Methods and compositions for peptide synthesis

Details Methods and compositions for peptide synthesis Methods and compositions for peptide synthesis

2002-03-29

2004-07-27

Horlick, Kenneth R. (Department: 1637)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

Synthesis of peptides

C435S069100, C530S300000, C530S333000, C530S350000, C530S402000, C530S412000

Reexamination Certificate

active

06767993

ABSTRACT:

1. INTRODUCTION
The present invention relates, first, to methods for the synthesis of peptides, in particular peptides referred to herein as T-1249 (SEQ ID NO:1) and T-1249-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 can act as intermediates in the synthesis of the peptides of interest (e.g., T-1249). The present invention still further relates to groups of such peptide intermediate fragments which can be utilized together to produce full-length T-1249 and T-1249-like peptides. The present invention still further relates to methods for the purification of peptides, in particular T-1249 and T-1249-like peptides, and the individual peptide fragments which act as intermediates in the synthesis of the subject peptides.
2. 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; PCT Publication Nos. WO 94/28920; WO 96/19495. As these peptides come to be used extensively, for example as therapeutics, the need arises for an ability to synthesize them 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-1249.
3. SUMMARY OF THE INVENTION
The present invention relates, first, to methods for the synthesis of peptides, in particular peptides referred to herein as T-1249 (SEQ ID NO:1) and T-1249-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-1249 or a T-1249-like peptide on a solid support, coupling the protected fragments in solution to form a protected T-1249 or T-1249-like peptide, followed by deprotection of the side chains to yield the final T-1249 or T-1249-like peptide. A preferred embodiment of the methods of the invention involves the synthesis of a T-1249 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-1249). 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
SEQUENCE OF
PEPTIDE
T-1249
NO.
AMINO ACID SEQUENCE
(SEQ. ID NO:1)
1
WQEWEQKITALL
(SEQ ID NO:2)
 1-12

2
EQAQIQQEKNEYEL
(SEQ ID NO:3)
13-26

3
QKLDKWASLWEW
(SEQ ID NO:4)
27-38

4
QKLDKWASLWEWF
(SEQ ID NO:5)
27-39

5
EQAQIQQEKNEYELQKLDKWASLWEWF
(SEQ ID NO:6)
13-39

6
WQEWEQKITALLEQAQIQQEKNEYEL
(SEQ ID NO:7
 1-26

7
EQAQIQQEKNEYELQKLDKWASLWEW
(SEQ ID NO:8)
13-38
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 1-6, as designated in Table 2 below.
TABLE 2
Corresponding
Numbered Amino
Acid Sequence of
T-1249
Group
Amino Acid Sequence
(SEQ. ID NO:1)
1
WQEWEQKITALL
(SEQ ID NO:2)
 1-12

EQAQIQQEKNEYEL
(SEQ ID NO:3)
13-26

QKLDKWASLWEW
(SEQ ID NO:4)
27-38

2
WQEWEQKITALL
(SEQ ID NO:2)
 1-12

EQAQIQQEKNEYEL
(SEQ ID NO:3)
13-26

QKLDKWASLWEWF
(SEQ ID NO:5)
27-39

3
WQEWEQKITALL
(SEQ ID NO:2)
 1-12

EQAQIQQEKNEYELQKLDKWASLWEWF
(SEQ ID NO:6)
13-39

4
WQEWEQKITALL
(SEQ ID NO:2)
 1-12

EQAQIQQEKNEYELQKLDKWASLWEW
(SEQ ID NO:8)
13-38

5
WQEWEQKITALLEQAQIQQEKNEYEL
(SEQ ID NO:7)
 1-26

QKLDKWASLWEW
(SEQ ID NO:4)
27-38

6
WQEWEQKITALLEQAQIQQEKNEYEL
(SEQ ID NO:7)
 1-26

QKLDKWASLWEWF
(SEQ ID NO:5)
27-39
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-1249 and T-1249-like peptides to be manufactured for the first time on a large scale with high throughput and high yield. In particular, in accordance with the methods of the invention, T-1249 and T-1249-like peptides may be synthesized on a scale of one or more kilograms. It has been found that by selecting the specific T-1249 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 an 0.5-fold excess (about 1.5 equivalents) of amino acid in the solid phase synthesis of the peptide fragments of the invention. This reduction in the amount of amino acid and reagents makes the methods of the invention suitable for large scale synthesis of T-1249 and T-1249-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 >0.5 mmol per gram of solid phase resin. This loading significantly enhances throughput over the loading range of 0.25 to 0.35 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 be synthesized in high purity and yield.
The impurity profile of T-1249 and T-1249-like peptides which are synthesized in the solution phase according to the methods of the invention by coupling of the high purity peptide fragments produced according to the invention consists of fragments that did not couple, and contains significantly lower levels of closely related deletion analogues than T-1249 and T-1249-like peptides synthesized according to conventional techniques, e.g., solely solid phase synthesis. Accordingly, T-1249 and T-1249-like peptides produced according to the invention are much easier to purify than those produced according to conventional techniques. In particular, T-1249 produced according to the methods of the present invention may be easily purified to >90% purity in single pass chromatography. For example, in accordance with the methods of the invention, T-1249 can be purified in amounts of 400 g or more using a 5 inch column. In contrast, T-1249 prepared using conventional solid phase synthesis (SPPS) is very difficult to purify, requiring multiple pass chromatography. By way of example, purification of T-1249 prepared by SPPS typically results in 10 grams or less of purified material from an 8 inch column.
The Examples presented in Section 9 below, demonstrate such combinatorial syntheses of T-1249 full-length peptides. The T-1249 and T-1249-like peptides and intermediates may be produced on a scale of one or more kilograms by the methods of the invention.
The present inventors have also unexpectedly found that peptides

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