Note: Descriptions are shown in the official language in which they were submitted.
133~631
Process and Intermediates for GRF Peptide
Field of the Invention
This invention relates to an improved process for
preparing an active fragment of human growth hormone
releasing factor (hGRF). More specifically, this
invention relates to an efficient process for
preparing the amidated fragment of hGRF containing
the first 29 amino acids of the N-terminal portion
thereof, and to intermediates for use in the
process.
Background of the Invention
hGRF is a linear peptide of 44 amino acids, having an
amidated C-terminus. Structure activity studies have
shown that amino acids can be deleted for the
C-terminal portion of hGRF without loss of intrinsic
activity; see, for example. N. Ling et al., Biochem.
Biophys. Res. Commun., 123, 854 (1984). Such studies
have led to the conclusion that the amidated fragment
containing the first 29 amino acids of the N-terminal
portion, which is the product of the process of this
invention, is a preferred active fragment since it
retains much of the in vitro and in vivo activity of
hGRF. This fragment, which according to convention
is designated as hGRF(1-29)NH2 has the following
structure:
H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-
Yal-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-
29
Ile-Met-Ser-Arg-NH
_~ 2
1339~31
The importance and potential use of growth hormone
releasing factors, and their active fragments
(including human as well as those from other
species), have been well documented; for example, see
F.X. Coude et al., Trends in Biotechnology, 2, 83
(1984), and A.M. Felix et al., Annu. Rep. Med.
Chem., 20, 185 (1985). These peptides stimulate the
release of growth hormone (GH). Thus the peptides
are indicated for treating GH deficiencies and for
augmenting the desirable effects of GH. More
explicitly, the peptides, including hGRF(1-29)NH2, or
their therapeutically acceptable salts, are useful
for treating growth related disorders due to
insufficient production of endogeneous GH in animals,
for example prepubertal growth hormone deficiency in
humans; for healing wounds; for improving milk
production in dairy herds, such as cows and goats;
for improving the quality of meat in meat-producing
animals (i.e. increasing the ratio of meat to fat);
for increasing wool growth; and for improving feed
efficiency in meat-producing animals and dairy cows.
The peptides also can be used diagnostically to
evaluate pituitary function.
Accordingly, there is a need for an efficient process
to prepare hGRF(1-29)NH2.
A number of preparations of hGRF(1-29)NH2 has been
reported; see, for example, Felix et al., supra. The
previously reported syntheses can be classified
either as solid phase synthesis or as solution phase
synthesis. A typical example of solid phase
synthesis of hGRF(1-29)NH2, wherein amino acids are
coupled serially to a solid support is described by
Ling et al., supra. A typical example of solution
synthesis, wherein peptide fragments are coupled in
1~39~3 L
solution is described by K. Ono et al., European
patent application 193,910, published September 10,
1986 . Sol id phase synthesis of hGRF(1-29) NH2 is
practical only for preparing experimental quantities
of the peptide. Processes based on the solution
methods for preparing GRF, or its active analogs, see
Ono et al., supra, or J. Diaz et al., US patent
4,707,541, November 17, 1987, are more amenable to
large scale production; however, they require
numerous operati ons and use l arge amounts of
solvents.
The present process has the features of being simple,
rapid and avoids the use of obnoxious chemicals. It
efficiently and economically produces hGRF(1-29)NH2
on a commercial scale and with a purity of greater
than 98%. By the particular choice of reaction
conditions and highly pure intermediate fragments,
the process yields the desired peptide free of
significant racemization and troublesome by-products.
