and concentrated in vacuo.
i` Residue is shaken with water (500 ml.~ and ethyl acetate (500 ml.) to
~r~, ' separate ethyl acetate layer. ~queous layer is again extracted with
ethyl acetate ~200 ml.). ~thyl acetate layer is combined, and indivi~
. .dually washed with 1 N HCl, water, and 5% sodlum bicarbonate solution
and water in this order. After dehydration with anhydrous magnesium
sulfate, the organic layer is concentrated in vacuo. Residue is
crystallized from ethyl ether-n-hexane to obtain crude material
.. ; (19.6 g.). This crude material is recrystallized from the same solvent
to give BOC-Thr(Bzl)Tyr(Bzl)-Pro-OBzl (14.4 g.: yield 54.9%).
Rf2 = 0.67.
(13) Preparation of BOC-Thr(Bzl)-Tyr(Bzl)-Pro-OH:
BOC-Thr(Bzl)-Tyr(Bzl)-Pro-OBzl (14.2 g.) ls dissolved in TH~ ~30 ml.),
and lN NaOH solution (23 ml.) is added dropwise for 15 minutes at -5C.
- After stirring for 4 hours at room temperature, pH is adjusted to 7
with 1 N HCl additlon, then mixture concentrated ln vacuo to remove THF.
Water is added to a~ueous layer and after washing with ethyl ether, pH
of the water layer ls adJusted to pH2 by adding 1 N MCl, then extracted
" ' ''
- 24 -
.. ... .
.. ...
,; '` ' st i ~ '
,
,
,
.
104~0~38
twice with ethyl acet~te. ~thyl acet~te layer ~S washed with wa~er and
dried by anhydrous magnesium sulfate and concentrated in vacuo. Residue
is crystallized from ethyl ether-n-hexane to obtain crude material
(12 g~). This crude material i5 dissolved in ethyl acetate, ethyl
ether added thereto and the solution is set aslde to crystallize by
gradual addition of n-hexane to give BOC-Thr(Bzl)-Tyr(Bzl)-Pro-OH
~10.5 g.: yield 84%). m.p. 134 - 136C. TLC: one spot, Rfl=0.68,
- Rf4=0.63.
Elemental analysis (C H N O ):
; 10 C% H% N% ~-
found: 66.85 6.80 6.68
; .
calculated:67.35 6.88 6.37
(14) Preparation of BOC-Thr(Bzl)-Tyr(Bzl)-Pro-Arg(Tos)-Thr(Bzl)-Asp(OBzl)-
; Val-Gly-Ala-Gly-Thr(Bzl)-Pro-NH2: ~ -
.,:
AOC-Arg(Tos)-Thr(Bzl)-Asp(OBzl)-~al-Gly-Ala-Gly-Thr(Bzl)-Pro-NH2 (6.0 g.) ~ -
is added in TFA (20 ml.) at -5C., stirred for 30 minutes and con- ~ ~
centrated under reduced pressure. Residue is treated with ethyl ether -
and dried in vacuo over NaOH. D~E (20 ml.) is added and after adjusting
the pH to 4.5 by addition of triethylamine at -5C,, HOBT (851 mg.) and
BCC-Thr(Bzl)-Tyr(Bzl)-Pro-OH (4.2 g.) are added and WSC (1.2 ml.) is
` added dropwise at -5C. Stirring ls carried for 1 hour at -5C. and
for one night at room temperature. Methanol (300 ml.) and ethyl ether
(100 ml.) are added to the reaction mixture, and precipitated material
is filtered and recrystallized three times with methanol-ethyl ether,
DMF-ethyl ether and methanol-ethyl ether, in this order, to obtain
Boc-Thr(Bzl)-Tyr(Bzl)-pro-Arg(Tos)-Thr(Bzl)Asp(oBzl)-val-Gly-Ala-G
Thr(Bzl)-Pro-NH2 (5.8 g., yield: 71.3%).
m.p.: 186.5 - 190C. (decomp.~. TLC: one spot (solvent systems 2 and 5).
~ 28_ -15.86 (c=l, DMF)-
:
:,,............. .: : , . : -:
i
::
1041088
Elemental analysiS (ClooHl28Nl6o22s 2 2 )
C% H% N% S%
,.
found: 61.44 6.63 11.551.73
- calculated: 61.68 6.68 11.511.65
Amino acid analysis: NH3 1.60, Arg 0.92 (1), Asp 1.04 (1), Thr 2.1 (3),
Pro 2.08 (2), Gly 1.86 (2), Val 1.00 (1), Tyr
, .
;~
'` 0.91 (1).
(15) Preparation of Boc-Gln-Thr(Bzl)-Tyr(Bzl)-pro-Arg(Tos)-Thr(Bzl)A
(OBzl)-Val-Gly-Ala-Gly-Thr(Bzl)-Pro-NH2:
BOC-Thr(Bzl)-Tyr(Bzl)-Pro-Arg(Tos)-Thr(Bzl)-Asp(OBzl)-Val-Gly-Ala-Gly-
Thr(Bzl)-Pro-NH2 (5.4 g.) is added to TFA (20 ml.) at -5C., the
.
mixture stirred for 30 minutes, and concentrated ln vacuo. Residue is
treated with ethyl ether and dried over NaOH in vacuo. DME (18 ml.)
::
- is added and then triethylamine is added to adjust pH to 7.5 and HOBT
(100 mg.) and BOC-Gln-ONP (1.54 g.) are added, followed by stirring
for 2 days at room temperature. During stirring, the pH is maintained
:,:''` . '
, at 7.5 by addition of N-methylmorpholine. Thereafter, BOC-Gln-ONP
(o.2 g.) is added and pH adjusted to 7.0 with stirring for overnight.
.
After the reaction, a large amount of water ls added in the reaction
mixture to precipitate the product, which is recrystallized three times
from methanol-ethyl ether to obtain BOC-Gln-Thr(8zl)-Tyr(Bzl)-Pro-~rg-
(Tos)-Thr(Bzl)-Asp(OBzl)-~al-Gly-Ala-Gly-Thr(8zl)-Pro-NH2 (4.8 g.,
yield: 82.9%). m.p.: 178 - 182 C. Rf2 ~ 0.67, ~7 D =-15.19 (c=l,DMF).
Elemental analysis (C105H136N18024S 2
C% H% N% S%
found: 59.92 6.5612.05 1.47
;- calculated: 59.98 6.71 11.99 1.53
(16) Preparation of BOC-Lys(DIP)-Leu-OBzl: ~
BOC-Lys(DIP)-OH (38.9 g.) and H-Leu-OBzi.TosOH (43.3 g.) are dissolved `
in dichloromethane (200 ml.). WSC (20 ml.) is added thereto at -5C.
_ 26 -
.' ~ ' _
. . ;- . . ~
. '
10410~38
"
and stirred at -5C. for 1 hour and at room temperature for one night.
Reaction mixture is concentrated in vacuo and residue is extracted with
ethyl acetate, then washed with 1 N HC1, 5% sodium bicarbonate solution
and water, in this order. After drying with anhydrous magnesium sulfate,
i the extract is concentrated in vacuo and crystalline precipitate is
recrystallized from ethyl acetate-n-hexane to obtain BOC-Lys(DIP)-Leu- ;
OBzl (52 g., yield 88.1%).
m.p.: 100 - 102C. TLC: one spot, Rfl = 0.53.
