Note: Descriptions are shown in the official language in which they were submitted.
2~24~
Process Intermediates for Producinq Glucaqon
Field of the Invention
This invention relates to an improved
process for preparing human glucagon (hereinafter
referred to as "glucagon"), and to intermediates
used in the process.
Backqround of the Invention
Glucagon, a peptide hormone composed of 29
amino acids, is produced in the alpha cells of
islets of Langerhans in the pancreas of a mammal,
including man. The hormone regulates
glycogenolysis and gluconeogenesis. It can act
as a hyperglycemic agent, increasing blood
glucose concentration by activating hepatic
glycogenolysis. Hence, glucagon is indicated for
the treatment of insulin-induced hypoglycemia;
see "Martindale, The Extra Pharmacopocia", 29th
ed., J.E.F. Reynolds, Ed., The Pharmaceutical
Press, London, UK, 1989, p. 1574.
Glucagon has the following structure:
5 10
H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-
15 20
Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-
25 29
Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH
The preparation of glucagon by chemical
synthesis has been r~ported on several occasions.
The first successful synthesis was accomplished
by E. WUnsch and G. Wendlberger, Chem. Ber., 101,
3659 (1968); see also E. WUnsch and
G. Wendlberger, US patent 3,642,763, issued
February 15, 1972. The latter investigators used
2~)2~
the classical method of peptide synthesis
involving the condensation of fragments in
solution (i.e. the solution phase peptide
synthesis). In 1975, the Protein Synthesis Group
of the Shanghai Institute of Biochemistry
reported the preparation of glucagon by a
combination of solution phase and solid phase
methods of synthesis; see Scientia Sinica, 18,
745 (1975) and Chem. Abstr., 86, 90223c (1977).
A solid phase synthesis was first reported by S.
Mojsov and R.B. Merrifield, Biochemistry, 20,
2950 (1981). Other reports of the synthesis of
glucagon include:
J.E. Shields and E.L. Smithwick, US patent
3,887,538, issued June 3, 1975;
M. Fujino et al., Chem. Pharm. Bull., 26,
539 (1978);
B.F. Lundt et al., Res. Discl., 181, 246
(1979), see also Chem. Abstr., 91, 57515e (1979);
M. S. Verlander et al., US patent 4,351,762,
issued September 28, 1982;
S. Mojsov and R.B. Merrifield, Eur. J.
Biochem., 145, 601 (1984); and
J.P. Tam and R.B. Merrifield, US patent
4,507,230, issued March 26, 1985.
Although some of the aforementioned
processes can produce glucagon in a reasonable
yield and purity, a facile and reliable synthesis
for producing glucagon on a commercial scale is
needed.
The present process can be accomplished
simply and rapidly to produce large quantities of
glucagon. By the particular choice of starting
materials, intermediates and reaction conditions,
the present process produces glucagon
~ ~4~ 5 5 3
effectively, free of significant racemization
and troublesome byproducts, and with a purity
of greater than 98%. The process is
distinguished from the aforementioned prior art
processes by the serial coupling of two key
fragments to a glucagon(23-29) fragment
attached to a solid support via the side chain
carboxyl of Asp at position 28.
Sllmm~ry of the Invention
The process of this invention for
preparing glucagon comprises:
(a)coupling a glucagon(15-22) fragment of
formula 1
X-Asp(W1)-Ser(W2)-Arg(W3)-Arg(W3)-Ala-Gln-
Asp(W1)-Phe-OH
wherein X is an ~-amino protective group, W1 is
a protective group for the ~-carboxyl of Asp,
w2 is a protective group for the hydroxyl of
Ser and W3 is a protective group for the
guanidino group of Arg, to a glucagon(23-29)-
resin of formula 2
H-Val-Gln-Trp(W4)-Leu-Met(O)-Asp(Q)-Thr(W5)-O-Y
?
