Note: Descriptions are shown in the official language in which they were submitted.
~~ - 2 - ~ B
RAN 4105/35
Thymosin ~1 is a heat stable, highly acidic polypeptide
compound of 28 amino acid residues of the following sequence:
5 1O
H3C-CO-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-
15 20
Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-
25 ~ -
Val-Val-Glu-Glu-Ala-Glu-Asn-OH.
'- ,,'
This peptide which is a potent immunopotentiating agent
was isolated from thymosin fraction S by Goldstein et al. by a ~`combination of ion-exchange chromatography and gel filtration
(Proc. Natl. Acad. Sci. USA 74, 725-729 (1977)).
. ' `: .
The present invention relates to a synthesis of thymosin
al or its ~Asn2]-analogue and their pharmaceutically acceptable
salts by removing the protecting groups from a protected `
octacosapeptide of the sequence:
: .
H3C-CO-Ser(Rl)-X-Ala-Ala-Val-Asp(OR2)-Thr(R3)-Ser(Rl)- ;
Ser(Rl)-Glu(OR4)-Ile-Thr(R3)-Thr(R3)-Lys(R )-
Asp(OR )-Leu-Lys(R5)-Glu(OR4)-Lys(R )-Lys(R )-
Glu(OR )-Val-Val-Glu(OR4)-Glu(OR )-Ala-Glu(OR )~
Asn-OR ; , (I)
.
wherein X is Asn or Asp(OR2); -~
R is a conventional protecting group for the
hydroxyl group of the serine residue;
Mez/15.2.1978 -
, `;
,..................................... ' ' ' ~ '' '. ,
,
.
R , R and R are conventional carboxyl
group protecting groups;
R3 is a conventional protecting group for the
hydroxyl group of threonine and
R is a conventional protecting group for the
~-amino group of the lysine residue,
and, if desired, convertin~ the compound obtained into a
pharmaceutically acceptable salt.
Examples of Rl are benzyl, acetyl, banzoyl, tert.-butyl,
trityl, 4-bromobenzyl, 2,6-dichlorobenzyl and benzyloxycar-
bonyl with benzyl being preferred. Examples of R , R4 and~R6
are aryl groups particularly phenyl or phenyl substituted with
lower alkyl, halo, nitro, mercapto or substituted mercapto such
as methylthio; aralkyl groups such as benzyl or benzyl sub-
stituted with methoxy, halo or nitro; lower alkyl groups such
as methyl, ethyl, tert.-butyl and tert.-amyl; substituted lower
alkyl groups such as 2-haloethyl, ~,~-dimethylaminoethyl and
cyanomethyl; benzhydryl and phenacyl groups with benzyl being
preferred. Examples of R3 are benzyI, acetyl, benzoyl, tert.-
butyl, trityl, 2,6-dichlorobenzyl, 4~bromobenzyl and benzyloxy~
; carbonyl with benzyl being preferred. Examples of R5 are
benzyloxycarbonyl which may be optionally substituted in the~
aromatic ring such as by 4-chloro, 2-bromo, 4-bromo, 2,4-
dichloro, 4-nitro, 4-methoxy, 3,5-dimethoxy, 4-methyl, 2,4,6-
trimethyl, 4-phenylazo, 4-(4-methoxyphenylazo), 2-(N,N~
dimethylcarbamido)~and 2-nitro-4,5-dimethoxy, urethane type
`~ protecting groups such as 4-toluenesulfonylethyloxycarbonyl,~
: , ,
:
:
: . , . : . `
~, ~
-- 4 --
9-fluorenylmethyloxycarbonyl and related base cleavable groups,
5-benzisoxazolylmethyleneoxycarbonyl, methylthio- and methyl-
sulfonylethyloxycarbonyl, isonicotinyloxycarbonyl, haloethyloxy-
carbonyl, diisopropylmethyloxycarbonyl, benzhydryloxycarbonyl,
isobornyloxycarbonyl, dinitrodiphenylmethyloxycarbonyl, tert.-
butyloxycarbonyl, tert.-amyloxycarbonyl, adamantyloxycarbonyl,
cyclopentyloxycarbonyl, methylcyclobutyloxycarbonyl, methyl-
cyclohexyloxycarbonyl, 2-arylisopropyloxycarbonyl groups such .-
as 2-(p-biphenylyl)-isopropyloxycarbonyl, 2-(4-pyridyl)-iso-
propyloxycarbonyl and related nitrogen containing urethane
groups; acyl groups such as formyl, trifluoroacetyl, phthaloyl,
benzenesulfonyl, acetoacetyl, chloroacetyl, 2-nitrobenzoyl,
4-toluenesulfonyl, sulfenyl groups such as benzenesulfenyl,
o-nitrophenylsulfenyl and related sulfenyl groups, and aryl-
lower alkyl groups such as diphenylmethyl and triphenylmethyl
with the benzyloxycarbonyl group being preferred. :
Removal of the protecting groups from the protected
: octacosapeptide of sequence I is readily accomplished by .... ~
procedures known per se, such as, for example by treatment with
~ 20 anhydrous acid such as ~ydrogen fluoride preferably in the
-~ presence of anisole.
; ~ ~ In accordance with the present invention the protected
octacosapeptide of sequence I can be synthesized by condensation
of a protected tetradecapeptide of the sequence
'
.
- , : , , .
: . : .
. ' ' , " .: . . ' '
~` - 5 ~
H3C-CO-Ser(R )-X-Ala-Ala-Val-Asp(OR )-Thr(R3)-Ser(R )-
Ser(R )-Glu(OR )-Ile-Thr(R )-Thr(R3)-Lys(R5)-OR II
with a protected tetradecapeptide of the sequence
H-Asp(OR )-Leu-Lys(R5)-Glu(OR )-Lys(R )-Lys(R )-
Glu(OR )-Val-Val-Glu(OR )-Glu(OR )-Ala-Glu(OR4)-
Asn-OR6 ~ III ;
. ~ '.
Rl R2 R3 R4 R5 and R6 are defined
as above and R7 is hydrogen or an activating group.
The aforesaid coupling reaction can be carried out
utilizing procedures well known in solution phase peptide
synthesis. Thus, for example, the amino terminal tetradeca-
peptide can be reacted with l-hydroxybenzotriazole (HOBT) and
dicyclohexylcarbodiimide (DCC) to yield the activated ester
which is then reacted with the carboxyl terminal tetradeca-
peptide to yield the desired octacosapeptide in protected form.
~ ' .'- .
The protected tetradecapeptides can be prepared in
accordance with methods well-known in the art.
The strategy employed in the chemical synthesis of the
protected carboxyl terminal tetradecapeptide III~ tX ~ Asp~OBzi)]
was as follows:
~ ~ '
, .
. ~, ' ' . '. ' '
, ~ . ' ~' ,
6 ~
H-Glu(OBzl)-OH was first coupled to Boc-Ala-OSu to give
the protected dipeptide fragment Boc-Ala-Glu(OBzl)-OH which was
then condensed with HCl H-Asn-OBzl via the DCC/HOSu procedure
of Wunsch and Drees, Chem. Ber. 99, 110 (1966). The hydro-
chloride salt of asparagine benzyl ester was prepared fromBoc-Asn-OBzl which in turn was synthesized from commercially
available Boc-Asn-OH and benzyl bromide using the cesium salt
of the amino acid. The Boc-protecting group was removed by a
30 minutes treatment with 4N HCl in dry THF.
Reaction between H-Glu(OBzl)-OH and Boc-Glu(OBzl)-OSu
produced Boc-Glu(OBzl)-Glu(OBzl)-OH as a colorless clear oil.
It was subsequently utilized in the synthesis of the protected
pentapeptide Boc-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl in
a DCC/HOSu mediated fragment condensation using HCl H-Ala-
Glu(OBzl)-Asn-OBzl that was derived from Boc-Ala-Glu(OBzl)-Asn-
OBzl upon 4N HCl/THF treatment. The aforesaid protected penta-
peptide was obtained in good yiel~ as a crystalli~e pure
material.
For the preparation of the protected octapeptide Boc-
Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl,
the required protected tripeptide Boc-Glu~OBzl)-Val-Val-OE was
first prepared. Boc-Val-OSu was allowed to react with free
valine to provide Boc-Val-Val-OH whlch on deblocking with ~N~
HCl in THF followed by rèaction with Boc-Glu(OBzl)-OSu yielded
- . .
the desired tripeptide which was crystallized as cyclohexyl- ~
.
amine salt Boc-Glu(OBzl)-Val-Val-OH CHA. The cyclohexylamine
: :
~ .
' . . '. , , . : ' . : '
.
.. : .
. ~, . . : : . . . .. .
salt was converted to the free acid and was then coupled by
DCC in the presence of HOSu to HCl H-Glu(OBzl)-Glu(OBzl)-Ala- -
Glu(OBzl)-Asn-OBzl that was derived from Boc-Glu(OBzl)-
Glu(OBzl)-Pla-Clu(OBzl)-~sn-OBzl on treatment with PCl in ~RF. The ~rotected
octapeptide Bcc-Glu(OBzl)-~Jal-~.7al-Clu(OBzl)-Glu(OBzl)-~la-~lu(~Bzl)-Asn-oBzl
was obtained in Purified for~ as an amornhous sOlia. It was de~rotected bv
hydro ~ olysis follcwed by treatment with trifluoroacetic acid in the usual
nHnner to give the free octape~tide ~lu-Val-~.7al-C~u-C-lu-Pla-Glu-Asn. This pro-
duct was purified by ion-exchan~e column chrc~ato~raphv to vield material h~
~eneous on thin layer chro~a~a,Dhv and paper electro~horesis.