(17) Preparation of BOC-His-Lys(DIP)-Leu-OBzl: ~ -
BOC-Lys(DIP)-Leu-OBzl (17.8 g.) is added at -5C. in T~A (110 ml.).
. .
After 40 minutes stirring, the reaction mixture is concentrated in vacuo.
Residue is dried over NaOH in vacuo. THF (150 ml.) is added thereto,
; and triethylamine and UOBT (4.9 g.) further added at -5C., followed by -
adding BOC-His(Tos)-OH (14.8 g.) and WSC (6.6 ml.). The pH is finally ~ ~-
adjusted to 4.0, and the mlxture stirred for one hour at -5C. and for
; overnight at room temperature. After reaction is completed, mixture is
i., :
concentrated in vacuo, ethyl acetate added, and washed with 1 N HC1,
1 N sodium bicarbonate solution and water, in this order. The organic
:
layer is dehydrated with anhydrous magnesium sulfate and then con-
centrated in vacuo. The concentrate is chromatographed using silica
gel with developer of chloroform-ethanol-ethyl acetate (5 : 2 : 5).
The eluted fractions are checked by silica gel thin layer chromatography
using the same solvent system and the active fraction collected. The
collected fraction is concentrated ln vacuo and recrystallized from
i ethyl acetate-n-hexane to obtaln BOC-His-Lys(DIP)-Leu-OBzl (19.7 g.).
~ m.p.: 214 - 216.5C. (decomp.).
- TLC: one spot, Rf4=0.16, ~7 D =-26.3 (c=l,DMF).
- (18) Preparation of BOC-Leu-His-Lys(DIP)-Leu-OBzl:
TFA (30 ml.) is added in BOC-His-Lys(DIP)-Leu-OBzl (8.7 g.~ at -5C.
and the mixture is stirred for 30 minutes, then concentrated in vacuo.
,,. . ~ . ~ . ,,
.:~
_ 27 - -
~ ' '' ; ~
- 10410~8
Oily residue is dried over NaOH in vacuo~ D~E (10 ~1.) ls ~dded and
pH adjusted to 7.5 at -5 C. with addit~on of triethylamine. Thereafter,
HOBT (1 g.) and BOC-Leu-OSU (4.7 g.) are added, and the mixture stirred
for 30 minutes at the same temperature and for 3 days at room temperature.
A large amount of water is added to the reaction mixture, precipitated
material is filtered and dissolved in ethyl acetate, and N,N-dimethyl-
amino 1,3-propanediamine (1 ml.) is added thereto and the mixture
stirred for 30 minutes. Ethyl acetate layer is washed with lN HCl, lN
sodium bicarbonate solution and water, in this order. After drying
with anhydrous sodium sulfate, the organic layer is concentrated in
,:
, vacuo. The thus obtained powdery substance is chromatographed using a
silica gel column. Developing with chloroformethanol-ethyl acetate
(5 : 2 : 5), the eluate ls checked by silica gel TLC. The fractions
containing the product are collected and concentrated in vacuo and
recrystallized from ethyl acetate, obtaining BOC-Leu-His-Lys(DIP)-Leu-
OBzl (7.3 g., yield 72.7%). m.p.: 94 - 88.5C. Rf2=0.37, Rf4=0.59,
[c~] DO =-28.5 (c=2, DMF).
(19) Preparation of BOC-Leu-His-Lys(DIP)-Leu-OH: ;
BOC-Leu-His~Lys(DIP)-Leu-OBzl (16.2 g.) is dissolved in methanol
(300 ml.) and hydrogenated in the presence of 5% palladium/carbon for
; 10 hours. After reaction, the catalyst is removed and the filtrate is
concentrated in vacuo. Residue is dissolved in methanol and the pre-
cipitated gel-like substance is filtered. This is recrystallized from
methanol-ethyl ether containing a small amount of chloroform to obtain
BOC-Leu-His(DIP)-Leu-OH (12.5 g., yield: 86.8%). Rf2=0.18.
(20) Preparation of BOC-Leu-~is-Lys(DIP)-Leu-Gln-Thr(Bzl)-Tyr(Bzl)-Pro-
Arg(Tos)-Thr(Bzl)-Asp(OBzl)-Val-Gly-Ala-Gly-Thr(Bzl)-Pro-NH2:
TFA (30 ml.) is added at -5C. to BOC-Gln-Thr(Bzl)-Tyr(Bzl)-Pro-Arg(Tos)-
Thr(Bzl)-Asp(OBzl)-Val-Gly-Ala-Gly~Thr(Bzl)~Pro-NH2 (4.5 g,), mixture
stirred for 45 minutes and concentrated in vacuo. Residue is treated
.~ ~
~ 28 -
;-,~ ;~
,~
.
.. 1041088
.
; with ethyl ether and thereafter dried over NaOH in vacuo. DMF (16 ml.)
. is added thereto and the pH adjusted to 6.0 by adding triethylamlne at
~; -5C; then BOC-Leu-His-Lys(DIP)-Leu-OH (2.2 g.), HOBT (386 mg.) and
WSC (0.52 ml.) are added. The pH is adjusted further by adding HOBT
z to 5.5 - 6.0 and the mixture stirred at room temperature for overnight.
~thyl acetate and ethyl ether are added to the reaction mixture, and
- the precipitated material is filtered. This is treated twice with DMF,
ethyl acetate, methanol and ethyl ether to obtain BOC-Leu-His-Lys(DIP)-
Leu-Gln-Thr(Bzl)-Tyr(Bzl)-Pro-Arg(Tos)-Thr(Bzl)-Asp(OBzl)-Val-Gly-Ala-
Gly-Thr(Bzl)-Pro-NH2 (6.0 g.).
m.p.: 162 - 166C. (decomp.). TLC: one spot, Rf3P0.38, Rf5-0.20,
[~ D =-18-20 (c=l, DMF).
Elemental analysis (Cl37Hl9lN25o3os- 2 2 )
C%- H% N% S%
found: 59.83 7.22 12.791.22
calculated: 59.947.20 12.761.17
Amino acid analysis: Lys 0.97 (1), His 0.96 (1), NH3 2.34, Arg 1.00 (1),
Asp 1.02 (1), Thr 2.43 (3), Glu 1.09 (l), Pro 1.96 (2) Gly 2.00 (2),
Ala 0.93 (1) Val 1.10 (1), Leu 1.94 (2?, Tyr 0.90 (1).
(21) BOC-Glu(OBzl)-Leu-His-Lys(DlP)-Leu-Gln-Thr(Bzl)-Tyr(Bzl)-Pro-Arg-
(Tos)-Thr(Bzl)-Asp(OBzl)-Val-Gly-Ala-Gly-Thr(Bzl)-Pro-NH2:
Heptadecapeptideamide ~16 - 32] obtained in (20) is added to TFA (25 ml.)
at -5C., mixture stirred for 40 minutes and concentrated in vacuo.
Residue is treated with ethyl ether and dried in vacuo over NaOH.