wherein W4 is a protective group for the
aromatic nitrogen atom of Trp, W5 is protective
group for the hydroxyl of Thr, Q is a
benzhydrylamine type resin and Y is a carboxyl
protective group, to obtain a glucagon(15-29)-
resin of formula 3
X-Asp(W1)-Ser(W2)-Arg(W3)-Arg(W3)-Ala-Gln-
Asp(W1)-Phe-Val-Gln-Trp(W4)-Leu-Met(O)-Asp(Q)-
Thr(W5)-O-Y 3
~,,,v~
1 ~
20~2 ~5~3
wherein Q, W1 to W5 inclusive, X and Y are as
defined above,
(b)selectively removing the a-amino protective
group of the glucagon~15-29)-resin to obtain the
corresponding glucagon(15-29)-resin of formula 3
wherein X is hydrogen,
(c)coupling a glucagon(7-14) fragment of formula
X-Thr (W5) -Ser(W2)-Asp(W1)-Tyr(W6)-Ser(W2)-Lys (W7) -
Tyr(W )-Leu-OH 4
wherein Wl, W2, W5 and X are as defined herein, W
is a protective group for the hydroxyl of Tyr and
W7 iS a protective group for the ~-amino group of
Lys, with the last-named glucagon(15-29)-resin
to obtain a glucagont7-29)-resin of formula 5
X-Thr(W )-Ser(W)-Asp(W1)-Tyr(W6)-Ser(W2)-Lys (W7)
Tyr(W )-Leu-Asp(W1)-Ser(W2)-Arg (W3) -Arg (W3) -Ala-
Gln-Asp(W)-Phe-Val-Gln-Trp(W)-Leu-Met(O)-Asp(Q)-
Thr (W5 ) -O-Y 5
wherein Q, W1 to W7 inclusive, X and Y are as
defined above,
(d) stepwise coupling the required a-amino
protected amino acids to the glucagon(7-29)-
resin to obtain a glucagon(1-29)-resin of formula
X-His(W )-Ser(W2)-Gln-Gly-Thr (W5) -Phe-Thr(W )-
Ser(W )-Asp(W1)-Tyr(W6)-Ser(W2)-Lys (W7) -Tyr(W6)-
Leu-Asp(W )-Ser(W )-Arg~W )-Arg(W )-Ala-Gln-
Asp(W )-Phe-Val-Gln-Trp(W )-Leu-Met(O)-Asp(Q)-
Thr(W )-O-Y 6
wherein Q, W1 to W7 inclusive, X and Y are as
defined above and w8 is a protective group for
the imidazole ring of His,
2 0 ~ 5
(e)selectively removing the a-amino protective
group of the glucagon(1-29)-resin to obtain the
corresponding glucagon(1-29)-resin of formula 6
wherein X is hydrogen, and
(f)deprotecting the last-named glucagon(1-29)-
resin to obtain glucagon.
Also included within the scope of this
invention are the glucagon(15-22) fragment of
formula 1, the glucagon(23-29)-resin of formula
2, the glucagon(15-29)-resin of formula 3 wherein
X is hydrogen or an a-amino protective group, the
glucagon(7-14) fragment of formula 4, the
glucagon(7-29)-resin of formula 5 and the
glucagon(1-29)-resin of formula 6 wherein X is
hydrogen or an a-amino protective group.
Details of the Invention
The term "residue" with reference to an amino
acid means a radical derived from the
corresponding a-amino acid by eliminating the
hydroxyl of the carboxy group and one hydrogen
of the a-amino group. The term "amino acid
residue" can include radicals derived from side
chain protected amino acids.
In general, the abbreviations used herein for
designating the amino acids and the protective
groups are based on recommendations of the IUPAC-
IUB Commission on Biochemical Nomenclature, see
Biochemistry, 11, 1726-1732 (1972). For
instance, His, Trp, Gln, Ala, Gly, Arg, Asp, Phe,
Ser, Leu, Asn, Thr, Lys, Val, Met, Met(O) and Tyr
represent the "residues" of L-histidine, L-
tryptohane, L-glutamine, L-alanine, glycine, L-
arginine, L-aspartic acid, L-phenylalanine, L-
20~ ~3~
serine, L-leucine, L-asparagine, L-threonine, L-
lysine, L-valine, L-methionine, L-methionine
sulfoxide and L-tyrosine, respectively.
