U.S. patent application number 10/342014 was filed with the patent office on 2004-12-30 for use of exendins and agonists thereof for the treatment of gestational diabetes mellitus.
Invention is credited to Hiles, Richard A., Prickett, Kathryn S..
Application Number | 20040266670 10/342014 |
Document ID | / |
Family ID | 23261070 |
Filed Date | 2004-12-30 |
United States Patent
Application |
20040266670 |
Kind Code |
A9 |
Hiles, Richard A. ; et
al. |
December 30, 2004 |
Use of exendins and agonists thereof for the treatment of
gestational diabetes mellitus
Abstract
Methods for treating gestational diabetes which comprise
administration of an effective amount of an exendin or an exendin
agonist, alone or in conjunction with other compounds or
compositions that lower blood glucose levels.
Inventors: |
Hiles, Richard A.; (San
Diego, CA) ; Prickett, Kathryn S.; (San Diego,
CA) |
Correspondence
Address: |
ARNOLD & PORTER LLP (18528)
555 TWELFTH ST, NW
WASHINGTON
DC
20004
US
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Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 0023871 A1 |
February 5, 2004 |
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Family ID: |
23261070 |
Appl. No.: |
10/342014 |
Filed: |
January 13, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10342014 |
Jan 13, 2003 |
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09323867 |
Jun 1, 1999 |
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6506724 |
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Current U.S.
Class: |
514/1.1 |
Current CPC
Class: |
A61K 38/2278 20130101;
A61P 3/10 20180101; Y10S 514/866 20130101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 038/17 |
Claims
We claim:
1. A method for treating gestational diabetes mellitus in a subject
comprising administering to said subject a therapeutically
effective amount of an exendin or an exendin agonist.
2. The method according to claim 1 wherein said exendin or exendin
agonist is administered continuously.
3. The method according to claim 1 wherein said administration is
by injection.
4. The method according to claim 3 wherein the injection is a
subcutaneous injection.
5. The method according to claim 1 wherein about 1 .mu.g-30 .mu.g
to about 1 mg of the exendin or exendin agonist is administered per
day.
6. The method according to claim 1 wherein about 1 .mu.g-30 .mu.g
to about 500 .mu.g of the exendin or exendin agonist is
administered per day.
7. The method according to claim 1 wherein about 1 .mu.g-30 .mu.g
to about 100 .mu.g of the exendin or exendin agonist is
administered per day.
8. The method according to claim 1, wherein about 3 .mu.g to about
50 .mu.g of the exendin or exendin agonist is administered per
day.
9. The method of claim 1 wherein said subject is human.
10. A method for reducing blood glucose level of a subject having
gestational diabetes mellitus comprising administering to said
subject a therapeutically effective amount of an exendin or an
exendin agonist.
11. The method according to any of claims 1-10 wherein said exendin
is exendin-3.
12. The method according to any of claims 1-10 wherein said exendin
is exendin-4.
13. The method according to any of claims 1-10 wherein said exendin
agonist is selected from the group consisting of exendin-4 acid,
exendin-4 (1-30), exendin-4 (1-30) amide, exendin-4 (1-28) amide,
.sup.14Leu, .sup.25Phe exendin-4 amide, and .sup.14Leu, .sup.25Phe
exendin-4 (1-28) amide.
14. The method according to any of claims 1-10 further comprising
administering a therapeutically effective amount of one or more
compounds selected from the group consisting of an insulin and an
amylin agonist.
15. The method according to any of claims 1-10 wherein said exendin
agonist is an exendin agonist according to Formula I.
16. The method according to any of claims 1-10 wherein said exendin
agonist is an exendin agonist according to Formula II.
17. The method according to any of claims 1-10 wherein said exendin
agonist is an exendin agonist according to Formula III.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods for treating
gestational diabetes mellitus comprising administration of an
effective amount of an exendin or an exendin agonist alone or in
conjunction with other compounds or compositions that affect blood
glucose control, such as an insulin or an amylin agonist.
Pharmaceutical compositions for use in the methods of the invention
are also disclosed.
BACKGROUND
[0002] The following description summarizes information relevant to
the present invention. It is not an admission that any of the
information provided herein is prior art to the presently claimed
invention, nor that any of the publications specifically or
implicitly referenced are prior art to that invention.
[0003] Gestational Diabetes Mellitus Gestational diabetes mellitus
("GDM") is a disorder associated with elevated circulating plasma
glucose. Although the diagnostic criteria for GDM have been the
subject of controversy for decades, it was defined by the Third
Workshop Conference on Gestational Diabetes Mellitus as
carbohydrate intolerance of varying severity with onset or first
recognition during pregnancy, irrespective of the glycemic status
after delivery. Metzger (ed.) Proceedings of the Third
International Workshop Conference on Gestational Diabetes Mellitus,
Diabetes 40(Suppl. 2), 1991. Despite advances in clinical
management of GDM, there are problems associated with GDM which
persist, including elevated rate of perinatal morbidity and
elevated rate of malformations in newborns. Persson et al.,
Diabetes and Pregnancy, In International Textbook of Diabetes
Mellitus, Second Edition, John Wiley & Sons 1997 (Alberti et
al. Eds.). For example, it has been reported that, when the mean
blood glucose level is greater than 105 mg/dl, there is a greater
risk for the P development of large-for-gestational age ("LGA")
infants when compared with a control population. Id. Additional
reported consequences of untreated GDM include an increased
incidence of macrosomia, respiratory distress syndrome, and other
abnormalities of fetal metabolism. Langer, Am. J. Obstet Gynecol.
176:S186, 1997; American Diabetes Association: Self-Monitoring of
Blood-Glucose Consensus Statement, Diabetes Care 17:81-82,
1994("ABA Consensus Statement"); Coetzee & Jackson, S. Afr.
Med. J. 56:467-475, 1979. It has been clearly established by those
in the field that tight glycemic control can serve as the primary
prevention of fetal disease relating to GDM. Drexel et al.,
Diabetes Care 11:761-768, 1988; Roversi et al., Diabetes Care
3:489-494,.1980; Langer & Mazze, Am. J. Obstet Gynecol.
159:1478-1483, 1988; Langer et al., Am. J. Obstet Gynecol.
161:646-653, 1989). GDM results in a greater incidence of
intrauterine death or neonatal mortality. Position Statement
American Diabetes Association: Gestational Diabetes Mellitus,
Diabetes Care 21 (Suppl. 1):S60-61, 1998. GDM pregnancies are at an
increased risk for fetal macrosomia and neonatal morbidities
including neural tube defects, hypoglycemia, hypocalcemiea,
hypomagnsemia, polycythemia and hyperbilirubinemia and subsequent
childhood and adolescent obesity. Siccardi, Gestational Diabetes.
Other complications to the woman include increased rates of
cesarean delivery, hypertensive disorders including preeclamsia and
urinary tract infections.
[0004] It has been reported that approximately 4% of all
pregnancies (135,000 cases annually) are complicated by GDM,
however, it has been estimated that the incidence may range from 1%
to 14% of all pregnancies, depending on the population and
diagnostic tests employed. ADA Consensus Statement, supra.
[0005] Normally during pregnancy, fasting plasma levels of insulin
gradually increase to reach concentrations that are approximately
twice as high in the third trimester as they were outside of
pregnancy. Women with gestational diabetes mellitus ("GDM") have
fasting insulin levels comparable to or higher than those of normal
pregnant women with the highest levels seen in women with GDM who
are obese. Insulin secretion also increases gradually in pregnancy
and also reaches a maximum during the third trimester. However, the
relative increase in secretion is significantly smaller in women
with GDM than in normal glucose tolerant ("NGT") women. The
first-phase insulin response in NGT women is significantly higher
than in GDM women; second phase insulin response was similarly
increased during pregnancy in both groups. This finding is
consistent with the finding that GDM women have a later time of
peak insulin concentration during an oral glucose tolerance test
than do NGT women. Consistent with this observation, the insulin
response per unit of glycemic stimulus is significantly higher in
NGT women than in GDM women (90% and 40%, respectively). The fact
that glucose tolerance deteriorates in both normal and GDM
pregnancies while at the same time, insulin secretion increases
indicates a decrease in insulin sensitivity. Comparative results
from an intravenous glucose tolerance test and a hyperinsulinemic,
euglycemic clamp showed a sensitivity decrease during pregnancy in
both groups of 50-60%, but GDM women had a slightly lower
sensitivity. In another study using radioactive glucose, turnover
of glucose and amino acids in GDM women was comparable to NGT women
only when insulin concentrations 3-5 fold higher in the GDM group
were used. Thus, it appears that GDM is due to a combination of
diminished insulin sensitivity and an impaired ability to increase
insulin secretion and has, in fact, many features in common with
type 2 diabetes. Normal or near normal glycemic control returns
upon parturition.
[0006] Clinical Diagnosis:
[0007] It is common clinical practice to screen women for elevated
glucose and glucose intolerance between weeks 24 and 28 of
gestation, especially women with any one the following four
characteristics: age .gtoreq.25; race/ethnicity of Hispanic, Native
American, Asian, African-American or Pacific Islander origin; obese
or a family history of diabetes. In addition, women with previous
pregnancies with complications due to a large weight fetus/neonate
are usually tested. In some medical centers all pregnant-women are
tested. Indeed, certain investigators have found that historical
risk factors account for only roughly half of the women known to
have GDM. Carr, Diabetes Care 21(Suppl. 2):B14-B18, 1998.
Additionally, there is some reported evidence that advancing
maternal age is associated with increased incidence of GDM. Id.
[0008] The clinical diagnosis is generally based on a multi-step
process. The evaluation is most typically performed by measuring
plasma glucose 1 hour after a 50-gram oral glucose challenge test
in either the fasted or the unfasted state. If the value in the
glucose challenge test is .gtoreq.140 mg/dl, a 3-hr 100 g oral
glucose tolerance test is done. If two or more of the following
criteria are met, the patient is considered in need of glycemic
control: fasted venous plasma .gtoreq.105 mg/dl, venous plasma
.gtoreq.190 mg/dl. at 1 hr, venous plasma .gtoreq.165 mg/dl at 2 hr
or venous plasma .gtoreq.145 mg/dl at 3 hr. Williams et al.,
Diabetes Care 22: 418-421, 1999. Variations of this test are also
used by some. See, e.g., Coustan, Gestational Diabetes In Diabetes
in America, 2d ed. National Institutes of Health Publication No.
95-1468, 1995.
[0009] Current Clinical Therapy:
[0010] The current therapeutic approach for GDM is to control
plasma glucose for the remainder of the gestation (i.e., the third
trimester through parturition). GDM has many features in common
with type 2 diabetes. The endocrine (impaired insulin secretion)
and metabolic (insulin resistance) abnormalities that characterize
both forms of diabetes are similar. In general, pregnancy is
characterized by increases in both insulin resistance and insulin
secretion. Women with GDM fail to respond with increased insulin to
the decrease in insulin sensitivity.
[0011] A significant correlation has been shown to exist between
late-stage gestational maternal glucose levels and preeclamsia,
macrosomia, Cesarean section delivery and phototherapy for
hyperbilirubinemia. Sermer et al., Diabetic Care 21 (Suppl.
2):B33-B42, 1998. It has also been determined that the length of
hospitalization of the new mother and the length of time the
neonate spent in the nursery could be correlated to the degree of
elevation of plasma glucose in the pregnant woman. Id. Tallarigo,
et al. reported a striking rise in the risk of fetal macrosomia
(9.9 vs. 27.5%) and preeclamsia/Cesarean sections (19.9 vs. 40.0%)
in women with abnormal glucose tolerance when compared to NGT
women. Tallarigo et al., N. Engl. J. Med. 315:989-992, 1986.
[0012] Thus, the goals for therapy of GDM are to achieve and
maintain as near normal glycemia as feasible with a special
emphasis to keep postprandial glucose concentrations within the
normal range. Optimal therapeutic strategies are safe and
efficacious in achieving a metabolic balancing without creating
complications, which may include ketosis and/or hypoglycemia.
Jovanovic, Diabetes Care 21(Suppl. 2):B131-B137, 1998. The initial
therapeutic approach is through. diet. Jovanovic-Peterson &
Peterson, J. Am. Coll. Nutr. 9:320-325, 1990.
[0013] If diet or diet and exercise are not effective (i.e.,
failure is fasting glucose .gtoreq.105 mg/dl and/or a 2-hr
postprandial plasma glucose of .gtoreq.120 mg/dl on 2 or more
occasions within a 1- to 2-week period), then insulin therapy
(preferably, human insulin) is considered appropriate. ADA Position
Statement, supra.
[0014] Oral glucose-lowering agents are not recommended during
pregnancy. Kuhl et al., Diabetic Care 21 (Suppl. 2): B19-B26, 1998.
Although sulfonylureas are used in the treatment of type 2 diabetes
due to their activity in increasing insulin sensitivity these
agents are contraindicated for use in GDM. Jovanovlic, Diabetes
Care 21 (Suppl. 2):B131-B137, 1998. See also Kahn & Shechter,
Insulin, Oral Hypoglycemic Agents, and the Pharmacology of the
Endocrine Pancreas, In Goodman & Gilman's The Pharmacological
Basis of Therapeutics (8.sup.th ed. 1993 Goodman Gilman et al.
eds.). Oral hypoglycemic drugs traverse the placenta, and may cause
prolonged severe hypoglycemia in the newborn. Persson et al.,
supra.
[0015] The difficulties with, and the highly variable approaches to
insulin therapy in GDM have been reviewed, for example, by Langer,
et al. Langer, Diabetes Care 21(Suppl.2):B91-B98, 1998. The
problems commonly associated, with insulin therapy in a
non-pregnant population remain when used in the treatment of GDM.
They are determination of the proper dose, maintenance of good
glucose control through each 24-hr period, possible hypoglycemia
and weight gain. Hypoglycemia can result when insulin is
administered to control postprandial plasma glucose, but the fetus
demands for energy in the presence of excess insulin later causes
the glucose level to drop to a hypoglycemic level. This
physiological state can be dangerous to both the mother and the
fetus. Excess weight gain is undesirable in any pregnancy. Another
problem with insulin therapy is the day-to-day and week-to-week
variability in glucose control vs. insulin dose.
[0016] Thus, it can be appreciated that an effective means to treat
gestational diabetes remains a major challenge and a superior
method of treatment would be of great utility. Such a method, and
compounds and compositions which are useful therefor, have been
invented and are described and claimed herein.
[0017] Exendins and Exendin Agonists
[0018] Exendins are peptides that were first isolated form the
salivary secretions of the Gila-monster, a lizard found in Arizona,
and the Mexican Beaded Lizard. Exendin-3 is present in the salivary
secretions of Heloderma horridum, and exendin-4 is present in the
salivary secretions of Heloderma suspectum (Eng, J., et al., J.
Biol. Chem., 265:20259-62, 1990; Eng., J., et al., J. Biol. Chem.,
267:7402-05, 1992). The exendins have some sequence similarity to
several members of the glucagon-like peptide family, with the
highest homology, 53%, being to GLP-1[7-36]NH.sub.2 (Goke, et al.,
J. Biol. Chem., 268:19650-55,. 1993). GLP-1[7-36]NH.sub.2, also
known as proglucagon[78-107] and most commonly as "GLP-1," has an
insulinotropic effect, stimulating insulin secretion from
pancreatic .beta.-cells; GLP-1 also inhibits glucagon secretion
from pancreatic .alpha.-cells (Orskov, et al., Diabetes, 42:658-61,
1993; D'Alessio, et al., J. Clin. Invest., 97:133-38, 1996). GLP-1
is reported to inhibit gastric emptying (Williams B. et al., J Clin
Endocrinol Metab 81 (1): 327-32, 1996; Wettergren A, et al., Dig
Dis Sci 38 (4): 665-73, 1993), and gastric acid secretion.
(Schjoldager B T, et al., Dig Dis Sci 34 (5): 703-8, 1989;
O'Halloran D J, et al., J Endocrinol 126 (1): 169-73, 1990;
Wettergren A, et al., Dig Dis Sci 38 (4): 665-73, 1993).
GLP-1[7-37], which has an additional glycine residue at its carboxy
terminus, also stimulates insulin secretion in humans (Orskov, et
al., Diabetes, 42:658-61, 1993). A transmembrane G-protein
adenylate-cyclase-coupled receptor believed to be responsible for
the insulinotropic effect of GLP-1 is reported to have been cloned
from a .beta.-cell line (Thorens, Proc. Natl. Acad. Sci. USA
89:8641-45 (1992)).
[0019] Exendin-4 potently binds at GLP-1 receptors on
insulin-secreting .beta.TC1 cells, at dispersed acinar cells from
guinea pig pancreas, and at parietal cells from stomach; the
peptide is also said to stimulate somatostatin release and inhibit
gastrin release in isolated stomachs (Goke, et al., J. Biol. Chem.
268:19650-55, 1993; Schepp, et al., Eur. J. Pharmacol., 69:183-91,
1994; Eissele, et al., Life Sci., 55:629-34, 1994). Exendin-3 and
exendin-4 were reported to stimulate cAMP production in, and
amylase release from, pancreatic acinar cells (Malhotra, R., et
al., Regulatory Peptides,41:149-56, 1992; Raufman, et al., J. Biol.
Chem. 267:21432-37, 1992; Singh, et al., Regul. Pept. 53:47-59,
1994). The use of exendin-3 and exendin-4 as insulinotrophic agents
for the treatment of diabetes mellitus and the prevention of
hypoglycemia has been proposed (Eng, U.S. Pat. No. 5,424,286).
[0020] C-terminally truncated exendin peptides such as
exendin-4[9-39], a carboxyamidated molecule, and fragments 3-39
through 9-39 have been reported to be potent and selective
antagonists of GLP-1 (Goke, et al., J. Biol. Chem., 268:19650-55,
1993; Raufman, J. P., et al., J. Biol. Chem. 266:2897-902, 1991;
Schepp, W., et al., Eur. J. Pharm. 269:183-91, 1994;
Montrose-Rafizadeh, et al., Diabetes, 45(Suppl. 2):152A, 1996).
Exendin-4[9-39] is said to block endogenous GLP-1 in vivo,
resulting in reduced insulin secretion. Wang, et al., J. Clin.
Invest., 95:417-21, 1995; D'Alessio, et al., J. Clin. Invest.,
97:133-38, 1996). The receptor apparently responsible for the
insulinotropic effect of GLP-1 has reportedly been cloned from rat
pancreatic islet cell (Thorens, B., Proc. Natl. Acad. Sci. USA
89:8641-8645, 1992). Exendins and exendin-4[9-39] are said to bind
to the cloned GLP-1 receptor (rat pancreatic .beta.-cell GLP-1
receptor (Fehmann H C, et al., Peptides 15 (3): 453-6, 1994) and
human GLP-1 receptor (Thorens B, et-al., Diabetes 42 (11): 1678-82,
1993). In cells transfected with the cloned GLP-1 receptor,
exendin-4 is reportedly an agonist, i.e., it increases cAMP, while
exendin [9-39] is identified as an antagonist, i.e., it blocks the
stimulatory actions of exendin-4 and GLP-1. Id.
[0021] Exendin-4[9-39] is also reported to act as an antagonist of
the full length exendins, inhibiting stimulation of pancreatic
acinar cells by exendin-3 and exendin-4 (Raufman, et al., J. Biol.
Chem. 266:2897-902, 1991; Raufman, et al., J. Biol. Chem.,
266:21432-37, 1992). It is also reported that exendin [9-39]
inhibits the stimulation of plasma insulin levels by exendin-4, and
ownership with the present invention and are hereby incorporated by
reference.
[0022] Additionally, exendins have been found to suppress glucagon
secretion (U.S. Provisional Application No. ______, entitled,
"Methods for Glucagon Suppression," filed Apr. 30, 1999, docket no.
242/168, which enjoys common ownership with the present invention
and is hereby incorporated by reference).
[0023] Exendin [9-39] has been used to investigate the
physiological relevance of central GLP-1 in control of food intake
(Turton, M. D. et al. Nature 379:69-72, 1996). GLP-1 administered
by intracerebroventricular injection inhibits food intake in rats.
This satiety-inducing effect of GLP-1 delivered ICV is reported to
be inhibited by ICV injection of exendin [9-391, (Turton, supra).
However, it has been reported that GLP-1 does not inhibit food
intake in mice when administered by peripheral injection (Turton,
M. D., Nature 379:69-72, 1996; Bhavsar, S. P., Soc. Neurosci.
Abstr. 21:460 (188.8), 1995).
SUMMARY OF THE INVENTION
[0024] The present invention concerns the surprising discovery that
exendins and exendin agonists do not cross the placenta, and yet
have a profound and prolonged effect on blood glucose, rendering
them ideal agents for the treatment of gestational diabetes
mellitus.
[0025] The present invention is directed to novel methods for
inhibits the somatostatin release-stimulating and gastrin
release-inhibiting activities of exendin-4 and GLP-1 (Kolligs, F.,
et al., Diabetes, 44:16-19, 1995; Eissele, et al., Life Sciences,
55:629-34, 1994).
[0026] Methods for regulating gastrointestinal motility using
exendin agonists are described and claimed in U.S. application Ser.
No. 08/908,867, filed Aug. 8, 1997, entitled, "Methods for
Regulating Gastrointestinal Motility," which application is a
continuation-in-part of U.S. application Ser. No. 08/694,954, filed
Aug. 8, 1996, which enjoys common ownership with the present
invention and is hereby incorporated by reference.
[0027] Methods of reducing food intake using exendin agonists are
described and claimed in U.S. application Ser. No. 09/003,869,
filed Jan. 7, 1998, entitled, "Use of Exendin and Agonists Thereof
for the Reduction of Food Intake," claiming the benefit of
Provisional Application Nos. 60/034,905, filed Jan. 7, 1997,
60/055,404, filed Aug. 7, 1997, 60/065,442 filed Nov. 14, 1997, and
60/066,029 filed Nov. 14, 1997. These applications also enjoy
common ownership with the present invention and are hereby
incorporated by reference.
[0028] Exendins have also been found to have inotropic and diuretic
effects. International Application No. PCT/US99/02554, filed Feb.
5, 1999, 1998, claiming the benefit of Provisional Application No.
60/075,122, filed Feb. 13, 1998. These applications also enjoy
common treating gestational diabetes mellitus comprising the
administration of an exendin, for example, exendin-3 [SEQ ID NO. 1:
His Ser Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly
Ala Pro Pro Pro Ser], or exendin-4 [SEQ ID NO. 2: His Gly Glu Gly
Thr Phe Thr Ser Asp Leu Ser Lys Gln Met. Glu Glu Glu Ala Val Arg
Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro
Pro Ser], or other compounds which effectively bind to the receptor
at which exendin exerts its actions which are beneficial in the
treatment of gestational diabetes mellitus.
[0029] In a first aspect, the invention features a method of
treating gestational diabetes mellitus in a subject comprising
administering to the subject a therapeutically effective amount of
an exendin or an exendin agonist. By an "exendin agonist" is meant
a compound that mimics the effects of exendin in the treatment of
gestational diabetes mellitus by binding to the receptor or
receptors where exendin causes one or more of these effects.
Exendins and exendin agonists should be especially beneficially in
the treatment of GDM because, due to their actions to inhibit
gastric emptying, administration of such compounds should not
result in increased weight gain. Additionally, in animal and human
studies to date, administration of exendins and exendin agonists
have not resulted in an increased incidence of hypoglycemia.
[0030] Exendin agonist compounds include exendin acids, for example
exendin-3 acid and exendin-4 acid. Preferred exendin agonist
compounds include those described in International Application No.
PCT/US98/16387, entitled, "Novel Exendin Agonist Compounds," filed
Aug. 6, 1998, claiming the benefit of U.S. Provisional Patent
Application Serial No. 60/055,404, entitled, filed Aug. 8, 1997;
International Application No. PCT/US98/24220 entitled, "Novel
Exendin Agonist Compounds," filed Nov. 13, 1998, claiming priority
on U.S. Provisional Patent Application Serial No. 60/065,442, filed
Nov. 14, 1997; and International Application No. PCT/US98/24273
entitled, "Novel Exendin Agonist Compounds," filed Nov. 13, 1998,
claiming priority on United States U.S. Provisional Patent
Application Serial No. 60/066,029, filed Nov. 14, 1997; all of
which enjoy common ownership with the present application and all
of which are incorporated by this reference into the present
application as though fully set forth herein. Additional preferred
exendin agonist compounds are those described and claimed in U.S.
Provisional Application Serial No. ______, entitled, "Modified
Exendins and Exendin Agonists," filed Apr. 30, 1999, docket no.
242/040, which enjoys common ownership with the present application
and which is incorporated by this reference into the present
application as though fully set forth herein.
[0031] By "gestational diabetes mellitus" or "GDM" is meant any
degree of glucose intolerance with onset or first recognition
during pregnancy.
[0032] Thus, in a first embodiment, the present invention provides
a method for treating gestational diabetes in a subject comprising
administering to said subject a therapeutically effective amount of
an exendin or an exendin agonist. Preferred exendin agonist
compounds include those described in International Application Nos.
PCT/US98/16387, PCT/US98/24220, and PCT/US98/24273, which have been
incorporated by reference in the present application. Preferably,
the subject is a vertebrate, more preferably a mammal, and most
preferably a human woman. In preferred aspects, the exendin or
exendin agonist is administered parenterally, more preferably by
injection. In a most preferred aspect, the injection is a
peripheral injection. Preferably, about 1 .mu.g-30 .mu.g to about 1
mg of the exendin or exendin agonist is administered per day. More
preferably, about 1-30 .mu.g to about 500 .mu.g, or about 1-30
.mu.g to about 50 .mu.g of the exendin or exendin agonist is
administered per day. Most preferably, about 3 .mu.g to about 50
.mu.g of the exendin or exendin agonist is administered per
day.
[0033] In one preferred aspect, the exendin or exendin agonist used
in the methods of the present invention is exendin-3. In another
preferred aspect, said exendin is exendin-4. Other preferred
exendin agonists include exendin-4 (1-30) [SEQ ID NO 6: His Gly Glu
Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg
Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly], exendin4 (1-30) amide
[SEQ ID NO 7: His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln
Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly
Gly-NH.sub.2], exendin-4 (1-28) amide [SEQ ID NO 40: His Gly Glu
Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg
Leu Phe Ile Glu Trp Leu Lys Asn-NH.sub.2], .sup.14Leu, .sup.25Phe
exendin-4 amide [SEQ ID NO 9: His Gly Glu Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu
Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser-NH.sub.2],
.sup.14Leu, .sup.25Phe exendin-4 (1-28) amide [SEQ ID NO 41: His
Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala
Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH.sub.2], and .sup.14Leu,
.sup.22Ala, .sup.25Phe exendin-4 (1-28) amide [SEQ ID NO 8: His Gly
Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala Val
Arg Leu Ala Ile Glu Phe Leu Lys Asn-NH.sub.2].
[0034] In the methods of the present invention, the exendins and
exendin agonists may be administered separately or together with
one or more other compounds and compositions that exhibit a long
term or short-term blood glucose control action, including, but not
limited to other compounds and compositions that comprise an
insulin or an amylin agonist. Suitable amylin agonists include, for
example, [.sup.25,28,29Pro-]-human amylin (also known as
"pramlintide," previously referred to as "AC-137," and, referred to
in its acetate salt form by its trademark SYMLIN.TM. (pramlintide
acetate), as described in "Amylin Agonist Peptides and Uses
Therefor," U.S. Pat. No. 5,686,511, issued Nov. 11, 1997, and
salmon calcitonin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 depicts the amino acid sequences for certain exendin
agonist compounds useful in the present invention [SEQ ID NOS
9-39].
[0036] FIG. 2 depicts concentrations of exendin-4 (AC2993) in
plasma and amniotic fluid of rats after 21 .mu.g subcutaneous
injection.
[0037] FIG. 3 depicts concentrations of exendin-4 (AC2993) in
plasma and amniotic fluid of rats after 210 .mu.g subcutaneous
injection.
DETAILED DESCRIPTION OF THE INVENTION
[0038] Exendins and exendin agonists are useful as described herein
in view of their pharmacological properties. Activity as exendin
agonists can be indicated by activity in the assays described
below. Effects of exendins or exendin agonists in treating
gestational diabetes can be identified, evaluated, or screened for,
using the methods described in the Examples below, or other methods
known in the art for determining effects on blood glucose
control.
[0039] Exendin Agonist Compounds
[0040] Exendin agonist compounds are those described in
International Application No. PCT/US98/16387, filed Aug. 6, 1998,
entitled, "Novel Exendin Agonist Compounds," which claims the
benefit of U.S. Provisional Application, No. 60/055,404, filed Aug.
8, 1997, including compounds of the formula (I) (SEQ ID NO. 3]:
1 Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Gly Thr Xaa.sub.4 Xaa.sub.5
Xaa.sub.6 Xaa.sub.7 Xaa.sub.8 Ser Lys Gln Xaa.sub.9 Glu Glu Glu Ala
Val Arg Leu Xaa.sub.10 Xaa.sub.11 Xaa.sub.12 Xaa.sub.13 Leu Lys Asn
Gly Gly Xaa.sub.14 Ser Ser Gly Ala Xaa.sub.15 Xaa.sub.16 Xaa.sub.17
Xaa.sub.18-Z
[0041] wherein Xaa.sub.1 is His, Arg or Tyr; Xaa.sub.2 is Ser, Gly,
Ala or Thr; Xaa.sub.3 is Asp or Glu; Xaa.sub.4 is Phe, Tyr or
naphthylalanine; Xaa.sub.5 is Thr or Ser; Xaa.sub.6 is Ser or Thr;
Xaa.sub.7 is Asp or Glu; Xaa.sub.8 is Leu, Ile, Val, pentylglycine
or Met; Xaa.sub.9 is Leu, Ile, pentylglycine, Val or Met;
Xaa.sub.10 is Phe, Tyr or naphthylalanine; Xaa.sub.11 is Ile, Val,
Leu, pentylglycine, tert-butylglycine or Met; Xaa.sub.12 is Glu or
Asp; Xaa.sub.13 is Trp, Phe, Tyr, or naphthylalanine; Xaa.sub.14,
Xaa.sub.15, Xaa.sub.16 and Xaa.sub.17 are independently Pro,
homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine,
N-alkylpentylglycine or N-alkylalanine; Xaa.sub.18 is Ser, Thr or
Tyr; and Z is --OH or --NH.sub.2; with the proviso that the
compound is not exendin-3 or exendin-4.
[0042] Preferred N-alkyl groups for N-alkylglycine,
N-alkylpentylglycine and N-alkylalanine include lower alkyl groups
preferably of 1 to about 6 carbon atoms, more preferably of 1 to 4
carbon atoms. Suitable compounds include those listed in FIG. 10
having amino acid sequences of SEQ. ID. NOS. 9 to 39.
[0043] Preferred exendin agonist compounds include those wherein
Xaa.sub.1 is His or Tyr. More preferably Xaa.sub.1 is His.
[0044] Preferred are those compounds wherein Xaa.sub.2 is Gly.
[0045] Preferred are those compounds wherein Xaa.sub.9 is Leu,
pentylglycine or Met.
[0046] Preferred compounds include those wherein Xaa.sub.13 is Trp
or Phe.
[0047] Also preferred are compounds where Xaa.sub.4 is Phe or
naphthylalanine; Xaa.sub.11 is Ile or Val and Xaa.sub.14,
Xaa.sub.15, Xaa.sub.16 and Xaa.sub.17 are independently selected
from Pro, homoproline, thioproline or N-alkylalanine. Preferably
N-alkylalanine has a N-alkyl group-of 1 to about 6 carbon
atoms.
[0048] According to an especially preferred aspect, Xaa.sub.15,
Xaa.sub.16 and Xaa.sub.17 are the same amino acid reside.
[0049] Preferred are compounds wherein Xaa.sub.18 is Ser or Tyr,
more preferably Ser.
[0050] Preferably Z is --NH.sub.2.
[0051] According to one aspect, preferred are compounds of formula
(I) wherein Xaa.sub.1 is His or Tyr, more preferably His; Xaa.sub.2
is Gly; Xaa.sub.4 is Phe or naphthylalanine; Xaa.sub.9 is Leu,
pentylglycine or Met; Xaa.sub.10 is Phe or naphthylalanine;
Xaa.sub.11 is Ile or Val; Xaa.sub.14, Xaa.sub.15, Xaa.sub.16 and
Xaa.sub.17 are independently selected from Pro, homoproline,
thioproline or N-alkylalanine; and Xaa.sub.18 is Ser or Tyr, more
preferably Ser. More preferably Z is --NH.sub.2.
[0052] According to an especially preferred aspect, especially
preferred compounds include those of formula (I) wherein: Xaa.sub.1
is His or Arg; Xaa.sub.2 is Gly; Xaa.sub.1 is Asp or Glu; Xaa.sub.4
is Phe or napthylalanine; Xaa.sub.5 is Thr or Ser; Xaa.sub.6 is Ser
or Thr; Xaa.sub.7 is Asp or Glu; Xaa.sub.8 is Leu or pentylglycine;
Xaa.sub.9 is Leu or pentylglycine; Xaa.sub.10 is Phe or
naphthylalanine.; Xaa.sub.11 is Ile, Val or t-butyltylglycine;
Xaa.sub.12 is Glu or Asp; Xaa.sub.13 is Trp or Phe; Xaa.sub.14,
Xaa.sub.15, Xaa.sub.16, and Xaa.sub.17 are independently Pro,
homoproline, thioproline, or N-methylalanine; Xaa.sub.18 is Ser or
Tyr: and Z is --OH or --NH.sub.2; with the proviso that the
compound does not have the formula of either SEQ. ID. NOS. 1 or 2.
More preferably Z is --NH.sub.2. Especially preferred compounds
include those having the amino acid sequence of SEQ. ID. NOS. 9,
10, 21, 22, 23, 26, 28, 34, 35 and 39.
[0053] According to an especially preferred aspect, provided are
compounds where Xaa.sub.9 is Leu, Ile, Val or pentylglycine, more
preferably Leu or pentylglycine, and Xaa.sub.13 is Phe, Tyr or
naphthylalanine, more preferably Phe or naphthylalanine. These
compounds will exhibit advantageous duration of action and be less
subject to oxidative degradation, both in vitro and in vivo, as
well as during synthesis of the compound.
[0054] Exendin agonist compounds also include those described in
International Application No. PCT/US98/24210, filed Nov. 13, 1998,
entitled, "Novel Exendin Agonist compounds," which claims the
benefit of U.S. Provisional Application No. 60/065,442, filed Nov.
14, 1997, including compounds of the formula (II) [SEQ ID NO.
4]:
2 Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Gly Xaa.sub.5 Xaa.sub.6 Xaa.sub.7
Xaa.sub.8 Xaa.sub.9 Xaa.sub.10 Xaa.sub.11 Xaa.sub.12 Xaa.sub.13
Xaa.sub.14 Xaa.sub.15 Xaa.sub.16 Xaa.sub.17 Ala Xaa.sub.19
Xaa.sub.20 Xaa.sub.21 Xaa.sub.22 Xaa.sub.23 Xaa.sub.24 Xaa.sub.25
Xaa.sub.26 Xaa.sub.27 Xaa.sub.28-Z.sub.1; wherein
[0055] Xaa.sub.1 is His, Arg or Tyr;
[0056] Xaa.sub.2 is Ser, Gly, Ala or Thr;
[0057] Xaa.sub.3 is Asp or Glu;
[0058] Xaa.sub.5 is Ala or Thr;
[0059] Xaa.sub.6 is Ala, Phe, Tyr or naphthylalanine;
[0060] Xaa.sub.7 is Thr or Ser;
[0061] Xaa.sub.8 is Ala, Ser or Thr;
[0062] Xaa.sub.9 is Asp or Glu;:
[0063] Xaa.sub.10 is Ala, Leu, Ile, Val, pentylglycine or Met;
[0064] Xaa.sub.11 is Ala or Ser;
[0065] Xaa.sub.12 is Ala or Lys;,
[0066] Xaa.sub.13 is Ala or Gln;
[0067] Xaa.sub.14 is Ala, Leu, Ile, pentylglycine, Val or Met;
[0068] Xaa.sub.15 is Ala or Glu;
[0069] Xaa.sub.16 is Ala or Glu;
[0070] Xaa.sub.17 is Ala or Glu;
[0071] Xaa.sub.19 is Ala or Val;
[0072] Xaa.sub.20 is Ala or Arg;
[0073] Xaa.sub.21 is Ala or Leu;
[0074] Xaa.sub.22 is Ala, Phe, Tyr or naphthylalanine;
[0075] Xaa.sub.23 is Ile, Val, Leu, pentylglycine,
tert-butylglycine or Met;
[0076] Xaa.sub.24 is Ala, Glu or Asp;
[0077] Xaa.sub.25 is Ala, Trp, Phe, Tyr or naphthylalanine;
[0078] Xaa.sub.26 is Ala or Leu;
[0079] Xaa.sub.27 is Ala or Lys;
[0080] Xaa.sub.28 is Ala or Asn;
[0081] Z.sub.1 is --OH,
[0082] --NH.sub.2
[0083] Gly-Z.sub.2,
[0084] Gly Gly-Z.sub.2,
[0085] Gly Gly Xaa.sub.31-Z.sub.2,
[0086] Gly Gly Xaa.sub.31 Ser-Z.sub.2,
[0087] Gly Gly Xaa.sub.31 Ser Ser-Z.sub.2,
[0088] Gly Gly Xaa.sub.31 Ser Ser Gly-Z.sub.2,
[0089] Gly Gly Xaa.sub.31 Ser Ser Gly Ala-Z.sub.2,
[0090] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36-Z.sub.2,
[0091] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36
Xaa.sub.37-Z.sub.2 or
[0092] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37
Xaa.sub.38-Z.sub.2;
[0093] Xaa.sub.31, Xaa.sub.36, Xaa.sub.37 and Xaa.sub.38 are
independently Pro, homoproline, 3Hyp, 4Hyp, thioproline,
N-alkylglycine, N-alkylpentylglycine or N-alkylalanine; and
[0094] Z.sub.2 is --OH or --NH.sub.2;
[0095] provided that no more than three of Xaa.sub.3, Xaa.sub.5,
Xaa.sub.6, Xaa.sub.8, Xaa.sub.10, Xaa.sub.11, Xaa.sub.12,
Xaa.sub.13, Xaa.sub.14, Xaa.sub.15, Xaa.sub.16, Xaa.sub.17,
Xaa.sub.19, Xaa.sub.20, Xaa.sub.21, Xaa.sub.24, Xaa.sub.25,
Xaa.sub.26, Xaa.sub.27 and Xaa.sub.26 are Ala. Preferred N-alkyl
groups for N-alkylglycine, N-alkylpentylglycine and N-alkylalanine
include lower alkyl groups preferably of 1 to about 6 carbon atoms,
more preferably of 1 to 4 carbon atoms.
[0096] Preferred exendin agonist compounds include those wherein
Xaa.sub.1 is His or Tyr. More preferably Xaa.sub.1 is His.
[0097] Preferred are those compounds wherein Xaa.sub.2 is Gly.
[0098] Preferred are those compounds wherein Xaa.sub.14 is Leu,
pentylglycine or Met.
[0099] Preferred compounds are those wherein Xaa.sub.25 is Trp or
Phe.
[0100] Preferred compounds are those where Xaa.sub.6 is Phe or
naphthylalanine; Xaa.sub.22 is Phe or naphthylalanine and
Xaa.sub.23 is Ile or Val.
[0101] Preferred are compounds wherein Xaa.sub.31, Xaa.sub.36,
Xaa.sub.37 and Xaa.sub.38 are independently selected from Pro,
homoproline, thioproline and N-alkylalanine.
[0102] Preferably Z.sub.1 is --NH.sub.2.
[0103] Preferable Z.sub.2 is --NH.sub.2.
[0104] According to one aspect, preferred are compounds of formula
(II) wherein Xaa.sub.1 is His or Tyr, more preferably His;
Xaa.sub.2 is Gly; Xaa.sub.6 is Phe or naphthylalanine; Xaa.sub.14
is Leu, pentylglycine or Met; Xaa.sub.22 is Phe or naphthylalanine;
Xaa.sub.23 is Ile or Val; Xaa.sub.31, Xaa.sub.36, Xaa.sub.37 and
Xaa.sub.38 are independently selected from Pro, homoproline,
thioproline or N-alkylalanine. More preferably Z.sub.1 is
--NH.sub.2.
[0105] According to an especially preferred aspect, especially
preferred compounds include those of formula (II) wherein:
Xaa.sub.1 is His or Arg; Xaa.sub.2 is Gly or Ala; Xaa.sub.3 is Asp
or Glu; Xaa.sub.5 is Ala or Thr; Xaa.sub.6 is Ala, Phe or
nephthylalaine; Xaa.sub.7 is Thr or Ser; Xaa.sub.8 is Ala, Ser or
Thr; Xaa.sub.9 is Asp or Glu; Xaa.sub.10 is Ala, Leu or
pentylglycine; Xaa.sub.11 is Ala or Ser; Xaa.sub.12 is Ala or Lys;
Xaa.sub.13 is Ala or Gln; Xaa.sub.14 is Ala, Leu or pentylglycine;
Xaa.sub.15 is Ala or Glu; Xaa.sub.16 is Ala or Glu; Xaa.sub.17 is
Ala or Glu; Xaa.sub.19 is Ala or Val; Xaa.sub.20 is Ala or Arg;
Xaa.sub.21 is Ala or Leu; Xaa.sub.22 is Phe or naphthylalanine;
Xaa.sub.23 is Ile, Val or tert-butylglycine; Xaa.sub.24 is Ala, Glu
or Asp; Xaa.sub.25 is Ala, Trp or Phe; Xaa.sub.26 is Ala or Leu;
Xaa.sub.27 is Ala or Lys; Xaa.sub.28 is Ala or Asn; Z.sub.1 is
--OH, --NH.sub.2, Gly-Z.sub.2, Gly Gly-Z.sub.2, Gly Gly
Xaa.sub.31-Z.sub.2, Gly Gly Xaa.sub.31 Ser-Z.sub.2, Gly Gly
Xaa.sub.31 Ser Ser-Z.sub.2, Gly Gly Xaa.sub.31 Ser Ser Gly-Z.sub.2,
Gly Gly Xaa.sub.31 Ser Ser Gly Ala-Z.sub.2, Gly Gly Xaa.sub.31 Ser
Ser Gly Ala Xaa.sub.36-Z.sub.2, Gly Gly Xaa.sub.31 Ser Ser Gly Ala
Xaa.sub.36 Xaa.sub.37-Z.sub.2, Gly Gly Xaa.sub.31 Ser Ser Gly Ala
Xaa.sub.36 Xaa.sub.37 Xaa.sub.38-Z.sub.2; Xaa.sub.31, Xaa.sub.36,
Xaa.sub.37 and Xaa.sub.38 being independently Pro homoproline,
thioproline or N-methylalanine; and Z.sub.2 being --OH or
--NH.sub.2; provided that no more than three of Xaa.sub.3,
Xaa.sub.5, Xaa.sub.6, Xaa.sub.8, Xaa.sub.10, Xaa.sub.11,
Xaa.sub.12, Xaa.sub.13, Xaa.sub.14, Xaa.sub.15, Xaa.sub.16,
Xaa.sub.17, Xaa.sub.19, Xaa.sub.20, Xaa.sub.21, Xaa.sub.24,
Xaa.sub.25, Xaa.sub.26, Xaa.sub.27, and Xaa.sub.28 are Ala.
Especially preferred compounds include those having the amino acid
sequence of SEQ. ID. NOS. 40-61.
[0106] According to an especially preferred aspect, provided are
compounds where Xaa.sub.14 is Leu, Ile, Val or pentylglycine, more
preferably Leu or pentylglycine, and Xaa.sub.25 is Phe, Tyr or
naphthylalanine, more preferably Phe or naphthylalanine. These
compounds will be less susceptive to oxidative degration, both in
vitro and in vivo, as well as during
[0107] Synthesis of the Compound.
[0108] Exendin agonist compounds also include those described in
International Patent Application No. PCT/US98/24273, filed Nov. 13,
1998, entitled, "Novel Exendin Agonist Compounds," which claims the
benefit of U.S. Provisional Application No. 60/066,029, filed Nov.
14, 1997, including compounds of the formula (III) [SEQ ID NO.
5]:
3 Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Xaa.sub.4 Xaa.sub.5 Xaa.sub.6
Xaa.sub.7 Xaa.sub.8 Xaa.sub.9 Xaa.sub.10 Xaa.sub.11 Xaa.sub.12
Xaa.sub.13 Xaa.sub.14 Xaa.sub.15 Xaa.sub.16 Xaa.sub.17 Ala
Xaa.sub.19 Xaa.sub.20 Xaa.sub.21 Xaa.sub.22 Xaa.sub.23 Xaa.sub.24
Xaa.sub.25 Xaa.sub.26 Xaa.sub.27 Xaa.sub.28- Z.sub.1; wherein
[0109] Xaa.sub.1 is His, Arg, Tyr, Ala, Norval, Val or Norleu;
[0110] Xaa.sub.2 is Ser, Gly, Ala or Thr;
[0111] Xaa.sub.3 is Ala, Asp or Glu;
[0112] Xaa.sub.4 is Ala, Norval, Val, Norleu or Gly;
[0113] Xaa.sub.5 is Ala or Thr;
[0114] Xaa.sub.6 is Phe, Tyr or naphthylalanine;
[0115] Xaa.sub.7 is Thr or Ser;
[0116] Xaa.sub.8 is Ala, Ser or Thr;
[0117] Xaa.sub.9 is Ala, Norval, Val, Norleu, Asp or Glu;
[0118] Xaa.sub.10 is Ala, Leu, Ile, Val, pentylglycine or Met;
[0119] Xaa.sub.11 is Ala or Ser;
[0120] Xaa.sub.12 is Ala or Lys;
[0121] Xaa.sub.13 is Ala or Gln;
[0122] Xaa.sub.14 is Ala, Leu, Ile, pentylglycine, Val or Met;
[0123] Xaa.sub.15 is Ala or Glu;
[0124] Xaa.sub.16 is Ala or Glu;
[0125] Xaa.sub.17 is Ala or Glu;
[0126] Xaa.sub.19 is Ala or Val;
[0127] Xaa.sub.20 is Ala or Arg;
[0128] Xaa.sub.21 is Ala or Leu;
[0129] Xaa.sub.22 is Phe, Tyr or naphthylalanine;
[0130] Xaa.sub.23 is Ile, Val, Leu, pentylglycine,
tert-butylglycine or Met;
[0131] Xaa.sub.24 is Ala, Glu or Asp;
[0132] Xaa.sub.25 is Ala, Trp, Phe, Tyr or naphthylalanine;
[0133] Xaa.sub.26 is Ala or Leu;
[0134] Xaa.sub.27 is Ala or Lys;
[0135] Xaa.sub.28 is Ala or Asn;
[0136] Z.sub.1 is --OH,
[0137] --NH.sub.2,
[0138] Gly-Z.sub.2,
[0139] Gly Gly-Z.sub.2,
[0140] Gly Gly Xaa.sub.31-Z.sub.2,
[0141] Gly Gly Xaa.sub.31 Ser-Z.sub.2,
[0142] Gly Gly Xaa.sub.31 Ser Ser-Z.sub.2,
[0143] Gly Gly Xaa.sub.31 Ser Ser Gly-Z.sub.2,
[0144] Gly Gly Xaa.sub.31 Ser Ser Gly Ala-Z.sub.2,
[0145] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36-Z.sub.2,
[0146] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37
-Z.sub.2,
[0147] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37
Xaa.sub.38-Z.sub.2 or Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36
Xaa.sub.37 Xaa.sub.38 Xaa.sub.39-Z.sub.2; wherein
[0148] Xaa.sub.31, Xaa.sub.36, Xaa.sub.37 and Xaa.sub.38 are
independently Pro, homoproline, 3Hyp, 4Hyp, thioproline,
N-alkylglycine, N-alkylpentylglycine or N-alkylalanine; and,
[0149] Z.sub.2 is --OH or --NH.sub.2;
[0150] provided that no more than three of Xaa.sub.31 Xaa.sub.4,
Xaa.sub.5, Xaa.sub.6, Xaa.sub.8, Xaa.sub.9, Xaa.sub.10, Xaa.sub.11,
Xaa.sub.12, Xaa.sub.13, Xaa.sub.14, Xaa.sub.15, Xaa.sub.16,
Xaa.sub.17, Xaa.sub.19, Xaa.sub.20, Xaa.sub.21, Xaa.sub.24,
Xaa.sub.25, Xaa.sub.26, Xaa.sub.27 and Xaa.sub.28 are Ala; and
provided also that, if Xaa.sub.1 is His, Arg or Tyr, then at least
one of Xaa.sub.3, Xaa.sub.4 and Xaa.sub.9 is Ala.
[0151] Definitions
[0152] In accordance with the present invention and as used herein,
the following terms are defined to have the following meanings,
unless explicitly stated otherwise.
[0153] The term "amino acid" refers to natural amino acids,
unnatural amino acids, and amino acid analogs, all in their D and L
stereoisomers if their structure allow such stereoisomeric forms.
Natural amino acids include alanine (Ala), arginine (Arg),
asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine
(Gln), glutamic acid (Glu), glycine (Gly), histidine (His),
isoleucine (Ile), leucine (Leu), Lysine (Lys), methionine (Met),
phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr),
typtophan (Trp), tyrosine (Tyr) and valine (Val). Unnatural amino
acids include, but are not limited to azetidinecarboxylic acid,
2-aminoadipic acid, 3-aminoadipic acid, beta-alanine,
aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid,
6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid,
3-aminoisbutyric acid, 2-aminopimelic acid, tertiary-butylglycine,
2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid,
2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine,
homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline,
4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine,
N-methylglycine, N-methylisoleucine, N-methylpentylglycine,
N-methylvaline, naphthalanine, norvaline, norleucine, ornithine,
pentylglycine, pipecolic acid and thioproline. Amino acid analogs
include the natural and unnatural amino acids which are chemically
blocked, reversibly or irreversibly, or modified on their
N-terminal amino group or their side-chain groups, as for example,
methionine sulfoxide, methionine sulfone,
S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cystein- e sulfoxide
and S-(carboxymethyl)-cysteine sulfone.
[0154] The term "amino acid analog" refers to an amino acid wherein
either the C-terminal carboxy group, the N-terminal amino group or
side-chain functional group has been chemically codified to another
functional group. For example, aspartic acid-(beta-methyl ester) is
an amino acid analog of aspartic acid; N-ethylglycine is an amino
acid analog of glycine; or alanine carboxamide is an amino acid
analog of alanine.
[0155] The term "amino acid residue" refers to radicals having the
structure: (1) --C(O)--R--NH--, wherein R typically is --CH(R')--,
wherein R' is an amino acid side chain, typically H or a carbon
containing substitutent; or (2) 1
[0156] wherein p is 1, 2 or 3 representing the azetidinecarboxylic
acid, proline or pipecolic acid residues, respectively.
[0157] The term "lower" referred to herein in connection with
organic radicals such as alkyl groups defines such groups with up
to and including about 6, preferably up to and including 4 and
advantageously one or two carbon atoms. Such groups may be straight
chain or branched chain.
[0158] "Pharmaceutically acceptable salt" includes salts of the
compounds described herein derived from the combination of such
compounds and an organic or inorganic acid. In practice the use of
the salt form amounts to use of the base form. The compounds are
useful in both free base and salt form.
[0159] In addition, the following abbreviations stand for the
following:
[0160] "ACN" or "CH.sub.3CN" refers to acetonitrile.
[0161] "Boc", "tBoc" or "Tboc" refers to t-butoxy carbonyl.
[0162] "DCC" refers to N,N'-dicyclohexylcarbodiimide.
[0163] "Fmoc" refers to fluorenylmethoxycarbonyl.
[0164] "HBTU" refers to
2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluroni- um
hexaflurophosphate.
[0165] "HOBt" refers to 1-hydroxybenzotriazole monohydrate.
[0166] "homoP" or hpro" refers to homoproline.
[0167] "MeAla" or "Nme" refers to N-methylalanine.
[0168] "naph" refers to naphthylalanine.
[0169] "pG" or pGly" refers to pentylglycine.
[0170] "tBuG" refers to tertiary-butylglycine.
[0171] "ThioP" or tPro" refers to thioproline.
[0172] 3Hyp" refers to 3-hydroxyproline
[0173] 4Hyp" refers to 4-hydroxyproline
[0174] NAG" refers to N-alkylglycine
[0175] NAPG" refers to N-alkylpentylglycine
[0176] "Norval" refers to norvaline
[0177] "Norleu" refers to norleucine
[0178] Preparation of Compounds
[0179] The exendins and exendin agonists described herein may be
prepared using standard solid-phase peptide synthesis techniques
and preferably an automated or semiautomated peptide synthesizer.
Typically, using such techniques, an .alpha.-N-carbamoyl protected
amino acid and an amino acid attached to the growing peptide chain
on a resin are coupled at room temperature in an inert solvent such
as dimethylformamide, N-methylpyrrolidinone or methylene chloride
in the presence of coupling agents such as dicyclohexylcarbodiimide
and 1-hydroxybenzotriazole in the presence of a base such as
diisopropylethylamine. The .alpha.-N-carbamoyl protecting group is
removed from the resulting peptide-resin using a reagent such as
trifluoroacetic acid or piperidine, and the coupling reaction
repeated with the next desired N-protected amino acid to be added
to the peptide chain. Suitable N-protecting groups are well known
in the art, with t-butyloxycarbonyl (tBoc) and
fluorenylmethoxycarbonyl (Fmoc) being preferred herein.
[0180] The solvents, amino acid derivatives and
4-methylbenzhydryl-amine resin used in the peptide synthesizer may
be purchased from Applied Biosystems Inc. (Foster City, Calif.).
The following side-chain protected amino acids may be purchased
from Applied Biosystems, Inc.: Boc-Arg(Mts), Fmoc-Arg(Pmc),
Boc-Thr(Bzl), Fmoc-Thr(t-Bu), Boc-Ser(Bzl), Fmoc-Ser(t-Bu),
Boc-Tyr(BrZ), Fmoc-Tyr(t-Bu), Boc-Lys(Cl-Z), Fmoc-Lys(Boc)
Boc-Glu(Bzl), Fmoc-Glu(t-Bu), Fmoc-His(Trt), Fmoc-Asn(Trt), and
Fmoc-Gln(Trt). Boc-His(BOM) may be purchased from Applied
Biosystems, Inc. or Bachem Inc. (Torrance, Calif.). Anisole,
dimethylsulfide, phenol, ethanedithiol, and thioanisole may be
obtained from Aldrich Chemical Company (Milwaukee, Wis). Air
Products and Chemicals (Allentown, Pa.) supplies HF. Ethyl ether,
acetic acid and methanol may be purchased from Fisher Scientific
(Pittsburgh, Pa.).
[0181] Solid phase peptide synthesis may be carried out with an
automatic peptide synthesizer (Model 430A, Applied Biosystems Inc.,
Foster City, Calif.) using the NMP/HOBt (Option 1) system and tBoc
or Fmoc chemistry (see, Applied Biosystems User's Manual for the
ABI 430A Peptide Synthesizer, Version 1.3B Jul. 1, 1988, section 6,
pp. 49-70, Applied Biosystems, Inc., Foster City, Calif.) with
capping. Boc-peptide-resins may be cleaved with HF (-5.degree. C.
to 0.degree. C., 1 hour). The peptide may be extracted from the
resin with alternating water and acetic acid, and the filtrates
lyophilized. The Fmoc-peptide resins may be cleaved according to
standard methods (Introduction to Cleavage Techniques, Applied
Biosystems, Inc., 1990, pp. 6-12). Peptides may be also be
assembled using an Advanced Chem Tech Synthesizer (Model MPS 350,
Louisville, Ky.).
[0182] Peptides may be purified by RP-HPLC (preparative and
analytical) using a Waters Delta Prep 3000 system. A C4, C8 or C18
preparative column (10.mu., 2.2.times.25 cm; Vydac, Hesperia,
Calif.) may be used to isolate peptides, and purity may be
determined using a C4, C8 or C18 analytical column (5.mu.,
0.46.times.25 cm; Vydac). Solvents (A=0.1% TFA/water and B=0.1%
TFA/CH.sub.3CN) may be delivered to the analytical column at a
flowrate of 1.0 ml/min and to the preparative column at 15 ml/min.
Amino acid analyses may be performed on the Waters Pico Tag system
and processed using the Maxima program. Peptides may be hydrolyzed
by vapor-phase acid hydrolysis (115.degree. C., 20-24 h).
Hydrolysates may be derivatized and analyzed by standard methods
(Cohen, et al., The Pico Tag Method: A Manual of Advanced
Techniques for Amino Acid Analysis, pp. 11-52, Millipore
Corporation, Milford, Mass. (1989)). Fast atom bombardment analysis
may be carried out by M-Scan, Incorporated (West Chester, Pa.).
Mass calibration may be performed using cesium iodide or cesium
iodide/glycerol. Plasma desorption ionization analysis using time
of flight detection may be carried out on an Applied Biosystems
Bio-Ion 20 mass spectrometer. Electrospray mass spectroscopy may be
carried out on a VG-Trio machine.
[0183] Peptide compounds useful in the invention may also be
prepared using recombinant DNA techniques, using methods now known
in the art. See, e.g., Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2d Ed., Cold Spring Harbor (1989). Non-peptide
compounds useful in the present invention may be prepared by
art-known methods. For example, phosphate-containing amino acids
and peptides containing such amino acids, may be prepared using
methods known in the art. See, e.g., Bartlett and Landen, Biorg.
Chem. 14:356-377 (1986)
[0184] Compositions useful in the invention may conveniently be
provided in the form of formulations suitable for parenteral
(including intravenous, intramuscular and subcutaneous) or nasal or
oral administration. In some cases, it will be convenient to
provide an exendin or exendin agonist and another blood
glucose-controlling, plasma glucose-lowering agent, such as an
insulin, an amylin, an amylin agonist, in a single composition or
solution for administration together. In other cases, it may be
more advantageous to administer the additional agent separately
from said exendin or exendin agonist. A suitable administration
format may best be determined by a medical practitioner for each
patient individually. Suitable pharmaceutically acceptable carriers
and their formulation are described in standard formulation
treatises, e.g., Remington's Pharmaceutical Sciences by E. W.
Martin. See also Wang, Y. J. and Hanson, M. A. "Parenteral
Formulations of Proteins and Peptides: Stability and Stabilizers,"
Journal of Parenteral Science and Technology, Technical Report No.
10, Supp. 42:2S (1988).
[0185] Compounds useful in the invention can be provided as
parenteral compositions for injection or infusion. Preferred
formulations are those described and claimed in U.S. application
Ser. No. 60/116,380, entitled, "Novel Exendin Agonist Formulations
and Methods of Administration Thereof," filed Jan. 14, 1999, which
enjoys common ownership with the present application and which is
incorporated by this reference into the present application as
though fully set forth herein. They can, for example, be suspended
in an inert oil, suitably a vegetable oil such as sesame, peanut,
olive oil, or other acceptable carrier. Preferably, they are
suspended in an aqueous carrier, for example, in an isotonic buffer
solution at a pH of about 3.0 to 8.0, preferably at a pH of about
3.5 to 5.0. These compositions may be sterilized by conventional
sterilization techniques, or may be sterile filtered. The
compositions may contain pharmaceutically acceptable auxiliary
substances as required to approximate physiological conditions,
such as pH buffering agents. Useful buffers include for example,
sodium acetate/acetic acid buffers. A form of repository or "depot"
slow release preparation may be used so that therapeutically
effective amounts of the preparation are delivered into the
bloodstream over many hours or days following transdermal injection
or delivery.
[0186] The desired isotonicity may be accomplished using sodium
chloride or other pharmaceutically acceptable agents such as
dextrose, boric acid, sodium tartrate, propylene glycol, polyols
(such as mannitol and sorbitol), or other inorganic or organic
solutes. Sodium chloride is preferred particularly for buffers
containing sodium ions.
[0187] The claimed compositions can also be formulated as
pharmaceutically acceptable salts (e.g., acid addition salts)
and/or complexes thereof. Pharmaceutically acceptable salts are
non-toxic salts at the concentration at which they are
administered. The preparation of such salts can facilitate the
pharmacological use by altering the physical-chemical
characteristics of the composition without preventing the
composition from exerting its physiological effect. Examples of
useful alterations in physical properties include lowering the
melting point to facilitate transmucosal administration and
increasing the solubility to facilitate the administration of
higher concentrations of the drug.
[0188] Pharmaceutically acceptable salts include acid addition
salts such as those containing sulfate, hydrochloride, phosphate,
sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate,
ethanesulfonate, benzenesulfonate, p-toluenesulfonate,
cyclohexylsulfamate and quinate. Pharmaceutically acceptable salts
can be obtained from acids such as hydrochloric acid, sulfuric
acid, phosphoric acid, sulfamic acid, acetic acid, citric acid,
lactic acid, tartaric acid, malonic acid, methanesulfonic acid,
ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid,
cyclohexylsulfamic acid, and quinic acid. Such salts may be
prepared by, for example, reacting the free acid or base forms of
the product with one or more equivalents of the appropriate base or
acid in a solvent or medium in which the salt is insoluble, or in a
solvent such as water which is then removed in vacuo or by
freeze-drying or by exchanging the ions of an existing salt for
another ion on a suitable ion exchange resin.
[0189] Carriers or excipients-can also be used to facilitate
administration of the compound. Examples of carriers and excipients
include calcium carbonate, calcium phosphate, various sugars such
as lactose, glucose, or sucrose, or types of starch, cellulose
derivatives, gelatin, vegetable oils, polyethylene glycols and
physiologically compatible solvents. The compositions or
pharmaceutical composition can be administered by different routes
including intravenously, intraperitoneal, subcutaneous, and
intramuscular, orally, topically, transmucosally, or by pulmonary
inhalation.
[0190] If desired, solutions of the above compositions may be
thickened with a thickening agent such as methyl cellulose. They
may be prepared in emulsified form, either water in oil or oil in
water. Any of a wide variety of pharmaceutically acceptable
emulsifying agents may be employed including, for example, acacia
powder, a non-ionic surfactant (such as a Tween), or an ionic
surfactant (such as alkali polyether alcohol sulfates or
sulfonates, e.g., a Triton).
[0191] Compositions useful in the invention are prepared by mixing
the ingredients following generally accepted procedures. For
example, the selected components may be simply mixed in a blender
or other standard device to produce a concentrated mixture which
may then be adjusted to the final concentration and viscosity by
the addition of water or thickening agent and possibly a buffer to
control pH or an additional solute to control tonicity.
[0192] For use by the physician, the compositions will be provided
in dosage unit form containing an amount of an exendin or exendin
agonist, for example, exendin-3, and/or exendin-4, with or without
another glucosed-lowering agent. Therapeutically effective amounts
of an exendin or exendin agonist for use treating a subject with
gestational diabetes mellitus are those that lower blood glucose to
a desired level. As will be recognized by those in the field, an
effective amount of therapeutic agent will vary with many factors
including the age and weight of the patient, the patient's physical
condition, the blood glucose level and other factors.
[0193] The effective daily blood glucose controlling dose of the
compounds will typically be in the range of about 3 to 30 .mu.g to
about 1 mg/day, preferably about 1 to 30 .mu.g to about 500
.mu.g/day and more preferably about 1 to 30 .mu.g to about 100
.mu.g/day, most preferably about 3 .mu.g to about 50 .mu.g/day, for
a 70 kg patient, administered in a single or divided doses.
Preferred dosages are described in U.S. application Ser. No.
60/116,380, entitled, "Novel Exendin Agonist Formulations and
Methods of Administration Thereof," filed Jan. 14, 1999, which has
been incorporated by reference into the present application. A
preferred dose for twice daily administration is about 0.05 to
about 0.3 .mu.g per kilogram. The exact dose to be administered is
determined by the attending clinician and is dependent upon where
the particular compound lies within the above quoted range, as well
as upon the age, weight and condition of the individual, and the
mode of administration. Administration should begin shortly after
diagnosis of GDM and continue for the remainder of the gestation
(i.e., the third trimester through parturition). Administration may
be by injection, preferably subcutaneous or intramuscular.
Administration may also be by non-injectable routes, for example,
via the respiratory tract, the mouth and the gut. Orally active
compounds may be taken orally, however dosages should be increased
5-10 fold. Preferred methods of administration are described in
U.S. Application Ser. No. 60/116,380, entitled, "Novel Exendin
Agonist Formulations and Methods of Administration Thereof," filed
Jan. 14, 1999, which has been incorporated by reference into the
present application. Solid dosage forms, such as those useful for
oral, buccal, sublingual, intra-tracheal, nasal or pulmonary
delivery may be used. Additionally, preserved or unpreserved liquid
formulations or dry powder may be used
[0194] The optimal formulation and mode of administration of
compounds of the present application to a patient depend on factors
known in the art such as the particular disease or disorder, the
desired effect, and the type of patient. While the compounds will
typically be used to treat human subjects they may also be used to
treat similar or identical diseases in other vertebrates such as
other primates, farm animals such as swine, cattle and poultry, and
sports animals and pets such as horses, dogs and cats.
[0195] To assist in understanding the present invention, the
following Examples are included. The experiments relating to this
invention should not, of course, be construed as specifically
limiting the invention and such variations of the invention, now
known or later developed, which would be within the purview of one
skilled in the art are considered to fall within the scope of the
invention as described herein and hereinafter claimed.
EXAMPLE 1
[0196] Preparation of Amidated Peptide Having SEQ. ID. NO. 9
[0197] The above-identified peptide was assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.). In general,
single-coupling cycles were used throughout the synthesis and Fast
Moc (HBTU activation) chemistry was employed. However, at some
positions coupling was less efficient than expected and double
couplings were required. In particular, residues Asp.sub.9, Thr,
and Phe.sub.6 all required double coupling. Deprotection (Fmoc
group removal) of the growing peptide chain using piperidine was
not always efficient. Double deprotection was required at positions
Arg.sub.20, Val.sub.19 and Leu.sub.14. Final deprotection of the
completed peptide resin was achieved using a mixture of
triethylsilane (0.2 mL), ethanedithiol (0.2 mL), anisole (0.2 mL),
water (0.2 mL) and trifluoroacetic acid (15 mL) according to
standard methods (Introduction to Cleavage Techniques, Applied
Biosystems, Inc.) The peptide was precipitated in ether/water (50
mL) and centrifuged. The precipitate was reconstituted in glacial
acetic acid and lyophilized. The lyophilized peptide was dissolved
in water). Crude purity was about 55%.
[0198] Used in purification steps and analysis were Solvent A (0.1%
TFA in water) and Solvent B (0.1% TFA in ACN).
[0199] The solution containing peptide was applied to a preparative
C-18 column and purified (10% to 40% Solvent B in Solvent A over 40
minutes). Purity of fractions was determined isocratically using a
C-18 analytical column. Pure fractions were pooled furnishing the
above-identified peptide. Analytical RP-HPLC (gradient 30% to 60%
Solvent B in Solvent A over 30 minutes) of the lyophilized peptide
gave product peptide having an observed retention time of 14.5
minutes. Electrospray Mass Spectrometry (M): calculated 4131.7;
found 4129.3.
EXAMPLE 2
[0200] Preparation of Peptide Having SEQ. ID. NO. 10
[0201] The above-identified peptide was assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 5.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 25% to 75% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide gave
product peptide having an observed retention time of 21.5 minutes.
Electrospray Mass Spectrometry (M): calculated 4168.6; found
EXAMPLE 3
[0202] Preparation of Peptide Having SEQ. ID. NO. 11
[0203] The above-identified peptide was assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 1.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide gave
product peptide having an observed retention time of 17.9 minutes.
Electrospray Mass Spectrometry (M): calculated 4147.6; found
4150.2.
EXAMPLE 3
[0204] Preparation of Peptide Having SEQ. ID. NO. 12
[0205] The above-identified peptide was assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 1.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 35% to 65% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide gave
product peptide having an observed retention time of 19.7 minutes.
Electrospray Mass Spectrometry (M): calculated 4212.6; found
4213.2.
EXAMPLE 4
[0206] Preparation of Peptide Having SEQ. ID. NO. 13
[0207] The above-identified peptide was assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 1.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 50% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide gave
product peptide having an observed retention time of 16.3 minutes.
Electrospray Mass Spectrometry (M): calculated 4262.7; found
4262.4.
EXAMPLE 5
[0208] Preparation of Peptide Having SEQ. ID. NO. 14
[0209] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 1.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4172.6
EXAMPLE 6
[0210] Preparation of Peptide Having SEQ. ID. NO. 15
[0211] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 1.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4224.7.
EXAMPLE 7
[0212] Preparation of Peptide Having SEQ. ID. NO. 16
[0213] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 1.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4172.6
EXAMPLE 8
[0214] Preparation of Peptide Having SEQ. ID. NO. 17
[0215] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 1.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4186.6
EXAMPLE 9
[0216] Preparation of Peptide Having SEQ. ID. NO. 18
[0217] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 1.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4200.7
EXAMPLE 10
[0218] Preparation of Peptide Having SEQ. ID. NO. 19.
[0219] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 1.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4200.7
EXAMPLE 11
[0220] Preparation of Peptide Having SEQ. ID. NO. 20
[0221] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 1.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4202.7.
EXAMPLE 12
[0222] Preparation of Peptide Having SEQ. ID. NO. 21
[0223] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 1.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4145.6.
EXAMPLE 13
[0224] Preparation of Peptide Having SEQ. ID. NO. 22
[0225] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 1.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
f carried out to determine the retention time of the product
peptide. Electrospray Mass Spectrometry (M): calculated 4184.6.
EXAMPLE 14
[0226] Preparation of Peptide Having SEQ. ID. NO. 23
[0227] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 1.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4145.6.
EXAMPLE 15
[0228] Preparation of Peptide Having SEQ. ID. NO; 24
[0229] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 1.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4224.7.
EXAMPLE 16
[0230] Preparation of Peptide Having SEQ. ID. NO. 25
[0231] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin; deprotected and purified in a similar way to Example 1.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4172.6.
EXAMPLE 17
[0232] Preparation of Peptide Having SEQ. ID. NO. 26
[0233] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 1.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4115.5.
EXAMPLE 18
[0234] Preparation of Peptide Having SEQ. ID. NO. 27
[0235] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 1.
Used in analysis are Solvent A (0.1% TFA in.water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to,60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4188.6.
EXAMPLE 19
[0236] Preparation of Peptide Having SEQ. ID. NO. 28
[0237] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 1.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4131.6.
EXAMPLE 20
[0238] Preparation of Peptide Having SEQ. ID. NO. 29
[0239] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 1.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4172.6.
EXAMPLE 21
[0240] Preparation of Peptide Having SEQ. ID. NO. 30
[0241] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 1.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4145.6.
EXAMPLE 22
[0242] Preparation of Peptide Having SEQ. ID. NO. 31
[0243] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 1.
Additional double couplings are required at the thioproline
positions 38, 37, 36 and 31. Used in analysis are Solvent A (0.1%
TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC
(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the
lyophilized peptide is then carried out to determine the retention
time of the product peptide. Electrospray Mass Spectrometry (M):
calculated 4266.8.
EXAMPLE 23
[0244] Preparation of Peptide Having SEQ. ID. NO. 32
[0245] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55.mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 1.
Additional double couplings are required at the thioproline
positions 38, 37 and 36. Used in analysis are Solvent A (0.1% TFA
in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC
(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the
lyophilized peptide is then carried out to determine the retention
time of the product peptide. Electrospray Mass Spectrometry (M):
calculated 4246.9.
EXAMPLE 24
[0246] Preparation of Peptide Having SEQ. ID. NO. 33
[0247] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 1.
Additional double couplings are required at the homoproline
positions 38, 37, 36 and 31. Used in analysis are Solvent A (0.1%
TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC
(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the
lyophilized peptide is then carried out to determine the retention
time of the product peptide. Electrospray Mass Spectrometry (M) :
calculated 4250.8.
EXAMPLE 25
[0248] Preparation of Peptide Having SEQ. ID. NO. 34
[0249] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 1.
Additional double couplings are required at the homoproline
positions 38, 37, and 36. Used in analysis are Solvent A (0.1% TFA
in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC
(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the
lyophilized peptide is then carried out to determine the retention
time of the product peptide. Electrospray Mass Spectrometry (M):
calculated 4234.8.
EXAMPLE 26
[0250] Preparation of Peptide Having SEQ. ID. NO. 35
[0251] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 1.
Additional double couplings are required at the thioproline
positions 38, 37, 36 and 31. Used in analysis are Solvent A (0.1%
TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC
(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the
lyophilized peptide is then carried out to determine the retention
time of the product peptide. Electrospray Mass Spectrometry (M):
calculated 4209.8.
EXAMPLE 27
[0252] Preparation of Peptide Having SEQ. ID. NO. 36
[0253] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 1.
Additional double couplings are required at the homoproline
positions 38, 37, 36 and 31. Used in analysis are Solvent A (0.1%
TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC
(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the
lyophilized peptide is then carried out to determine the retention
time of the product peptide. Electrospray Mass Spectrometry (M):
calculated 4193.7.
EXAMPLE 28
[0254] Preparation of Peptide Having SEQ. ID. NO. 37
[0255] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example l.
Additional double couplings are required at the N-methylalanine
positions 38, 37, 36 and 31. Used in analysis are Solvent A (0.1%
TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC
(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the
lyophilized peptide is then carried out to determine the retention
time of the product peptide. Electrospray Mass Spectrometry (M):
calculated 3858.2.
EXAMPLE 29
[0256] Preparation of Peptide Having SEQ. ID. NO. 38
[0257] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved
from-the resin, deprotected and purified in a similar way to
Example 1. Additional double couplings are required at the
N-methylalanine positions 38, 37 and 36. Used in analysis are
Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN).
Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over
30 minutes) of the lyophilized peptide is then carried out to
determine the retention time of the product peptide. Electrospray
Mass Spectrometry (M): calculated 3940.3.
EXAMPLE 30
[0258] Preparation of Peptide Having SEQ. ID. NO. 39
[0259] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 1.
Additional double couplings are required at the N-methylalanine
positions 38, 37, 36 and 31. Used in analysis are Solvent A (0.1%
TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC
(gradient 30% to 60% Solvent B in Solvent A over 30 minutes of the
lyophilized paptide is then carried out to determine the retention
time of the product peptide. Electrospray Mass Spectrometry (M):
calculated 3801.1.
EXAMPLE 31
[0260] Preparation of C-Terminal Carboxylic Acid Peptides
Corresponding to the Above C-Terminal Amide Sequences.
[0261] The above peptides of Examples 1-5 to 30 are assembled on
the so called Wang resin (p-alkoxybenzylalacohol resin (Bachem,
0.54 mmole/g)) using Fmoc-protected amino acids (Applied
Biosystems, Inc.), cleaved from the resin, deprotected and purified
in a similar way to Example 1. Used in analysis are Solvent A (0.1%
TFA in water) and Solvent B (0.1% TFA in ACN). Analytical RP-HPLC
(gradient 30% to 60% Solvent B in Solvent A over 30 minutes) of the
lyophilized peptide is then carried out to determine the retention
time of the product peptide. Electrospray Mass Spectrometry
provides an experimentally determined (M).
EXAMPLE 32
[0262] Preparation of Peptide Having SEQ ID NO. 7
4 [SEQ. ID. NO. 7] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys
Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly
Gly-NH.sub.2
[0263] The above amidated peptide was assembled on
4-(2'-4'-dimethoxypheny- l)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using.
Fmoc-protected amino acids (Applied Biosystems, Inc.). In general,
single-coupling cycles were used throughout the synthesis and Fast
Moc (HBTU activation) chemistry was employed. Deprotection (Fmoc
group removal) of the growing peptide chain was achieved using
piperidine. Final deprotection of the completed peptide resin was
achieved using a mixture of triethylsilane (0.2 mL), ethanedithiol
(0.2 mL), anisole (0.2 mL), water (0.2 mL) and trifluoroacetic acid
(15 mL) according to standard methods (Introduction to Cleavage
Techniques, Applied Biosystems, Inc.) The peptide was precipitated
in ether/water (50 mL) and centrifuged. The precipitate was
reconstituted in glacial acetic acid and lyophilized. The
lyophilized peptide was dissolved in water). Crude purity was about
75%.
[0264] Used in purification steps and analysis were Solvent A (0.1%
TFA in water) and Solvent B (0.1% TFA in ACN). The solution
containing peptide was applied to a preparative C-18 column and
purified (10% to 40% Solvent B in Solvent A over 40 minutes).
Purity of fractions was determined isocratically using a C-18
analytical column. Pure fractions were pooled furnishing the
above-identified peptide. Analytical RP-HPLC (gradient 30% to 50%
Solvent B in Solvent A over 30 minutes) of the lyophilized peptide
gave product peptide having an observed retention time of 18.9
minutes. Electrospray Mass Spectrometry (M): calculated 3408.0;
found 3408.9.
EXAMPLE 33
[0265] Preparation of Peptide Having SEQ ID NO. 40
5 [SEQ. ID. NO. 40] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys
Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn-NH.sub.2
[0266] The above amidated peptide was assembled on
4-(2'-4'-dimethoxypheny- l)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 40% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide gave
product peptide having an observed retention time of 17.9 minutes.
Electrospray Mass Spectrometry (M): calculated 3294.7; found
3294.8.
EXAMPLE 34
[0267] Preparation of Peptide Having SEQ ID NO. 41
6 [SEQ. ID. NO. 41] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys
Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0268] The above-identified amidated peptide was assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 29% to 36% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide gave
product peptide having an observed retention time of 20.7 minutes.
Electrospray Mass Spectrometry (M): calculated 3237.6; found
3240.
EXAMPLE 35
[0269] Preparation of Peptide Having SEQ ID NO. 42
7 [SEQ. ID. NO. 42] His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys
Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0270] The above amidated peptide was assembled on
4-(2'-4'-dimethoxypheny- l)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 36% to 46% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide gave
product peptide having an observed retention time of 15.2 minutes.
Electrospray Mass-Spectrometry (M): calculated 3251.6; found
3251.5.
EXAMPLE 36
[0271] Preparation of Peptide Having SEQ ID NO. 43
8 [SEQ. ID. NO. 43] His Gly Glu Gly Ala Phe Thr Ser Asp Leu Ser Lys
Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0272] The above amidated peptide was assembled on
4-(2'-4'-dimethoxypheny- l)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis were Solvent A (0.1% TFA in water) and:Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 36% to 46% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide gave
product peptide having an observed retention time of 13.1 minutes.
Electrospray Mass Spectrometry (M): calculated 3207.6; found
3208.3.
EXAMPLE 37
[0273] Preparation of Peptide Having SEQ ID NO. 44
9 [SEQ. ID. NO. 44] His Gly Glu Gly Thr Ala Thr Ser Asp Leu Ser Lys
Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0274] The above amidated peptide was assembled on
4-(2'-4'-dimethoxypheny- l)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 35% to 45% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide gave
product peptide having an observed retention time of 12.8 minutes.
Electrospray Mass Spectrometry (M): calculated 3161.5; found
3163.
EXAMPLE 38
[0275] Preparation of Peptide Having SEQ ID NO. 45
[0276] His Gly Glu Gly Thr Phe Thr Ala Asp Leu Ser Lys Gln Leu Glu
Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH.sub.2 [SEQ.
ID. NO. 45]
[0277] The above-identified amidated peptide was assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 36% to 46% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide gave
product peptide having an observed retention time of 15.2 minutes.
Electrospray Mass Spectrometry (M): calculated 3221.6; found
3222.7.
EXAMPLE 39
[0278] Preparation of Peptide Having SEQ ID NO. 46
10 [SEQ. ID. NO. 46] His Gly Glu Gly Thr Phe Thr Ser Asp Ala Ser
Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0279] The above-identified amidated peptide was assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 34% to 44% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide gave
product peptide having an observed retention time of 14.3 minutes.
Electrospray Mass Spectrometry (M): calculated 3195.5; found
3199.4.
EXAMPLE 40
[0280] Preparation of Peptide Having SEQ ID NO. 47
11 [SEQ. ID. NO. 47] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ala
Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0281] The above-identified amidated peptide was assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using,
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 38% to 48% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide gave
product peptide having an observed retention time of 15.7 minutes.
Electrospray Mass Spectrometry (M): calculated 3221.6; found
3221.6.
EXAMPLE 41
[0282] Preparation of Peptide Having SEQ ID NO. 48
12 [SEQ. ID. NO. 48] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Ala Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0283] The above-identified amidated peptide was assembled on
4-(21-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 38% to 48% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide gave
product peptide having an observed retention time of 18.1 minutes.
Electrospray Mass Spectrometry (M): calculated 3180.5; found
3180.9.
EXAMPLE 42
[0284] Preparation of Peptide Having SEQ ID NO. 49
13 [SEQ. ID. NO. 49] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Ala Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0285] The above-identified amidated peptide was assembled on
4-2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 36% to 46% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide gave
product peptide having an observed retention time of 17.0 minutes.
Electrospray Mass Spectrometry (M): calculated 3180.6; found
3182.8.
EXAMPLE 43
[0286] Preparation of Peptide Having SEQ ID NO. 50
14 [SEQ. ID. NO. 50] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Ala Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0287] The above-identified amidated peptide was assembled on
4-(21-4,-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino:acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 32% to 42% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide gave
product peptide having an observed retention time of 14.9 minutes.
Electrospray Mass Spectrometry (M): calculated 3195.5; found
3195.9.
EXAMPLE 44
[0288] Preparation of Peptide Having SEQ ID NO. 51
15 [SEQ. ID. NO. 51] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Ala Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0289] The above-identified amidated peptide was assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 37% to 47% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide gave
product peptide having an observed retention time of 17.9 minutes.
Electrospray Mass Spectrometry (M): calculated 3179.6; found
3179.0.
EXAMPLE 45
[0290] Preparation of Peptide Having SEQ ID NO. 52
16 [SEQ. ID. NO. 52] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Ala Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0291] The above-identified amidated peptide was assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 37% to 47% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide gave
product peptide having an observed retention time of 14.3 minutes.
Electrospray Mass Spectrometry (M): calculated 3179.6; found
3180.0.
EXAMPLE 46
[0292] Preparation of Peptide Having SEQ ID NO. 53
17 [SEQ. ID. NO. 53] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Ala Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0293] The above-identified peptide was assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 37% to 47% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide gave
product peptide having an observed retention time of 13.7 minutes.
Electrospray Mass Spectrometry (M): calculated 3179.6; found
3179.0.
EXAMPLE 47
[0294] Preparation of Peptide Having SEQ ID NO. 54
18 [SEQ. ID. NO. 54] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Ala Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0295] The above-identified amidated peptide was assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 35% to 45% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide gave
product peptide having an observed retention time of 14.0 minutes.
Electrospray Mass Spectrometry (M): calculated 3209.6; found
3212.8.
EXAMPLE 48
[0296] Preparation of Peptide Having SEQ ID NO. 55
19 [SEQ. ID. NO. 55] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Ala Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0297] The above-identified amidated peptide was assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis were Solvent A (0.1% TFA in-water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 38% to 48% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide gave
product peptide.having an observed retention time of 14.3 minutes.
Electrospray Mass Spectrometry (M): calculated 3152.5; found
3153.5.
EXAMPLE 49
[0298] Preparation of Peptide Having SEQ ID NO. 56
20 [SEQ. ID. NO. 56] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Arg Ala Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0299] The above-identified amidated peptide was assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 35% to 45% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide gave.
product peptide having an observed retention time of 12.1 minutes.
Electrospray Mass Spectrometry (M): calculated 3195.5; found
3197.7.
EXAMPLE 50
[0300] Preparation of Peptide Having SEQ ID NO. 57
21 [SEQ. ID. NO. 57] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Ala Phe Leu Lys
Asn-NH.sub.2
[0301] The above-identified amidated peptide was assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 38% to 48% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide gave
product peptide having an observed retention time of 10.9 minutes.
Electrospray Mass Spectrometry (M): calculated 3179.6; found
3180.5.
EXAMPLE 51
[0302] Preparation of Peptide Having SEQ ID NO. 58
22 [SEQ. ID. NO. 58] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Ala Leu Lys
Asn-NH.sub.2
[0303] The above-identified amidated peptide was assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 32% to 42% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide gave
product peptide having an observed retention time of 17.5 minutes.
Electrospray Mass Spectrometry (M): calculated 3161.5; found
3163.0.
EXAMPLE 52
[0304] Preparation of Peptide Having SEQ ID NO. 59
23 [SEQ. ID. NO. 59] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Ala Lys
Asn-NH.sub.2
[0305] The above-identified amidated peptide was assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 32% to 42% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide gave
product peptide having an observed retention time of 19.5 minutes.
Electrospray Mass Spectrometry (M): calculated 3195.5; found
3199.
EXAMPLE 53
[0306] Preparation of Peptide Having SEQ ID NO. 60
24 [SEQ. ID. NO. 60] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Ala
Asn-NH.sub.2
[0307] The above-identified amidated peptide was assembled on A
over 30 minutes) of the lyophilized peptide gave product peptide
having an observed retention time of 22.8 minutes. Electrospray
Mass Spectrometry (M): calculated 3194.6; found 3197.6.
EXAMPLE 55
[0308] Preparation of Peptide Having SEQ ID NO. 62
25 [SEQ. ID. NO. 62] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro-NH.sub.2
[0309] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4099. 6.
EXAMPLE 56
[0310] Preparation of Peptide Having SEQ ID NO. 63
26 [SEQ. ID. NO. 63] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro-NH.sub.2
[0311] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA-resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4042.5.
EXAMPLE 57
[0312] Preparation of Peptide Having SEQ ID NO. 64
27 [SEQ. ID. NO. 64] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly Gly Pro Ser Ser Gly Ala Pro Pro-NH.sub.2
[0313] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4002.4
EXAMPLE 58
[0314] Preparation of Peptide Having SEQ ID NO. 65
28 [SEQ. ID. NO. 65] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
Gly Gly Pro Ser Ser Gly Ala Pro Pro-NH.sub.2
[0315] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1%, TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3945.4.
EXAMPLE 59
[0316] Preparation of Peptide Having SEQ ID NO. 66
29 [SEQ. ID. NO. 66] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly Gly Pro Ser Ser Gly Ala Pro-NH.sub.2
[0317] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3905.3.
EXAMPLE 60
[0318] Preparation of Peptide Having SEQ ID NO. 67
30 [SEQ. ID. NO. 67] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
Gly Gly Pro Ser Ser Gly Ala Pro-NH.sub.2
[0319] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3848.2.
EXAMPLE 61
[0320] Preparation of Peptide Having SEQ ID NO. 68
31 [SEQ. ID. NO. 68] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly Gly Pro Ser Ser Gly Ala-NH.sub.2
[0321] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3808.2.
EXAMPLE 62
[0322] Preparation of Peptide Having SEQ ID NO. 69
32 [SEQ. ID. NO. 69] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
Gly Gly Pro Ser Ser Gly Ala-NH.sub.2
[0323] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3751.1.
EXAMPLE 63
[0324] Preparation of Peptide Having SEQ ID NO. 70
33 [SEQ. ID. NO. 70] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly Gly Pro Ser Ser Gly-NH.sub.2
[0325] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3737.1.
EXAMPLE 64
[0326] Preparation of Peptide Having SEQ ID NO. 71
34 [SEQ. ID. NO. 71] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
Gly Gly Pro Ser Ser Gly-NH.sub.2
[0327] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3680.1.
EXAMPLE 65
[0328] Preparation of Peptide Having SEQ ID NO. 72
35 [SEQ. ID. NO. 72] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly Gly Pro Ser Ser-NH.sub.2
[0329] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3680.1
EXAMPLE 66
[0330] Preparation of Peptide Having SEQ ID NO. 73
36 [SEQ. ID. NO. 73] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
Gly Gly Pro Ser Ser-NH.sub.2
[0331] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3623.0.
EXAMPLE 67
[0332] Preparation of Peptide Having SEQ ID NO. 74
37 [SEQ. ID. NO. 74] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly Gly Pro Ser-NH.sub.2
[0333] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M) calculated: 3593.0
EXAMPLE 68
[0334] Preparation of Peptide Having SEQ ID NO. 75
38 [SEQ. ID. NO. 75] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
Gly Gly Pro Ser-NH.sub.2
[0335] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3535.9
EXAMPLE 69
[0336] Preparation of Peptide Having SEQ ID NO. 76
39 [SEQ. ID. NO. 76] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly Gly Pro-NH.sub.2
EXAMPLE 70
[0337] Preparation of Peptide Having SEQ ID NO. 77
40 [SEQ. ID. NO. 77] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
Gly Gly Pro-NH.sub.2
[0338] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3448.8
EXAMPLE 71
[0339] Preparation of Peptide Having SEQ ID NO. 78
41 [SEQ. ID. NO. 78] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
Gly Gly-NH.sub.2
[0340] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3351.7.
EXAMPLE 72
[0341] Preparation of Peptide Having SEQ ID NO. 79
42 [SEQ. ID. NO. 79] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly-NH.sub.2
[0342] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3351.8.
EXAMPLE 73
[0343] Preparation of Peptide Having SEQ ID NO. 80
43 [SEQ. ID. NO. 80] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
Gly-NH.sub.2
[0344] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Aralytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then,
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3294.7.
EXAMPLE 74
[0345] Preparation of Peptide Having SEQ ID NO. 81
44 [SEQ. ID. NO. 81] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly Gly tPro Ser Ser Gly Ala tPro tPro tPro-NH.sub.2
[0346] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Double couplings are required at residues 37,36 and 31. Used in
analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA
in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in
Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4197.1.
EXAMPLE 75
[0347] Preparation of Peptide Having SEQ ID NO. 82
45 [SEQ. ID. NO. 82] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly Gly Pro Ser Ser Gly Ala tPro tPro tPro-NH.sub.2
[0348] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Double couplings are required at residues 37, 36 and 31. Used in
analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA
in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in
Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4179.1.
EXAMPLE 76
[0349] Preparation of Peptide Having SEQ ID NO. 83
46 [SEQ. ID. NO. 83] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly Gly NMeala Ser Ser Gly Ala Pro Pro-NH.sub.2
[0350] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Double couplings are required at residues 36 and 31. Used in
analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA
in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in
Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3948.3.
EXAMPLE 77
[0351] Preparation of Peptide Having SEQ ID NO. 84
47 [SEQ. ID. NO. 84] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Tie Glu Trp Leu Lys Asn
Gly Gly NNeala Ser Ser Gly Ala NMeala Nmeala-NH.sub.2
[0352] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Double couplings are required at residues 36 and 31. Used in
analysis are Solvent A (0.1% TFA in water) and Solvent 2 (0.1% TFA
in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in
Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3840.1.
EXAMPLE 78
[0353] Preparation of Peptide Having SEQ ID NO. 85
48 [SEQ. ID. NO. 85] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly Gly hPro Ser Ser Gly Ala hPro hPro-NH.sub.2
[0354] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Double couplings are required at residues 36 and 31. Used in
analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA
in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in
Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4050.1.
EXAMPLE 79
[0355] Preparation of Peptide Having SEQ ID NO. 86
49 [SEQ. ID. NO. 86] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly Gly hPro Ser Ser Gly Ala hPro- NH.sub.2
[0356] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
A double coupling is required at residue 31. Used in analysis are
Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN).
Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over
30 minutes) of the lyophilized peptide is then carried out to
determine the retention time of the product peptide. Electrospray
Mass Spectrometry (M): calculated 3937.1.
EXAMPLE 80
[0357] Preparation of Peptide Having SEQ ID NO. 87
50 [SEQ. ID. NO. 87] Arg Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly Gly Pro Ser Ser Gly Ala-NH.sub.2
[0358] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3827.2.
EXAMPLE 81
[0359] Preparation of Peptide Having SEQ ID NO. 88
51 [SEQ. ID. NO. 88] His Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly Gly-NH.sub.2
[0360] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3394.8.
EXAMPLE 82
[0361] Preparation of Peptide Having SEQ ID NO. 89
52 [SEQ. ID. NO. 89] His Gly Glu Gly Thr Naphthylala Thr Ser Asp
Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu
Lys Asn-NH.sub.2
[0362] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3289.5.
EXAMPLE 83
[0363] Preparation of Peptide Having SEQ ID NO. 90
53 [SEQ. ID. NO. 90] His Gly Glu Gly Thr Phe Ser Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Iie Glu Trp Leu Lys
Asn-NH.sub.2
[0364] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN) Analytical RP-HPLC (gradient 30% to 60% Solvent B
in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3280.7.
EXAMPLE 84
[0365] Preparation of Peptide Having SEQ ID NO. 91
54 [SEQ. ID. NO. 91] His Gly Glu Gly Thr Phe Ser Thr Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn-NH.sub.2
[0366] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3294.7.
EXAMPLE 85
[0367] Preparation of Peptide Having SEQ ID NO. 92
55 [SEQ. ID. NO. 92] His Gly Glu Gly Thr Phe Thr Ser Glu Leu Ser
Lys Gln Met Ala Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn-NH.sub.2
[0368] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 3 0% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3250.7.
EXAMPLE 86
[0369] Preparation of Peptide Having SEQ ID NO. 93
56 [SEQ. ID. NO. 93] His Gly Glu Gly Thr Phe Thr Ser Asp pentyigly
Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0370] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide,
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3253.5.
EXAMPLE 87
[0371] Preparation of Peptide Having SEQ ID NO. 94
57 [SEQ. ID. NO. 94] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Naphthylala Ile Glu Phe Leu
Lys Asn-NH.sub.2
[0372] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3289.5.
EXAMPLE 88
[0373] Preparation of Peptide Having SEQ ID NO. 95
58 [SEQ. ID. NO. 95] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe tButylgly Glu Trp Leu
Lys Asn-NH.sub.2
[0374] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3183.4.
EXAMPLE 89
[0375] Preparation of Peptide Having SEQ ID NO. 96
59 [SEQ. ID. NO. 96] His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Asp Phe LeU Lys
Asn-NH.sub.2
[0376] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3237.6.
EXAMPLE 90
[0377] Preparation of Peptide Having SEQ ID NO. 97
60 [SEQ. ID. NO. 97] His Gly Glu Gly Thr Phe Thr Ser Asp Ala Ser
Lys Gln Leu Glu Glu Glu Ala Vai Arg Leu Phe Ile Glu Phe Leu Lys Asn
Gly Gly Pro Ser Ser-NH.sub.2
[0378] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3637.9.
EXAMPLE 91
[0379] Preparation of Peptide Having SEQ ID NO. 98
61 His Gly Glu Gly Thr Phe Thr Ser Asp Ala Ser Lys Gln Met Glu
[SEQ. ID. NO. 98] Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn Gly-NH.sub.2
[0380] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3309.7.
EXAMPLE 92
[0381] Preparation of Peptide Having SEQ ID NO. 99
62 His Gly Glu Gly Thr Phe Thr Ser Asp Ala Ser Lys Gln Met Glu
[SEQ. ID. NO. 99] Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn Gly Gly hPro Ser Ser Gly Ala hPro hPro-NH.sub.2
[0382] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g), using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 37.
Double couplings are required at residues 36 and 31. Used in
analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA
in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in
Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3711.1.
EXAMPLE 93
[0383] Preparation of C-Terminal Carboxylic Acid Peptides
Corresponding to the Above C-Terminal Amide Sequences for SEQ ID
NOS. 7, 40-61, 68-75, 78-80 and 87-96
[0384] Peptides having the sequences of SEQ ID NOS. 7,: 40-61,
68-75, 78-80 and 87-96 are assembled on the so called Wang resin
(p-alkoxybenzylalcohol resin (Bachem, 0.54 mmole/g)) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (.0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry provides an experimentally
determined (M).
EXAMPLE 94
[0385] Preparation of C-Terminal Carboxylic Acid Peptides
Corresponding to the Above C-Terminal Amide Sequences for SEQ ID
NOS. 62-67, 76, 77 and 81-86
[0386] Peptides having the sequences of SEQ ID NOS. 62-67, 76, 77
and 81-86 are assembled on the 2-chlorotritylchloride resin
(200-400 mesh), 2% DVB (Novabiochem, 0.4-1.0 mmole/g)) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 32.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry provides an experimentally
determined (M).
EXAMPLE 95
[0387] Preparation of Peptide Having SEQ ID NO. 100
63 Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu
[SEQ. ID. NO. 100] Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0388] The above amidated peptide was assembled on
4-(2'-4'-dimethoxypheny- l)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.). In general,
single-coupling cycles were used throughout the synthesis and Fast
Moc (HBTU activation) chemistry was employed. Deprotection (Fmoc
group removal) of the growing peptide chain was achieved using
piperidine. Final deprotection of the completed peptide resin was
achieved using a mixture of triethylsilane (0.2 mL), ethanedithiol
(0.2 mL), anisole (0.2 mL), water (0.2 mL,) and trifluoroacetic
acid (15 mL) according to standard methods (Introduction to
Cleavage Techniques, Applied Biosystems, Inc.) The peptide was
precipitated in ether/water (50 mL) and centrifuged. The
precipitate was reconstituted in glacial acetic acid and
lyophilized. The lyophilized peptide was dissolved in water). Crude
purity was about 75%.
[0389] Used in purification steps and analysis were Solvent A (0.1%
TFA in water) and Solvent B (0.1% TFA in ACN).
[0390] The solution containing peptide was applied to a preparative
C-18 column and purified (10% to 40% Solvent B in Solvent A over 40
minutes). Purity of fractions was determined isocratically using a
C-18 analytical column. Pure fractions were pooled furnishing the
above-identified peptide. Analytical RP-HPLC (gradient 30% to 60%
Solvent B in Solvent A over 30 minutes) of the lyophilized peptide
gave product peptide having an observed retention time of 19.2
minutes. Electrospray Mass Spectrometry (M): calculated 3171.6;
found 3172.
EXAMPLE 96
[0391] Preparation of Peptide Having SEQ ID NO. 101
64 His Gly Ala Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu
[SEQ. ID. NO.' 101] Glu Glu Ala Val Arg Leu Phe Tie Giu Phe Leu Lys
Asn-NH.sub.2
[0392] The above amidated peptide was assembled on
4-(2'-4'-dimethoxypheny- l)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 36% to 46% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide gave
product peptide having an observed retention time of 14.9 minutes.
Electrospray Mass Spectrometry (M): calculated 3179.6; found
3180.
EXAMPLE 97
[0393] Preparation of Peptide Having SEQ ID NO. 102
65 His Gly Glu Ala Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu
[SEQ. ID. NO. 102] Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0394] The above amidated peptide was assembled on
4-(2'-4'-dimethoxypheny- l)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 37% to 47% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide gave
product peptide having an observed retention time of 12.2 minutes.
Electrospray Mass Spectrometry (M): calculated 3251.6; found
3253.3.
EXAMPLE 98
[0395] Preparation of Peptide Having SEQ ID NO. 103
66 His Gly Glu Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Leu Glu
[SEQ. ID. NO. 103] Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0396] The above amidated peptide was assembled on
4-(2'-4'-dimethoxypheny- l)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis were Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 35% to 45% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide gave
product peptide having an observed retention time of 16.3 minutes.
Electrospray Mass Spectrometry (M): calculated 3193.6; found
3197.
EXAMPLE 99
[0397] Preparation of Peptide Having SEQ ID NO. 104
67 Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu
[SEQ. ID. NO. 104] Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn-NH.sub.2
[0398] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3228.6.
EXAMPLE 100
[0399] Preparation of Peptide Having SEQ ID NO. 105
68 His Gly Ala Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu
[SEQ. ID. NO. 105] Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn-NH.sub.2
[0400] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3234.7.
EXAMPLE 101
[0401] Preparation or Peptide Having SEQ ID NO. 106
69 His Gly Glu Ala Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu
[SEQ. ID. NO. 106] Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn-NH.sub.2
[0402] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3308.7.
EXAMPLE 102
[0403] Preparation of Peptide Having SEQ ID NO. 107
70 His Gly Glu Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Met Glu
[SEQ. ID. NO. 107] Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn-NH.sub.2
[0404] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3250.7.
EXAMPLE 103
[0405] Preparation of Peptide Having SEQ ID NO. 108
71 [SEQ. ID. NO. 108] His Gly Glu Gly Thr Phe Thr Ser Asp Ala Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn-NH.sub.2
[0406] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3252.6.
EXAMPLE 104
[0407] Preparation of Peptide Having SEQ ID NO. 109
72 [SEQ. ID. NO. 109] Ala Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn-NH.sub.2
[0408] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3200.6.
EXAMPLE 105
[0409] Preparation of Peptide Having SEQ ID NO. 110
73 [SEQ. ID. NO. 110] Ala Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0410] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3143.5.
EXAMPLE 106
[0411] Preparation of Peptide Having SEQ ID NO. 111
74 [SEQ. ID. NO. 111] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn-NH.sub.2
[0412] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55; mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3214.6.
EXAMPLE 107
[0413] Preparation of Peptide Having SEQ ID NO. 112
75 [SEQ. ID. NO. 112] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0414] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3157.5.
EXAMPLE 108
[0415] Preparation of Peptide Having SEQ ID NO. 113
76 [SEQ. ID. NO. 113] Ala Gly Asp Gly Ala Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn-NH.sub.2
[0416] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3184.6.
EXAMPLE 109
[0417] Preparation of Peptide Having SEQ ID NO. 114
77 [SEQ. ID. NO. 114] Ala Glu Asp Gly Ala Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0418] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product,
peptide. Electrospray Mass Spectrometry (M): calculated 3127.5.
EXAMPLE 110
[0419] Preparation of Peptide Having SEQ ID NO. 115
78 [SEQ. ID. NO. 115] Ala Gly Asp Gly Thr NaphthylAla Thr Ser Asp
Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu
Lys Asn-NH.sub.2
[0420] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the, resin, deprotected and purified in a similar way to Example
95. Used in analysis are Solvent A (0.1% TFA in water) and Solvent
B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60%
Solvent B in Solvent A over 30 minutes) of the lyophilized peptide
is then carried out to determine the retention time of the product
peptide. Electrospray Mass Spectrometry (M): calculated 3266.4.
EXAMPLE 111
[0421] Preparation of Peptide Having SEQ ID NO. 116
79 [SEQ. ID. NO. 116] Ala Gly Asp Gly Thr Naphthylala Thr Ser Asp
Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu
Lys Asn-NH.sub.2
[0422] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3209.4.
EXAMPLE 112
[0423] Preparation of Peptide Having SEQ ID NO. 117
80 [SEQ. ID. NO. 117] Ala Gly Asp Gly Thr Phe Ser Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn-NH.sub.2
[0424] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3200.6.
EXAMPLE 113
[0425] Preparation of Peptide Having SEQ ID NO. 118
81 Ala Gly Asp Gly Thr Phe Ser Ser Asp Leu Ser Lys Gln Leu Glu
[SEQ. ID. NO. 118] Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0426] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3143.5.
EXAMPLE 114
[0427] Preparation of Peptide Having SEQ ID NO. 119
82 Ala Gly Asp Gly Thr Phe Thr Ala Asp Leu Ser Lys Gln Met Glu
[SEQ. ID. NO. 119] Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn-NH.sub.2
[0428] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3198.6.
EXAMPLE 115
[0429] Preparation of Peptide Having SEQ ID NO. 120
83 Ala Gly Asp Gly Thr Phe Thr Ala Asp Leu Ser Lys Gln Leu Glu
[SEQ. ID. NO. 120] Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0430] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3141.5.
EXAMPLE 116
[0431] Preparation of Peptide Having SEQ ID NO. 121
84 Ala Gly Asp Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Met Glu
[SEQ. ID. NO. 1211] Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn-NH.sub.2
[0432] The above-identified peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3170.6.
EXAMPLE 117
[0433] Preparation of Peptide Having SEQ ID NO. 122
85 Ala Gly Asp Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Leu Glu
[SEQ. ID. NO. 122] Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0434] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3113.5.
EXAMPLE 118
[0435] Preparation of Peptide Having SEQ ID NO. 123
86 Ala Gly Asp Gly Thr Phe Thr Ser Glu Leu Ser Lys Gln Met Glu
[SEQ. ID. NO. 123] Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn-NH.sub.2
[0436] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids: (Applied Biosystems, Inc.), cleaved
from the resin, deprotected and purified in a similar way to
Example 95. Used in analysis are Solvent A (0.1% TFA in water) and
Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to
60% Solvent B in Solvent A over 30 minutes) of the lyophilized
peptide is then carried out to determine the retention time of the
product peptide. Electrospray Mass Spectrometry (M): calculated
3228.6.
EXAMPLE 119
[0437] Preparation of Peptide Having SEQ ID NO. 124
87 Ala Gly Asp Gly Thr Phe Thr Ser Glu Leu Ser Lys Gln Leu Glu
[SEQ. ID. NO. 124] Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0438] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3171.6.
EXAMPLE 120
[0439] Preparation of Peptide Having SEQ ID NO. 125
88 Ala Gly Asp Gly Thr Phe Thr Ser Asp Ala Ser Lys Gln Met Glu
[SEQ. ID. NO. 125] Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn-NH.sub.2
[0440] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3172.5.
EXAMPLE 121
[0441] Preparation of Peptide Having SEQ ID NO. 126
89 Ala Gly Asp Gly Thr Phe Thr Ser Asp Ala Ser Lys Gln Leu Glu
[SEQ. ID. NO. 126] Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0442] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3115.4.
EXAMPLE 122
[0443] Preparation of Peptide Having SEQ ID NO. 127
90 Ala Gly Asp Gly Thr Phe Thr Ser Asp Pentylgly Ser Lys Gln [SEQ.
ID. NO. 127] Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu
Lys Asn-NH.sub.2
[0444] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3230.4.
EXAMPLE 123
[0445] Preparation of Peptide Having SEQ ID NO. 128
91 [SEQ. ID. NO. 128] Ala Gly Asp Gly Thr Phe Thr Ser Asp Pertylgly
Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0446] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3198.6.
EXAMPLE 124
[0447] Preparation of Peptide Having SEQ ID NO. 129
92 [SEQ. ID. NO. 129] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ala
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn-NH.sub.2
[0448] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3141.5.
EXAMPLE 125
[0449] Preparation of Peptide Having SEQ ID NO. 130
93 [SEQ. ID. NO. 130] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ala
Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0450] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3157.5.
EXAMPLE 126
[0451] Preparation of Peptide Having SEQ ID NO. 131
94 [SEQ. ID. NO. 131] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Ala Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn-NH.sub.2
[0452] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3100.4.
EXAMPLE 127
[0453] Preparation of Peptide Having SEQ ID NO. 132
95 [SEQ. ID. NO. 132] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Ala Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0454] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3157.6.
EXAMPLE 128
[0455] Preparation of Peptide Having SEQ ID NO. 133
96 [SEQ. ID. NO. 133] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Ala Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn-NH.sub.2
[0456] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3100.5.
EXAMPLE 129
[0457] Preparation of Peptide Having SEQ ID NO. 134
97 [SEQ. ID. NO. 134] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Ala Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0458] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3100.5.
EXAMPLE 130
[0459] Preparation of Peptide Having SEQ ID NO. 135
98 [SEQ. ID. NO. 135] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Ala Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn-NH.sub.2
[0460] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3154.5.
EXAMPLE 131
[0461] Preparation of Peptide Having SEQ ID NO. 136
99 [SEQ. ID. NO. 136] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Ala Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0462] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3115.5.
EXAMPLE 132
[0463] Preparation of Peptide Having SEQ ID NO. 137
100 [SEQ. ID. NO. 137] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Pentylgly Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu
Lys Asn-NH.sub.2
[0464] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids. (Applied Biosystems, Inc.), cleaved
from the resin, deprotected and purified in a similar way to
Example 95. Used in analysis are Solvent A (0.1% TFA in water) and
Solvent B (0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to
60% Solvent B in Solvent A over 30 minutes) of the lyophilized
peptide is then carried out to determine the retention time of the
product peptide. Electrospray Mass Spectrometry (M): calculated
3212.4.
EXAMPLE 133
[0465] Preparation of Peptide Having SEQ ID NO. 138
101 [SEQ. ID NO. 138] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Pentylgly Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu
Lys Asn-NH.sub.2
[0466] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3173.4.
EXAMPLE 134
[0467] Preparation of Peptide Having SEQ ID NO. 139
102 [SEQ. ID. NO. 139] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Ala Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn-NH.sub.2
[0468] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3156.6.
EXAMPLE 135
[0469] Preparation of Peptide Having SEQ ID NO. 140
103 [SEQ. ID. NO. 140] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Ala Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0470] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3099.5.
EXAMPLE 136
[0471] Preparation of Peptide Having SEQ ID NO. 141
104 [SEQ. ID. NO. 141] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Ala Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn-NH.sub.2
[0472] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3156.6.
EXAMPLE 137
[0473] Preparation of Peptide Having SEQ ID NO. 142
105 [SEQ. ID. NO. 142] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Ala Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0474] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3099.5.
EXAMPLE 138
[0475] Preparation of Peptide Having SEQ ID NO. 143
106 [SEQ. ID. NO. 143] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Ala Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn-NH.sub.2
[0476] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A 0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3156.6.
EXAMPLE 139
[0477] Preparation of Peptide Having SEQ ID NO. 144
107 [SEQ. ID. NO. 144] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Ala Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0478] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3099.5.
EXAMPLE 140
[0479] Preparation of Peptide Having SEQ ID NO. 145
108 [SEQ. ID. NO. 145] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Ala Arg Leu Phe Ile Glu Trp Leu Lys
Asn-NH.sub.2
[0480] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent SA over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3186.6.
EXAMPLE 141
[0481] Preparation of Peptide Having SEQ ID NO. 146
109 [SEQ. ID. NO. 146] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Ala Arg Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0482] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3129.5.
EXAMPLE 142
[0483] Preparation of Peptide Having SEQ ID NO. 147
110 [SEQ. ID. NO. 147] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Ala Leu Phe Ile Glu Trp Leu Lys
Asn-NH.sub.2
[0484] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes). of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3129.5.
EXAMPLE 143
[0485] Preparation of Peptide Having SEQ ID NO. 148
111 [SEQ. ID. NO. 148] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Ala Leu Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0486] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3072.4.
EXAMPLE 144
[0487] Preparation of Peptide Having SEQ ID NO. 149
112 [SEQ. ID. NO. 149] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Ala Phe Ile Glu Trp Leu Lys
Asn-NH.sub.2
[0488] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3172.5.
EXAMPLE 145
[0489] Preparation of Peptide Having SEQ ID NO. 150
113 [SEQ. ID. NO. 150] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Arg Ala Phe Ile Glu Phe Leu Lys
Asn-NH.sub.2
[0490] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3115.5.
EXAMPLE 146
[0491] Preparation of Peptide Having SEQ ID NO. 151
114 [SEQ. ID. NO. 151] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Naphthylala Ile Glu Trp Leu
Lys Asn-NH.sub.2
[0492] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3266.4.
EXAMPLE 147
[0493] Preparation of Peptide Having SEQ ID NO. 152
115 [SEQ ID. NO. 152] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu GLu Ala Val Arg Leu Naphthylala Ile Glu Phe Leu
Lys Asn-NH.sub.2
[0494] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3209.4.
EXAMPLE 148
[0495] Preparation of Peptide Having SEQ ID NO. 153
116 [SEQ. ID. NO. 153] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Val Glu Trp Leu Lys
Asn-NH.sub.2
[0496] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3200.6.
EXAMPLE 149
[0497] Preparation of Peptide Having SEQ ID NO. 154
117 [SEQ. ID. NO. 154] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Val Glu Phe Leu Lys
Asn-NH.sub.2
[0498] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3143.5.
EXAMPLE 150
[0499] Preparation of Peptide Having SEQ ID NO. 155
118 [SEQ. ID. NO. 155] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe tButylgly Glu Trp Leu
Lys Asn-NH.sub.2
[0500] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3216.5.
EXAMPLE 151
[0501] Preparation of Peptide Having SEQ ID NO. 156
119 [SEQ. ID. NO. 156] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe tButylgly Glu Phe Leu
Lys Asn-NH.sub.2
[0502] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3159.4.
EXAMPLE 152
[0503] Preparation of Peptide Having SEQ ID NO. 157
120 [SEQ. ID. NO. 157] Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Asp Trp Leu Lys
Asn-NH.sub.2
[0504] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3200.6
EXAMPLE 153
[0505] Preparation of Peptide Having SEQ ID NO. 158
121 Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu
[SEQ. ID. NO. 158] Glu Glu Ala Val Arg Leu Phe Ile Asp Phe Leu Lys
Asn-NH.sub.2
[0506] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3143.5.
EXAMPLE 154
[0507] Preparation of Peptide Having SEQ ID NO. 159
122 Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu
[SEQ. ID. NO. 159] Glu Glu Ala Val Arg Leu Phe Ile Glu Ala Leu Lys
Asn-NH.sub.2
[0508] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3099.5.
EXAMPLE 155
[0509] Preparation of Peptide Having SEQ ID NO. 160
123 Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu
[SEQ. ID. NO. 160] Glu Glu Ala Val Arg Leu Phe Ile Glu Ala Leu Lys
Asn-NH.sub.2
[0510] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3081.4.
EXAMPLE 156
[0511] Preparation of Peptide Having SEQ ID NO. 161
124 Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu
[SEQ. ID. NO. 161] Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Ala Lys
Asn-NH.sub.2
[0512] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3172.5.
EXAMPLE 157
[0513] Preparation of Peptide Having SEQ ID NO. 162
125 Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu
[SEQ. ID. NO. 162] Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Ala Lys
Asn-NH.sub.2
[0514] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3115.5.
EXAMPLE 158
[0515] Preparation of Peptide Having SEQ ID NO. 163
126 Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu
[SEQ. ID. NO. 163] Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Ala
Asn-NH.sub.2
[0516] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3157.5.
EXAMPLE 159
[0517] Preparation of Peptide Having SEQ ID NO. 164
127 Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu
[SEQ. ID. NO. 164] Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Ala
Asn-NH.sub.2
[0518] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray mass Spectrometry (M): calculated 3100.4.
EXAMPLE 160
[0519] Preparation of Peptide Having SEQ ID NO. 165
128 Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu
[SEQ. ID. NO. 165] Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Ala-NH.sub.2
[0520] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3171.6.
EXAMPLE 161
[0521] Preparation of Peptide Having SEQ ID NO. 166
129 Ala Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu
[SEQ. ID. NO. 166] Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Ala-NH.sub.2
[0522] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3114.5.
EXAMPLE 162
[0523] Preparation of Peptide Having SEQ ID NO. 167
130 Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu
[SEQ. ID. NO. 167] Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro-NH.sub.2
[0524] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4033.5.
EXAMPLE 163
[0525] Preparation of Peptide Having SEQ ID NO. 168
131 His Gly Ala Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu
[SEQ. ID. NO. 168] Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro-NH.sub.2
[0526] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3984.4.
EXAMPLE 164
[0527] Preparation of Peptide Having SEQ ID NO. 169
132 His Gly Glu Ala Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu
[SEQ. ID. NO. 169] Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro-NH.sub.2
[0528] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4016.5.
EXAMPLE 165
[0529] Preparation of Peptide Having SEQ ID NO. 170
133 His Gly Glu Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Met Glu
[SEQ. ID. NO. 170] Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn Gly Gly Pro Ser Ser GJy Ala Pro-NH.sub.2
[0530] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3861.3.
EXAMPLE 166
[0531] Preparation of Peptide Having SEQ ID NO. 171
134 Ala Gly Glu Gly Thr Phe Thr Ser Asp Ala Ser Lys Gln Leu Glu
[SEQ. ID. NO. 171] Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn Gly Gly Pro Ser Ser Gly Ala Pro-NH.sub.2
[0532] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3746.1.
EXAMPLE 167
[0533] Preparation of Peptide Having SEQ ID NO. 172
135 Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu
[SEQ. ID. NO. 172] Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn Gly Gly Pro Ser Ser Gly Ala-NH.sub.2
[0534] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3742.1.
EXAMPLE 168
[0535] Preparation of Peptide Having SEQ ID NO. 173
136 His Gly Ala Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu
[SEQ. ID. NO. 173] Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn Gly Gly Pro Ser Ser Gly Ala-NH.sub.2
[0536] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3693.1.
EXAMPLE 169
[0537] Preparation of Peptide Having SEQ ID NO. 174
137 His Gly Glu Ala Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu
[SEQ ID. NO. 174] Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn Gly Gly Pro Ser Ser Gly-NH.sub.2
[0538] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3751.2.
EXAMPLE 170
[0539] Preparation of Peptide Having SEQ ID NO. 175
138 His Gly Glu Gly Thr Phe Thr Ser Ala Leu Ser Lys Gln Met Glu
[SEQ. ID. NO. 175] Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn Gly Gly Pro Ser Ser-NH.sub.2
[0540] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3634.1.
EXAMPLE 171
[0541] Preparation of Peptide Having SEQ ID NO. 176
139 Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu
[SEQ. ID. NO. 176] Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn Gly Gly Pro Ser-NH.sub.2
[0542] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3526.9.
EXAMPLE 172
[0543] Preparation of Peptide Having SEQ ID NO. 177
140 His Gly Ala Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu
[SEQ. ID. NO. 177] Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn Gly Gly Pro Ser-NH.sub.2
[0544] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3477.9.
EXAMPLE 173
[0545] Preparation of Peptide Having SEQ ID NO. 178
141 [SEQ. ID. NO. 178] His Gly Glu Ala Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly Gly Pro-NH.sub.2
[0546] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3519.9.
EXAMPLE 174
[0547] Preparation of Peptide Having SEQ ID NO. 179
142 [SEQ. ID. NO. 179] His Gly Glu Gly Thr Phe Thr Ser Ala Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
Gly Gly-NH.sub.2
[0548] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3307.7.
EXAMPLE 175
[0549] Preparation of Peptide Having SEQ ID NO. 180
143 [SEQ. ID. NO. 180] Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn
Gly-NH.sub.2
[0550] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3186.5.
EXAMPLE 176
[0551] Preparation of Peptide Having SEQ ID NO. 181
144 [SEQ. ID. NO. 181] His Gly Ala Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly Gly tPro Ser Ser Gly Ala tPro tPro tPro-NH.sub.2
[0552] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Double couplings are required at residues 37, 36 and 31. Used in
analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA
in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in
Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4121.1.
EXAMPLE 177
[0553] Preparation of Peptide Having SEQ ID NO. 182
145 [SEQ. ID. NO. 182] His Gly Glu Ala Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly Gly Pro Ser Ser Gly Ala tPro tPro tPro-NH.sub.2
[0554] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Double-couplings are required at residues 37, 36 and 31. Used in
analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA
in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in
Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4173.2.
EXAMPLE 178
[0555] Preparation of Peptide Having SEQ ID NO. 183
146 [SEQ. ID. NO. 183] His Gly Glu Gly Thr Phe Thr Ser Ala Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly Gly NMeala Ser Ser Gly Ala NMeala NMeala-NH.sub.2
[0556] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Compound 1.
Double couplings are required at residues 36 and 31. Used in
analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA
in ACN) . Analytical RP-HPLC (gradient 30% to 60% Solvent B in
Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3796.1.
EXAMPLE 179
[0557] Preparation of Peptide Having SEQ ID NO. 184
147 [SEQ. ID. NO. 184] Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly Gly hPro Ser Ser Gly Ala hPro- NH.sub.2
[0558] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
A double coupling is required at residue 31. Used in analysis are
Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN).
Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over
30 minutes) of the lyophilized peptide is then carried out to
determine the retention time of the product peptide. Electrospray
Mass Spectrometry (M): calculated 3871.1.
EXAMPLE 180
[0559] Preparation of Peptide Having SEQ ID NO. 185
148 [SEQ. ID. NO. 185] His Gly Ala Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly Gly Pro Ser Ser Gly Ala-NH.sub.2
[0560] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3750.2.
EXAMPLE 181
[0561] Preparation of Peptide Having SEQ ID NO. 186
149 [SEQ. ID. NO. 186] His Gly Asp Ala Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly Gly-NH.sub.2
[0562] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 3408.8.
EXAMPLE 182
[0563] Preparation of Peptide Having SEQ ID NO. 187
150 [SEQ. ID. NO. 187] Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser-NH.sub.2
[0564] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4120.6.
EXAMPLE 183
[0565] Preparation of Peptide Having SEQ ID NO. 188
151 Ala Gly Ala Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu
[SEQ. ID. NO. 188] Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys
Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser-NH.sub.2
[0566] The above-identified amidated peptide is assembled on
4-(2'-4'-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide
norleucine MBHA resin (Novabiochem, 0.55 mmole/g) using
Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from
the resin, deprotected and purified in a similar way to Example 95.
Used in analysis are Solvent A (0.1% TFA in water) and Solvent B
(0.1% TFA in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent
B in Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry (M): calculated 4005.5.
EXAMPLE 184
[0567] Preparation of C-Terminal Carboxylic Acid Peptides
Corresponding to the Above C-Terminal Amide Sequences for Peptides
Having SEQ ID NOS. 100-166, 172-177, 179-180 and 185-188
[0568] C-terminal carboxylic acid peptides corresponding to
amidated having SEQ ID NOS. 100-166, 172-177, 179-180 and 185-188
are assembled on the so called Wang resin (p-alkoxybenzylalcohol
resin (Bachem, 0.54 mmole/g)) using Fmoc-protected amino acids
(Applied Biosystems, Inc.), cleaved from the resin, deprotected and
purified in a similar way to that described in Example 95. Used in
analysis are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA
in ACN). Analytical RP-HPLC (gradient 30% to 60% Solvent B in
Solvent A over 30 minutes) of the lyophilized peptide is then
carried out to determine the retention time of the product peptide.
Electrospray Mass Spectrometry provides an experimentally
determined (M).
EXAMPLE 185
[0569] Preparation of C-Terminal Carboxylic Acid Peptides
Corresponding to the Above C-Terminal Amide Sequences for Peptides
Having SEQ ID NOS. 167-171, 178 and 181-184
[0570] C-terminal carboxylic acid peptides corresponding to
amidated SEQ ID NOS. 167-171, 178 and 181-184 are assembled on the
2-chlorotritylchloride resin (200-400 mesh), 2% DVB (Novabiochem,
0.4-1.0 mmole/g)) using Fmoc-protected amino acids (Applied
Biosystems, Inc.), cleaved from the resin, deprotected and purified
in a similar way to that described in Example 95. Used in analysis
are Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN).
Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent A over
30 minutes) of the lyophilized peptide is then carried out to
determine the retention time of the product peptide. Electrospray
Mass Spectrometry provides an experimentally determined (M).
EXAMPLE 186
[0571] Evaluation of Ability to Cross Placenta
[0572] I. Introduction
[0573] The purpose of this experiment was to determine whether this
exendin-4, when delivered to the maternal circulation, is
transported across the placenta and is detectable in amniotic fluid
or fetal blood.
[0574] II. Materials and Methods
[0575] Animals:
[0576] Female Harlan Sprague Dawley rats (age 12 weeks, 17-21 days
pregnant, approximately 300 grams) were housed at
22.8.+-.0.8.degree. C. in a 12:12 hour light dark cycle. All
experiments were performed during the light cycle. Animals were
given free access to food and water until the start of the
experiment.
[0577] Sample Collection:
[0578] Rats were anesthetized with 5% halothane and then maintained
with 2% halothane during the surgical procedures.
[0579] Body temperature was measured and controlled using a
thermistor probe/controller (Model 73A, YSI, Yellow Springs, Ohio)
and a heated operating table. Blood was collected from the tail
vein immediately prior to a subcutaneous injection of exendin-4
(AC2993 Amylin Pharmaceuticals, Inc.) or vehicle (100.mu.l 0.15M
NaCl) at t=0. At t=30 minutes, when plasma concentrations following
a subcutaneous injection have been found to be maximal, another
blood sample was taken. Immediately thereafter, a midline
laparotomy was made to expose the uterine horns. Fluid was
collected from the individual amniotic sacs by aspiration through a
16 g needle into a syringe. The amniotic fluids from individual
fetuses were pooled from a given rat, but fluids from each rat were
kept separate. Fetal blood was collected by heart puncture with a
28 g microfine needle and aspirated into a syringe. Amniotic fluid
and fetal blood samples were collected within 10 minutes of when
the laparotomy was made. (t=30-40 min.). All blood and fluid
samples were centrifuged. The plasma or supernatant was stored at
-70.degree. C. until assayed.
[0580] Treatment Groups:
[0581] There were 2 treatment groups:
[0582] Group A: Rats receiving exendin-4 dissolved at 21 .mu.g/100
.mu.l in 0.15M NaCl n=4.
[0583] Group B: Rats receiving exendin-4 dissolved at 21 .mu.g/100
.mu.l in 0.15M NaCl n=5.
[0584] III. Results
[0585] Exendin-4 was not detected in any of the baseline samples,
taken at t=0, when measured by a specific IRMA
(immuno-radio-metric-assay) which has a LLQ (low limit of
quantitation) of 15 pM. At t=30 plasma levels of exendin-4 in the
mother rats that received 21 .mu.g exendin-4 were 16.47 nM.+-.2.45.
Values obtained from amniotic fluid (6.1.+-.5.3 pM) and fetal blood
(12.7.+-.6.5 pM) were 2700-fold and 1300-fold less than those in
plasma and were generally below the lower limit of quantitation of
the assay (FIG. 2). Similar results were obtained with the rats
receiving 210 .mu.g exendin-4 where plasma levels in the mother
rats at t=30 were 232.16 nM.+-.63.45 (FIG. 3). Values obtained from
amniotic fluid (18.3.+-.9.3 pM) and fetal blood (16.9.+-.13.8 pM)
were 12,680-fold and 13,750-fold less than those in plasma and were
undetectable in over half of the samples.
[0586] IV. Discussion
[0587] The placenta is the organ responsible for nutrient and waste
exchange between the fetus and the mother. Maternal and fetal
circulations are separated by an epithelial layer that allows or
denies diffusion or carrier mediated transport of substances across
the interface. The risk of adverse effects on the fetus can be
related to the extent to which the drug enters the fetal
circulation. The data obtained here indicate that, even with high
injected doses, which may exceed the per-kilogram doses
administered to humans by up to 3000-fold, little or no exendin-4
appeared in the fetal circulation or amniotic fluid. Six out of 15
measurements were above the lower limit of quantitation, and in 9
of 15, exendin-4 was undetectable. In those samples in which
exendin-4 was measurable, its presence may have been due to
contamination from maternal blood (which need be present only at
1:1,000-1:10,000 to be measurable). Such contamination is possible
following laparotomy of the dam and puncture of the fetus.
[0588] Various modifications of the invention in addition to those
shown and described herein will become apparent to those skilled in
the art from the foregoing description and fall within the scope of
the following claims.
* * * * *