DMF (13 ml.) is added and pH ls ad~usted to 6.5 by addition of triethyl-
amine at -5 C. HOBT (270 ml.) and BOC-Glu(OBzl)-OSU (1.74 g.) are
added thereto, the pH ad~usted to 7 by addition of N-methylmorpholine
and the mixture stirred for 2 days at room temperature. ~urther BOC-
, Glu(OBzl)-OSU (870 mg.) is added the pH ad~usted to 7.0 and the mixture
stirred for 2 days. ~ ~-
29
A~ .
:
. ' ' ', - ':,: . . , , : .,
: - . , :, . , . , :
- :, : ,, , '.... ': . ~ : . '
10410~1
Ethyl acetate, methanol and ethyl ether are added to
the reaction mixture to precipitate a yellow product whlch is filtered.
DMF, ethyl ether and ethyl acetate are added to obtain the precipitate.
; This is treated with active carbon in hot methanol, and the ethyl ether
added to precipitate the product. Recrystallization from methanol-
ethyl ether gives the product (5.13 g., yield: 87.8%)
m.p.: 184 - 192C. Rf4=0.56
(22) Preparation of BOC-Gln-Glu(OB~l)-Leu-His-Lys~DIP)-Leu-Gln-Thr(Bzl)-
Tyr(Bzl)-Pro-Arg(Tos)-Thr(Bzl)-Asp(OBzl)-Val-Gly-Ala-Gly-Thr(Bzl)-
; 10 Pro-NH2:
Octadecapeptideamide obtained in step (21) [15 - 32~ (4.96 g.) is added
to TFA (35 ml.) at -5 C., the mixture stirred for 40 minutes and con-
centrated in vacuo. Residue is treated with ethyl ether, then dried
`~ in vacuo over NaOH. DMF (15 ml.), is added and the pH adjusted to 7.0
by addition of triethylamine at -5 C. ~fter adding HOBT (70 mg.) and
BOC-Gln-ONP (940 mg.), pH is adjusted to pH 7.0 by adding N-methyl-
~ morpholine and mixture stirred for one night at room temperature.
; BOC-Gln-ONP (320 mg.) is further added thereto and mixture stirred again
for one night.
To the reaction mixture is added a large amount of water.
i The precipitate is collected by filtration, and is then recrystallized
twice with methanol-ethyl ether and once with DMF-methanol-ethyl ether
and hot methanol to obtain the product (4.03 g., yield: 77.8%).
m.p.: 217 - 227C. (decomp.). TLC: one spot (solvent system 3).
~] 28 = -18.06 (c=l, DMF).
Elemental analysis (C154H212N28O35 2
C% H% N%
found:59.49 7.01 12.57
calculated: 59.63 7.08 12.65
- 30 -
1(~410~ :
.
Amino acid analysis: Lys 0.93 ~1), His 0.92 (1), Arg 0.85 (1), Asp 1.04
(1), Thr 2.43 (3), Glu 3:15 (3), Pro 1.92 (2), Gly 2.0 (2), Ala 0.84 (1),
Val 1,10 (1), Leu 2.00 (2), Tyr 0.74 (1).
(23) Preparation of BOC~Leu-Ser(Bzl)-OH:
H-Ser(Bzl)-OH (11.7 g.) is dissolved in a mlxed solution of sodium
carbonate (6.4 g.), water (50 ml.) and dioxane (10 Tlll.). BOC-Leu-OS~
(16.4 g.) dissolved in dioxane-water (3 : 1) (20 ml.) is added thereto
and stirred for one night at room temperature. After adjusting the pH
to 7 by adding 6 N HCl, the mixture is concentrated in vacuo. Residue
:
is mixed with ethyl acetate and 1 N HC 1 and the separated ethyl acetate
layer is washed with 1 N HC1 and water, dehydrated with magnesium
,. ::
sulfate, and concentrated in vacuo and crystallized from ethyl acetate-
i n-hexane. Recrystallization from ethyl acetate-n-hexane gives BOC-Leu-
Ser (Bzl)-OH (18.5 g., yield: 92.5%). m.p.: 82 - 85.5C. TLC: one spot, - -
Rfl = 0.44. ~] DO = ~6.9 (C=2, DMF).
(24) Preparation of BOC-Lys(DIP)-Leu-Ser(Bzl)-OH:
, TFA (90 ml.) is added to BOC-Leu-Ser(Bzl)-OH (18.0 g.) at -5 C., the
mixture stirred for 40 min. and concentrated in vacuo. Ethyl ether
is added to the residue to precipitate the product, which 1s dried
over NaOH in vacuo. This is dissolved in pyridine (40 ml.), and
HOBT (1 g.) and BOC-Lys(DIP)-OSU (21.O g.) added at -5C, and the
mixture stirred for 5 days at room temperature.
- A large amount of water is added to the reaction
- mixture, and the formed precipitate is filtered, dried and dissolved
in chloroform, then washed with 1 N sodium carbonate solution, 1 N
HC1 and water, in this order. After dehydration with anhydrous
- magnesium sulfate, the mixture is concentrated and the thus obtained ~-
gel-like substance is sub~ected to column chromatography using silica
gel. Developing with chloroform-ethanol-ethyl acetate (5 : 2 : 5), the
eluted fraction is checked by silica gel TLC using same developing system.
., ~
- 31 - ~
;.'' .~/~ ~
., . : ~ .
, ~- ' " '. ' .
10410~
The required fraction is c~llected and concentrated in ~cuo. Residue
is recrystallized from chloroform-ethyl ether to obtain BOC~Lys(DIP)- ~;
Leu-Ser(Bzl)-OH (12 g., yield: 40.3%). m.p.: 178.5 - 183.0 (decomp.).
Rf4=0.53, [~] D8= -7.1 (c=1, DMF).
Further chromatography is conducted using methanol on
the same column hereinabove to obtain the substance (9 g., yield:
30.2%).
(25) Preparation of BOC-Lys(DIP)-Leu-Ser(Bzl)-Gln-Glu(OBzl)-Leu-His-
,,,
- Lys(DIP)-Leu-Gln-Thr(Bzl)-Tyr(Bzl)-Pro-Arg(Tos)Thr(Bzl)-Asp(OBzl)-
Val-Gly-Ala-Gly-Thr(Bzl)-Pro-NH2:
TFA (35 ml.) is added at -5C. to the nonadecapeptideamide [14 -32]
obtained above in step (22) (3.81 g.), stirred for 40 minutes and
concentrated in vacuo. Resldue is treated with ethyl ether and dried
~- over NaOH in vacuo. DME (15 ml.) is added thereto, the pH of the
~; solution adjusted to pH 5 by addition of triethylamine, then HOBT
(220 mg.), BOC-Lys(DIP)-Leu-Ser(Bzl)-OH (1.10 g.) and WSC (0.3 ml.)
are added thereto at -5 C. and the mixture stirred for one hour,
following which the mixture is stirred overnight at room temperature.
HOBT (34 mg.), BOC-Lys(DIP)-Leu-Ser(Bzl)-OH (185 mg.) and WSC ~0.046 ml.)
are then added and the mixture stirred for 7 hours at room temperature.