The term "photosensitive spacer" or
"photolabile spacer", designated by the symbol
"S", as used herein in connection with the
preparation of the glucagon(15-22) and
glucagon(7-14) fragments described hereinafter,
when incorporated into a peptide-resin system,
links the first amino acid building block to the
resin by orthogonal covalent bonds; the unit or
spacer being further characterized in that the
bond between the spacer and the first amino acid
residue can be cleaved by photolysis to afford
the peptide (or the first amino acid residue)
with a C-terminal carboxyl. For examples of such
spacers, see D.H. Rich and S.K. Gurwara, Canadian
patent 1,108,348, issued September 1, 1981; J.P.
Tam et al., J. Amer. Chem. Soc., 102, 6117
(1980); F.S. Tjoeng and G.A. Heavner, J. Org.
Chem., 48, 355 (1983); and J. Gauthier, Canadian
patent application, SN 547,394, filed
September 21, 1987. When utilized herein, the
spacer is first attached to the resin to give the
solid support of formula J-S-P wherein J is
bromo, chloro or iodo, and S is a photosensitive
spacer and P is a resin. Preferred spacers are
represented by the formulae
-CH(CH3)CO ~ OCH2CO- and
-CH(CH3)CO ~ CH2CO- .
when the resin is one of the benzhydrylamine
type, and -CH(CH3)CO- when the resin is one of
the styrene divinylbenzene type.
2024~5~
. .
The term "benzhydrylamine type resin", as used
herein, means a benzhydrylamine resin of the type
commonly employed in solid phase peptide
synthesis ~SPPS). Such resins include
benzhydrylamine resin (BHA) and 4-
methylbenzhydrylamine resin.
Turning to the process of this invention, one
feature is the protection of labile side chain
groups of the various amino acid residues with
suitable protective groups to prevent a chemical
reaction from occurring at those sites until
after the completion of the stepwise coupling to
produce the glucagon(1-29)-resin of formula 6.
Preferred protective groups for amino acids
with labile side chain groups are tosyl (Tos) for
His and Arg, benzyl (Bzl) for Ser and Thr,
cyclohexyloxy (OChl), benzyloxy (OBzl) or 2,6-
dichlorobenzyloxy (O-Cl2Bzl) for Asp, 2,6-
dichlorobenzyl for Tyr, 2-chlorobenzyloxycarbonyl
(ClZ) for Lys and formyl (For) for Trp. In a
broader sense, the oxygen of methionine sulfoxide
is a protective group for Met.
Another common feature is the protection of
the a-amino group of an amino acid while the free
carboxyl group of that reactant is coupled with
the free a-amino group of the second reactant;
the a-amino protective group being one that can
be selectively removed to allow the subsequent
coupling step to take place at the amino group
from which the protective group is removed. A
preferred a-amino protective group, which is
represented herein for the disclosed process by
the symbol "X", is t-butyloxycarbonyl (Boc).
2~3~5
A preferred carboxyl protective group is
benzyl.
Turning now to the starting materials for the
process. Two of the starting materials, i.e. the
glucagon(15-22) fragment of formula 1 and the
glucagon(7-14) fragment of formula 4, are
prepared readily in a high state of purity from
corresponding protected peptide-resins from which
the glucagon fragments are cleaved by photolysis.
This manner of generation of these individual
peptide fragments enables one to purify important
intermediates products before further coupling,
thus decreasing the chances of carrying
undesirable impurities through to the final
product.