For the synthesis of the protected undecapèptide Boc-
Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-
Ala-Glu(OBzl)-Asn-OBzl, the required tripeptide fra~ment was
synthesized starting from Boc-Lys(Z)-OSu and H-Lys(Z)-OH. The
dipeptide Boc-Lys(Z)-Lys(Z)-OH thus obtained was treated with
4N HCl in THF and the ensuing salt HCl ~ H-Lys(Z)-Lys(Z)-OH ~ -
was then allowed to react with Boc-Glu(OBzl)-OSu to provide th~
desired tripeptide Boc-Glu~OBzl)-Lys(Z)-Lys(Z)-OH. The tri-
peptide was then activated with DCC and HOSu according to the
procedure of Weygand et al., Z. Naturforsch~ 21b, 426 (1966),
and the solution of the active tripeptide ester Boc-Glu(OBzl)-
Lys(Z)-Lys(Z)-OSu generated in situ was combined with the
trifluoroacetate salt of H-Glu(OBzl)-Val-Val-Glu(OBzl)-
GlutOBzl)-Ala-Glu(OBzl)-Asn-OBzl derived from the corresponding
blocked octapeptide by a 30 minutes treatment with TFA. Upon
addition of a small amount of a base the desired protected
undecapeptide Boc-Glu(OBzl)-Lys(Z)-Lys(Z~-Glu(OBzl)-Val-Val-
Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl was thus obtained.
.
......
.,. , ' ~
~ '
36~
8 - ~
Deprotection of the protected undecapeptide Boc-Glu(OBzl~- -
Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-
Glu(OBzl)-Asn-OBzl with anhydrous hydrofluoric acid provided
the free undecapeptide Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-
Asn which was homogeneous on paper electrophoresis after ion-
exchange column chromatography.
The synthesis of the protected tetradecapeptide followed
a similar pattern. Boc-Leu-OSu was coupled to H-Lys(Z)-OH to
provide Boc-Leu-Lys(Z)-OH. After removal of the N -Boc-group
with 4N HCl in THF and reaction with Boc-Asp(OBzl)-OSu the
protected tripeptide Boc-Asp(OBzl)-Leu-Lys(Z)-OH was obtained
as a crystalline pure solid. It was converted into the active
ester Boc-Asp(OBzl)-Leu-Lys(Z)-OSu and condensed with the tri-
fluoroacetate salt of H-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-
Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl obtained from
TFA treatment of the corresponding blocked undecapeptide. The
desired product Boc-Asp-(OBzl)-Le;u-Lys(%)-Glu(OBzl)-Lys(~)-
Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-
OBzl was obtalned in good yield. Thin layer chromatography
indicated that the product was homogeneous. The free tetra-
decapeptide Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-
Glu-Asn was obtained from the protected compound by anhydrous
~; hydrofluoric acid treatment and purification on an ion exchange
column.
- : , . ,. . -
' ~
_ 9 ~
In a similar way the protected amino terminal tetradeca-
peptide H3C-CO-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp (OBæl)-
Thr(Bzl)-Ser (Bzl) -Ser (Bzl)-Glu(OBzl) -Ile-Thr (Bzl) -Thr(Bzl)-
Lys(Z)-OH was assembled from an acetyl tetrapeptide fragment, a
hexapeptide, and another tetrapeptide using procedures well
known in the peptide synthesis art. For the synthesis of the
amino terminal acetyl tetrapeptide, H3C-CO-Ser(Bzl)-O~zl was
prepared from H3C-CO-Ser(Bzl) -OH and benzyl bromide via the
cesium salt of the amino acid. Hydrazinolysis of H3C-CO-Ser(Bzl)-
OBzl gave H3C-CO-Ser(Bzl)-HNNH2 as a pure crystalline solid in
good yield. Deblocking of Boc-Ala-Ala-OH afforded the dipeptide
hydrochloride salt HCl H-Ala-Ala-OH. Coupling of this dl-
peptide with Boc-Asp(OBzl)-OSu provided the protected tri-
; peptide Boc-Asp(OBzl)-Ala-Ala-OH which was isolated as the
dicyclohexyl amine salt. Removal of the amino protecting group
and condensation with H3C-CO-Ser(~Bzl)-HNN~2 via the azide
procedure of Honzl and Rudinger, Collection Czech. Chem. Commun.
26, 2333 (1961),gave the partially protected tetrapeptide
~' ~
H3C-Ser(Bzl)-Asp(OBzl)-Ala-Ala-OH which on reaction with
equlvalent amounts of hydrazine mediated by DCC in the presence
of HOBT produced the deslred intermediate H3C-CO-Ser(Bzl)-
Asp(OBzl)-Ala-Ala-HNNH2.
For the synthesis of the protected hexapeptide Boc~Val-~
Asp(oBzl)-Thr(Bzl)-ser(Bzl~-ser(Bzl)-Glu(oBzl)-HNNH2~ a process
involving the stepwise elongation of the peptide chain from
the C-terminal end was adopted. Thus, H-Glu(OBzl)-OH was
coupled with Boc-Ser(Bzl)-OSu to form the protected dipeptide~
..
.
Boc-Ser(Bzl)-Glu(OBzl)-OH which on removal of the Boc-group and
further reaction with Boc-Ser(Bzl)-OSu gave rise to the tri-
peptide Boc-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH. Deprotection of
the amino group followed by condensation of the resultant tri-
peptide salt HCl H-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH with Boc-
Thr(Bzl)-OSu yielded the protected tetrapeptide Boc-Thr(Bzl)-
Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH which on treatment with HCl in
THF removed the Boc-group and further reaction with Boc-
Asp(OBzl)-OSu resulted in the formation of the protected
pentapeptide Boc-Asp(OBzl)-Thr(szl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-
OH. The Boc-group of this compound was removed by HCl
treatment and the ensuing product HCl H-Asp(OBzl)-Thr(Bzl)-
Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH was subsequently coupled to
Boc-Val-OSu to give Boc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-
Ser(Bzl)-Glu(OBzl)-OH as a crystalline pure material.
The -carboxyl group on the terminal glutamic acid residue
of the aforesaid hexapeptide was then specifically converted
into the hydrazide ~unction by the reaction with an equivalent
amount of hydrazine using DCC as coupling agent in the presence
of HOBT. The desired protected hexapeptide hydrazide-Boc-Val-
Asp(OBzl)-Thr(szl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-HNNH2 was iso-
lated as pure crystalline solid in a reasonable yield.
For the synthesis of the protected tetrapeptide Boc-Ile-
~ Thr(Bzl)-Thr~Bzl)-Lys(Z)-OH, a similar stepwlse procedure using
the N-hydroxysuccinimide active ester procedure of Anderson
~ ~ .
~ et al., J. Amer. Chem. Soc. 86, 1839 (1964),was utilized.
.. , : . - , : .
.. . ...
.. , : .
Reaction between Boc-Thr(Bzl)-OSu and H-Lys(Z)-OH gave the
dipeptide Boc-Thr(Bzl)-Lys(Z)-OH as an oil which was deprotected
at the ~-amino end and allowed to react with Boc-Thr(Bzl)-OSu
to provide the tripeptide Boc-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH as a
crystalline solid. Removal of the Boc-group and reaction of
the resultant material HCl ' H-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH
with Boc-Ile-OSu yielded the desired protected tetrapeptide
Boc-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH as a crystalline pure
compound after chromatography on a silica gel column. This
tetrapeptide fragment was then deprotected at the amino term-
inal and condensed with the protected hexapeptide Boc-Val-
Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-HNNH2 by the
azide method to produce the protected decapeptide Boc-Val-
Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-lle-Thr(Bzl)-
Thr(Bzl)-Lys(Z)-OH in good yield.
Removal of the Boc-group frc~m the decapeptide compound with
TFA and subsequent coupling with the N-terminal tetrapeptide
Ac-Ser(Bzl)-Asp(08zl)-Ala-Ala-HNNH2 through the azide procedure~
resulted in formation of the required protected tetradeca-
peptide H3C-CO-Ser(Bzl)-AsptOBzl)-Ala-Ala-Val-Asp(OBzl)-
Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-
Lys(Z)-OH. For the final coupling, this acetyl tetradecapeptide
was activated with DCC and HOBT and the ensuing active ester
was then allowed to react with TFA H-Asp(OBzl)-Leu-Lys(Z)-
Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-
Ala-Glu(OBzl)-Asn-OBzl which was derived from the corresponding
: blocked compound on treatment with TFA to give the protected
- ~
.
.
-
- 12 -
acetyl octacospeptide H3-CO-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-
Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-
Thr(Bzl)-Lys(Z)-Asp(OBzl)-Leu-Lys(Z)-Glu(OBzl)-Lys(Z)-Lys(~)-
Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl.
Treatment with anhydrous HF removed all the protecting groups
and purification on ion-exchange chromatography yielded thymosin
al .
In lil;e manner there was ohtained ~rom the protected tetra-
decapeptide H3C-CO-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp(OBzl)- :-
Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-
Lys(Z)-OH the free tetradecapeptide H3C-CO-Ser-Asp-Ala-Ala-Val- . .
Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-OR.
The synthesis of [Asn2]-thymosin al followed the same`
pattern as the synthesis of thymosin ~1' All of the inter- ` ~.
mediates used were the same except the N-terminal acetyl tetra-
peptide hydrazide H3C-CO-Ser(Bzl)-Asn-Ala-Ala-HNNH2. For the
synthesis of this compound, Boc-Ala-Ala-OH was first converted
into Boc-Ala-Ala-OBzl which on selective removal of the Boc-
group with HCl gave the dipeptide ester salt HCl H-Ala-Ala-
OBzl. The dipeptide was then coupled with Boc-Asn-OH using the
DCC/HOBT procedure of Ronig and Geiger, Chem. Ber. 103, 788
(1970),to give the protected tripeptide ester Boc-Asn-Ala-Ala-
OBzl which was treated with HCl in THF to remove the Boc-group..
