Branched building units for synthesizing cyclic peptides

Details Branched building units for synthesizing cyclic peptides Branched building units for synthesizing cyclic peptides

1999-02-08

2001-02-06

Borin, Michael (Department: 1631)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

Peptides with at least one nonpeptide bond other than a...

C530S332000, C530S333000

Reexamination Certificate

active

06184345

ABSTRACT:

TECHNICAL FIELD
The present invention relates to a method for preparing branched building units for use in the synthesis of backbone cyclized peptides, and to the building units themselves.
BACKGROUND OF THE INVENTION
Biologically active peptides are conformationally restricted through cyclization to achieve metabolic stability, to increase potency, to confer or improve receptor selectivity, and to control bioavailability. The use of medium and long-range cyclization to convert natural bioactive peptides into potential peptidomimetic drugs has been prompted by the ability to control these important pharmacological characteristics. Furthermore, the structural constraints on a peptide brought about by cyclization also enhance conformational homogeneity and facilitate conformational analysis. See, e.g., Kessler, H.
Angew. Chem. Int. Ed. Eng.
21:512 (1982). Thus, cyclization may give insight into the biologically active conformations of linear peptides provided that their biological activities and selectivities are maintained.
Common modes of long-range (or global) peptide cyclization include side-chain to side-chain, end-group to end-group, and side-chain to end-group, all of which require the alteration or removal of residue side chains. See, e.g., Manesis, N. J., and Goodman, M.
Org. Chem.
52:5331 (1987). Backbone cyclization, wherein a connection is made between the N
&agr;
and/or C
&agr;
atoms of a peptidic backbone, does not require such disruption of the peptide's natural structure because only the hydrogens of the peptide bond are affected. The hydrogens are replaced by &ohgr;-functionalized chains that can be interconnected, connected to residue side-chains, or connected to the ends of a peptide to form the desired cyclic peptide. Backbone cyclization can thus stabilize a peptide's bioactive conformation and protect against its enzymatic degradation without altering its side chains.
Although different methods of backbone cyclization exist, a preferred method uses dipeptide building blocks. Gilon and coworkers have disclosed backbone to side-chain and backbone to C-terminus peptide cyclization using lactam and disulfide bridges formed from such building blocks. This was done with the use of N-aminoalkyl amino acids obtained either by the alkylation of amino, carboxy or thiol alkyl amines with triflates of &agr;-hydroxy acids, or by the nucleophilic substitution of alkylene diamines. U.S. Pat. No. 5,723,575 and Gilon et al.,
J. Org. Chem.
57: 5687-5692 (1992) (collectively “Gilon”).
In the first method of synthesizing N-aminoalkyl amino acids, a diamine is reacted with an &agr; bromo acid to provide an &ohgr; amine which is then selectively protected. Variation of the protecting group provides a building unit suitable for Boc chemistry peptide synthesis. In the second method of synthesizing these building units, a selectively protected diamine is reacted with chloroacetic acid to provide a protected glycine derivative suitable for Fmoc peptide synthesis.
In order to take advantage of the facile nucleophilic displacement of carboxylic acid substituents, both synthetic methods described by Gilon require the reaction of a molecule of formula Halides-CH(R)—CO—OR′ (wherein Halides represents a halogen leaving group) and an amine. The amine bears an alkylidene chain that is terminated by another amine, as shown in Scheme (I):
The terminating nitrogen atom of the resulting building unit will be contained by the moiety used to form the bridging chain of a cyclized peptide.
In a molecule where R is other than hydrogen, there is a high tendency to eliminate H-Halides under basic conditions. And because the secondary amine formed by the addition reaction is a better nucleophile than the primary amine of the diamine reactant, double alkylation products may form. This side reaction reduces the yield of the method shown in Scheme (I) to such an extent that it cannot be used for the practical production of building units based on amino acids other than glycine. Gilon, however, does not suggest backbone cyclization building units having end-group moieties that are not amine, and so only provides compounds useful in backbone to side-chain and backbone to C terminus peptide cyclization.
Other workers have described alternative backbone cyclization building blocks. For example, lactam bridges have been formed with the protected building block HN(CH
2
COOBu
t
)Phe, although the synthesis of bradykinin analogues using the Boc protected building block was reportedly hindered by low yields and undesired double couplings. Reissman et al.
Biomedical Peptides, Proteins
&
Nucleic Acids
1:51-6 (1994). Increased efficiency was obtained, however, when the protecing group N,O-bis(trimethylsilyl)-acetamide was used. The cyclic peptides made with the building block, which contain N-alkylamide bonds, were reported to be unstable under acidic conditions typical of solid phase peptide synthesis.
The synthesis of backbone cyclized peptides using glycine-based building blocks has also been reported by Zuckermann et al.
J. Am. Chem. Soc.
114: 10646-10647 (1994). This synthetic approach, which is limited to the solid phase preparation of N-substituted glycine oligomers consists of two steps: first, a resin bound secondary amine is alkylated; and second, a side-chain is introduced into the polypeptide by nucleophilic displacement of a halogen with an excess of a primary amine.
A more general method for the synthesis of backbone cyclized peptides is disclosed by Kaljuste et al.
Int. J. Peptide Protein Res.
43: 505-511 (1994). By utilizing amino acid aldehydes, this method allows the formation of branched building units on a solid support, but requires that both the backbone and the branching chain of the resulting cyclic peptide contain reduced peptide bonds. The method is further limited by synthetic and storage problems associated with amino acid aldehydes, the relatively slow rates of alkylation of the reduced peptide bonds formed during the reaction, and the residue formed from sequence-specific side reactions that can occur during the reductive alkylation of reduced peptide bonds. Additional purification and racemization problems are also reported.
As made clear above, methods of peptide backbone cyclization have been constrained by the limited variety of building blocks. For example, the use of similar building block protecting groups can form hydrophobic clusters along the side chains of a growing peptide which can reduce coupling rates, reaction efficiencies and yields. Furthermore, some protecting groups can cause the racemization of terminal amino acids during the synthesis of a cyclic peptide. A third drawback of many building blocks is that cyclic structures formed from them degrade under the acidic and/or basic conditions typical of solid phase synthetic methods.
There thus exists a need for a larger variety of building units suitable for the synthesis of cyclic peptides, and a facile and efficient method of synthesizing such building units. There further exists a need for building units that are stable under solid phase synthetic conditions.
SUMMARY OF THE INVENTION
Accordingly, the present invention relates to building units suitable for the synthesis of backbone cyclized peptides. The building units of this invention have structures of Formula (I):
X—AA
m
—U—AA
p
—Y  (I)
wherein AA
m
and AA
p
are residues of natural or synthetic amino acids. U refers to C
&agr;
ON[(CH
2
)
n+1
W] or &PSgr;(C
&agr;
H
2
N)[CO(CH
2
)
q
W], in which C
&agr;
is a carbon atom and is the a carbon of AA
m
if m>0, with respect to N. W is NH—X or COY, and X is H, Boc, Z, Fmoc, or Alloc while Y is the hydroxyl group of a carboxylic acid moiety or a carboxylic acid protecting group. The letter m refers to an integer from 0 to 10; n is a number from 0 to 6; p is a number from 1 to 10; and q is a number from 1 to 6. The present invention also is directed to methods of making building units of Formula (I).
The invention more specifically is directed to a me

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