More specifically, a convenient and practical
photochemical resin for the preparation of
glucagon fragments of formulae 1 and 4 is
obtained by modifying commercially
benzyhydrylamine (BHA) resin or 4-methyl-
benzhydrylamine resin by attaching a photo-
sensitive spacer thereto. Preferred spacers have
been noted previously. A practical and efficient
photochemical resin for the present purpose is 4-
(2-chloropropionyl)phenoxyacetyl BHA resin.
To initate the preparation of the fragments of
formula 1 and 4, a first amino acid is coupled to
the photolabile resin. The preparation of the
amino acid-resin is exemplified as follows: An a-
amino protected amino acid, e.g. Na-Boc-Phe-OH or
Na-Boc-Leu-OH, is coupled to a solid support of
formula J-S-P wherein J is bromo, chloro or iodo
and S and P are as defined herein, e.g. 4-(2-
chloropropionyl)phenoxyacetyl BHA-resin, in the
20~8~
presence of potassium fluoride or cesium chloride
to give the corresponding solid support having an
a-amino protected amino acid linked thereto.
Thereafter, the a-amino protective group of the
latter resin derivative is removed to give the
desired amino acid-resin with a free amino group.
The preceding amino acid-resin, in which the
amino acid residue is either Phe or Leu, is
utilized to prepare respectively a protected
peptide resin in which the peptide portion
corresponds to that of the desired fragments of
formulae 1 and 4. This transformation of the
amino acid resin is accomplished by stepwise
coupling thereto in the desired order the
appropriate a-amino protected amino acids. (For
a recent review of solid phase synthesis, see
J.M. Stewart and J.D. Young, "Solid Phase Peptide
Synthesis", 2nd ed, Pierce Chemical Company,
Rockford, Illinois, USA, 1984.) More explicitly,
the coupling of the amino acid residues is
achieved by using dicyclohexylcarbodiimide
~optionally adding 1-hydroxybenzotriazole, 3-
hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine or
N-hydroxysuccinimide) as the coupling agent, or
by employing the "mixed anhydride" activated form
of the a-amino protected acids. Another useful
coupling agent is benzotriazol-1-yloxytri-
(dimethylamino)phosphonium hexafluorophosphate
(BOP), described by B. Castro et al., Tetrahedron
Letters, 14, 1219 (1975). Each a-amino protected
amino acid or protected fragment is introduced
into the reaction system in a relatively slight
excess (1.5 to 2 molar equivalents). The success
of the coupling reaction at each stage is
monitored by the ninhydrin reaction as described
by E. Kaiser et al., Anal. Bioch., 34, 595
20î48~
(1970). Removal of the a-amino protective group
completes the coupling cycle. In the instance
where the a-amino protective group is a t-
butyloxycarbonyl, trifluoroacetic acid in
methylene chloride is used to remove the
protective group.
The cleavage of the protected peptide-resins
so obtained to give the desired fragments of
formulae 1 and 4 is achieved by photolysis. The
photolysis is accomplished by dissolving or
suspending the protected peptide-resin in a
photolytically stable liquid medium; for example,
dioxane, dimethylformamide, methanol, ethanol or
N-methylpyrrolidine; purging the solution or
suspension of the peptide-resin with argon or
nitrogen to remove any dissolved oxygen; and then
irradiating the suspension or solution with
photolytically effective ultraviolet light. In
practice, irradiation at a wavelength of 350 nm
has been found to be very effective. In this
manner, the glucagon(15-22) fragment of formula
1 and the glucagon(7-14) fragment of formula 4
are obtained with a high degree of purity.