The resultant product HCl ~ H-Asn-Ala-Ala-OBzl was then
condensed (DCCjHoBT procedure) wIth H3C-CO-Ser(Bzl)-OH ~ DCHA
. to af~ord the desired protected tetrapeptide ~3C-CO-Ser(Bzl)-
_,
." , , i .
. .
. .
.
- 13 -
: '
Asn-Ala-Ala-OBzl.
The corresponding hydrazide H3C-CO-Ser(Bzl)-Asn-Ala-Ala-
HNNH2 was obtained in good yield on hydrazinolysis of this
compound. Fragment condensation between the hydrazide and the
decapeptide TFA ~ H-Val-Asp(OBzl) -Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-
Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z~-OH produced the
protected tetradecapeptide H3C-CO-Ser(Bzl)-Asn-Ala-Ala-Val-
Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-
Thr(Bzl)-Lys (Zl-OH. This compound was then counled to the
deblocked C-terminal tetradecapeptide TFA salt discussed above
to give the corresponding protected [Asn2]-thymosin al. Removal ~ -
of all the protecting groups by treatment with anhydrous HF
followed by column ion-exchange chromatographic purification
gave the desired [Asn2]-thymosin al while the same procedure
with the protected tetradecapeptide H3C-CO-Ser(Bzl~-Asn-Ala-
Ala-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)--Ser(~zl~-Glu(OBzl)-Ile-
Thr(Bzl)-Thr(Bzl)~Lys(Z)-OH yielded the acetyla~e2 tetradecape~tide
H3C-CO-Ser-Asn-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-
Thr-Lys-OH.
In a further aspect of this invention it has been found
that severàl of the in~ermediate peptides used in the above
synthesis of thymosin al and [Asn~]-thymosin ul have activity
in the regulation, differentiation and function of T-cells,
namely
Glu-Val-Val-Glu-Glu-A~a-Glu~Asn;
Glu-Lls-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn;
:
; :. -
.... .. ~ .
2~
14 -
Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn; :
H3C-CO-Ser-Asn-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-
Thr-Lys; and
H3C-CO-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-
Thr-Lys.
:
Thymosin al, [Asn ]-thymosin al, or the aforesaid novel
octa-, undeca- or tetradecapeptides which form a part of the
present invention, ànd their pharmaceutically acceptable salts
,
may be administered to warm blooded mammals by parenteral
application either intravenously, subcutaneously or intra-
muscularly. These compounds are potent immunopotentiating agents
with a daily dosage in the range of about 1 to 100 mg/kg of
body weight per day for intravenous administration. Obviously
the required dosage will vary with the particular condition
being treated, the severity of the condition and the duration of
treatment. A suitable dosage form for pharmaceutical use is 1 mg
o lyophilized thymosin al, [Asn2]-thymosin al, or one of the
aforesaid peptide fragments thereof to be reconstituted prior~to~
use by the addition of sterile water or saline. ;
Also included within the scope of the present invention;are
the pharmaceutically acceptable salts of thymosin a1, [Asn2]~
` thymosin al, and the aforesaid peptide fragments thereof.
Suitable salts include the sodium and potasslum salts or salts~ith a strong
orgamc base such as ~ùanidine. In addition, the counter ions of.~these
cations such as the~chloride, bromide, sulfate, phosphate,
~ maleate, acetate, citrate, benzoate, succinate, malate, ascorbate~
: ~ ` ; '
:
-
,. : . ` : '
. ,
,, ,
.
`` - 15 ~
and the like, may be included in the preparation.
- Abbreviations used herein have the following meaning:
Boc =t-butyloxycarbonyl; Bzl= benzyl; DCC= dicyclohexvlcarbodi-
imide; DMF= dimethylformamide; THF= tetrahydrofuran; HOSu=
N-hydroxysuccinimide; Triton B= 40~ methanolic solùtion of
trimethylbenzylammonium hydroxide; Nr~l= N-methylmorpholin; CHA=
cyclohexylamine; DCHA= dicyclohexylamine; Z= benzyloxycarbonyl;
DMSO= dimethyl sulfoxide; TFA= trifluoroacetic acid; TLC=
thin layer chromatography; Et3N= triethylamine; HOBT= l-hydroxy-
benzotriazole.
The following Examples describe in detail the synthesis
of.thymosin al and [Asn2~-thymosin al.
While specific protecting groups have been employed in
describing the synthesis of thymosin al and [Asn2]-thymosin a
. it is within the skill of the art~to utilize equivalent
protecting groups in such synthesis.
` ` .'
:
: :
:,, :
: -
,
~ - 16 - ~ 3~
Example 1
A.a) Boc-Asn-OH (11.0 g, 47.5 mmol) was dissolved in 200 ml of
MeOH and 20 ml of water was added. The solution was titrated to
pH 7.0 with a 20% aq. solution of Cs2C03 (ca. 55 ml). The mixture
was evaporated to dryness and the residue reevaporated twice '-
from DMF (120 ml each, 45C). The white solid obtained was then
stirred with 8.9 g of benzyl bromide (52 mmol) in 120 ml DMF for
6 hours. On evaporation to dryness and treatment with a large
volume of water, the product solidified immediately. It was col-
lected by filtration, dissolved in ethyl acetate, washed withwater, dried over Na2S04, evaporated to a solid mass and cry-
stallized ~rom ethyl acetate with petroleum ether.
~ield 13.8 g (90.3%)of Boc-Asn-OBzl; m.p. 120-122 C; [a]D =
-17.29 (c = 1, DMF).
Boc-Asn-OBzl (13.7 g, 42.4 mmol) was dissolved in 80 ml
of THF and treated with 500 ml of 4N HCl in THF. The mixture was
left standin~ for 45 minutes during which time some product
began to preclpitate. On treatment with 1000 ml of ether, a white
solid material ~ormed immediately. The Product was filtered
washed with èther and dried over NaOH pellets in vacuo. Yield:
10.3 g (94%)of HC1 H-Asn-OBzl; m.p. 122-126 C; [a]D5= + 6.82.
b~ H-Glu(OBzl)-OH (7.0 g, 29.5 mmol) was finely ground in a
mortar and pestle and then stirred with 8.88 g (32.3 mmol) of
Boc-Ala-OSu for 48 hours in 250 ml DMF in the presence of 6 ml
- 25 NMM. Some more NM~I was added to maintain the reaction slightly
~; basic during the reaction. The solvent was evaporated and the
~:
..... ~, .. .. . .
,
`''' ' , '~' ~ ' ~, ' "'
. . . ~
.: - - . .
: '' . . :' ~ ", ', ' '.
: ,
~ 17 ~
residue partitioned between 300 ml ethyl acetate and 500 ml
H20 containing 2 ml of 10% H2S04. The organic layer was then
washed three times with water, dried over Na2S04 and evapo-
rated to dryness. The product was taken up in a small volume
of ether and treated with a large volume of petroleum ether. A
white amorphous solid was obtained which was homogeneous on
TLC. Yield: 11.0 g (91.5%) of Boc-Ala-Glu(OBzl)-OH; m.p. 84-
88C; C~]2D =8.0S (c=l, D~).
Boc-~la-Glu(OBzl)-OH (10.4 g, 25.4 mmol), HCl-~H-Asn-
OBzl (6.56 g, 25.4 mmol) and HOSu (5.9 g, 50.8 mmolj were dis-
solved in DMF (250 ml, 0C). DCC (5.7 g, 27.6 mmol) was added
followed immediately by Et3N (3.5 ml). The mixture was stirred
at 0C ~or 2 hours and then at 25C for 4~ hours during which
period some more Et3N was added from time to time to maintain
the reaction slightly basic. The insoluble by-products formed
were ~iltered off and the filtrate evaporated to dryness. The
residual oily material solidi~ied on treatment with water. The
crude product was taken up in CHC13, washed with water (3x),
dried over Na2S04 and evaporated to a smaller volume. Som~ soli~
formed at this stage was filtered off (heavily contaminated with
dlcyclohexylurea) and the filtrate treated with petroleum ether.
A crystalline product was obtained. Yield: 8.0 g (51.4~) of
Boc-Ala-Glu(OBzl)-Asn-OBzl; m.p. 102-105C; [~1 5 = 12.5
~ (c = 1, DMF).
: :
c) H-Glu~OBzl)-OH (4.74 g, 20 mmol) was ground in a mortar
and pestle and stirred with Boc~Glu(OBzl)-OSu (0.7 g, 20 mmol)
ln DMF for 36 hours in the presence of 3.6 ml NMM. The ensulng
- , . ~ .: ., . ,. : ..
-. ~ ' ' : . '. ' .. ,. . : .:"':
~r 18
solution was evaporated to a syrup and treated with water. The
oily precipitate was taken up in ethyl acetate, washed succes-
sively with 5~ HOAc and water (3x), dried over Na2S04 and eva-
porated to dryness yielding 14.03 g of a clear oil. It was left
standing submerged under petroleum ether. The residual oily
Boc Glu(OBzl)-Glu(OBzl)-OH weighed 10.2 g (9o.o~). TLC indlcated
that the product was homogeneous. [a]D = ~7 59 (c = 1, DMF).
d) Boc-Ala-Glu(OBzl)-Asn-OBzl (28.2 g; 46 mmol) was treated
with 1.1 liter of 4N HCl in THF for 1 hour. Eva~oration of the
solvent and excess acid left an oil which was evaporated twice
more with fresh THF. The residual oil turned into a solid when
treated with a large ~olume of ether. Tha solid HCl~H-Ala-Glu
(OBzl)-Asn-OBzl was stirred with Boc-Glu(OBzl) Glu(OBzl)-OH
(25.6 g, 46 mmol), HOSu (10.6 g,'~92 mmol) and DCC (10.9 ~, 53
mmol) in DMF (540 ml) at 0C for 1 hour and then at 25C for 48
hours. Et3N was added to maintain the reaction sli~htl~ basic
over the entire period of time (ca,16 ml Et3N total) . The i~solu-
ble by-products formed were filtered off and the filtrate eva-
porated to dryness. mhe crude product was dissolved~in CHC13,
washed with water (3x), dried over Na2S04 and evaporated to
dryness. The product solidified when treated with petroIeum ether.
