Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 15 to 23 amino acid residues in defined sequence
Reexamination Certificate
1998-03-23
2001-08-28
Low, Christopher S. F. (Department: 1654)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
15 to 23 amino acid residues in defined sequence
C530S327000, C530S328000, C530S329000, C530S333000, C514S013800, C514S014800, C514S015800, C514S016700
Reexamination Certificate
active
06281331
ABSTRACT:
INTRODUCTION
The present invention relates, first, to methods for the synthesis of peptides, in particular T-20 (also referred to as “DP-178”; SEQ ID NO:1) and T-20-like peptides. Such methods utilize solid and liquid phase synthesis procedures to synthesize and combine groups of specific peptide fragments to yield the peptide of interest. The present invention further relates to individual peptide fragments which act as intermediates in the synthesis of the peptides of interest (e.g., T-20). The present invention still further relates to groups of such peptide intermediate fragments which can be utilized together to produce full length T-20 and T-20-like peptides.
BACKGROUND
Recently, a large number of peptides have been identified which exhibit an ability to inhibit fusion-associated events, and, importantly, also exhibit potent antiviral activity. See, for example, U.S. Pat. Nos. 5,464,933; 5,656,480 and PCT Publication No. WO 96/19495T-20. As these peptides to extensively be used, as therapeutics, for example, the need arises for an ability to synthesize in large scale quantities.
While techniques exist for peptide synthesis, (see, e.g., Mergler et al., 1988, Tetrahedron Letters 29:4005-4008; Mergler et al., 1988, Tetrahedron Letters 29:4009-4012; Kamber et al. (eds), “Peptides, Chemistry and Biology, ESCOM, Leiden, 1992, 525-526; and Riniker et al., 1993, Tetrahedron Letters 49:9307-9320) no techniques currently exist which can be utilized for large scale, economical production of easily purified peptides such as T-20 and T-20-like peptides.
SUMMARY OF THE INVENTION
The present invention relates, first, to methods for the synthesis of peptides, in particular T-20 (also referred to as “DP-178”; SEQ ID NO:1) and T-20-like peptides. Such methods utilize solid and liquid phase synthesis procedures to synthesize and combine groups of specific peptide fragments to yield the peptide of interest. Generally, the methods of the invention comprise synthesizing specific side-chain protected peptide fragment intermediates of T-20 or a T-20-like peptide on a solid support, coupling the protected fragments in solution to form a protected T-20 or T-20-like peptide, followed by deprotection of the side chains to yield the final T-20 or T-20-like peptide. A preferred embodiment of the methods of the invention involves the synthesis of a T-20 peptide having an amino acid sequence as depicted in SEQ ID NO:1.
The present invention further relates to individual peptide fragments which act as intermediates in the synthesis of the peptides of interest (e.g., T-20). The peptide fragments of the invention include, but are not limited to, those having amino acid sequences as depicted in Table 1 below:
TABLE 1
CORRESPONDING
NUMBERED AMINO ACID
PEPTIDE NO.
AMINO ACID SEQUENCE
SEQUENCE OF T-20
1
YTSLIHSL
(SEQ ID NO:2)
1-8
2
YTSLIHSLIEESQNQ
(SEQ ID NO:3)
1-15
3
YTSLIHSLIEESQNQQ
(SEQ ID NO:4)
1-16
4
YTSLIHSLIEESQNQQEK
(SEQ ID NO:5)
1-18
5
IEESQNQ
(SEQ ID NO:6)
9-15
6
IEESQNQQ
(SEQ ID NO:7)
9-16
7
QEKNEQELLELDKWASLWNW
(SEQ ID NO:8)
16-35
8
QEKNEQELLELDKWASLWNWF
(SEQ ID NO:9)
16-36
9
EKNEQEL
(SEQ ID NO:10)
17-23
10
EKNEQELLEL
(SEQ ID NO:11)
17-26
11
EKNEQELLELDKWASLWNWF
(SEQ ID NO:12)
17-36
12
NEQELLELDKWASLWNW
(SEQ ID NO:13)
19-35
13
NEQELLELDKWASLWNWF
(SEQ ID NO:14)
19-36
14
LELDKWASLWNW
(SEQ ID NO:15)
24-35
15
LELDKWASLWNWF
(SEQ ID NO:16)
24-36
16
DKWASLWNW
(SEQ ID NO:17)
27-35
17
DKWASLWNWF
(SEQ ID NO:18)
27-36
18
EKNEQELLELDKWASLWNW
(SEQ ID NO:19)
17-35
The present invention still further relates to particular groups of peptide fragments which act as intermediates in the synthesis of the peptide of interest. The groups of peptide fragments according to the invention include Groups
1
-
20
, as designated in Table 2 below.