The glucagon(23-29)-resin of formula 2 is
another requisite starting material for the
present process. This peptide-resin serves as
the solid support system for the process. In
contrast to previously reported solid phase
syntheses of glucagon which use a C-terminus
attached resin, the present process uses as a
starting material this particular glucagon(23-
29)-resin in which a benzhydrylamine-type resin
is attached to the side chain carboxyl of Asp at
position 28. The use of this glucagon(23-29)-
resin with its different point of attachment of
2 0 ~
the resin provides the present process with some
very practical advantages. More particularly, it
has been realized that with the use of the
peptide-resin of formula 2, the subsequent
couplings of fragments and amino-acid residues
according to the present process are completed
rapidly and effectively with as little as 1.5
molar equivalents of activated ester or
symmetrical anhydride (no double coupling
required). In practice, the glucagon(23-29)
resin, prepared by SPPS from N-(9-
fluorenylmethyl-oxycarbonyl)-Asp(BHA resin)-
Thr(Bzl)-OBzl, has been found to be a good choice
of starting material.
With reference to the process, the two
fragments of formula 1 and 4 are coupled
successively and in proper order with the
glucagon(23-29)-resin of formula 2, via the
glucagon(15-29)-resin of formula 3, to yield the
glucagon(7-29)-resin of formula 5. The above
noted coupling techniques are employed
effectively and smoothly.
Thereafter, the glucagon(7-29)-resin of
formula 5 is coupled stepwise, and in the order
of the amino acid sequence of glucagon, with the
remaining amino acid residues, using the coupling
conditions described hereinbefore, to yield the
glucagon(1-29)-resin of formula 6 in which X is
hydrogen.
Subsequent deprotection of the latter peptide
resin, followed by standard purification
techniques, yields pure glucagon. The
deprotection can be achieved with hydrogen
fluoride, preferably in the presence of p-cresol
2~24~5~
and p-thiocresol in dimethyl sulfide, which
simultaneously removes the side chain protective
groups and cleaves the peptide residue from the
resin. An efficient method of deprotection
(using HF) of the latter peptide resin is the low
high procedure described by S. Mojsov and R.B.
Merrifield, Eur. J. Biochem., 145, 601 (1984).
The following examples illustrate further this
invention. Abbreviations used in the examples
include Boc: t-butyloxycarbonyli But:t-butyl;
CH2Cl2: methylene chloride; DCC: N,N/-dicyclo-
hexylcarbodiimide; DIEA: diisopropylethylamine;
DMF: dimethylformamide; Et2O:diethyl ether;
EtOAc: ethyl acetate; EtOH: ethanol; Fmoc: 9-
fluorenylmethyloxycarbonyl; HOBT: 1-
hydroxybenzotriazole; MeOH: methanol and TFA:
trifluoroacetic acid. Solution percentages are
calculated on a volume/volume basis unless stated
otherwise. Temperatures refer to the centigrade
scale. The following terms are trademarks: Pyrex
and Vydac.
Example 1
Preparation of4-(2-chloropropionyl)phenoxyacetyl
BHA-resin
4-(2-Chloropropionyl)phenoxyacetic acid
(8.35 g, 34.5 mmoles) and HOBT (4.66 g,
34.5 mmoles) were dissolved separately in DMF
(2 x 40 ml). The two solutions were mixed and
the resulting mixture was cooled at 0 ~ for 20
min. A solution of DCC in CH2Cl2 (27.5 ml,
1.256 mmoles/ml) was added to the solution. The
~ mixture of activated acid was stirred for 30 min.at 0 ~. The free base of benzhydrylamine copoly
(styrene-1% divinylbenzene) resin (200-400 mesh,
202~5
50.0 g, amine content = 0.46 mmole/g) was
generated with DIEA in CH2Cl2. The resulting
resin was stirred in CH2C12 (900 ml). The above
noted mixture of activated acid was added in one
portion to the stirred resin. The resulting
mixture was stirred for 20 h at room temperature.
The resin was collected by filtration, washed
with DMF (3X), MeOH (3X), CH2Cl2 (3X), EtOH (3X)
and finally dried to constant weight in a vacuum
oven to yield 54.3 g of resin. The Kaiser test,
E. Kaiser et al., Anal. Biochem., 34, 595 (1970),
was negative indicating no starting material.
