Note: Descriptions are shown in the official language in which they were submitted.
1 18 1~2
BACKGROUND AND PRIOR ART
Mammalian spermatozoa have been known to be antigenic
for many years. More recently, it has been demonstrated
that mammalian sperm contain an antigenic enzyme, which
is known as the C4 isozyme of lactate dehydrogenase
(LDH-X, LDH-C4). LDH-C~ has been isolated in pure
crystalline form from mouse testes. Goldberg (1972)
J. Biol. Chem. 247:2044-2048. The enzyme has a molecular
weight of 140,000 and is composed of four identical C
subunits. The amino acid sequence and three-dimensional
structure o LDH-C4 has been studied and partially
determined by a number of investigators. See Musick et al
(1976) J. Mol. Biol. 104:659-668; and Wheat et al (1977)
Biochem. & Biophys. Res. Comm., 74, No. 3:1066-1077.
Wheat et al determined the sequence of the essential thiol
peptide from amino acid 159 to 171, and found this to be
nearly identical to essential thiol peptides from other
vertebrate LDH isozymes.
In 1974, Dr. Erwin Goldberg re~iewed the effects of
immunization with LDH-X (LDH-C4) on fertility, and advanced
the possibility that "by using a defined macromolecular
constituent of sperm it becomes possible to elucidate its
primary structure in terms of amino acid sequence, to map
specifically the antigenic determinant(s) responsible for
inducing infertility, and then to construct synthetic
peptides containing these determinants. Possessing the
capability for ~ynthesizing a molecule with such properties,
makes the immunologic~l approach to fertility control
~;
7'12
feasible." Karolinska Symposia on Research Methods in
Reproductive Endocrlnolgy, 7th Symposia: Immunological
Approaches to Fertility Control, Geneva, 1974 202-222.
However, such synthetic antigenic peptides remained a goal
and not achievement, although their theoretical desirability
has been recognized. In 1979, Dr. Erwin Goldberg summarized
the state of the art as follows:
"In conclusion, and on a pxactical basis, L
immunotherapy for birth control requires more than
effectiveness, specificity, reversibility, and
absence of systemic side reaction. Rather large
amounts of the antigen must be available in
unequivocally pure form. This condition probably
cannot be met by a natural product enzyme antigen
from sperm or testes. Rather, contraceptive
technology requires a synthesizable peptide fragment
retaining antigencity and provoking a response
which impairs fertility. Completion of the
structural analysis of LDH-C should allow mapping
_L antigenic determinants an~ synthesis of such
peptides for use in a new contraceptive technology."
"Recent advances in ReProduction and Reaulation of
Fërtility," G.P. Talwar, ediior, Elsevier/North
Holland Biomedical Press ~1979).
SUMMARY OF INVENTION
It has now been discovered that antigenic peptides
can be prepared by synthesizing a linear sequence of 9
to 14 amino acids including the sequence: arginine-methionine-
valine-serine-glycine-glutamine-threonine~arginine-leucine.
A11 of the amino acids used to prepare these peptide
compounds are in their L-form with the exception of
glycine. The arginine is at the N-terminal and the leucine
is at the C terminal, or in the C-terminal end portion.
Althouyh not known with certainty, it is believed that
the foregoiny sequence of nine amino acids corresponds
7 ~ ~ 1
to amino acids 101 to 109 of LDH-C4. This is contrary to
a recently published tentative sequence. Musick et al
(1979) J. Biol~ Chem., 254, No. 16: 7621-7623. The other
compounds respectively are believed to correspond to the
101-lll, 101-112, 101-113, 101-114, and 101-115 se~uence
of LDH-C4, contrary to Musick et al. In the sequence
numbering convention used, there is no amino acid 104.
DESCRIPTION OF IMVENTION
The present invention relates to novel antigenic
linear peptides having chain length from 10 to 14 amino
acids. These peptides all include the N-t~rminal sequence:
N-Arg-Met-Val-Ser-Gly-Gln-Thr-Arg-Leu
More specif:ically the invention generically comprises
the following five peptide compounds.
