Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Synthesis of peptides
Reexamination Certificate
2000-01-28
2002-11-05
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Synthesis of peptides
C530S333000, C525S054100, C525S054110, C523S466000, C423S335000
Reexamination Certificate
active
06476191
ABSTRACT:
TECHNICAL FIELD
The invention relates to solid phase syntheses, and more particularly to solid phase synthesis on a synthetic support that is volatilized upon the cleavage of the synthesized material from the support.
BACKGROUND OF THE INVENTION
The preparation of compounds using a solid phase approach was first described by Merrifield in 1963 [Merrifield, 1963,
J. Am. Chem. Soc.,
85:2149-2154. Since this initial seminal concept, in which a polystyrene solid phase was used to prepare peptides, a wide range of different solid supports have been used (i.e., polyamides [Atherton et al., 1975,
J. Am. Chem. Soc.,
97:6584-6585], porous glass [Parr et al., 1974.
Justus Liebigs Ann. Chem.,
pp. 655-666] and microchip quartz [Fodor et al., 1991,
Science,
251:767-773]). While useful, these solid phase supports all require a final cleavage step, in which the compounds (peptides, peptidomimetics, oligonucleotides, small organic molecules, various heterocycles, and the like) are cleaved from the solid phase, then separated from the spent solid support.
Where the compound of interest can be used in an immobilized manner (i.e., it remains on the solid support in its final use and/or manifestation), then the remaining solid support may not be problematic, and in fact may be useful for certain assays. However, in the majority of cases, the compound of interest has to be used in solution and therefore has to be separated from its solid support. Significant time and/or cost savings would be realized if the removal of the solid phase material did not have to be accomplished in a separate step following cleavage of the desired compound from the solid support (typically by filtration or centrifugation). The invention disclosed hereinafter provides one solution to the problem of separating the spent solid support from the desired synthesized material.
BRIEF SUMMARY OF THE INVENTION
The present invention contemplates solid phase synthesis on a solid support in which the desired product is left behind following cleavage from and vaporization of the solid. Thus, a solid phase synthesis method is contemplated in which at least one reagent is coupled to a solid phase support. A plurality of reactions is carried out upon the solid phase-coupled reagent to form a solid phase-coupled reaction product, and the reaction product is cleaved from the solid phase support to form a cleaved product. The improvement in this otherwise standard synthesis is that the solid phase support is reacted to form a volatile compound(s) that is separated from the cleaved product by vaporization as by distillation. The desired cleaved product is preferably recovered and the solid support is absent due to its volatilization.
A particularly preferred solid support is silica. Cleavage of the product from the solid support and formation of the volatile compound is typically carried out in a single step, although separate steps can be used.
The present invention has several benefits and advantages.
One benefit is the simplicity in reaction steps that are carried out in that the usual filtering step required in prior solid phase syntheses is not required.
An advantage of a contemplated method is that losses of desired product that can occur because of entrapment of the desired product within the usual spent solid support or filter do not occur.
Another benefit is that the usual final extraction step(s) to remove the product from the solid support required in prior solid phase syntheses after cleavage from the solid support is not required here.
Still further benefits and advantages of the contemplated invention will be apparent to the skilled worker from the disclosure that follows.
DETAILED DESCRIPTION OF THE INVENTION
A solid phase synthetic method is contemplated in which the usual solid phase synthetic steps are carried out in the synthesis of a peptide, peptidomimetic, glycopeptide, oligonucleotide, small organic molecules, or heterocyclic product as noted hereinafter. The improvement here lies in the separation of the cleaved product from the solid support by conversion of the solid phase support into a volatile material that is separated from the desired reaction product by vaporization so that the usually used filtration or extraction separation of the desired product from the spent solid phase support is unnecessary.
Thus, taking a solid phase peptide synthesis as exemplary, at least one reagent such as a side chain- and N-protected amino acid is coupled to the solid support. A plurality of reactions is carried out on that solid phase-coupled reagent such as N-de-protection, coupling of another side chain- and N-protected amino acid, and N-de-protecting the resulting product to form a solid phase-coupled reaction product. Any side chain protecting groups present are removed, and the link between solid support and desired product is broken to form a cleaved product. A volatile compound is formed from the spent solid support. In preferred practice for peptide synthesis, HF is used to remove any side chain protecting groups present, cleave the product from the solid support and form the volatile compound(s) from the spent solid support.
As used herein, the material formed on the solid phase support and bonded thereto is referred to as a “reaction product”. The reaction product can have protecting groups bonded to it or those protecting groups can be removed.
The “cleaved product” is that material obtained upon breaking of the bond between the solid phase support and the reaction product. The cleaved product is typically free of protecting groups but need not be so. In addition, the cleaved product is typically protonated, although protonation is not a defining feature of a cleaved product.
A “spent solid support” is the material remaining after cleavage of the desired reaction product from the support. As discussed below, the solid support is preferably converted into a volatile compound concomitantly with formation of the cleaved product. In that preferred case, there is usually no spent solid support.
For example, porous glass has been used as a solid support to prepare a peptide with cleavage of the desired product effected by reaction of the solid phase-bound peptide with methanol and triethylamine. [Parr et al., 1974,
Justus Liebigs Ann. Chem.,
pp. 655-666.] Contrarily, using a contemplated method, the porous glass can be completely transformed by liquid or gaseous hydrogen fluoride into volatile silicon tetrafluoride (SiF
4
, bp: −86° C.) that can be warmed or a vacuum applied to effect separation, as compared to use of a reagent that cleaves the compound from the support followed by filtration of the spent solid support from the desired compound as was carried out by Parr et al. Use of a contemplated method leaves the desired compound in the reaction container, with the porous glass solid support volatilized away as SiF
4
.
This concept greatly facilitates the production of individual compounds or mixtures of compounds, or the large scale production of individual compounds, arrays of compounds, or combinatorial libraries of mixtures [Plunkett et al., 1995,
J. Org. Chem.,
60:6006-6007; Houghten, 1985,
Proc. Natl. Acad. Sci. USA,
82:5131-5135; Houghten et al., 1991,
Nature,
354:84-86; Pinilla et al., 1992,
BioTechniques
13: 901-905; Ostresh et al., 1994,
Proc. Natl. Acad. Sci. USA,
91: 11138-11142; Dooley et al., 1994,
Science,
266:2019-2022; and Eichler et al., 1995,
Molecular Medicine Today
1:174-180]. In addition, when working with mixtures of compounds, the risk of losing part of the compounds during the separation process of the solid phase (filtration or centrifugation) is minimized.
The present invention also contemplates the use of so-called non-cleavable linkers in connection with such volatilizable solid supports. A non-cleavable linker is a linker that remains bonded to the cleaved product, but is cleaved from the support. This use leads, after cleavage, to a modified compound (compound attached to linker) that can be of interest
Houghten Richard A.
Moran Michael J.
Pascal Jeanick H.
Low Christopher S. F.
Lukton David
Mixture Sciences, Inc.
Welsh & Katz Ltd.
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