Example 2
BOC-AMINO-ACID RESINS:
a) Preparation of 4-[2-(Boc-phenylalanyl)-
propionyl]phenoxyacetyl BHA-resin
4-[2-Chloropropionyl)phenoxyacetyl]-BHA resin
of Example 1 (80.0 g, amine content: 0.70 mmole/g
of resin) was stirred in dry DMF (1600 ml) for 10
min to allow the resin to swelli then potassium
fluoride (29.23 g) and Boc-Phe-OH (59.4 g) were
added in portions to the mixture. The reaction
mixture was stirred for 24 h at 60 ~ and then
filtered. The collected resin was washed three
times each with DMF, H2O, dioxane, MeOH, CH2C12
and EtOH. The resin was dried to constant weight
in a vacuum oven to afford free-flowing granules
(90.0 g).
Titration with the picric acid indicated an
amino content of 0.47 mmoles/g for phenylglycine.
202485~
14
b) P r e P a r a t i o n o f 4 - [ 2 - ( B o c -
leucyl)propionyl]phenoxyacetyl BHA-resin
The title compound was obtained by following
the procedure of example 2a and replacing Boc-
Phe-OH with Boc-Leu-OH.
Example 3
PROTECTED FRAGMENTS:
a) Preparation of protected qlucaqon(15-22)
fraqment:
The Boc-amino acid resin of Example 2a
(15.0 g, amine content: 0.47 mmole/g) was used to
form the glucagon(15-22) fragment attached to the
resin by a modification of the solid phase
techniques of R.B. Merrifield, J. Amer. Chem.
Soc., 85, 2149 (1963). The selected Boc-amino
acids were added to the growing peptidyl-resin
chain by the DCC-HOBT activated acid method which
comprised adding DCC (2 equiv.) in CH2C12 to a
cold solution of HOBT (2 equiv.) and the selected
Boc-amino acid (2 equiv.) in DMF, stirring the
mixture at 0 ~ for 30 min., and adding the
mixture to a suspension of the growing peptidyl
resin in CH2C12. The coupling protocol consisted
of i) deprotection with 45% TFA in CH2Cl2 (twice
for 5 min, once for 25 min) (ii) neutralization
with 5% DIEA in CH2C12 (twice for 3 min) and (iii)
coupling by the DCC-HOBT or activated ester
method. Intermediate washes were done
successively with CH2C12, 50% isopropanol in
CH2Cl2, isopropanol and CH2Cl2. The coupling
reactions were monitored by the Kaiser test
supra, and the fluorescamine test, see A.M. Felix
and M.H. Jimenez, Anal. Biochem., 52, 377 (1973).
The time required to complete the coupling
2024~
. .
reaction ranged from 4 to 24 h. The final
product was washed with DMF, CH2C12, isopropanol,
CH2Cl2 and EtOH, and then dried under vacuum to
give 25 g (86% yield) of the peptidylresin 4-[2-
(Boc-Asp(OChl)-Ser(Bzl)-Arg(Tos)-Arg(Tos)-Ala-
Gln-Asp(OChl)-Phe)propionyl]phenoxyacetyl BHA-
resin.
The latter peptidylresin was subjected to
photolysis as follows: The peptidylresin (25g)
was suspended in a mixture of DMF (7.5 l) and
EtOH (3.9 l) in a Pyrex vessel. The suspension
was purged with argon. While being subjected to
a continuous stream of argon, the suspension was
stirred and irradiated at 0 ~ at a wavelength of
350 nm for 70 h. The suspension was filtered.