(P-l) N-Arg-Met-Val-Ser-Gly-Gln-Thr-Arg-Leu-C,
(P-2) N-Arg-Met-Val-Ser-Gly-51n-Thr-Arg-Leu-Asp-C,
(P-3) N-Arg-Met-Val-Ser-Gly-Gln-Thr-Arg-Leu-Asp-Leu-C,
(P-4) N-Arg-Met-Val-Ser-Gly-Gln-Thr-Arg-Leu-Asp-Leu-Leu-C,
(P-5) N-Arg-Met-Val-Ser-Gly-Gln-Thr-Arg-Leu-Asp-Leu-Leu-Gln-C, and
(P-6) N-Arg-Met-Val-Ser-Gly-Gln-Thr-Arg-Leu-Asp-Leu-Leu-Gln-Arg-C,
wherein the letter "N" designates the N-terminal amino
acids, while the letter "C" designates the C-terminal
amino acids. Gly represents glycine, and Arg, Met, Val,
5er, Gln, Thr, Leu, and Asp respectively represent
~ 1~17~2
the L-amino acid forms arginine, methionine, valine, serine,
glutamine, threonine, leucine, and aspartic acid.
The peptide compounds of the present invention P-l,
P-2, P-3, P-4, P-5, and P-6 can be synthesized from their
constituent amino acids. For example, the synthesis can
be carried out by the Merrifield solid phase method, as
described in J.A.C.S. 85:2149-2154 (1963). This solid
phase method for synthesizing sequences of amino acids is
also described in Stewart and Young, Solid Phase Peptide
Synthesis (W. H. Freeman and Co., San Francisco, 1969),
pages 1-4. In this procedure, the C-terminal amino acid,
such as arginine for the P-l compound of this invention
is attached to chloromethylated polystyrenedivinylbenzene
copolymer beads~ Each subsequent amino acid, with suitable
protecting group, is then added sequentially to the
growing chain. For example, as described in the Merrifield
article, the protective group may be a carbobenzoxy group.
By the procedure of coupling, depro ection, and coupling
of the next amino acid, the desired amino acid sequence
and chain length can be produced. As a final step, the
protective group is removed from the N-terminal amino
acid such as from the N-terminal arginine, and then is
cleaved from the resin, using a suitable reagent, such
as trifluoroacetic acid and hydrogen bromide. Since
this synthesis procedure is well known, it is not believed
that it will be necessary to further describe it herein.
The peptide of this invention can be prepared by this
synthesis procedure for use in reducing the fertility
of mammals.
7 ~ ~
To utilize the antigenic peptides o this invention
(P-l to P-6) in the form of fertility reducing vaccines,
the peptide used is conjugated to a carrier molecule,
which is preferably a protein which itself elicits an
antigenic response and which can be safely administered.
For e~ample, the selected peptide can be coupled to
tetanus toxoid for administration by intramuscular injection.
For example, a mixture of l~Mole tetanus toxoid, 60~Mole
antigenic peptide, and 18 millimoles 1-ethyl-3-(3 dimethyl
aminopropyl) carbodiimide hydrochloride reacted in water
(pH 6) for 12 hours at room temperature and 24 hours at
4C. gives a product containing 3.5 moles of peptide/
mole of tetanus toxoid. Excess reactants can be removed
by dialysis or gel filtration. See Pique et al ,
Immunochemistry, 15: 55-60 (1978). Alternatively, the
peptide may be coupled using bisdiazotized benzidine
(Bassiri et al, Endocrinology, 90: 722 (1972)) or
glutaraldehyde.
For intramuscular injection, the coupled peptide
may be suspended in a sterile isotonic sallne solution,
or other conventional vehicle, and, if desired, an
adjuvant may be included. A preferred use of such a
vaccine is for administration to human females. Anti-
bodies will be formed, which will appear in the oviduct
fluids and thereby achieve a significant reduction in
fertility. For this purpose, the amount to be administered
will range from about 1 to 10 milligrams (mg) of the
antigenic peptide.
17~2
The peptide compounds of this inventlon and their
method of preparation are further illustrated by the
following examples.