Recrystallization fro~ isopropan~l ylelded 28.9 g (59.8~o) Of Boc-
Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl; m,p. 169-175 C;
[a]D = -11.78 (c = 1, D~F).
Boc-Glu(OBzl)-Glu(OBzl)-Ala-G1u(OBzl)-Asn-OBzl (3.9 ~,
3.48 mmol) was treated with 15 ml 4N HCl in THF for 30 minutes.
Some c~yst~lline pro~uct started to form. Ether (210 ml) was
.
~, - ' ' '.''', :
.; . .
.~ , ,:
. . . ,' . :, ' - :, .. : , : . : , : .,. . . - ~ . , -
. ,, : .- . .. - :. : : .
added and the precipitated solid was collected and washed with
ether. The crude material was crystallized from MeOH and ether.
Yield: 2.58 g (75.196) of HCl-H-Glu(OBzl)~Glu(OBzl)-Ala-Glu
(OBzl)-Asn-OBzl; m.p. 148-151 C; [~]D = -3.65 ~c = 1, D~F).
e) Boc-Val-OSu (12.6 g, 40 mmol)- and H-Val-OH (4.68 g, 40
mmol) were condensed in DMF (250 ml) for 96 hours in the pre-
sence of 2 ml Et3N. More Et3N was added when needed to maintain
the reaction slightly basic. The remaining insoluble material
was filtered off and the filtrate evaporated to dryness (45C).
The residue was partitioned ~etween ether and dilute H2S04 `
(ca~1%) and the orqanic layer washed with water (3x), dried over
Na2S04 and evaporated to a foamy glass. The product was crystal-
lized from ether and petroleum ether. Yield: 12.2 g (96.496) of
Boc-Val-Val-OH; m.p. 155-158C; [a]25= ~1.10 (c = 1, DMF).
Boc-Val-Val-OH (40.5 g, 128 mmol) was treated with 1.8 1
of 4N HCl in THF for 60 minutes. 'Evaporation to remove excess
acid and solvent followed by treatment with ether provided
34.5 g of HCl~H-Val-Val-OH as a white amo:~phous powder. It was
treated with Boc-Glu(OBzl)-OSu (55.6 g, 128 mmol~ in 1 liter
DME` for 24 hours in the presence of 54 ml Et3N. The reaction
mixture was filtered to remove some insoluble material and the
filtrate evaporated to dryness. The remaining oil~ residue was
taken up in EtOAc (1''.51) and washed with 5~ HOAc (2x) followed
by water (3x). The organic la~er was dried (Na2S04)~and evspo-
rated to dryness to give~a`colorless clear oil which did not cry-
stallize. It was thus dissolved in 3.2 1 of ether and treated
`~', with CHA (17 ml) until the pH of the mixture was 7.5. The~solid
.. ~ ', .: . : . , -: ~ ::: : .~:
- 20 ~
salt obtained was collected and recrystalli~ed from MeOH and
ether. Yield: 58.9 g (72.7%) of Boc-Glu(OBzl)-Val-Val-OH~CHA;
m.p~ 158-160C; [a]D =33.41 (c = 1, MeOH).
Boc-Glu(OBzl)-Val-Val-OH~CHA (1.69 g, 2.66 mmol) was
suspended in water (40 ml) and ethyl acetate (40 ml) in a sepa-
ratory funnel when 4 ml of 1 M H2S04 was added. After vigorous
shaking, the solid dissolved and the organic layer was washed
several times with water, dried over Na2S04 and evaporated to an
oil (1.45 g). The free tripeptide thus obtained was then con-
densed with 2.58 g of HCl H-Glu~OBzl)-Glu (OBzl)-Ala-Glu(OBzlj-
Asn-OBzl (2.61 mmol) in 15 ml DMF in the presence of HOSu
(0.612 g, 5.32 mmol), NMM (0.3 ml, 2.66 mmol) and DCC (0.63,
3.06 mmol) during 1 hour at 0C and 60 hours at 25C. More NM~1
was added when needed to maintain the reaction slightly basic.
An insoluble by-product ~ormed was f~iltered:~of~ and ~~
the filtrate evaporated to dryness (45C). The remaining oily
residue solidified when treated with water. The crude solid was
dissolved in D~ (50 ml) and precipitated with MeOH (300 ml).
Yield: 2.25 g ~58.7~) of B~c-Glu(OBzl)-Val-Val-Glu(OBzl)-
Glu(OBzl)-Ala-C-lu(OBzl)-Asn-OBzl; m.D. 277-280C; [a]D = -12.43
(c = 1, DMF).
This product (0.72 g, 0.49 mmol) was hydrogenated over
5% Pd/BaS04 10.5 g) for 3 hours at 3.4 atm in a mixture of 40 ml
DMF/30 ml MeOH/ 2 ml H20. The mixture was then filtered and the
filtrate evaporated to dryness. It was subsequently treated with
5 ml of TFA for 30 minutes and the residue obtained after eva-
poration of the acid was triturated several times with ather.
The ensuing white solid was taken up in water (20 ml~ and lyo-
.
, 1; -- '1; '
, ~ . - , ;.
r, . :
,
phili~ed to give 0.47 g of crude product. The compound was
loaded on a 3 x 32 cm column of a strongly basis polystyrene
resin (Bio-Rad AGl-X 2) e~uilibrated with pH 8.1 ammonium ace-
tate buffer (2~ HOAc made to p~ 8.1 with NH3). The column was
eluted successively with 200 ml each of 0.025 M pH 5.5 N~140Ac,
0.025 M HOAc, 0.05 M HOAc, 0.1 M HOAc, 0~25 M HOAc, 0.5 M HOAc,
0.75 M HOAc, 1 1~ HOAc. Fractions of 12 ml were collected and 'he
eluate from each tube was monitored by TLC. The fractions con-
taining the desired material (tubes 225-229) were pooled and
lyophilized twice to yield 0.223 g (48.1%) of pure Glu-Val-Val- ~.
Glu-Glu-Ala-Glu-Asn which ~tas ho~o~eneous on ~C and paper !
electrophoresis.
f) Boc-Lys~Z)-OH ~15 ~, 39.5 mmol) was stirred with HOSu
(5.8 g, 50.5 mmol) and DCC ~8.66 g, 42 mmol) in THF (250 ml)
for 3 hours. An insoluble b~-product was filtered off and the
filtrate evaporated to dryness. The residual syrup (24.2 g) w~s
treated ~Jith iso-pro~anol (150 ml) and petroleum ether (150 ml)
to yield an oily product (21 g) which failed to crystallize.
The crude active ester Boc-hys(Z)-OSu was thus used ~or conden~
sation with H-I,ys(Z)-OH ~10.6 g, 38 mmol) in D~IF (250 ml) for
72 hours in the presence of 5.5 ml Et3N. More Et3~ was added
occasionally in order to maintain the stirred reaction mixture
slightly basic. Some small quantity of undissolved material was
tnen iltered o~f and the filtrate evaporaied to dr~ness ~4SC~.
?5 The ramaining oil~ residue was treated with 1 liter of 5~ HOAc.
The product precipitated was extracted into ethyl acetate and
ths organic phase washed with water, dried over Na~S04 and eva-
-~ * Trademark
". .
, ~ , . .: . ' '. . ' . ~ ' .
:-
- 22 -
porated to an oil. It was crystallized from ethyl acetate (300
ml) containing DCHA (10 ml) as a salt. Recrystallization fro~
MeOH and ether yielded 22.7 g (72.5%) of Boc-Lys(Z)-Lys(Z)-OH- -
DCHA; m.p. 160-162C; [~]D5= -2.21 (c = 1, ~eOH).
Boc-Lys(Z)-Lys(Z)-OH DCHA (10 g, 12.14 mmol) was parti-
tioned between EtOAc (1 liter) and 0.1 N H2S04 (1 liter). The
organic layer was then washed with water (3x), dried over Na2S04
and evaporated to dryness (7.9 g). ~he free acid, Boc-TJys(Z)-
Lys(Z)-OH, thus obtained was treated with freshly prepared
4N HCl in THF for 30 minutès The solvent and the excess acid
was evapora~ed (30O) and the residue re-evaporated twice with
THF. The remaininy residue solidified when treated with ether.
The salt HCl-H-Lys(Z)-Lys(Z)-OH was collected by filtration and
washed several times with ether to yield 6.7 g of white powder.
It was dissolved in DMF (70 ml), chilled in an ice-bath and - ~.
treated with Et3N (1.63 ml) followed b~ Boc-Glu(OBzl)-OSu (5.54 q,
12.76 mmol). The mixture was stirred at 0C for 1 hour and
then at 25C for 24 hours. More Et3N was added durin~ this time
to maintain the reaction at approximately pH 7.5. A few ml o
acetic acid was added to make the reaction acidic (pH 3.5) and
the solvent removed by evaporation. The ensuing residue was taken
up in EtOAc, washed with water (3x), dried over ~la2SO~ and eva-
; ~ porated to dryness when the product began to solidify. It was
triturated in ether and recrystallized from eth~l acetate.
Yield: 7.26 g (69.5~)~ of ~oc-Glu(OBzl)-Lys(Z)-Lys(Z)-OH; m.p.
153-155 C; [~] 5 = -2.71 ( c = 1, THF).
~.
- . . : ~ . -: .
.. . .. . ..
- 23 -
g) Boc-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-GlutOBzl)-
Asn-OBzl (1.7 g, 1.16 mmol) was treated with TFA (24 ml) for 30
minutes. After evaporation of the excess acid (30C) the residue
was triturated with ether. The powder obtained was washed thoro-
ughly with ether and petroleum ether and dried over NaOH in
vacuo to give the trifluoroacetate salt of the octapeptide
(1.71 g). The active ester Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-OSu was `
then generated in situ by stirring Boc-~lu(OBzl)-L~s(Z)-Lys(Z)-
OH (0.99~ g, 1.16 mmol), HOSu (0.16 g, 1.4 mmol) and DCC (0.274
g, 1.33 mmol) in 15 ml DMF at 0C for 3 hours. To this solùtion
containing the tripeptide active ester, the octapeptide salt
CF3COOH-H-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu~OBzl)-
~sn-OBzl (1.71 g) was added together with 0.2 ml of Et3N. A few
more drops of Et3N and DMF (15 ml) were added and the mixture
was stirred fbr 3 da,ys at 25C. A gelatinous semi-solid formed.
It was acidified with acetic acid and treated with water. The
whi'te solid precipitate was collected and washed '(H20, MeOH,
ether) to yield 2.25 g of crude product melting at 310-313C.
It was dissolved in DMF and precipitated with MeOH. Yield: '
1.75 g t68.3%) of Boc-Glu(OBzl~-Lys(Z)-Lys(Zj-Glu(OBzl)-Val~
Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzlj-Asn-OBzl; m.~. 314-
316 C; [~]D = 13~.68 (c = l,,DMSO); homogeneous on TLC.
: Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-G;u(Obzl)- ~ :
Glù(OBzl)-Alà-Glu(OBzl)-Asn-OBzl (0.5 g, 0.226 mmol) was dls- ~ -
solved in ml of TFA and stirred with 15 ml of HF at O C for 15
minutes. After evaporation of excess acid (0C), the residue
~,~ was dissolved in 5~ aqueous HOAc, washed with ether (3x), evapo- ;
. . , ., , . : . . : , :- ~ :
. .
- . . ::::
3~
rated to a smaller volume and lyophilized to yield 0.34 g of
crude product. It was chromatographed on the ion-exchange
column as described above for the octapeptide to give 0.13 g
(42.1~) of pure Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn;
[~]D = -85.65 (c = 1, H20).
h) Boc-Leu-OSu ~4.0 g, 12.2 mmol) and H-L~s(Z)-OH (3.42 g,
12.2 mmol) were condensed in DMF (75 ml) during 48 hours in the
presence of Et3N (1.7 ml). The reaction pH was maintained at 7.5
by addition of Et3N periodically as usual. The remaining inso-
luble material was filtered off and the filtrate evaporated to
dryness. The ensuing foamy glass was dissolved in ether (200 ml)
and the mixture treated with 3 ml of DCHA to produce crystalline
material which was collected, washed with ether and recrystalli-
zed from MeOH and ether. Yield: 5.7 g (69.5~) of Boc-Leu-Lys(Z)-
OH~DCHA; m.p. 140-142C; ~a]D5= -7.20 (c = 1, MeOH).
Boc-Leu-Lys(Z)-OH~DCHA (2.97 g, 4.4 mmol) Was converted
; into the free acid (partitioned between EtOAc and 0.1 N H2S04)
and the colorless oil~obtained (2.2 g) Was treated ~with 4N HCl
in THF (40 ml) for 30 minutes. The excess acid and the solvent
~;~ 20 were`evaporated (30C) and the residue treated with ether. The
remaini.ng oil was dlssolved in ether and evaporated twlce more
with ~resh ether. The residue was then stirred with Boc-Asp(OBzl)-
OSu (1.85 g, 4.4 mmol)~ in the presence of Rt3N (1.85 ml)
overnlght. The reaction mixture was then evaporated to dryness
giving an oily residue which was taken up in ethyl acetate, ;
washed with water (3x), dried over Na2S04 and evaporated to dry-
, ~ ~ ` `'.
.
. , , : .
: . ., - : :
ness again. The crude product thus obtainèd was crystallized
from ethyl acetate and petroleum ether and yielded 1.52 g
(49.6%) of pure Boc-Asp(OBzl)-Leu-Lys(Z)-OH; m.p. 109-111 C;
[~]D = -16.14 (c = 1, D~IF).
Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-
Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl (1.2 g, O.545 mmol) was trea-
ted with 35 ml TFA for 30 minutes. The excess acid was quickly
evaporated and the residue triturated with ether several times
to g1ve 1.2 g of undecapeptide TFA-salt as a white powder. It
was dissolved in a mixture of DMF (5 ml) and DMSO (2 ml) and~ -
treated with Boc-Asp(OBzl)-Leu-Lys(Z)-OSu generated in situ by
stirring Boc-Asp~OBzl)-Leu-Lys(Z)-OH (0.381 g, 0.545 mmol) with
HOSu (0.126 g, l.lmmol) and DCC (0.124 g, 0.599 mmol) in 3 ml
DMF at 0C for 3 hours. The mixture containing the tripeptide
lS active ester and undecapeptide was stirred at 0C for 2 hours
and then at 25C for 3 days, during which period Et3N was added
~rom tlme to time in order to maintain the pH slightly basic.
A gelatineous substance formed. It was triturated with 5% HOAc
and the resulting white solid was filtered and washed with ~
water, MeOH and ether to give 1.28 g of crude material melting
at 325-326C. Reprecipitation from DMF/DMSO (lO ml/5 ml) and~
MeOH (230 ml) yielded 1.22 g (80.2%) of pure Boc-Asp(OBzl)-
Leu-Lys(Z)-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu
.
(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl; m.p. 326-327 C;
[iD =-15.71 (c = 1, DMF/DMSO). ~ :
This product (1.128 g, 0.404 mmoI) was mixed with 7 ml of
-~ anisole and treated ~ith 25 ml anhydrous HF at 0C for 15 mi-
,
:
. . . . : : . .
: '.. . ~ ~ ' . "i` ` . ,
.
~ ~ .
.
- 26 -
nutes. The excess acid was evaporated (0C) and the remaining
residue partitioned between ether and water. The water layer
was washed twice with ether, evaporated to 1/2 the original
volume and lyophilized to provide 0.69 g of crude material. It
was chromatographed in the manner described above for the octa-
peptide. The material eluted at tubes 101-120 was collected and
lyophilized to give 0.25 g of product which was shown to be
slightly contaminated with minor impurities. It was thus re-
chromatographed on the same column to give 0.155 g t22%) of
pure Asp-Leu-Lys~Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn.--~
Paper electrophoresis indicated that it was homogeneous.
t~]25= -86.27 (c = 1, 0.1 N HCl).
B.a) H-Ser(Bzl)-OH (18.6 g, 95.4 mmol) was dissolved in 45 ml
Triton B, evaporated to dryness, and the residue re-evaporated
twice with DMF (100 ml each). The residue was then stirred with
AcOSu (16.9 g, 95.4 mmol) in 150 ml D~F for 20 hours. N-Methyl-
morpholin was added from time to time in order to maintain the
;~ ~ reactlon slightyl basic. The solv~ent was removed and extraction
of the product into EtOAc was followed by washin~ with small
volumes of 10~ HOAc, and H20 (the product is water soluble, use
a small volume of H20). Drying over Na2S04 and evaporation to
drvness again ~ave a clear oil (14.5 g, Ac-Ser(Bzl)-OH). The
compound failed to crystallize. It was thus dissolved in a mix-
ture of 300 ml o MeOH and 30 ml of H20, titrated to pH 7.0
with 20% Cs2C03 and evaporated to a solid mass. The salt was re-
evaporated twice more with DMF and stirred with benzyl bromide
. '
.
- .. .'
~ , ' `
27
(15.4 g, 91 mmol) in 250 ml DMF for 18 hours. On evaporation
of the solvent, the residue was taken up in H20 (600 ml) and the
oily product formed was extracted into EtOAc. It'was washed with
H20~ dried over Na2S04 and evaporated to a syrup which on see-
ding crystallized immediatelv. It was recrystallized from EtOAcand petroleum¦ether to yield 10.42 g ~32.2% overall) of H3C~X~
Ser(Bzl)-OBzl; m.p. 89~91C.
H3C-CO-Ser(Bzl)-OBzl(2.2 ~, 6.7~ m~ol) was dissolved in 75
ml EtOH and stirred ~ently with 5 ml of H2NNH2 overni~ht. Some
insoluble matter precipitated was filtered off and the filtrate~
was evaporated to an oil which solidified when treated with
ether. The product was recrystallized from a small volume of
EtOH and ether to yield 1.40 g (82.8~) of H3CO-~Ser(Bzl)-HNNH2,
m.p. 128-130 C; ~a]D = ~5.80 (c = 1, MeOH).
b) L-Alanine (3.57 g, 40 mmoL) was dissolved in 18.8 ml of
; Triton B t40 mmol), evaporated to dryness, and the oily residue
re-evaporated twlce with DMF (30 ml each). The salt obtained was
~tirred with 11.45 g~of Boc-Ala-OSu (40 mmol) in 40 ml DMF, with
;~ -4 ml of N~ added, for 20 hours. The solvent was removed and ~
the residue taken up in 10% HOAc (100 ml). The product was extrac-
ted into EtOAc (4xlOO ml), washed twice with a small volume of
H20, dried over Na2SO~, evaporated to a small volume, and trea-
ted with petroleum ether until cloudiness developed. A crystal-
line product formed on stora~e in the refrigerator overni~ht.
Yleld: 8.2 g (76.3%) of Boc-Ala-Ala-oEl; m.p. 115-118 C.
~ . ` ' ' ~ ` " ' ' ' ~ ' ' ` ' ' ` :' ''
:
- 28 - ~3~
Boc-Ala-Ala-OH (36.2 g, 139 mmol) was treated with 3 li
of 4 N HCl in THF for 30 minutes. Evaporation and work-up
as usuaI gave an oily mass which solidified when treated with
ether. The product was recrystallized from methanol with ether
to yield 9.1 g (33.3%) of HCI~H-Ala-Ala-OH; m.p. 209-211C.
HCl-H-Ala-Ala-OH (2.36 g, 12 mmol) was dissolved ln 20
ml of DMF, chilled in an ice-bath, and treated with 1.68 ml of~
Et3N (12 mmol) followed by Boc-Asp(Oszl)-OSu (12 mmol). The
mixture was stirred gently at 0C for 2 hours and then at ~5C
overnight during which time one more equivalent of Et3N (12
mmol) was added, in small proportions, maintaining the reaction
pH near 8Ø A few ml of HOAc was added and the acidified mix-
ture evaporated to dryness. The pr.oduct formed was extracted
into EtOAc, washed with H20 (3 times), dried over Na2S04, and
evaporated to an oily residue (6 g). It was dissolved in EtOAc
and titrated to pH 8.0 with DCHA. The crystalline salt precipi-
tated and was recrystallized from iso-propanol and petroleum;~
ether to yield 5.1 g (65.7~) of Boc-Asp(OBzl)-Ala-Ala-OH~DCHA;~ ~ -
~ m.p. 138-141; [~]D5=-13.33 (c = 1, MeOH).
; ~ ~ Boc-Asp(OBzl)-Ala-Ala-OH~DCHA (3.5 g, 5.4 mmol) was
partitioned between 500 ml EtOAc and'350 ml H20 containing 10
ml of 10~ H2S04. The aqueous layer was extracted once more with
EtOAc (250 ml) and the comblned EtOAc laver washed twice w1th
H20, dried over Na~S04, and evaporated to dryness, leaving a
glassy solid o~ Boc-Asp(OBzl)-Ala-Ala-OH (2.5 g). This material
. ~ ....... - ~
. . - - ~ ~: .: ;
'' ~
. . . ,: . ; ~ . . ~: , .
-.. - , . ~, :
. ~.. .
- 29 -
was treated with 200 ml of freshly prepared 4 N HCl in THF for
30 minutes, evaporated at 32C to a svrup, and re-evaporated
twice more with THF. The oily residue solidified when treated
with ether. This HCl~H-Asp(OBzl)-Ala-Ala-OH (1.93 g, 4.83
mmol) was then used in the next reaction involving azidecoupling
with H3C-CO-Ser(Bzl)-N~I that was Drenared from 1.24 q H3C-CO-~er
(Bzl)-HNNH2 (4.9 mmol) in 25 ml DMF (-25C) with 7.42 ml of
3.3 N HC1 in THF (24.5 mmol) and 0.99 ml of i-amylnitrite
(7.35 mmol) stirred at -30C for 30 minutes. The azide solution ~ -
prepared was cooled down to -35C, mixed with 4.1 ml of Et3N
and then treated with the white powder of HCl H-Asp(OBzl)-Ala-
Ala-OH (1.93 g) prepared above. The mixture was stirred at
-20C for 30 minutes and then at 4C for 2 darvs. Some more E~3N
was added to keep the reaction slightly basic. Work-up as usual
gave a crystalline mass which was recrystallized from THF and
petroleum ether to yield 1.85 g (65.6%) of H3C-CO-Ser(Bzl)-
Asp(OBzl)-Ala-Ala-OH; m.n. 167-170C; [a]D5--13.91 (c=l, D~SO).
.
H3C-CO-Ser(~zl)-Asp(OBzl)-Ala-Ala-~E~ (0.825 q, 1.41 m~ ~s
dissolved in 4 ml DMF and chilled to 0C in an ice-bath. To the
solution, H2NNH2 (54~3 mg; 1.69 mmol) was added folIowed by
HOBT~H20 (0.475 g, 3.10 mmol) and DCC (0.32 g, 1.55 mmol). The
mixture was adjusted to pH 7.5 with NMM and stirred at 0C for
2 hours followed by`17 hours at 25C. The reaction became a gel
during this time. It was diluted with MeOH and the solid
material remaining was collected on a suction filter and washed
thoroughly with MeOH, ether and petroleum ether to ~ive a
.
: ~ . .. ..
- . ., ~ . .: . .:
- 30 - ~ '~ ~3~
material melting at 229-232C. The product was then precipi-
tated from DMF and MeOH to yield 0.51 g (61.0~) of H3C-CO-Ser(Bzl)-
Asp(OBzl)-Ala-Ala-HNNH2; m.p. 230-232C; [à]25= -17.94~ ~c - 1,
DMSO).
c) H-Glu(OBzl)-OH (39.4 g, 166 mmol) was stirred with Boc-
Ser(Bzl)-OSu (65.0 g, 166 mmol) in 900 ml DMF overnight in the
presence of Et3N (2.3 ml, 165 mmol). More Et3N was added during
this time in order to maintain the reaction slightl~ basic. The
clear solution was evaporated to dryness and the oily residue
partitioned between EtOAc (1.5 liters) and 5% HOAc (2 liters).
The organic layer was washed with H20 (2 times), dried over
Na2S04, and concentrated to a clear oil (90.0 g) which was
taken up in 3 liters of ether and treated with 25 ml of cyclo-
hexylamine. The solid formed was recrystallized from MeOH and
ether. Yield: 76.2 g (74.8~) of Boc-Ser(Bzl)-Glu(OBzl)-OH-CHA;
m.p. 154-156.5C; ~a]25= +6.32 (c = 1, MeOH).
Boc-Ser(Bzl~-Glu(OBzl)-OH-CHA (76.2 g 124 mmol~ was sus-~
` pended in a mixture of 1.5 liters each of H20 and EtOAc. To this
mixture, 10~ H2S04 was added until it became acidic (~H ca. 2.5)
and the solid dissolved. The organic layer containing the dipep-
tide free acid was~washed with H20 (2 times), dried, and evapo-
;~ rated to dryness leavlng a clear oil (68.5 g). It was treated
with 3 liters of freshly prepared 4.1 N HCl in THF for 45 minu-
tes and evaporated to an oily residue which was re-evaporated
twice more with THF. The residue (HCl~H-Ser(Bzl)-Glu(OBzl)-OHj
~~
was dissolved in DMF (500 ml), chilled to 0C, and treated with
: :
- , . - : :
,
- 31 -
Boc-Ser(Bzl)-OSu (48.66 g, 124 mmol), followed immediately by
27 ml of Et3N. The mixture was stirred overnight at 25C during
which time more Et3N was added occasionallv in order to main-
tain the reaction slightly basic. Some small quantities of in-
soluble matters ~lere removed by filtration and the filtrate was eva-
porated to an oil which was taken up in EtOAc, washed with 5~ -~
HOAc and H20, dried over Na2S04 and evaoorated a~ain to drvness.
The product was crystallized from EtOAc with oetroleum ether.
Yield: 71.8 q ~83.7%) of Boc-Ser(Bzl)-Ser(Bzl)-C-lu(OBzl)-OH;
m.p. 112-11'C; ra]D = +17.91 (c - 1, TH~).
Boc-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH (71.6 g, 104 mmol)
was treated with 2.7 liters o~ freshly prepared 3.9 N HCl in
TH~ for 45 minutes. The mixture was evaporated to dryness and
the residue evaporated twice more wit:h THF to give a solid mass `
~59.3 g; m.p. 161-165C). It was collected and washed with
ether and stirred in 500 ml of DMF with Boc-Thr(Bzl)-OSu ~38.2 g,
94 mmol) in the presence of Et3N (25 ml) at 0C for 1 hour and
then at 25C for 15 hours. More Et3N (14.5 ml) was added in se-
veral portions during this time to maintain the reaction
sli~htly basic. Some insoluble matter formed was filtered off
and the filtrate evaporated to an oil which was dissolved in
EtOAc (1.5 liters), washed with 5% HOAc, H20 (2x), dried over
Na2S04 and evaporated to a solid mass. The nroduct was re-
crystallized from EtOAc and petroleum ether to yield 64.8 g(78.1%) of Boc-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH; m.p.
'. ~ '
, . . . .
. ' ' . . , ., '- , . . . .. `
~3~
115-118 C; [~]D = + 11.64 (c = 1, DMSO).
Boc-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH (54.5 g,
61.7 mmol) was treated with HCl in THF (1.5 liters; 4.1 N)
and worked up as usual to aive HCl H-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-
Glu(OBzl)-OH (46.3 g, 56.6 mmol) as a white powder. It
was then stirred in DMF t500 ml) with Boc-Asp(OBzl)-OSu (23.7 g,
56mmol) at 0C for 2 hours in the resence of Et3N (16 ml).
The mixture was further stirred at 25C for 15 hours during
which time additional 7.4 ml of Et3N was added.The pro~t was wor-
ked up as usual and crystallized from CH2C12 and petroleum
ether. Yield: 50.35 g (82.7%) of Boc-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-
Ser(Bzl)-Glu(OBzl)-OH; m.p. 111-113C; ~a]D = +7.21
( c = 1, DMSO).
, Boc-Asp(OBzl)-Thr(Bzl)-Ser(Bæl)-Ser(Bzl)-Glu(OBzl)-OH
(50.0 g, 46 mmol) was deprotected with HCl (4.15 N) in THF
and worked up as usual to-give 45.4 g of white solid. It was
dissolved in THF (1.5 liters) and treated with ether (7 liters).
On standing at 0C overnight a white solid powder was obt~alned~
(44.0 g, m.p. 179-184C). Part of this material, HCl-H-As~
(OBzlj-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH (43.7 g, 42.7
mmol),was then dissolved in 500 ml of DMF, cooled to 0C, and
treated with Boc-Val-OSu (15.4 g, 49 mmol) and Et3N (10 ml).
:
~;~ The`mixture was stirred for 15 hours during which time more
Et3N (7.9 ml) was added ln several portions maintalning the
~ 25 ~ reaction slightly basic. The insoluble matter was removed by
`~ ' :
~ , :
.
. .
.~
.
_~ 3 ~3~
filtration and the filtrate evaporated to dryness. The oily
residue was dissolved in CH2C12, washed with 5% HOAc, H20, dried
over Na2S04, and evaporated to a smaller volume (0.5 liter)
when treated with petroleum ether. The product crystallized
slowly during overnight standing. It was recrystallized from
THF and iso-propanol to yield 26.3 g (51.4%) of Boc-Val-Asp(OBzl)-
Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-~.lu(OBzl)-OH; m.n. 174-
177 C; [a]D = +0.84 (c = 1, THF).
Boc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(oszl)- OH
(13.0 g; 10.94 m~ol) was dissolved in Dr~ (50 ml), cooled
to 0C, and treated with H2NNH2 (0.421~; 13.14 mmol), HOBT
(3.688 g; 24.1 mmol), and DCC (2.48 g; 12.04 mmol). NMM was
then added until the reaction showed pH 7.5. The mixture was
stirred for 18 hours and filtered to remove the insoluble by-
products. The filtrate was evaporc~ted to dryness and the residue
treated with H20. The solid formecl was collected and crystal-
lized from DMF and iso-proPanol to yield 8.7 g (66.4%) of Boc-
Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-HNNH2; m.p.
215-218C; [a]D5= +7.62 (c = 1, DMSO).
d) Boc-Thr(Bzl)-Lys(Z)-OH (14.0 g, 24.5 mmol) was treated
ith 500 ml of- 4.0 N HC1 in THF for 30 minutes, evaporated to
dryness, and re-evaporated twice with fresh ~HF. ~he oilv residue
solidified when treated with ether. The dried powder (11.4 g,
21.6 mmol) of the dipeptide hydrochloride salt was then dis-
solved in 140 ml DMF, cooled to 0C, and treated with Boc-Thr
''
'' ', '- ' , ~ . ' '' .,'' '' '' . ' '' ' '',:' ....... : ' ':
: . . .. ~ .,, : . ~ , , .
(Bzl)~OSu (8.8 g, 21.6 mmol) followèd ~y 3 ~ -~ ~t3N. A few
drops of Et3N was added to maintain a slightly basic condition
while the mixture was stirred for an additional 24 hours at
~ 25C. It was acidified with 5 ml of HOAc and then diluted with
a large volume of water. The solid crude product precipitated
was collected, dissolved in EtOAc, washed with H20, dried over
Na2S04, and evaporated to dryness, leaving a glassv solid mass.
Crvstallization from FtOAc and ~etroleum ether yielded 13.~ q
(83.7%) of Boc-Th~(Bzl~-Thr(Bzl)-Lys(Zj-OH; m.p. 110-112C;
[a] D =+19 . 45 (c = 1, EtOAc).
Boc-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH (41. 5 g, 54 mmol) was
treated with 500 ml of freshly prepared 3.55 M HCl in THF for
25 minutes and evaporated to a s~rup which was re-evaporated
twice with fresh T~IF. The oily re~lidue solidified when treated
with ether. It was collected and washed with ether to give 37.4
of crude hydrochloride salt of the tripeptide, dissolvecl in
500 ml of DMF, chilled to 0C, and treated with Boc-Ile-OSu
~17.4 g, 53 mmol),followed by 16 ml of Et3N. The mixture was
stirred at 25C overnight during which time more Et3N was added
in small portions (6.2 ml total) to maintain a slightly basic
condition. The resultant mixture was filtered and the fil-
trate evaporated to an oil which was extracted into EtOAc, was-
hed with 5% HOAc, H20, dried over Na2S04, and evaporated to
give a yellowish oil. It was crystallized from EtOAc and petro-
leum ether. The crude solid thus obtained (39.6 g, mp 140-
142C~ was found to be contaminated with several minor impu-~
: :
`
- 35 -
'
rities. The material was then chromatographed on a silica gel
column (70-230 mesh, 4.7 x 67 cm) using CHC13/~eOEI (95:5, v/v) as
eluent. The fractions containing the desired product (moni-
tored by TCl) were pooled and evaporated to give an oily pro-
duct which was crystallized from CHC13 and petroleum ether.Yield: 19.1 g (41.2~) of Boc-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH;
m.p. 144-146C; ~a]25= +2.40 (c = 1, CHC13).
Boc-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH (0.439 g, 0.496
mmol) was treated with 4 N HCl in THF for 30 minutes and worked
up as usual to give 0.39 g of HCl~H-Ile-Thr(Bzl)-Thr(Bzl)-
Lys(Z)-OEI. Boc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu
(OBzl)-HNNH2 (0.59 g, 0.492 mmol) was then dissolved in 6 ml of
DMF, cooled to -25C, and treated with 0.57 ml of 4.3 N HCl in
THF (2.46 mmol) followed immediatel~v b~ 0.1 ml of i-amylnitrite
(0.74 mmol). A~ter stirring at -20- -25C or ~ minutes, the
temperature was lowered to -35C when 0.42 ml of Et3N and HCl-
H-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-oH (0.39 g, prepared above)were
added. The mixture was stirred at -20C for 30 minutes and then
at 4C for 48 hours during which time Et3N was added ~rom time -
to tlme in order to keep the pH at about 7.5. The mixture was
then diluted with 250 ml of 5% HOAc and the solid product for-
med was collected and washed with H20, MeOH, ether, and dried
over NaOH pellets in vacuo to give 0.82 g of crude material
(m.p. 244-254C). It was dissolved in DMSO and precipitated by
addition of MeOH~ Yield: 0.698 g (81.7~) of Boc-Val-Asp(OBzl)-
Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl) Thr(Bzl)-
Lys(Z)-OH; m.p. 268-271 C.
. : . .... -.. . .
. : -. : : . -, . ., . ~:
- 36 -
H3C-CO-SerCBzl2-AspCOBzl2-Ala-Ala-HNNH2 ~0.408 g r 0.68
mmol2 suspended in 10 ml DMF was cooled to -20C and treated
with freshly prepared 5.43 N HCl in THF (0.627 ml, 3~4 mmol)
followed by 10~ i-amylnitrite in DMF (1.39 ml, 1.03 mmol). After
stirring ~or 30 minutes, it was cooled down to -30C when Et3N
(0.476 ml, 3.4 mmol) was added followed by the TFA salt of the
decapeptide H-Val-Asp(:OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-
Ile-Thr(Bzll-Thr(Bzl~Lys~Z)-OH (1.334 g, 0.68 mmol).
The mixture was stirred at -20C for 30 minutes and then at
4C for 5 days during which time more Et3N and DMSO were added
in order to maintain the reaction at a slightly basic condi-
tion and to keep the gel from forming. The reaction mixture was
poured into 5% HOAc (300 ml) and th~ solid precipitate formed
was collected, washed with H2O, MeO~I, ether, and dried to give
1.49 g of material melting at 296-299C. The product was repre-
cipitated from DMSO with MeOH. Yielcl: 1.40 g (85.37%) of H3C-CO-
Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp(C)Bzl)-Thr(Bzl)-Ser(Bzl)-
Ser(Bzll-Glu(OBzl~-Ile-Thr(Bzl)-Thr~Bzl)-Lys(Z)-OH; ~a~ D5= +6.37
(c = 1, DMSO~.
The tetradecapeptide obtained (1.35 ~, 0.558 mmol) was
stirred with HOBT~;H2O (0.188 g, 1.23 mmol) for a few minutes in
a mixture of 15 ml each of DMF and DMSO. The mixture was then
cooled in an ice-bath when DCC (0.126 ~, 0.614 mmol) was added
and stirring continued for 24 hours at the same tempera-
ture. In a separate flask Boc-Asp(OBzl)-Leu-Lys(Z)-Glu(OBzl)-
Lys(~Z~-Lys(Z2-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-
I~
-
,
:
-
': ~ ~ ` `
'
,
.
- 37 ~
Asn-OBzl (4.0 g, 1.43 mmol~ ~as treated with 40 ml of
TFA for 25 minutes and the TFA salt of the ensuing tetradecapep-
tide precipitated quickly with addition of a large volume of
ether. The solid formed was collected and washed thoroughly
with ether to give 3.74 g of white powder (TFA salt of C-ter-
minal tetradecapeptide~. Part of this material (1.567 g,
0.5583 mmol) was added to the active ester derived from the
amino terminal tetradecapeptide as prepared above in a DMF-
DMSO mixture. A few drops of NMM was added to bring the pH of
tne reaction to 7.5-8.0, and the stirring was continued for l
hour at 0C and then 5 days at 25C. The reaction mixture was then
poured into 1.5 1 of 5% acetic acid~ The precipitated product
was washed thoroughly with H2O, MeOH, DMF, MeO~ and ether to
give 2.21 g of the desired product, H3C-CO-Ser(Bzl)-Asp(OBzl)-
Ala-~la-~al-Asp(pBzl~-Thr(Bzl)-Ser(~zl)-Ser(Bzl)-Thr(Bzl)-Ile-
Thr(Bzl)-Thr(Bzl~-LystZ~-Asp(OBzl)-l,eu-Lys(Z)-Glu(OBzl)-Lys(Z)-
Lys(Z)-Glu(OBzl~-VaI-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-
Asr-OBzl, melting above 300C.