The filtrate was concentrated to dryness under
reduced pressure at room temperature. The
residual oil was triturated with anhydrous Et2O
to give a white solid. The solid was collected,
washed with anhydrous Et2O and dried under vacuum
over P2O5 to give 10 g of the corresponding
protected N-terminal, free C-terminal carboxyl
segment, Boc-Asp(Chl)-Ser(Bzl)-Arg(Tos)-Arg(Tos)-
Ala-Gln-Asp(OChl)-Phe-OH (100% yield, 98.7% pure
by HPLC).
b) Preparation of protected qlucaqon(7-14)
fraqment
The peptidylresin, 4-[2-(Boc-Thr(Bzl)-
Ser(Bzl)-Asp(OChl)-Tyr(Cl2Bzl)-Ser(Bzl)-Lys(ClZ)-
Tyr(Cl2Bzl)-Leu)propionyl]phenoxyacetyl BHA-
resin (28.3 g), was obtained in a 95% yield in
the same manner as described for the previous
peptidylresin using the Boc-amino acid resin of
Example 2b (15g, amine content: 0.49 mmole/g).
2024~
16
Subsequent photolysis of the instant peptidyl
resin (28.3 g) in the same manner afforded 11.0 g
of the corresponding protected N-terminal, free
C-terminal carboxyl segment, Boc-Thr(Bzl)-
Ser(Bzl)-Asp(OChl)-Tyr(Cl2Bzl)-Ser(Bzl)-Lys(ClZ)-
Tyr(Cl2Bzl)-Leu-OH (82% yield, 97.5% pure by
HPLC).
c) Preparation of protected qlucaqon(28-29)
fraqment
i) H-Thr(OBzl)-OBzl: A mixture of H-Thr-OH
(90.5 g), p-toluenesulfonic acid monohydrate
(188 g), benzyl alcohol (760 ml) and toluene
(1520 ml) was heated at reflux temperature (with
azeotropic distillation of H2O) for 19 h. The
reaction mixture was cooled and diluted with
EtOAc (1000 ml). Aqueous Na2CO3 (0.5 N) was added
until the pH of the mixture was 9. The phases
were separated. The organic phase was washed
with fresh aqueous Na2CO3 and water and dried
(MgSO4). The addition of oxalic acid (92 g) in
MeOH (200 ml) to the organic phase gave a
precipitate. After the mixture had been stored
at 4 for 24 h, the precipitate was collected
and washed with hexane and EtOH.
Recrystallization of the precipitate from EtOH
gave H-Thr(Bzl)-OBzl as a hemioxalate salt (40 g,
mp 163-164 ~).
ii) Fmoc-Asp(OBu )-Thr(Bzl)-OBzl: DDC (16.09 g) in
EtOAc (100 ml) was added to a suspension of Fmoc-
Asp(OBut)-OH (32.1 g) and N-hydroxysuccinimide
(9.35 g) in EtOAc at O . The mixture was
stirred at 0 ~ for 1.5 h and then at 20-22 ~ for
3.5 h. H-Thr(Bzl)-OBzl (obtained from 33.4 g, of
its corresponding hemioxalate salt) in EtOAc
20~48~S
17
(100 ml) was added dropwise to the mixture at
0 ~. The resultant mixture was stirred 18 h at
room temperature (20 - 22 ~) and then filtered.
The filtrate was concentrated to dryness. The
oily residue (80 g) was placed on a silica gel
column (25 cm x 9 cm, 650 g of SiOz). The column
was eluted at 2 psi with EtOH-hexane (1:3).
Fraction size was 400 ml. Fractions 6 to 23 were
pooled and concentrated. Addition of hexane to
the concentrate precipitated the title compound
(41.2 g, mp 83-85 ~).
iii) Fmoc-Asp-Thr(Bzl)-OBzl: Fmoc-Asp(Bu )-
Thr(Bzl)-OBzl (200 mg) was added to 25% TFA in
CH2Cl2 (10 ml) at 0~. The stirred mixture was
allowed to come to room temperature over a period
of 0.5 h. The mixture was evaporated to dryness.
the solid was washed with hexane and dried to
give the title compound (150 mg, mp 90-95 ~).
Example 4
Preparation of Glucaqon(1-29)-resin
By following the procedure of Example 3, the
title compound was prepared. Namely, Fmoc-Asp-
Thr(Bzl)-OBzl of Example 3c (1.6 g) was coupled
with BHA resin (2.0 g) by the DCC-HOBT method.