Again, the same amount of HOBT, BOC-Lys(DIP)-Leu-Ser(Bzl)-OH and ~SC
are added thereto and the mixture stirred for one night.
After reaction is completed, a large amount of water is
added to the reaction mixture, the precipitated product is treated
twice with methanol-ethyl ether, treated with hot ethanol and centri-
fuged five times to obtain the required product (2.26 g., yield: 50.1%).
m.p.: 228 C. (decomp.). Rf3=0.54.
Elemental analysis (C184H260N32041S 2 )
:.
- - 32 -
~''
::
.: :: : ~:
~0410~38
.
C~ H% N%
found: 60.53 7.47 11.98
- calculated: 60.64 7.30 12.30
Amino acid sequence No. 1 - 10: Preparation of
BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-OH:
Solution of WSC (34 g.) in dichloromethane (50 ml.) is added dropwise
to a suspension of BOC-Leu-OH (46.2 g.), HOBT (1 g.) and H-Gly-OBzl.-
; TosOH (74 g.) in DMF (100 ml.) and dichloromethane (200 ml.) withstirring at -5 C. for 1 hour. After one hour, the solution is stirred
overnight at room temperature. The reaction mixture is concentrated
'.; in vacuo to remove dichloromethane. To DMF layer is added water andextracted with ethyl acetate (1 1.), and again the same solvent
' (500 ml.). Ethyl acetate layer is washed with 1 N HCl, water, 5%
;~ sodium bicarbonate and water, in this order and dehydrated with sodium
sulfate (anhydrous), then concentrated in vacuo to obtain oily BOC-Leu-
Gly-OBzl (82 g.). Rfl = 0.68.
- (27) Preparation of BOC-Val-Leu-Gly-OBzl:
TFA (70 ml.) is added to BOC-Leu-Gly-OBzl (76 g.) at -5 C., stirred
, . .
for 30 minutes and concentrated in vacuo. Residue is dried over NaOH
- 20 in vacuo. DMF (200 ml.) is added thereto and pH is adjusted to 6.5 by
addition o~ triethylamine (about 50 ml.) at -5C. BOC-Val-OSU (l9 g.)
~- and HOBT (2 g.) are then added. The reaction mixture is adjusted to; pH 6 by adding N-methylmorpholine, and stirred for 4 days at room
temperature. During the stirring, the pH of the mixture is maintained
at pH 6 by adding N-methylmorpholine. A large amount of water is
;; added and extracted with ethyl acetate. N,N-dimethylamino-1,3-propane
diamine (1 ml.) is added to the extract, and the mixture stirred for
30 minutes. The extract is washed with water, 1 N HCl, 5% sodium bi-
carbonate and water in this order and dried with anhydrous magnesium
; 30 sulfate. Ethyl acetate is removed, and the ~product is crystallized
;' - ~.
- 33 -
,,
.. .
104~088
....
from n-hexane to obtain BOC-Val-Leu-Gly-OBzl (90 g., yield: 94%).
.
;; m.p.: 119 - 121C.
Elemental Analysis (C25H39N306)
C% H% N%
;' found:62.73 8.34 8.85
c . .,
calculated: 62.86 8.25 8.80
.~'' -
~;- (28) Preparation of BOC-Cys(Mbzl)-Val-Leu-Gly-OBzl:
TFA (150 ml.) is added at -5C. to BOC-Val-Leu-Gly-OBzl (47.8 g.), the
~; mixture stirred for 20 minutes and concentrated in vacuo. Benzene is
1 added to the residue and removed azeotropically, and dried over NaOH
in vacuo. The material is dissolved in DMF (200 ml.), the pH adjusted
~ to 7 by adding triethylamine, and BOC-Cys(Mbzl)OSU (5 7 g.) and HOBT
; (3g.) are added and the mixture stirred for 2 days. After reaction,
N,N-dimethylamino-1,3-propane diamine (0.5 ml.) is added and the
mixture stirred for 1 hour. Water is then added in excess and extracted
with ethyl acetate. Ethyl acetate layer is washed with 1 N HCl, water,
~.-.................................................................... :
; 5% sodium bicarbonate solution, 5% sodium carbonate solution and water,
~ then dried by anhydrous magnesium sulfate and concentrated in vacuo.
. . .
Residue is crystallized from ethyl acetate-benzene-n-hexane to obtain
. 20 BOC-Cys(Mbzl)-Val-Leu-Gly-OBzl (53 g., yield: 75.7%). Rf6 = 0.36.
- m.p.: 141 - 143C. C~D1 = -43.8 (c=1.7, EtOH).
Elemental analysis (C36H52N408S):
C% H% N%
found: 61.56 7.60 8.19
calculated: 61.68 7.49 7.99
(29) Preparation of BOC-Thr(Bzl)-Cys(Mbzl)-Val-Leu-Gly-OBzl:
TFA (150 ml.) is added to BOC-Cys(Mbzl)-Val-Leu-Gly-OBzl (52 g.) at
`` -5C., mixture stirred for 30 minutes and concentrated in vacuo.
- Residue is treated with ethyl ether containing n-hexane to powderize
' 30 and dried over NaOH in vacuo. DMF (200 ml.) is added thereto, ad~usting
, -
.,'' .
- 34 -
, .
.. . .
08~
the pH to 6.5 by additlon o~ t~iethylamine, then HOBT C2g.) and
BOC-Thr(Bzl)-OSU (39 g.) are added, and the solution adjusted to pH 6
by addition of N-methylmorpholine, and stirred for 2 days at room
; temperature.
To the reactlon mixture is added 0.5 N HCl and pre-
cipitated material is filtered, washed with water and recrystallized
:: :
from 90% ethanol-ethyl acetate to obtain BOC-Thr(Bzl)-Cys(Mbzl)-Val-
Leu-Gly-OBzl (55 g., yield: 83.4%). m.p.: 190 - 194 C. (decomp.).
~ Rfl = 0-46 L~ D = -14.5 (c=1.5, DMF).
. lo Elemental analysis (C47H65N501o )
, C% H% N%
found: 62.98 7.47 7.92
j ~ calculated:63.26 7.36 7.85
(30) Preparation of BOC-Ser(Bzl)Thr(Bzl)-Cys(~bzl)-Val-Leu-Gly-OBzl:
'r.. '' TF~ (250 ml.) is added to BOC-Thr(Bzl)-Cys0Mbzl)-~al-Leu-Gly-OBzl~ (53.5 g.) at -5C., stirred ~or 30 minutes and concentrated in vacuo.
,, .
- Residue is treated with ethyl ether-n-hexane (1 : 1) to precipitate
'~ the product and dried over NaOH in vacuo. DMF (200 ml.) is added
thereto and adjusted to pH 6.5. HOBT (2 g.) and BOC-Ser(Bzl)-OSU
` 20 (28.2 g.) are added thereto, the pH adjusted to 6 by addition of N- -
methylmorpholine and mixture stisred for 2 days. -
"
0.5 N HCl (700 ml.) is added to the reaction mixture
',.- and filtered. The precipitate is washed with water and again preci-
.. . . .
pitated by adding ethanol (800 ml.), then washed with ethyl ether
and dried to obtain crude material (58 g.), which is recrystallized
from ethanol-ethyl acetate. BOC-Ser(Bzl)-Thr(Bzl)-Cys(Mbzl)-~al-Leu-
Gly-OBzl (49.0 g., yield: 76.5%). m.p.: 234 - 236 C. (decomp.).