Summary of the Invention
The process of this invention is directed to the
preparation of hGRF(1-29)NH2, represented by formula
1 :
H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-
Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-
Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-NH2
The process comprises:
(a) stepwise coupling the required amino acid
residues to the amino acid-resin of formula H-Gly-S-P
4 133~31
wherein S is a photosensitive spacer and P is a resin
to obtain the HGRF(10-15)-resin of formula 2
X-Tyr(W1)-Arg(W2)-Lys(W3)-Val-Leu-Gly-S-P 2
wherein X is an a-amino protective group, preferably
t-butyloxycarbonyl, W1 is a protective group for the
hydroxyl of Tyr, preferably benzyl or 2,6-dichloro-
benzyl, W is a protective group for the guanidino
group of Arg, preferably tosyl or nitro, W3is a
protective group for the ~-amino group of Lys,
preferably 2-chlorobenzyloxycarbonyl or tosyl, and S
and P are as described above; and cleaving the
hGRF(10-15)-resin by photolysis to obtain the
corresponding hGRF(10-15)0H fragment of formula 3
1 2 3
X-Tyr(W )-Arg(W )-Lys(W )-Val-Leu-Gly-OH 3
wherein X, W1, w2 and W3 are as described above;
(b) stepwise coupling the required amino acid
residues to the amino acid-resin of formula
H-Leu-S-P wherein S and P are as defined above to
obtain the hGRF(16-22)-resin of formula 4
X-Gln-Leu-Ser(W4)-Ala-Arg(W2)-Lys(W3)-Leu-S-P 4
wherein X is an ~-amino protective group, W2, W3, S
and P are as defined above, and W4 is a protective
group for the hydroxyl of Ser, preferably benzyl; and
cleaving the hGRF(16-22)-resin by photolysis to
obtain the corresponding hGRF(16-22)0H fragment of
formula 5
X-Gln-Leu-Ser(W4)-Ala-Arg(W2)-Lys(W3)-Leu-OH 5
in which X is an ~-amino protective group and W2, W3,
1339~31
and W are as defined above;
(c) coupling stepwise a hGRF(23-29)-resin of formula
H-Leu-Gln-Asp(W5)-Ile-Met-Ser(W4)-Arg(W2)-Q 6
wherei n w2 and W4 are protecti ve groups as defined
above, W is a protective group for the ~-carboxyl of
Asp, preferably benzyl, 2,6-dichlorobenzyl or
cyclohexyl, and Q is a benzhydrylamine type resin,
with the previously noted hGRF(16-22)0H fragment of
formula 5 and the hGRF(10-15)0H fragment of formula
3, to obtain a hGRF(10-29)-resin of formula 7
X-Tyr(W1)-Arg(W2)-Lys (W3)-Val-Leu-Gly-Gln-
Leu-Ser (W4)-Ala-Arg(W2)-Lys (W3)-Leu-Leu-Gln- 7
Asp(W5)-Ile-Met-Ser (W4)-Arg(W2)-Q
wherein X is an a-amino protective group and W1, W2,
W3, W4, W5 and Q are as defined above;
(d) sel ectively removing the a-amino protective group
of the hGRF-(10-29)-resin to obtain the corresponding
hGRF(10-29)-resin of formula 7 wherein X is
hydrogen;
(e) coupling stepwise the last-named hGRF(10-29)-res-
in with the required amino acid residues to obtain
t h e hGRF(1-29)-resin of formula 8
X-Tyr(Wl)-Ala-Asp(W5)-Ala-Ile-Phe-Thr(W6)-
Asn-Ser (W4)-Tyr-(W1)-Arg(W2)-Lys (W3)-Val-Leu-
Gly-Gln-Leu-Ser(W4)-Ala-Arg(W2)-Lys(W3)-Leu- 8
Leu-Gln-Asp(W5)-Ile-Met-Ser (W4)-Arg(W2)-Q
wherein X is an a-amino protective group, W1, W2, W3,
W4, W5 and Q are as def i ned above, and W6 is a
1339~31
protective group for the hydroxyl of Thr, preferably
benzyl; and
f) deprotecting the hGRF(1-29)-resin of formula 8 to
obtain hGRF(1-29)NH2.
The hGRF(10-15)-resin of formula 2 and the hGRF-
(16-22)-resin of formula 4 also are included within
the scope of this invention.
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
carboxyl 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 abbrevi ati ons used herei n 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,
Gln, Ala, Gly, Ile, Arg, Asp, Phe, Ser, Leu, Asn,
Thr, Lys, Val, Met and Tyr represent the "residues"
of L-glutamine, L-alanine, glycine, L-isol eucine,
L-arginine, L-aspartic acid, L-phenylalanine,
L-serine, L-leucine, L-asparagine, L-threonine,
L-lysine, L-valine, L-methionine and L-tyrosine,
respectively.
The term "photosensitive spacer" or "photolabile
spacer", designated by the symbol "S", as used
herein, means a divalent organic linking unit which,
7 1339631
when incorporated into the 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 exampl es of such spacers, see D.H Rich
and S.K. Gurwara, Canadian patent 1,108,348,
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 Q-S-P wherein Q is bromo,
chloro or iodo, and S and P are as defined herein.