The resulting peptide-resin was subjected to
deprotection (removal of Fmoc) in 45% TFA in
CH2Cl2; followed by coupling the resulting H-Asp-
(BHA resin)-Thr(Bzl)-OBzl with the appropriate
Boc-amino acids by the DCC-HOBT method. The
resulting glucagon(23-29)-resin, i.e. H-Val-Gln-
Trp(For)-Leu-Met(O)-Asp(BHA resin)-Thr(Bzl)-
OBzl, was coupled serially (DCC-HOBT method with
the protected glucagon(15-22) fragment of Example
3a and the protected glucagon(7-14) fragment of
2024~
Example 3b. Thereafter, the resulting protected
glucagon(7-29)-resin was coupled stepwise with
the appropriate Boc-amino acids [DCC-HOBT method,
except for Gly (Boc-Gly-OH) and His (Boc-
His(Tos)-OH) which were coupled by the DCC
symmetrical anhydride method].
The symmetrical anhydride method comprised
adding DCC (2 equiv.) in CH2Cl2 to the selected
Boc-amino acid (4 equiv.) in CH2Cl2 at 0
stirring the mixture at 0 ~ for 30 min, filtering
the mixture and adding the filtrate to a
suspension of the peptidyl resin in CH2Cl2.
Accordingly, the protected glucagon(1-29)-resin,
i.e. H-His(Tos)-Ser(Bzl)-Gln-Gly-Thr(Bzl)-Phe-
Thr(Bzl)-Ser(Bzl)-Asp(OChl)-Tyr(Cl2Bzl)-Ser(Bzl)-
Lys (ClZ) -Tyr (Cl2Bzl)-Leu-Asp (OChl) -Ser (Bzl) -
Arg(Tos)-Arg(Tos)-Ala-Gln-Asp(OChl)-Phe-Val-Gln-
Trp(For)-Leu-Met(O)-Asp(BHA resin)-Thr(Bzl)-OBzl
(7.10 g, 84% yield), was obtained.
Example 5
Preparation of Glucaqon
The preceding protected glucagon(1-29)-resin
(16.3 g) was mixed with p-cresol (6.0 g) and p-
thiocresol (2.0 g) in dimethylsulfide (56 ml).
The mixture was placed under nitrogen and cooled
to -70 ~. Anhydrous hydrogen fluoride (20 ml)
was distilled into the mixture. The mixture was
stirred at 0 ~ for 2 h under nitrogen, and then
concentrated under reduced pressure at 0 ~. The
residue was washed with EtOAc and dried. The
resulting solid was mixed with p-cresol (12 g).
Anhydrous hydrogen fluoride (120 ml) was
distilled into the mixture at -70 ~. Thereafter,
the mixture was stirred at 0 ~ for 45 min. The
2~ 5 ~
mixture was concentrated to dryness under
reduced pressure at 0~. The residue was washed
with EtOAc and then extracted with 5% aqueous
acetic acid. The extract was lyophized to give
s 2.91g (59% yield) of the crude product.
The crude product (1.4 g) was purified by
reversed-phase chromatography on a Pharmacia*
octadecasilyl-silica column (2.5 x 35 cm, C-18,
Vydac*, 10~ particle size) using a gradient of
0.06% TFA in H2O and 0.06% TFA in H2O: -
acetonitrile (1:1). The fractions comprising
the major peptide peak (W detection at 254 nm)
were pooled and freeze dried to give 0.49 g
lS (26% overall yield starting from the BHA resin)
of the title compound with a purity of greater
than 98% as indicated by analytical HPLC. The
hyperglycemic potency of the glucagon so
obtained was evaluated in rabbits according to
procedure described in the British
Pharmacopocia Appendix XIV, A-142 (1980). The
glucagon prepared by the present process was
found to be equipotent to the glucagon
standard.
* Trademark
.
