Synthesis of VIP analog

Details Synthesis of VIP analog Synthesis of VIP analog

1998-12-18

2001-11-13

Low, Christopher S. F. (Department: 1653)

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, C530S330000, C530S333000, C530S338000, C530S339000

Reexamination Certificate

active

06316593

ABSTRACT:

TECHNICAL FIELD
This invention relates to a novel process for the synthesis of vasoactive intestinal peptide analog Ac(-31)-NH2 from four protected peptide fragments.
BACKGROUND OF THE INVENTION
Vasoactive intestinal peptide (VIP) is a smooth muscle relaxant/bronchodilator which regulates airway mucus secretion and has anti-allergic and anti-inflammatory properties. Recent studies have resulted in the discovery of an analog of VIP which possesses enhanced metabolic stability and has increased receptor-binding properties. This VIP analog is the subject of a co-pending patent application U.S. Ser. No. 08/308,729.
To date, this VIP analog has been prepared using solid phase synthesis. The solid phase synthesis includes attaching an alpha-amino acid, protecting with, for example t-butyloxycarbonyl (Boc), by ester linkage, to a chloromethylated resin or a hydroxymethyl resin. More amino acids are added sequentially to the resin. The alpha amino Boc protection is removed under acidic conditions and the subsequent protected amino acids are coupled stepwise to obtain an intermediate, protected peptide-resin. Blocking groups are removed and the peptide is cleaved from the resin through multiple hydrogen fluoride cleavage reactions. Purification of the peptides occurs in two stages, a) size exclusion gel chromatography and b) preparative high performance liquid chromatography (HPLC). This multistep process is time consuming and results in inefficient recovery of the target peptide.
It is thus an object of the present invention to provide a relatively simple, more efficient and economic procedure for the synthesis of the VIP analog.
SUMMARY OF THE INVENTION
The present invention provides a novel process for the synthesis of a VIP analog, AC(1-31)-NH
2
, having the formula
from four protected peptide fragments. The novel process according to the present invention does not require prior preparative HPLC purification of the peptide fragments as is required when the analog is prepared by solid phase synthesis, nor does it require purification of the intermediates formed during the synthesis of the target cyclic VIP analog. The resulting product was purified in the final stage after assembly by a single pass via preparative HPLC.
DETAILED DESCRIPTION OF THE INVENTION
The method of the present invention for the synthesis of the cyclic VIP analog Ac-(1-31)-NH
2
comprises the repetitive assemblage of four protected peptide fragments: Fragment I, Fmoc-(26-31)-NH
2
(SEQ ID NO: 2); Fragment II, Fmoc-(19-25)-OH (SEQ ID NO:3); Fragment III, Fmoc-(9-18)-OH (SEQ ID NO:4); and Fragment IV, Ac-(1-8)-OH (SEQ ID NO:5), each of which is shown below.
The method of the present invention for the synthesis of a compound Ac-(1-31)-NH
2
(SEQ ID NO:1) by coupling four Fmoc protected peptide fragments, peptide Fragment I (SEQ ID NO:2), peptide Fragment II (SEQ ID NO:3), peptide Fragment III (SEQ ID NO: IV) and peptide Fragment IV (SEQ ID NO:5) comprises (a) deprotecting the Fmoc-protecting group of peptide Fragment I and coupling the deprotected peptide Fragment I with protected peptide Fragment II; (b) deprotecting the Fmoc-protecting group of the resulting peptide of step (a) and coupling it with protected Fragment III; (c) deprotecting the Fmoc-protecting group of the resulting peptide of step (b) and coupling it with protected Fragment IV; (d) deprotecting the resulting protected peptide of step (c) to yield deprotected Ac(1-31)-NH
2
.
The protected peptide fragments I-IV were selected on the basis of maximum coupling efficiency and minimal racemization of the product of each coupling reaction. Equivalent amounts of each fragment were used for each coupling reaction, providing an economic pathway to the target peptide. The intermediates formed after each coupling were used directly for subsequent coupling reactions without further purification.