Preferred spacers are represented by the formulae
-CH(CH3)CO~OCH2CO- and
-CH(CH3)CO~CH 2CO-,
when the resin is one of the benzhydrylamine type,
and -CH(CH3)CO- when the resin is one of the
styrene-divinylbenzene type.
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.
8 1~3~31
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 hGRF(1-29)resin of
formula 8. Another common feature is the protection
of the ~-amino group of an amino acid while the free
carboxyl group of that reactant is coupled with the
free ~-amino group of the second reactant; the
~-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.
Still another feature involves the preparation by
SPPS of protected peptide-resins, i.e. hGRF(10-15)-
resin of formula 2 and the hGRF(16-22)resin of
formula 4, from which the resin can be cleaved by
photolysis to give corresponding ~-amino protected
fragments with a free C-terminal carboxyl. These
protected fragments, i.e. the fragments of formulae 3
and 5, are generated in a form suitable for suc-
cessive coupling by SPPS methodology to the
hGRF(23-29)-resin of formula 6. This manner of
generation of the individual peptide fragments
enables one to purify important intermediate products
before coupling, thus decreasing the chances of
carrying undesirable impurities through to the final
product.
Two types of photochemical resins are employed to
generate the protected fragments. In one case, the
commercially available copoly(styrene-divinylbenzene)
resin is reacted with 2-chloropropionyl chloride in
the presence of aluminum chloride under Friedel-
Crafts conditions to obtain the photolabile resin
1:~39S31
2-chloropropionyl copoly(styrene-divinylbenzene). In
another case, the commercially available benzhydryl-
amine (BHA) resin or 4-methylbenzhydrylamine resin
was modified by attaching a photosensitive spacer
thereto. Preferred spacers have been noted
previously. A preferred photochemical resin of the
second type is 4-(2-chloropropionyl)phenoxyacetyl BHA
resin. The actual choice of one type of resin is
based on the optimized preparation of the various
fragments on respective resins.
To initate the preparation of the fragments of
formulae 3 and 5, 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-glycine, is coupled
to a solid support of formula Q-S-P wherein Q is
bromo, chloro or iodo and S and P are as defined
herein in the presence of potassium fluoride or
cesium chloride to give the corresponding soid
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 starting material with a
free amino group. Thus, the later amino acid-resin
serves as the solid component to elaborate the
desired fragment-resin by SPPS.
In practice, the desired fragments of formulae 3 and
5 are prepared by stepwise coupling in the desired
order the appropriate a-amino protected amino acids
to the growing peptide-resin using a modified form of
solid phase synthesis. (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
1339631
residues is achieved by using dicyclohexylcarbodii-
mide (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 agent is
benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate (BOP), described by B. Castro et
al., Tetrahedron Letters, 14, 1219 (1975). Subse-
quent coupling of the fragments is achieved using di-
cyclohexylcarbodiimide, dicyclohexylcarbodiimide/-
1-hydroxybenzotriazole or BOP. Each ~-amino
protected amino acid or protected fragment is
introduced into the reaction system in a relatively
low excess (two 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. Biochem., 34, 595 (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 effect deprotection.
The cleavage of the protected peptide-resins of
formulae 2 and 4 is achieved by photolysis. The
photolysis is accomplished by dissolving or suspend-
ing the protected peptide-resin in a photolytically
stable liquid medium; for example, dioxane, dimeth-
ylformamide, methanol, ethanol or N-methylpyrroli-
dine; purging the solution or suspension of the pept-
ide-resin with argon or nitrogen to remove any dis-
solved 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 fragments hGRF(10-15) of formula 3 and
hGRF(16-22)0H of formula 5 are obtained with a high
degree of purity. Noteworthy at this point is the
fact that the aforementioned procedure, based on the
- B
11 1339~31
use of photosensitive resins, yields highly pure
(295% pure) fragments having a protected N-terminal
amine and a free C-terminal carboxyl, rendering them
as ideal intermediates for eventual preparation of
high quality hGRF(l-Z9)NH2.
The two fragments of formulae 3 and 5 are now coupled
successively and in proper order with a hGRF(23-29)-
resin to yield the hGRF(10-29)-resin of formula 7.