Rfl = 54~ ~]D =-12.7 (c=l, DMF).
- 35 -
,,;,. ..
~,~'`','' ,
' . ' . ' ' ' ' ~ ., ,: ~ ' ' .
.. ~'' 10410~
(31) Preparation o~ Cbz-Asn-Leu-OBzl:
Cbz-Asn-OH (26.6 g.) and H-Leu-OBzl.TosOH C43.3 g.) are dissolved in
DMF 150 ml. HOBT (2g.) is added thereto and WSC (16.3 g.) ls added
dropwise at -10C. for 30 minutes, then mixture is stirred overnight
at room temperature. Water is added to the reaction mixture. The
product is precipltated and extracted with ethyl acetate. Extract is
washed with 1 N HCl, water, 5% sodium carbonate and water in this
order, dried with anhydrous magnesium sulfate and concentrated in
vacuo. Recrystallization from ethyl acetate-n-hexane gives Cbz-Asn-
Leu-OBzl (40 g., yield: 80%~. m.p.: 154 - 155.5C.
,~. .,:
Elemental analysis (C25H31N306):
C% H% N%
'':'
- found: 63.98 6.72 8.99
- calculated: 63.94 6.67 8.95
(32) Preparation of BOC~Ser(Bzl)-Asn-Leu-OH: -
Cbz-Asn-Leu-OBzl (30 g.) is dissolved in mixed solvent of methanol
(400 ml.), DMF (50 ml.), acetic acid (50 ml.) and water (100 ml.) at
warm temperature, and hydrogenzied in the presence of 5% palladium/carbon.
After reaction catalyst is removed and the filtrate is concentrated
in vacuo up to 150 ml. The solution is adjusted to pH 7 by adding
triethylamine, and HOBT (1 g.) and BOC-Ser(Bzl)-OSU (30 g.) added -
thereto and the mixture stirred for 2 days at room temperature. Water
is added to the reaction mixture to precipitate the product and
extracted with ethyl acetate. The extract is washed with 1 N HCl and
water, then dehydrated with anhydrous sodium sulfate and concentrated
in vacuo. ~esidue is crystallized from ethyl acetate-n-hexane to
obtain BOC-Ser(Bzl)-Asn-Leu-OH (30.8 g., yield: 92%).
m.p.: 176 - 178C. [~ D =~7-3 (c~0.9, DMF). !
- 36 -
': ~ 1
-- - . , , :
: ' ' ',, . ', . ' . ' ' ' , . .' ' , ~ :
10410~1B
(33) Preparation of BOC-Ser(Bzl)-Asn-Leu-Ser(Bzl)-Thr(Bzl)-Cys(Mbzl)-
Val-Leu-Gly-OBzl:
TFA (50 ml.) is added to BOC-Ser(Bzl)-Thr(Bzl)-Cys(Mbzl)-Val-Leu-Gly-
OBzl (10 g.) at -5C., the mixture stirred for 30 minutes and con- -
centrated in vacuo. Ethyl ether is added to the residue to precipitate
the product, which is dried over NaOH in vacuo. DMF (30 ml.) is added
.,
~ thereto and the pH adjusted to 5.5 by adding triethylamine. HOBT (1.3 g.)
; and BOC-Ser(Bzl)-Asn-Leu-OH (5.5 g.) are added thereto and ~SC (1.92 ml.)
is further added at -5C. The reaction mixture is stirred for 1 hour
~- 10 and allowed to stand overnight at room temperature. DMF ~20 ml.) is
added to the thus formed gel-like substance and the suspension formed
is added to 0.5 N HCl (200 ml.). The precipitated white substance is
;-:
filtered, washed with water, ethanol and ethyl ether, in this order,
and dried. The crude substance is treated by adding ethanol (100 ml.)
with heating. After cooling, the substance is dried to obtain BOC-Ser-
(Bzl)-Asn-Leu-Ser(Bzl)-ThrCBzl)-Cys(Mbzl)-Val-Leu-Gly-OBzl (12.5 g.,
yield: 85%). m.p.: 246 - 252 C.(decomp.). Rf2=0.68, [~ D = -18.96
(c=0.62, DMF).
(34) Preparation o~ BOC-Cys(Mbzl)-Ser(Bzl)-~sn-Leu-Ser(Bzl)-Thr(Bzl)-
; 20 Cys(Mbzl)-Val-Leu-Gly-OBzl:
TF~ (50 ml.) is added to BOC-Ser(Bzl)-Asn-Leu-Ser(Bzl)-Thr(Bzl)-Cys-
Mbzl)-~al-Leu-Gly-OBzl (10.0 g.) at -5C., the mixture stirred for 30
minutes and concentrated ln vacuo. Ethyl ether is added to the residue
to precipitate the material and dried over NaOH ln ~acuo. The dried
product is treated with DMF (30 ml.) and hexamethyl phosphoric triamide
(15 ml.) and the pH ad~usted to 6.5 by adding triethylamine at -5C.
HOBT (1 g.) and BOC-Cys(Mbzl)-OSU (4.5 g.) then added and reacted
therewith at room temperature for 2 days. During the reaction the pH
of the reaction mixture is maintained at pH 6 by addition of N-methyl-
morpholine.
- 37 -
,
~,;
, . . ..
~0410~8
~- ~ After adding ethyl acetate, the p~ecipitate is filtered
and heated in the presence of ethanol to obtain BOC-Cys(Mbzl)-Ser(Bzl)-
Asn-Leu-ser(Bzl)-Thr(Bzl)-cys(Mbzl)-val-Leu-Gly-oBzl (10.7 g., yield:
93%). m.p.: 247 - 253 C. (decomp.). Rf2 ~ 0-55~ [~7D8 = -17.01
(c = 0.53, DMF).
(35) Preparation of
r _~
- BOC-Cys-Ser-~sn-Leu-Ser-Thr-Cys-Val-Leu-Gly-OH:
BOC-Cys(Mbzl)-Ser(Bzl)-Asn-Leu-Ser(Bzl)~Thr~Bzl)-Cys(Mbzl)-Val-Leu-Gly-
OBzl (1.10 g.) is placed in a 300 ml. Erlenmyer flask, and under
cooling with dry_ice methanol,dried liquid ammonia is added. Then the
dry-ice bath is removed and metalllc sodium is added thereto until the
blue color has disappeared (about 20 seconds) with bubbling of dry
nitrogen gas therethrough. Excess sodlum ls decomposed by adding a
~- few drops of acetic acid, and the liquid ammonia is removed using an
aspirator for 2 hours. Thereafter, the product is dried over conc.