EXAMPLE I
Preparation of Linear Peptide
Axg-Met-Val-Ser-Gly-Gln-Thr-
Ar~-Leu-Asp~l~eu-Leu-Gln-Arg
Synthesis of the above peptide (P-6) can be carried
out employing solid phase techniques now well known in
the art. In a preferred procedure amino protected
arginine, representing the -COOH terminal group of the
above peptide, is coupled to a conventional solid phase
peptide synthesis resin such as chloromethyl polystyrene
cross-linked with 1 to 2% divinyl benzene. The amino
protPcting group is then selectively removed utilizing
a suitable xeagent whose nature will depend on the protecting
group used. In the preferred embodiment the t-butyloxycarbonyl
(Boc) group is utilized for amino group protection and
40~ trifluoroacetic acid in methylene chloride is the
selective deprotecting agent.
After deprotection, the arginine resin is treated
with protected glutamine, preferably N-Boc-Glutamine, and
dicyclohexylcarbodiimide in a malmer known per se as to
form a peptide bond between the free amino group of the
arginine residue and the carboxyl group of protected
glutamine.
1 1~17~
The cycle of deprotection and coupling with amino
acid derivatives and dicyclohexylcarbodiimide is then
repeated with the remaining amino acids in the sequence
order of the above peptide. Some of the amino acids
required side-chain blocking groups besides the alpha-
amino protection. Such amino acids and the blocking
groups are as follows:
Met(MBzl),Ser (oBzl),Arg(Tos),Asp(oBzl),Thr
(oBzl)
Where oBzl is benzyl, Tos is guanidino-p-Toluenesulfonyl
and MBzl is methoxybenzyl.
Completion of the synthesis provided the following
peptide coupled to the styrenedivinylbenzene copolymer
resin:
TFA-Arg(Tos)-Met(mBzl)-Val-Ser(oBzl)-Gly-Gln-Thr
(oBzl)-Arg(Tos)-Leu-Asp(oBzl)-Leu-Leu-Gln-Arg(Tos)-resin
Decoupling of the peptide from the resin is accomplished
by treatment with liquid hydrogen fluoride with concomittant
cleavage of all protecting groups to produce the desired
peptide.
I ~i7~1~
Attac~nent of N-Boc-Arginlne(Tos) to chloromethyl
resin was performed by the ceslum salt method. A sample
of chloromethyl resin (200 g.) containing 0.74 mmol
chloride per gram is treated with the cesium salt of
Roc-Arginine(Tos) resulting from the neutrallization of
Boc-Arginine(Tos) with cesium carbonate. About 38.6
grams of Boc-Arginine(Tos) is dissolved in 80~ methanol
and 20% water and adjusted to pH 7.0 with about 29 grams of
cesium carbonate. The resulting solution is dried on a
rotary evaporator, then dried three more times after
three addition~ of 100 milliliters of xylene. To the
cesium salt of Boc-Arginine(Tos) was added 200 g ams of
chloromethyl resin as above and sufficient 1-Methyl~2-
Pyrrolidinone to make about 1.90 liters total volume.
The resulting mixture is stirred at 55C. for 48 hours.
The resin was then washed extensively with methanol,
then water, then again with methanol. The resin was
air dried, then clried under vacumn. After cleavage
of Arginine from the resin with HF, amino acid analysis
gave one peak corresponding to 0.48 mmol/gm~
A sample of the resin just described (6.0g.) was
submitted to the following synthesis schedule: (1) Wash
with three 100 ml. portions of methylene chloride;
(2) removal of the Boc group with 40% TFA in methylene
chloride for a one minute wash and for a 20 minute reaction
time; (3) wash with three 100 ml. portions of methylene
chloride; (4) wash with two 100 ml portions of isopropanol;
(5) wash with three 100 ml. portions of methylene chloride;
(6) a one minute wash and ten min~ltes neutrallization
with 100 rnl. portions of 10~ triethylamine in methylene
'1 2
chloride; (7) wash with three portions of 100 ml. of methylene
chloride; (8) add 2.5 equivalents (7.2 mmol) of Boc amino
acid and 2.5 equivalents (7.2 mmol) of dicyclohexylcarbodiimide
in methylene chloride and shake for 2 hours; (9) wash with
three 100 ml. portions of methylene chloride; (10) wash
with two 100 ml. portions of isopropanol; (11) wash with
three 100 ml. portions of methylene chloride. The above
cycle was repeated for the following N protected amino
acids:
Boc-Gln Boc-Thr (oBzl)
Boc-Leu Boc-Gln
Boc Leu Boc Gly
Boc-Asp (oBzl) Boc-Ser (oBzl)
Boc-Leu Boc-Val
Boc-Arg (Tos) Boc-Met (mBzl)
Boc-Arg (Tos)
The protected peptide resin was submitted to deprotection
to give the TFA salt of the protected peptide resin. The
dried resin (5.88g) was stirred in the presence of 6 ml.
of anisole and 60 ml. of liquid HF at 0C for 1 hour. The
HF was removed by vacumn and the oily residue was washed
with two 50 ml. portions of ethyl ether. The peptide was
extracted from the resin by three 50 ml. portions of 1
molar acetic acid and the combined filtrates were lyophilized
- to give 2.27 grams of crude peptide. This was purified
by 250 transfers in a counter-current distribution apparatus.
The solvent system for the above fractionation was butanol:
acetic acid:water at 4:1:5 ratios.
Somewhat purified fractions from the counter-current
distribution apparatus were fwrther purified by column
chromatography on diethylaminoethylcellulose with a linear
gradient of 0.01 to 0.5 molar ammonium bicarbona-te -to
give 250 mg. of pure peptide.
--lO-
1 7 '1 2
Amino acid analysis of the pure peptide after acid
hydrolysis gave: ammonia 1.76, Arg 2.91, Asp 0.99, Thr 1.00,
Ser 0.99, Glu 2.081, Gly 1.01, Val 1.081, Met 0.97,
Leu 3.04. This peptide gave a single spot with a Rf of
0.62 on cellulose thin layer chromatography with a solvent
system of butanol:pyridine:acetic acid:water of 15:10:3:12,
and an rF of 0.53 with these same solvents
of 42:24:4:30.
EXA~PL~ II
The respective shorter chain sequence compounds of
the invention are prepared by the method of Example 1
starting with the respective C-terminal amino acids; leucine
fo,t the 9-sequence compounds P-l, aspartic acid for the
10-sequence compounds P-2, leucine for the ll-sequence
and 12-sequence compounds P-3 and P-4, and glutamine for
the 13-sequence compound P-5, ending all sequences, as
shown above, with the N-terminal arginine.
For example, the 9-sequence P-l compound is prepared
as follows:
Preparation of Linear Peptide
Arg~Met-Val-Ser-Gly-Gln-Thr-Arq-Leu
Synthesis of the above peptide can be carried out
employing solid phase techniques now well known in
the art. In a preferred procedure amino protected leucine,
--11--
I t817~2
representing the -COOH terminal group of the above peptide, I
is coupled to a conventional solid phase peptide synthesis
resin such as chloromethyl polystyrene cross-linked with
1 to 2~ divinyl benzene. The amino protecting group is
then selectively removed utilizing a suitable reagent
whose nature will depend on the protecting group used.
In the preferred embodiment the t-butyloxycarbonyl (Boc)
group is utilized for amino group protection and 40
trifluoroacetic acid in methylene chloride is the
selective deprotecting agent.
After deprotection, the leucine resin is treated
with protected arginine, preferably N-Boc-Nb-Tosylarginine,
and dicyclohexylcarbodiimide in a manner known per se as
to form a peptide bond between the free amino group of
the leucine residue and the carboxyl group of protected
arginine.
The cycle of deprotection and coupling with amino
acid derivatives and dicyclohexylcarbodiimide is then
repeated with the remaining amino acids in the sequence
order of the above peptide. Some of the amino acids
required side-chain blocking groups besides the alpha-
amino protection. Such amino acids and the blocking
groups are as follows:
Met(MBzl),Ser(oBzl),Arg(Tos),Thr(oBzl)
-l2-
1 ~17'1~
~here oBzl is benzyl, Tos i5 ~uanidino-p-Toluenesulfonyl
and MBzl is methoxybenzyl.
Completion of the synthesis provided the following
peptide coupled to the styrenedivinylbenzene copolymer
resin:
TFA-Arg(Tos)-Met (mB21) -Val-~er(oBzl)-Gly-Gln-Thr
(oBzl)-Arg(Tos)~Leu-resin
Decoupling of the peptide from the resin is accomplished
by treatment with liquid hydrogen fluoride with concomittant
cleavage of all protecting groups to produce the desired
peptide.
Attachment of N-Boc-leucine to chloromethyl resin
was performed by the cesi~n salt method. A sample
of chloromethyl resin (200 g.) containing 0.74 mmol
chloxide per gram is treated with the cesium salt of Boc-
leucine resulting from the neutrallization of Boc-leucine
with cesium carbonate. ~bout 38.6 grams of Boc-leucine
is dissolved in 80~ methanol and 20% water and adjusted
to p~ 7.0 with about 29 grams of cesium carbonate. The
resulting solution is dried on a rotary evaporator, then
dried three more times after three additions of 100
milliliters of xylene. To the cesium salt of Boc-leucine
was added 200 grams of chloromethyl resin as above and
sufficient l-Methyl-2-Pyrrolidinone to make about 1.90
liters total volume. The resulting mlxture is ~tirred
at 55~C. for ~8 hours. The resln was then washed
extensively with methanol, then water, then again with
methanol. The resin was air dried, then dried under
vacumn. Ater cleavage of leucine from the resin with HF,
3~ amino acid analysis gave one peak corresponding -to
~ 3 Ill(n~ n,
.... I I ~,
17~2
A sample of the resin just described (6.0g.) was
submitted to the ~ollowing synthesis schedule: (1) Wash
with three 100 ml. portions of methylene chloride;
(2) removal of the Boc group with 40~ TFA in methylene
chloride for a one minute wash and for a 20 minute
reaction time; (3) wash with three 100 ml. portions of
methylene chloride; (4) wash with two 100 ml portions
of isopropanol; (5) wash with three 100 ml. portions of
methylene chloride; (6) a one minute wash and ten minutes
neutrallization with 100 ml. portions of 10~ triethylamine
in methylene chloride; (7) wash with three portions of
100 ml. of methylene chloride; (8) add 2.5 equivalents
(7.2 mmol) of Boc amino acid and 2.5 equivalents
(7.2 mmol) of dicyclohexylcarbodiimide in methylene
chloride and shake for 2 hours; (9) wash with three 100
ml. portions of methylene chloride; (10) wash with
two 100 ml. portions of isopropanol; (11) wash with three
100 ml. portions of methylene chloride. The above cycle
was repeated for the foliowing N-protected amino acids:
Boc~Arg(Tos)
Boc-Thr(oBzl)
Boc-Gln
Boc-Gly
Boc-Ser(oBzl)
Boc-Val
Boc-Met(mBzl)
Boc-Arg(Tos)
The protected peptide resin was submitted to deprotection
to give the TFA salt of the protected peptide resin. The
dried resin (5.88g) was stirred in the presence of 6 ml.
of anisole and 60 ml. of liquid HF at 0C for 1 hour. The
HF was removed by vacumn and the oily residue was washed
1.18.~7~
with two 50 ml. portions o ethyl ether. The pep~ide was
extracted from the resin by three 50 ml. portions of 1
molar acetic acid and the combined filtrates were lyophilized
to give 2.27 grams of crude peptide. This was purified
by 250 transfers in a counter-current distribution apparatus.
The solvent system for the above fractionation was butanol:
acetic acid:water at 4:1:5 ratios~
Somewhat purified fractions from the counter current
distribution apparatus were further purified by column
chromatography on diethylaminoethylcellulose with a linear
gradient of O.01 to 0.5 molar ammonium bicarbonate to
give 250 mg. of pure peptide.
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