The protected octacosapeptide (2.21 g, 0.435 mmol) was
dissolved in 8 ml of TFA, mixed with 4 ml of anisole and stirred
with anhydrous HF at 0C for 30 minutes. The acids were removed
at 0C (vacuum distillation) and the solid residue which
remained was dissolved in 200 ml H2O, washed twice with ether,
evaporated to half of the volume and lyophilized to give l.I g
of crude product. It was purified by passlng through a
Sephadex ~ G-10 column (3 x 80 cm; 0.2M HOAc) and then a DEAE-
'.
., . :: . . : :
.
. .
38 ~
Sephadex~ column (3 x 75 cm) eluted with increasing concen-
trations of ammonium acetate (pH 7.0, 0.025M, 0.25~) followed by
dilute acetic acid. The fractions containing the desired mate-
rial were pooled and lyophilized to gike 0.281 g of H3C-CO-Ser-
Asp-Ala-Ala-Yal-Asp-Thr-Ser-Ser-Glu-Ile~Thr-Thr-Lvs-Asp-Leu-
Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn [Thymosin 1]
as an a~orphous white powder.
On acrylamide gel isoelectric focusing, the synthetic
material migrates identically as the natural thymosin al isola-
ted from bovine thymus gland (Proc. Natl. Acad. Sci.(IJSA),74, 725 (1977)).
Example 2
.
Boc-Ala-Ala-OH t7.23 g, 27.8 mmol) was dissolved in a mixture
of 200 ml MeOH and 20 ml H20. Cs2C03 solution ~20~ a~ueous)
WAS added until the pH reached 7.0 (ca. 30 ml) anfl the resultant
neutral solution was evaporated to drvness. The resi~ue~as re-eva-
~orated twice with DMF (150 ml) and the qelatineous solid that
remained was stlrred in 120 ml DMF with benzyl bromide (7.2 g,
42 mmol) for 15 hours. The solvent was then removed by evapo-
ration and the residue treated with 500 ml water. The product
precipitated as an oil which gradually solidified on standing.
It was taken up in EtOAc (400 ml), washed with H20 S3X),
dried over Na2S04 and evaporated to a syrup which on treatment
with petroleum ether began to crystallize. The product was recry-
.
, - - ,
,
.. ~,'' -, -,;
. , - .
~ 39 ~ ~ L3~
stallized from EtOAc and petroleum ether to yield 7.2 g
(73.8~) of Boc-Ala-Ala-OBzl; m.p. 71-i3 C.
Boc-Ala-Ala-OBzl (6.0g, 17.15 mmol) was treated with
380 ml of freshly prepared 4 N HCl in THF for 30 minutes. The
excess acid and solvent were evaporated off and the remaining
syrup was re-evaporated twice with fresh THF. The residue soli-
dified immediately when treated with ether. It was recrystalli-
zed from MeOH and ether to yield 4.30 g (76.4%) of HCl.H-Ala-
Ala-OBzl; [a]D5=-38.86 (c = 1, MeOH).
: .,
HCl~H-Ala-Ala-OBzl (4.25 g, 14.83 mmol) was dissolved
ln DMF (60 ml), cooled to 0C, and stirred with Boc-Asn-OH
(3.45 g, 14.83 mmol), HOBT (4.02 g, 28.6 mmol), NMM (2 ml) and
DCC (3.37 g, 16.35 mmol)at 0C for 2 hours and then at 25C for
20 hours. More NMM was added from time to time in order to
maintain the reaction slightly basic. The insoluble by-products
formed we~e filtered off and the filtrate ~as eva~orated to dryness.
~,
The oily residue solidi~ied in~nediately when treated with water.
It was taken up in EtOAc, washed with H20 (3 x), ~ried
over Na2S04 and evaporated to a smaller volume when the pro-
duct began to crystallize. Equal volume of petroleum ether
.
was added and the mixture was left to stand overnight. The
crude product was recrystallized from THF and petroleum ether
to y eld 5.2 g ~75.6%) of Boc-Asn-Ala-Ala-OBzl; m.p. 153-155C;
[cl]D = -55.61 (c -- 1, MeOH).
'~
.
- 40 ~
Boc-Asn-Ala-Ala-OBzl (2.2 g, 4.75 mmol) was treated with
- 220 ml of 4.0 N HCl in THF for 30 minutes. The excess acid and
solvent were evaporated off and the residue re-evaporated twice
with fresh THF. The oily product solidified when treated with
ether. It was triturated with more fresh ether and collected to
give a white powder (1.85 g). The hydrochloride salt thus obtai-
ned was dissolved in 40 ml D~ and chilled to 0C when H3C-CO-Ser-
Ser(Bzl)-OH. DCHA (1.97 ~, ~.75 mmol) was added.~fter stirring at
0C for 30 minutes, precipitation of DCHA~HCl was observed and
HOBT (1.19 g) was added followed by DCC `(1.08 g, 5.23 mmol).
The reaction was then adjusted to pH 7.5 with a few drops of
NMM and stixred at 0C for 2 hours and then at 25C overnight.
The insoluble by-product was removed by filtration and the fil-
trate evaporated to a slightly colored solid mass. It was was-
hed thoroughly with H20 and EtOAc to give a buff colored powder
which was crystallized from DMF (40 ml) and iso-propanol (500
ml). ~ield: 1.57 ~ (56.9%) of H3C-CO-Ser(Bæl)-~sn-Ala-~la-ORzl;
m.p. 213-215C; ~a]D5=-23.11 (c = 1, DMSO).
H3C-CO-Ser~Bzl)-Asn-Ala-Ala-OBzl (1.57 g, 2.69 mmol) was
dissolved in 20 ml DMF and stirred with 2 ml cf H2NNH2 for 18
hours. The solid product formed was collected and washed tho-
roughly with DMF, EtOH, and ether to yield 1.22 ~ (89.6~)
of H3C-CO-Ser(Bzl)-~sn-Ala-Ala-HNNH2; m.p. 262-26~C; [~]D =-26.7
(c = 1, D~1SO).
Boc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-
~ ~ .
. . .
~' .
- 41 - ~3~B
Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH (0.698 g, 0.358 mmol) was
treated with 10 ml of TFA for 30 minutes and the peptide salt
was precipitated with ether. It was collected on a suction
filter, washed with ether and aried to give 0.652 g-of material
.
(0.333 mmol as TFA salt). In a separate flask, H3C~X~Ser(Bzl)-Psn-
~la-Ala-HNNH2 (0.17 g, 0.335 mmol) was suspended in 7 ml DMF and
treated with 0.27 ml of 6.18 N HCl in THF at -20C. To the mix-
ture, 0.68 ml of 10% i-amvlnitrite in DMF was added and the so- -
lution stirred at the same temperature for 30 minutes. The tem-
10 perature was lowered to -30C when 0.234 ml of Et3N (1-67 mmoi)
was added followed hv the TFA salt of decapeptide (0.652 q)
prepared above. The ~.ixture was diluted with 3 ml of D~SO
at -20C and adjusted to a slightly basic condition (pH 7.5)
with a few drops of Et3M. It was stirred at -20C
15 for 30 minutes and then at 4 C for 5 days. More DMSO ~5 ml) and
Et3N were added during this pariod of time to maintain the
slightly basic conditlons and to keep the reaction from bè-
coming a gel. The entire solution was then poured into 5%
HOAc (250 ml) to give a white precipitate which was collected,
washed with H20,MeOH, ether, and dried. The crude product
(0.702 g; m.p. 290-291C) was reprecipitated from DMSO with
MeOH. Yield: 0.348 g ~42.0%) of H3C-CO-Ser(Bzlj-~sn-~la-~la-~7al-
Asp~OBzl)-Thr~Bzl)-Ser(Bzl)-Ser~Bzl~-Glu(OBzl)-Ile-Thr(Bzl)-Thr
~Bzl)-Lys(Z)-OH; m.p. 296-298 C (dec.); [a]D = ~3.77 ~c
DMSO).
''
The product obtained (0.866 g, O.373 mmol) was stirre~d
- ` ,
'' ' ' '' ~ .' . ' ` ''
- 42
~`
with ~OBT.H20 (0.126 g, 0.82 mmol) in a mixture of DMSO-
(8 ml) and D~ (6 ml) and chilled in an ice-bath. The mix-
ture was treated with DCC (0.085 g, 0.411 mmol) and then
stirred at 0 for 24 hours. It was then-mixed with theTFA salt of
the tetradecapeptide prepared as in Example lB.d)(1.05 g,
0.373 mmol) and 2 ml more of DMSO. A few drops of NMM were
added to bring the pH to 7.5-8.0 and the stirring was continued
at 0C for 1 hour and then at~25.~C~for 5 days. Work up as
described in Example lB.d) gave 1.5775 g of the fully protec-
ted octacosapeptide H3C-CQ-Ser(~zl)-Asn-Ala-Ala-~al-~.sp(OBzl)-
Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-
Lys(Z)-Asp(OBzl)-Leu-Lys(Z)-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-
Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl.
The fully protected octacosapeptide (1~57 g~ 0~32
lS mmol) was dissolved in 10 ml of TFA which contained 3 ml of
anisole. The mixture was stirxed with 45 ml of HF at 0C for 30
minutes and then worked up as described in Example lB.d). Puri-
fication on Sephade ~ G-10 and DEAE-Sephadex~ columns as des-
cribed above gave 0.283 g of H3C-C~-Ser-Asn-Ala-~la-Val-Asn-Thr-
Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-
Val-Glu-Glu-Ala-Glu-Asn [(Asn )-thymosin 1] as white amor-
phous product. The compound migrated at the position slightly
less acidic than natural thymosin ~1 on acrylamide gel isoelec-
tri~ focusing In agreement with dlffe~ence- in the structur-.
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