H2S04 in vacuo, and dissolved in D~ (200 ml.) and 0.5~ acetic acid
; (400 ml.). The mixture is ad~usted with aqueous ammonla to pH 6.8
;~ and decarbonated air is bubbled therethrough until the reaction of
; sodium nitropruside reagent becomes negative. After adjusting the pH
: 20 of the reaction mixture to pH 4.0 by addlng acetic acid, the mixture
~. .
is concentrated in vacuo up to about 150 ml. Ethyl acetate (500 ml)
is added thereto to effect crystallization by scratching the glass wall. -~
; Further ethyl acetate (500 ml.) is added and the mixture stored in an
ice-box. Organic solvent is removed by decantation and treated with
ethyl acetate to precipitate the powder containing inorganic salt
(3.76 g.). The powder is dissolved in a small amount of methanol-
water (1 : 1), charged on a column of CM-sephadex (4.4 x 60 cm.) and
chromatographed using methanol-water (1 : 1). The eluate is fractionated
each 10 ml. and the fraction No. 36 - 46 collected, which shows
absorption peak at 230 m~u. Methanol is removed off by vacuum
- 38 _
,,, ,~, .
.. .
. 10410~8
` . .
concentration and freeze dried to obtain the powder ~210 mg.). TLC:
one spot (solvent systems 2 and 8 on silica gel).
Amino acid composition : Asp 1.02 (1), Thr 0.90 (1), Ser 1.72 (2),
~!" Gly 1.00 (1), Cys/2 1.52 (2), Val 1.09 (1), Leu 2.00 (2).
:, .
Example 2
Production of
H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-
Leu-Gly-Lys-Leu-Ser-Gln-Glu~Leu-
His-Lys-Leu-Gln-Thr-Tyr~Pro-Arg-
~`~ Thr-Asp-Val-Gly-,Ala-Gly-Thr-Pro-NH2
"';
732 mg. of BOC-Lys(ClCbz)-Leu-Ser(Bzl)-Gln-Glu(OBzl)-Leu-His-Lys(ClCb7)-
~ Leu-Gln-Thr(Bzl)-Tyr(Bzl)-Pro-Arg(Tos)-Thr(Bzl)-Asp~OBzl)-
; Val-Gly-Ala-Gly-Thr(Bzl)-Pro-N~2
was dissolved in 3 ml. of TFA and 1 ml. of 4N HCl/dioxane at -5C. After
stirring for 35 minutes at room temperature, the solution was concentrated
in vacuo and ethyl ether was added thereto to form a precipitate. The
precipitate was dried over sodium hydroxide. The dried precipitate was
dissolved in 3 ml. of DMF, and the pH adjusted to 5.5 - 6 by adding
~- N-methylmorpholine at -5 C. To that :olution were added 3 mg. of HOBT,
100 mg. of DCC and 500 mg. of BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-
. ., ~.
Gly-OH (obtained by the same method as described in Example 1), and the
mixture stirred for 2 hours at 0 C. and overnight at room temperature.
After reaction, water was added to precipitate the product, and the thus
; fomred precipitate was collected by filtration, washed thoroughly with
, . . .
water and ethyl acetate, then dried in vacuo to obtain 700 mg. of the
crude product. The crude material was treated with hydrogen fluoride
; (35 ml.) at 0 C. for one hour in the presencé of 1.5 g. o~ phenol and
4.0 ml. of anisole. After distillation of hydrogen fluoride, the
,., ' .
- - 39 -
",~
',:
::
:
:
j: :
`; ~ 1041(~
residue was washed th~ee times with ethyl acet~te (30 ml.) and dried,
then dissolved in 200 ml. o~ 0.5 mole acetic acid. The solution was
passed through the column of Dowex 1 X 2 (acetate form) (2 X 20 cm.),
and the eluate was freeze dried to obtain the powder t451 mg. ) . 393 mg.
of this powder dissolved in 1 ml. of 0.1 N acetic acid was poured inco
~: .
the top of a column packed with Sephadex G-50 in 0.1 N acetic acid
(3.0 X 107 cm.), and eluted with 0.1 N acetic acid ~elution rate
10 ml./hour). Active fractions checked by bioassay were collected and
freeze dried to obtain the active powder (267 mg.).
The actlve powder dissolved in 0.1 N acetic acld (1 ml.)
was charged on a column packed with carboxymethyl cellulose in 0.01 mole
ammonium acetate (2.0 X 20 cm.), then was gradiently eluted with 500 ml.
of 0.01 mole ammonium acetate (pH 4.5) to 500 ml. of 0.15 mole ammonium `
acetate (pH 4.5). Each 6.7 ml. of eluted fraction was collected and
: the active fractions No. 109 - 126 checked by bioassay were freeze dried
to obtain the product as whlte powder. This powder (18 mg.) dissolved
in small amount of 0.1 mole acetic acid was poured into the top of a
column of Sephadex LH-20 (2 X 75 cm.), eluted with 0.1 mole acetic acid, -
. ..................................................................... .
fractioned each 5 ml. of eluate and the active fractions No. 13 - 17
` 20 collected which were freeze dried to obtain the active powder.
Rf=0.65 - 0.71,~] D0 ~ ~75 (c=l, 0.1 N formic acid).
Potency: 4200 MRC units/mg.
; Amino acid composition: Cys/2: 2.07 (2), Ser: 2.84 (3), Leu: 5.15 ~5), `
Thr: 3.50 (4), Val: 2.18 (2), Gly: 3.00 (3), Lys: 1.95 (2), Glu: 3.30 (3),
His: 0.96 (1), Tyr: 0.91 (1), Pro: 1.97 (20, Arg: 1.04 (1), Asp: 2.05 (2),
Ala: 0.98 (i).
A starting material is produced as follows: -
... , ~-.
.
- 30
- 40 -
. .
.' " ''~ .
.
104~0198
- Production o~ partial amino acid sequence 11 - 32:
BOC-Lys(ClCbz)-Leu-Ser(Bzl)-Gln-Glu(OBzl)-Leu-His-Lys(ClCbz)-Leu-
Gln-Thr(Bzl)-Tyr(Bzl)-Pro-Arg(Tos)-Thr~Bzl)-Asp(OBzl)-Val-Gly-Ala-
Gly-Thr(Bzl)-Pro-NH2
(1) Preparation of BOC-Lys(ClCbz)-Leu-OEt:
BOC-Lys(ClCbz)-OH.t-butylamine salt (7.32 g.) suspended
in ethyl acetate was washed three times with 1 N-HCl (100 ml.) and
water (100 ml.). After drylng with anhydrous magnesium sulfate, ethyl
acetate was distilled off. To the resldue were added dichloromethane
(60 ml.), H-Leu-OEt.HCl (2.93 g.) and HOBT (2.0 g.), WSC (2.75 ml.)
:; was further added at -5C. and mixture stirred for 1 hour, and further
- stirred overnight at room temperature. After reaction, dichloromethane
-- was removed in vacuo, and the residue was dissolved in ethyl acetate
,::
(150 ml.). The solution was washed with 1 N-HCl, 5% sodium bicarbonate
solution and water. After drying with anhydrous magnesium sulfate
:;~
followed by concentration in vacuo, the residue was recrystallized from
ethyl acetate-n-hexane to obtain 6.4 g. of BOC-Lys(ClCbz)-Leu-OEt
(Yield: 76.6%). m.p.: 76 - 79C.