Standard SPPS techniques are applied both for the
preparation of the hGRF(23-29)-resin and the
subsequent coupling of the two fragments to the
hGRF(23-29)-resin. BHA resin serves as a very
practical resin for the preparation of the hGRF-
(23-29)-resin. The conditions, which were described
previously for achieving in practice the coupling of
the amino acid residues for the preparation of the
fragments of formulae 3 and 5, apply likewise to the
preparation of the hGRF(23-29)-resin and its
subsequent coupling with the fragments to give the
hGRF(10-29)-resin.
Thereafter, the latter peptide-resin is coupled
stepwise and in the order of the amino acid sequence
of hGRF with the remaining amino acid residues, using
the coupling conditions described hereinbefore, to
yield hGRF(1-29)-resin.
Subsequent deprotection of the latter peptide resin
yields the desired hGRF(1-29)NH2. The deprotection
is readily achieved with hydrogen fluoride which
simultaneously removes the side chain protecting
groups and cleaves the peptide residue form the
resin.
The following examples illustrate further this
invention. Abbreviations used in the examples
12 1~39~31
...
include Boc: t-butyloxycarbonyl; TFA: trifluoroace-
ti c aci d; CH2Cl 2: methyl ene chl oride; DIEA:
diisopropylethylamine; DMF: dimethylformamide; EtOH:
ethanol; DCC: N,N'-dicyclohexylcarbodiimide; HOBT:
1-hydroxybenzotri azol e; and MeOH: methanol .
Sol uti on percentages are cal cul ated on a vol ume/vol -
ume basis unless stated otherwise. Temperatures
refer to the centigrade scale. The following terms
are trademarks : Pyrex , Sep Tech, and Vydac .
1339631
Example 1
RESINS:
a) Preparation of 2-chloropropionyl
copoly(styrene-1% divinylbenzene) resin
2-Chloropropionyl chloride (140ml, 183.12 9, 1.44
moles) was added to a suspension of aluminum chloride
(230.0 y, 1.72 moles) in 1,2-dichloroethane (1.6
liters). The mixture was stirred until complete
solution occurred. The solution was then added over
a 5 min period to a mechanically stirred suspension
of copoly(styrene-1% divinylbenzene) resin (200-400
mesh; 1 Kg). The reaction mixture was allowed to
stir at room temperature (20-22~) for 4h. The resin
was filtered, washed sequentially with MeOH (3x),
CH2Cl2 (3x), and EtOH (3x), and then dried to
constant weight in a vacuum oven. The dried material
(1.20 Kg) was ready for use.
b) Preparation of 4-(2-chloropropionyl)phenoxyacetyl
BHA-resin
4-(2-Chloropropionyl)phenoxyacetic acid (8.35 9, 34.5
mmoles) and HOBT (4.66 9, 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 (sty-
rene-1% divinylbenzene) resin (200-400 mesh, 50.0 9,
amine content = 0.46 mmole/g) was generated with DIEA
14 133g~31
in CH2Cl2. The resulting resin was stirred in CH2Cl2
(9OOml). The above noted mixture of activated acid
was added in one portion to the stirred resin. The
resulting mixture was stirred for 20h at room temper-
ature. 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 2-(Boc-glycyl)propionyl copoly-
(sty rene-1% divinylbenzene)
2-Chloropropionyl copoly(styrene-1% divinylbenzene)
resin of Example la (20-400 mesh, 100g) was stirred
in DMF at 60~ for lh. Boc-Gly-OH (56.09, 320 mmoles)
and anhydrous potassium fluoride (46.00 g, 70 mmoles)
were added in portions to the mixture. The resultant
mixture was stirred at 60~ for 24 h. The resin was
collected by filtration and washed three times each
with DMF, DMF-water, water, DMF-water, EtOH, CH2Cl2
and EtOH. The resin was dried in a vacuum oven to
yield 115.22 g of the title compound.
Titration with picric acid according to the method of
B.F. Gisin, Anal. Chem. Acta, 58, 248 (1972) indicat-
ed an amine content of 0.79 mmole/g resin for
glycine.
1339631
b) Preparation of 2-(Boc-leucyl)propionyl copoly(sty-
rene-1% divinylbenzene)
By following the procedure of section (a) of this
example but replacing Boc-Gly-OH with an equivalent
amount of Boc-Leu-OH, 2-(Boc-leucyl)propionyl
copoly(styrene-1% divinylbenzene) was obtained.
c) Preparation of 4-[2-(Boc-glycyl)propionyl]phe-
noxyacetyl BHA-resin
4-[2-Chloropropionyl)phenoxyacetyl]-BHA resin of Ex-
ample lb (15.0 g, 0.32 mmole/g resin was stirred in
dry DMF (160 ml) for lh to allow the resin to swell;
then potassium fluoride (2.53 g, 43.2 mmoles) and
Boc-Gly-OH (3.36 g, 19.2 mmoles) were added in por-
tions to the mixture. The reaction mixture was
stirred for 24h at 70~ and then filtered. The col-
lected resin was washed three times each with DMF,
DMF-H20, H20, dioxane-H20, dioxane, MeOH, CH2C12 and
EtOH. The resin was dried to constant weight in a
vacuum oven to afford free-flowing granules (14.90
9 ) -
Titration with the picric acid indicated an aminocontent of 0.28 mmoles/g for glycine.
16
1339~31
Example 3
PROTECTED FRAGMENTS:
a) Preparation of hGRH(16-22)-resin fragment,
2-(Boc-Gln-Leu-Ser(Bzl)-Ala-Arg(Tos)-Lys(2-ClZ)-
Leu)propionyl copoly(styrene-1% divinylbenzene),
and its subsequent cleavage to the corresponding
C-terminal carboxyl fragment hGRF(16-22)OH
The Boc-amino acid resin of Example 2b (618 9, amine
content: 0.805 mmole/g) was used to form the hGRF-
(16-22)-resin fragment by a modification of the solid
phase technique 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 (Lys, Arg, Ser, Gln)
or by the symmetrical anhydride method (Ala, Leu).
The DCC-HOBT method comprised adding DCC (2 equiv.)
in CH2Cl2 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 peptidyl resin in
CH2Cl2. 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. The coupling cycle consis-
ted of i) deprotection with 30% TFA in CH2Cl2 (twice
for 5 min, once for 25 min) (ii) neutralization with
5% DIEA in CH2Cl2 (twice for 3 min) and (iii) coupl-
ing by addition of symmetrical anhydride or activated
ester corresponding to the selected Boc-amino acid.
Intermediate washes were done successively with
17 1~396~1
CH2Cl2, 50~ isopropanol in CH2Cl2, isopropanol and
CH2Cl2. The coupling reactions were monitored by the
Kaiser tests and the fluorescamin test, see B.F.
Gisin, supra. The time required to complete the
coupling reaction ranged from 4 to 24h . The final
product was washed with DMF, CH2Cl2' isopropanol,
CH2Cl 2 and EtOH, and then dried under vacuum to give
1217.4 g of the hGRF(16-22)-resin (100% yield, 96%
pure by HPLC).
The latter peptidyl resin was subjected to photolysis
as follows: The peptidylresin (300g) 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 70h. The suspension
was filtered. The filtrate was concentrated to
dryness under reduced pressure at room temperature.
The residual oil was triturated with anhydrous Et20
to give a white solid. The solid was collected,
washed with anhydrous Et20 and dried under vacuum
over P205 to give 185 g of the corresponding pro-
tected N-terminal, free C-terminal carboxyl segment,
hGRF (16-22)OH, (100% yield, 95.4% pure by HPLC).
b) Preparation of hGRF(10-15)-resin fragment,
4-[2-(Boc-Tyr(2,6-diClBzl)-Arg(Tos)-Lys(2-ClZ)-Val-
Leu-Gly)propionyl]phenoxyacetyl BHA resin, and its
subsequent cleavage to the corresponding C-terminal
carboxyl fragment hGRF(10-15)OH
The hGRF(10-15)-resin was assembled with the
appropriate Boc-amino acids in the same manner as
described for the preceding peptidylresin, using the
symmetrical anhydride method (Leu ,Val ) and the
DCC-HOBT method (Lys, Arg,Tyr) and the Boc-Amino acid
~ -~ ~ .
18
1~9~31
resin of Example 2c (574 g, amine content: 0.45
mmole/g) as a starting material. Thus, the
hGRF(10-15)-resin (933 g) was obtained (100% yield).
The latter peptidylresin (466 g) was subjected to
photolysis in the manner described for the preceding
peptidylresin of Example 3a to give 161 g of the cor-
responding protected N-terminal, free C-terminal
carboxyl segment, hGRF(10-15)0H (87% yield, 97% pure
by HPLC).
In the same manner but replacing the Boc-amino acid
resin of Example 2c with an equivalent amount of
2-(Boc-glycyl)propionyl copoly(styrene-1% divinylben-
zene) of Example 2a, h-GRF(10-15)0H also was
obtained.
Example 4
Preparation of protected hGRF(1-29)-resin
By following the solid phase technique described in
Example 3, the fragment hGRF(23-29) was assembled on
a BHA resin. The appropriate Boc-amino acids were
coupled by the DCC-HOBT method (Arg, Ser, Gln) or by
the symmetrical anhydride method (Met, Ile, Asp,
Leu). The t-butylcarbocation scavenger, DL-methion-
ine (1% by volume of the scavanger in 30% TFA-CH2Cl2
solution), was used during Boc-deprotection following
the coupling of Met, Ile, Asp and Gln, cf. D.
Le-Nguyen et al., J. Chem. Soc. Perkin Trans., 1,
1915 (1987). On completion of coupling the Leu
residue to the growing peptide-resin, the resultant
h G R F ( 2 3 - 2 9 ) - r e s i n , i . e .
Boc-Leu-Gln-Asp(Chxl)-Ile-Met-Ser(Bzl)-Arg(Tos)-BHA,
was coupled serially (DCC-HOBT method) with the pro-
1 9 1 ~ 3 '~
tected fragments of hGRF(16-22)OH and hGRF(10-15)OH
of Examples 3a and 3b, respectively. (For the depro-
tection steps prior to the latter two couplings, and
for all subsequent deprotection steps of this examp-
le, DL-methionine was used as a scavenger.) There-
after, the resultant hGRF(10-29)-resin was coupled
stepwise with the appropriate Boc-amino acids, using
the DCC-HOBT method for coupling Ser, Asn and Tyr,
and the symmetrical anhydride method for Thr, Phe,
Ile, Ala, Met and Asp. Following a final deprotect-
ion (30% TFA in CH2Cl2 plus 1% by volume of DL-methi-
onine), the protected hGRF(1-29)-resin, i.e. H-Tyr-
(2,6-diClBzl)-Ala-Asp(Chxl)-Ala-Ile-Phe-Thr(Bzl)-
Asn-Ser(Bzl)-Tyr(2,6-diClBzl)-Arg(Tos)-Lys(2-ClZ)-
Val-Leu-Gly-Gln-Leu-Ser(Bzl)-Ala-Arg(Tos)-Lys(2-ClZ)-
Leu-Leu-Gln-Asp(Chxl)-Ile-Met-Ser(Bzl)-Arg(Tos)-BHA
(124.45 g, 91% yield) was obtained.
Example 5
Preparation of hGRF(1-29)NH2
A mixture of the preceding protected hGRF(1-29)-resin
(75.9g), DL-methionine (7.6 g) and anisole (distil-
led, 75ml) was placed under nitrogen (positive pres-
sure). The mixture was cooled at -80~ for 10 min
(dry ice-isopropanol bath). Anhydrous hydrogen
fluoride was distilled slowly into the cooled mixture
over a period of about 70 min. The total amount of
hydrogen fluoride added to the mixture was 750 ml.
The reaction mixture was stirred at 0~ for lh under
nitrogen. Thereafter, a stream of nitrogen was
passed immediately over the reaction vessel at 20-22~
until most of the hydrogen fluoride had evaporated.
The residue was dried under high vacuum for 100 min
1339631
and then extracted with TFA (700 ml). The extract
was concentrated to a small volume. Dilution of the
concentrate with Et20 afforded a solid. The solid
was collected, washed with Et20 and dried under high
vacuum to give 44.7 9 of the crude title compound
(TFA salt).
A portion of the latter crude product (4.0g) was
purified further by HPLC (Sep Tech 1200, 5 x 35 cm;
Vydac ODS, 15-20~ particle size; 300 nm; linear
gradient, 100ml/min, solvent A: 0.06% TFA in water,
solvent B: 0.06% TFA in acetonitrile). The
appropriate fractions were combined. Acetonitrile
was removed under vacuum. The residue was lyophi-
lized to give hGRF(1-29)NH2 (662 mg, 16.5% yield,
98.4% pure as indicated by HPLC).