TABLE 2
Corresponding
Numbered Amino
Acid Sequence of
Group
Amino Acid Sequence
T-20
1
YTSLIHSLIEESQNQQ
(SEQ ID NO:4)
1-16
EKNEQELLELDKWASLWNWF
(SEQ ID NO:12)
17-36
2
YTSLIHSLIEESQNQQ
(SEQ ID NO:4)
1-16
EKNEQELLEL
(SEQ ID NO:11)
17-26
DKWASLWNWF
(SEQ ID NO:18)
27-36
3
YTSLIHSLIEEQNQQ
(SEQ ID NO:4)
1-16
EKNEQELLEL
(SEQ ID NO:11)
17-26
DKWASLWNW
(SEQ ID NO:17)
27-35
4
YTSLIHSL
(SEQ ID NO:2)
1-8
IEESQNQ
(SEQ ID NO:6)
9-15
EKNEQELLELDKWASLWNWF
(SEQ ID NO:12)
17-36
5
YTSLIHSL
(SEQ ID NO:2)
1-8
IEESQNQ
(SEQ ID NO:6)
9-15
EKNEQELLEL
(SEQ ID NO:11)
17-26
DKWASLWNWF
(SEQ ID NO:18)
27-36
6
YTSLIHSL
(SEQ ID NO:2)
1-8
IEESQNQ
(SEQ ID NO:6)
9-15
EKNEQELLEL
(SEQ ID NO:11)
17-26
DKWASLWNW (SEQ ID NO:17)
27-35
7
YTSLIHSL
(SEQ ID NO:2)
1-8
IEESQNQQ
(SEQ ID NO:7)
9-16
EKNEQELLELDKWASLWNWF
(SEQ ID NO:12)
17-36
8
YTSLIHSL
(SEQ ID NO:2)
1-8
IEESQNQQ
(SEQ ID NO:7)
9-16
EKNEQELLEL
(SEQ ID NO:11)
17-26
DKWASLWNWF
(SEQ ID NO:18)
27-36
9
YTSLIHSL
(SEQ ID NO:2)
1-8
IEESQNQQ
(SEQ ID NO:7)
9-16
EKNEQELLEL
(SEQ ID NO:11)
17-26
DKWASWNW
(SEQ ID NO:17)
27-35
10
YTSLIHSLIEESQNQQ
(SEQ ID NO:4)
1-16
EKNEQEL
(SEQ ID NO:10)
17-23
LELDKWASLWNWF
(SEQ ID NO:16)
24-36
11
YTSLIHSLIEESQNQQ
(SEQ ID NO:4)
1-16
EKNEQEL
(SEQ ID NO:10)
17-23
LELDKWASLNWN
(SEQ ID NO:15)
24-35
12
YTSLIHSL
(SEQ ID NO:2)
1-8
IEESQNQ
(SEQ ID NO:6)
9-15
EKNEQEL
(SEQ ID NO:10)
17-23
LELDKWASLWNWF
(SEQ ID NO:16)
24-36
13
YTSLIHSL
(SEQ ID NO:2)
1-8
IEESQNQ
(SEQ ID NO:6)
9-15
EKNEQEL
(SEQ ID NO:10)
17-23
LELDKWASLWNW
(SEQ ID NO:15)
24-35
14
YTSLIHSL
(SEQ ID NO:2)
1-8
IEESQNQQ
(SEQ ID NO:7)
9-16
EKNEQEL
(SEQ ID NO:10)
17-23
LELDKWASLWNWF
(SEQ ID NO:16)
24-36
15
YTSLIHSL
(SEQ ID NO:2)
1-8
IEESQNQQ
(SEQ ID NO:7)
9-16
EKNEQEL
(SEQ ID NO:10)
17-23
LELDKWASLWNW
(SEQ ID NO:15)
24-35
16
YTSLIHSLIEESQNQ
(SEQ ID NO:3)
1-15
QEKNEQEKKEKDKWASLWNWF
(SEQ ID NO:9)
16-36
17
YTSLIHSLIEESQNQ
(SEQ ID NO:3)
1-15
QEKNEQELLELDKWASLWNW
(SEQ ID NO:8)
16-35
18
YTSLIHSLIEESQNQQEK
(SEQ ID NO:5)
1-18
NEQELLELDKWASLWNWF
(SEQ ID NO:14)
19-36
19
YTSLIHSLIEESQNQQEK
(SEQ ID NO:5)
1-18
20
NEQELLELDKWASLWNW
(SEQ ID NO:13)
19-35
YTSLIHSLIEESQHQQ
(SEQ ID NO:4)
1-16
EKNEQELLELDKWASLWNW
(SEQ ID NO:19)
17-35
This invention is based, in part, on the inventors' unexpected discovery that certain combinations of solid phase liquid phase synthetic reactions allow high purity T-20 and T-20-like peptides to be manufactured for the first time on a large scale with high throughput and high yield. It has been found that by selecting the specific T-20 peptide fragments of the invention for solid phase synthesis, the highly efficient coupling of solid phase techniques may be exploited without having to use the 3-, 4- or even 5-fold excess of amino acids and reagents that are normally required in solid phase synthesis. The methods of the invention use only about a 1.5-fold of amino acid in the solid phase synthesis of the peptide fragments of the invention. This cost-saving reduction in the amount of amino acid and reagents makes the methods of the invention suitable for large scale synthesis of T-20 and T-20-like peptides.
In addition, the inventors have surprisingly found that certain peptide fragments may be synthesized in the solid phase at a loading of about 0.8 to 1 mmol per gram of solid phase resin. This loading is significantly greater than the loading range of 0.25 to 0.4 mmol per gram of resin typically achieved in solid phase peptide synthesis. Moreover, the inventors have found that synthesizing selected peptide fragments in the solid phase using super acid sensitive resin produces peptide fragments of unusually high purity. Chromatographic techniques are not necessary to purify the peptide fragments produced according to the invention; the fragments are simply put through precipitation and/or trituration steps before use, or used as obtained directly from the resin. Use of a super acid sensitive resin allows the synthesized, protected peptides of the invention to be cleaved from the resin without concomitant removal of the side-chain protecting groups. This reduces impurities, and allows peptides comprising 10 amino acids or greater to
Bray Brian
Kang Myung-Chol
Lichty Maynard
Mader Catherine
Gupta Anish
Low Christopher S. F.
Pennie & Edmonds LLP
Trimeris, Inc.
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