(2) Preparation of BOC-Lys(ClCbz)-Leu-OH:
~ 20 To a solution of BOC-Lys(ClCbz)-Leu-OEt (6.13 g.) in
- ethyl acetate (20 ml.) was added 1 N-NaOH (12.1 ml.) at 0C. and the
mixture stirred for 2 hours at room temperature. Further 1 N-NaOH
(1.1 ml.) was added thereto and after one hour stirring the pH adjusted
to 7 and the mixture concentrated in vacuo. Residue was washed with
ethyl ether, the aqueous layer ad~usted to pH 3 and extracted three
times with ethyl acetate (100 ml.). After washing the ethyl acetate
layer with water (100 ml.) and drying with anhydrous sodium sulfate,
the product was concentrated in vacuo. Residue was dissolved in ethyl
ether (50 ml.), concentrated under xeduced pressure, and dried to
obtain BOC-Lys(clCbz)-Leu-OH as a powder (5.85 g.).
- 41 -
' ~;
- ' ' ` , ,.
,
:- 1 10410l9~
m.p.: ~46 - 60C. ~]D9 ~ -10.68 (cFl, PYY).
Elemental analysis [C25H38N307Cl. 3 H20~ :
C% H% N%
found: 56.25 7.35 8.00
; calculated: 56.21 7.31 7.87
(3) Preparation of BOC-Leu-His-OH:
H-His-OH.HCl ~3.83 g.) was dissolved with heating in
~; water (25 ml.). After cooling to room temperature, sodium bicarbonate
(1.84 g.) was added, and, further, HOBT (270 mg.), BOC-Leu-OSU (8.53 g.) ~
and THF (25 ml.) were added. 10 ml. of water was then added and the -
- mixture stirred overnight at room temperature. The pH of the reaction
...
mixture was adjusted to 7 with addition of aqueous sodium bicarbonate,
, ......................................................................... .
and BOC-Leu~OSU (1.3 g.) was again added and the mixture stirred.
~fter 6 hours, a further 1.3 g. was added and stirring continued over-
~, night. After reaction was completed by checklng with TLC, the reaction
mixture was concentrated in vacuo to remove organic solvent and the
residual aqueous layer was washed with ethyl acetate. The aqueous layer
. :,., ~
was adjusted to pH 5 and poured into the top of the column of HP-20
(2.3 X 18 cm.), followed by washing with water until the washing eluate
showed ninhydrin negative, then eluted with methanol. Eluate was con- -
centrated in vacuo and the residue was treated with ethyl ether. The
precipitate formed was recovered by filtration and reprecipitated twice
with methanoI-ethyl ether to obtain dried powder (3.94 g., Yield: 53.5%).
M.P.: 178 - 180 C. ~ D = -0.19 (c-1, DMF).
Elemental analysis: (C17Hz8N405- 3 H20)
C% H% N%
found: 53.39 .7.65 14.71
calculated: 53.65 7.79 14.73
''' , '' :,
~ 42 -
,~ ' ''' ~
.~, . ~
"'
:, - " ,. , , - . . ,: .. , . .:
~ `~
1041V~
(4) Preparation of BOC-Leu-Ser(Bzl)-OH:
~; H-Ser(Bzl) OH (11.7 g.) was dissolved in water (50 ml.)
and sodium bicarbonate (10.1 g.) and dioxane (10 ml.) added thereto.
BOC-Leu-OSU (16.4 g.) in dioxane (15 ml.) and DMF (5 ml.) were then
` added. After one night stirring, the pH of the reaction mixture was
; adjusted to pH 7, and the mixture concentrated in vacuo to removeorganic solvent. The aqueous layer was shaken with ethyl acetate
(100 ml.) and the organic layer was washed twice with 1 N-HCl and water.
The organic layer was dried with anhydrous magnesium sulfate, con-
centrated in vacuo and the residue was twice recrystallized from ethyl
acetate-n-hexane to obtain BOC-Leu-Ser(Bzl)-OH (16.1 g.) (Yield: 78.8%).
m.p.: 82 - 86C.
(5) Preparation of BOC-Lys(ClCbz)-Leu-Ser(Bzl)-OH:
- BOC-Leu-Ser(Bzl)-OH (4.1 g.) (10 m mol.) obtained in
the above step (4) was treated at -5C with T~A (25 ml.), the mixture
stirred for 25 minutes and therea~ter concentrated in vacuo. Ethyl
ether was added to the residue to precipitate the material, which was
dried over NaOH in vacuo.
The crystals were dissolved in DMF (8 ml.) and BOC-Lys-
(ClCbz)-ONP (8 g.) and HOBT (270 mg.) were added thereto. The pH of
the solution was adjusted to pH 8 with N-methyl morpholine then stirred
for overnight at room temperature. 1 N-HCl was added thereto and the
' precipitate was dissolved in chloroform which was washed three times
with 1 N-HCl, three times with 5~ sodium bicarbonate solution and three
times with water. ~fter drying with anhydrous magnesium sulfate, the
chloroform layer was concentrated in vacuo. The residue was three times
reprecipitated from ethyl acetate-n-hexane and a further three times
- reprecipitated from ethyl ether-n-hexane, then passed through the column
of silica gel (180 g.) which was ~ashed with ethyl acetate-benzene
(2 : 1) and eluted with methanol. The methanol eluate was concentrated
, .
' '"''' .'' ,
- ~. ~ ' . : -
: .
in vacuo. The residue was recrystallized from ethyl acetate-n-bexane
to obtain BOC-Lys(ClCbz)-Leu-Ser(Bzl)-OH (5.25 g.)
: ,:
(Yield: 74.5%). m.p.: 84 ~ 100C. (dec.). TLC: one spot.
Amino acid composition: Lys: 0.98 (1), Leu: 1.00 (1),
' Ser: 0.82 (1).
(6) Preparation of BOC-Lys(ClCbz)-Leu-Gln-Thr(Bzl)-Tyr(Bzl)-Pro-Arg(Tos)-
Thr(Bzl)-Asp(OBzl)-Val-Gly-Ala-Gly-Thr(Bzl)-Pro-NH~
~- BOC-Gln-Thr(Bzl)-Pro-Arg(Tos)-Thr(Bzl)-Asp(OBzl)-Val-
: ..
Gly-Ala-Gly-Thr(Bzl)-Pro-NH2 (7.65 g.) obtained in the same process as -
described in Example I, step (15) was dissolved in TFA (35 ml.) at
-5C., stirred for 30 min. and concentrated ln vacuo. Residue was
treated with diethyl ether and the precipitated material was dried ~-
over NaOH in vacuo. This was dissolved in DMF (25 ml.), added HOBT
(600 mg.), BOC-Lys(ClCbz)-Leu-OH (2.35 g.) obtained in (2) hereinabove
and WSC (0.69 ml.) at -5C. then stirred for one hour at -5C. and
thereafter stirred overnight at room temperature. The 1 N-HCl was
., - . .
, added to the reaction mixture to precipitate the material which was - '
~ reprecipitated twice with methanol-ethyl ether and dried to obtain the
! ~ product (8.0 g.).