The purity of each fragment produced after solid phase synthesis, as described herein, was from about 82% to about 97% after a single purification step as determined by analytical HPLC, and each fragment was used for the synthesis of the cyclic VIP analog without further purification.
More particularly the method for the synthesis of the purified compound of the formula Ac-(1-31)-NH
2
(SEQ ID NO:1) comprises: (a) deprotecting the Fmoc-protecting group of peptide Fragment I (SEQ ID NO:2) and coupling the deprotected peptide Fragment I with protected peptide Fragment II (SEQ ID NO:3) yielding protected intermediate peptide Fmoc(19-31)-NH
2
(SEQ ID NO:6); (b) deprotecting the Fmoc-protecting group of intermediate Fmoc(19-31)-NH
2
and coupling the deprotected intermediate Fmoc(19-31)-NH
2
with protected Fragment III (SEQ ID NO: IV) yielding protected intermediate peptide Fmoc(9-31)-NH
2
(SEQ ID NO:7); (c) deprotecting the Fmoc-protecting group of intermediate Fmoc(9-31)-NH
2
and coupling the deprotected intermediate Fmoc(9-31)-NH
2
with protected Fragment IV (SEQ ID NO:5) yielding protected intermediate peptide Ac-(1-31)-NH
2
; (d) deprotecting the protected peptide Ac(1-31)-NH
2
; and (e) purifying the deprotected peptide Ac(1-31)-NH
2
, for example, via preparative HPLC.
As used herein, the nomenclature used to define the peptides is that typically used in the art, wherein the amino group at the N-terminus appears to the left and the carboxyl group at the C-terminus appears to the right. By natural amino acids is meant one of the naturally occurring amino acids found in proteins, i.e., Gly, Ala, Val, Leu, Ile, Ser, Thr, Lys, Arg, Asp, Asn, Glu, Gln, Cys, Met, Phe, Tyr, Pro, Trp, and His. Where the amino acid has isomeric forms, it is the L form of the amino acid that is represented, unless otherwise expressly indicated.
The following abbreviations or symbols are used to represent amino acids in addition to those described elsewhere herein, protecting groups, solvents, reagents and the like.
Symbol
Meaning
Ac
Acetyl
Nle
Norleucine
Fm
9-Fluorenylmethyl
DIPEA
N,N-Diisopropylethylamine
DMF,
Dimethylformamide
The suffixes “—OH” and “—NH
2
” following “VIP” refer to the free acid and amide forms of the polypeptide, respectively. In the event neither suffix is used, the expression is intended to encompass both forms.
A cyclic peptide, as defined herein, is a peptide wherein the side chain carboxy terminus of one amino acid in the peptide is attached covalently to the side chain amino terminus of another amino acid in the peptide chain via formation of an amide bond. Several nomenclatures and symbols are utilized to represent a cyclic peptide. The following are examples:
a. cyclo(Lys
21
-Asp
25
)-Fmoc-Ala
19
-Lys(Boc)
20
-Lys
21
-Tyr(tBu)
22
-Leu
23
-Asn
24
-Asp
25
-OH;
b. Fmoc-(19-25)-OH;
c. Fmoc-[Ala
19
-Asp
25
]-VIP cyclo(21→25);
d. [Fmoc-(SEQ ID NO:3)-OH];
e. Fmoc-[Ala
19
-Asp
25
]-VIP cyclo (Lys
21
→Asp
25
);
f. [Fmoc-(SEQ ID NO:3)-OH];
The above structures (a-g), and the representation using the “(SEQ ID NO:)” each represent and define the same peptide having an amino acid sequence corresponding to a VIP peptide fragment in which an Fmoc group has been substituted for hydrogen at the N-terminus. Additionally, an amide bond has been formed between the side chain carboxyl of the lysine at position 21 and the side chain amine of aspartic acid at position 25, thus forming the cyclic peptide fragment. The above representations for the peptide structure are considered to be equivalent and interchangeable.
In the cyclic peptides of the present invention, the following configurations apply unless otherwise stated.
Amino Acid
Terminus of amino acid
in chain
bound to make cyclic peptide
Lys
&egr; amino &egr;
Asp
&bgr; cwarboxyl (&bgr; = beta)
Glu
&ggr; carboxyl (&ggr; = gamma)
The peptide fragments which comprise the VIP analog of the present invention may be readily synthesized by any known conventional procedure for the formation of a peptide linkage between amino acids. Such conventional procedures include, for example, any solution phase procedure permitting a condensation between the free alpha amino gr

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