20 (Yield: 87.3%). m.p.: > 164 C. (dec.), rc~7 D =-18.65 (c=l, DMF). -
Elemental analysis (C125H164N21028S
C% H% N%
found: 59.63 6.67 11.78
calculated: 59.75 6.62 11.71
(7) Preparation of BOC-Leu-His-Lys(ClCbz)-Leu-Gln-Thr(Bzl)-Tyr(Bzl)-
Pro-Arg(Tos)-Thr(Bzl)-Asp(OBzl)-Val-Gly-Ala-Gly-Thr(Bzl)-Pro-NH2:
BOC-Lys(ClCbz)-Leu-Gln-Thr(Bzl)-Tyr(Bzl)-Pro-Arg(Tos)-
` Thr(Bzl)-Asp(OBzl)-Val-Gly-Ala-Gly-Thr(Bzl)-Pro-NH2 (7.68 g.) obtained
in the above step (6) was dissolved in TFA (35 nl.) at -5C., stirred
- 30 for 30 minutes and concentrated in vacuo. Residue was dissolved in
'` . , :
- 44 -
.
. .
~ '
:., -.
I' ' , . ~ . ; :
~. ', ' ` , ' ', ',~
.:.
;::
10~101~8
DMF (20 ml.), ad~usted to pH 5 b~ add~ng trlethyla~ine, HOSU (430 mg.),
BOC-Leu-His-OH (1.37 g.) and WSCO (58 ml.) added at -5C. then stirred
"':
for one hour, and thereafter overnight at room temperature. 1 N-HCl was
added to the reaction mixture to precipitate the material which was
further reprecipitated from methanol-ethyl ether and repeatedly
reprecipitated from DMF-ethyl ether. The product was obtained by
drying. (8.30 g., Yield: 98.2%). m.p.: 167 ~ 175C. [~7 D =-17.45
(c=l, DMF).
~ Elemental analysis tC137H182N2530SC1-HC1-4H2)
-: 10 C% H% N%
found: 58.19 6.71 12.42
calculated: 58.04 6.79 12.35
(8) Preparation of BOC-Glu(OBzl)-Leu-His-Lys(ClCBz)-Leu-Gln-Thr(Bzl)-
Tyr(Bzl)-Pro-Arg(Tos)-Thr(Bzl)-Asp(OBzl)-Val-Gly-Ala-Gly-Thr(Bzl)-
Pro-NH2
Heptadecapeptideamide (amlno acid sequence No. 16 - 32)
(7.64 g.) obtained in the above step C7) was dissolved in TFA (35 ml.)
at -5C., stirred for 40 minutes and concentrated in vacuo. Residue
was dissolved in DMF (30 ml.), the pH adjusted to 7 by addition of N-
methylmorpholine, then HOBT (70 mg.) and BOC-Glu(OBzl)-OSU (1.82 g.)
were added thereto. The pH was again adjusted to pH 7 by addition of
N-methylmorpholine and stirred for 2 days at room temperature. 1 N-HCl
was added to the reaction mixture and the precipitate was recrystallized
twice from methanol-ethyl ether to obtain the product after drying.
(7.55 g., Yield: 92.5%). m.p.: 161 - 169C. (dec.).
Amino acid composition: His: 0.83 (1), Arg: 1.05 (1),
Asp: 1.08 (l), Thr: 2.52 (3), Glu: 2.06 (2), Pro: 2.16 (2), Gly: 2,00 (2),
Ala: 1.06 (1), Val: 1.10 (1), Leu: 1.82 (2), Tyr: 0.73 (1).
. '' ' ' ' , .
- 45 -
., ~,
' .: .'., ' ~ , ' :
.. .. . . .
~0410~
(9) Preparation of BOC-Gln-Glu(OBzl)~Leu~His-Lys~ClCbz)-Leu-Gln-Thr(Bzl)-
Tyr(Bzl)-Pro-Arg(Tos)-Thr(Bzl)-Asp(OBzl)-Yal-Gly-Ala-Gly-Thr(Bzl)-
Pro-NH2
The octadecapeptideamide (15 - 32) obtained in the
above step (8) (7.29 g.) was dissolved in TF~ (30 ml.) at -5 C.,
stirred for 45 minutes and concentrated in vacuo. Residue was dissolved
in DMF (15 ml.), the pH adjusted to 7 by addition of N-methylmorpholine,
HOBT (100 mg.) and BOC-Gln-ONP (1.4 g.), added then stirred for one
night at room temperature. 1 N-HCl was added to the reactlon mixture
lo and the precipitate was collected and washed with water. Reprecipita-
;~ tion was carried out three times to obtain dried product. (7.00 g.;
~ield: 91.5%). m.p.: 171 - 175 C. (dec.).
(10) Preparation of BOC-Lys(ClCbz)-Leu-Ser(Bzl)-Gln-Glu(OBzl)-Leu-His-
::
Lys~ClCbz)-Leu-Gln-Thr(Bzl)-Tyr~Bzl)-Pro-Arg(Tos)-Thr~Bzl)-Asp(OBzl)-
; Val-Gly-Ala-Gly-Thr(Bzl)-Pro-NH2:
The nonadecapeptide (14 - 32) obtained in the above step
(9) (6.70 g.) was dissolved in TF~ (30 ml.) at -5C., and 4 N-HCl/dioxane
; (2.2 ml.) added and stirred for 45 minutes at room temperature. Reaction
~ mixture was concentrated in vacuo and ethyl ether added to precipitate
. .
the material, which was washed thoroughly with ethyl ether and dried
over NaOH in vacuo.
The substance hereinabove was dissolved in DMF (15 ml.),
- the pH adjusted to 5.5 to 6 by adding N-methylmorpholine, HOBT (360 mg.),
BOC-Lys(ClCbz)-Leu-Ser(Bzl)-OH (1.86 g.) and DCC (540 mg-.j dissolved in
DMF (5 ml.) added, then mixture was stirred for one hour and overnight
at room temperature. 1 N-HCl was added and the precipitate formed was
collected by filtration. The ethanol insolub~e part of the precipitate
~ and the precipitated material obtained from addition of ethyl ether in
the filtrate were combined. Methanol-diethyl ether was added thereto
and the precipitate was repeatedly recrystallized from methanol-ethyl
,
- 46 -
: :;
' .0,,;
'~'. ,- ' ' ' . ~ ' .. , ' ' '
: ~, - ' ' '';
-
:
; 10410~8
ether to obtain the product C7.0 ~.).
(Yield: 87.6%). m.p.: 171 - 177C. (dec.). [~ D = -18.45 (c-l,DNF).
Elemental analysis (C184H242N32041S 2 2 2
C% H% N% C1%
- found: 58.43 6.65 11.83 2.64
calculated: 58.21 6.7411.81 2.80
Amino acid composition: Lys: 1.86 (2), His: 0.74 (1),
~- Arg: 0.97 (1), Asp: 1.04 (1), Thr: 2.55 (3), Ser: 0.53 (1), Glu: 2.85(3),
.~
Pro: 2.08 (2), Gly: 2.00 (2), Ala: 1.04 (1), Val: 1.04 (1), Leu: 2.67(3),
Tyr: 0.67 (1).
." ;- ' , .
.. ~,. :
,',', `' , .
~"
.''~ .
... .
, ,',: '
, .... .
;,. -.
. ...
.. .. . .
, , :
,'', `.~: ~ '
: ~
. ' ' ,
;~
- 47 -
,
. ,:
