Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Synthesis of peptides
Patent
1999-03-09
2000-02-22
Woodward, Michael P.
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Synthesis of peptides
530333, 530337, 530338, 422 99, 422103, 422129, 422238, 422239, A61K 3800, B01L 300, B01J 1900
Patent
active
060281723
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to solid phase peptide synthesis and, in particular, to a reactor system and method utilizing a novel solid phase reactor system in which solid phase peptide synthesis can be carried out on a large scale with increased outputs and yields over those currently available.
BACKGROUND ART
The broader applicability of our system and method to other liquid-solid reactions involving stepwise synthesis on solid substrates, such as the production of oligonucleotides, oligodeoxynucleotides, oligoribonucleotides, oligosaccharides, proteins, etc., will be apparent to those skilled in the art.
Solid phase peptide synthesis (SPPS) was developed by Merrifield in 1963. The basic procedure is well known. The SPPS method typically begins with a polymer gel, such as a partially chlorinated polystyrene cross-linked with divinyl benzene. The C-terminal of a protected amino acid is initially bound to the resin, for example, by means of a benzyl ester of the amino acid. Other binding agents may of course be used. The peptide is synthesized in sequence from the C-terminus with protected amino acids. The amino groups and all reactive side-chain functional groups of the amino acids must be protected by stable blocking groups in order to prevent undesirable side reactions. The blocking groups are selected such that the amino group may be deprotected without disturbing the side chain protecting groups or the link between the C-terminus and the resin. The amino group may be protected by, for example, boc (t-butoxy carbonyl) or fmoc (9-fluorenyl methoxy carbonyl) groups.
The peptide synthesis is typically conducted by the following procedure: The N-terminus of the resin-bound peptide (protected by boc) is deblocked in a solution of trifluoroacetic acid (TFA) in dichloromethane (DCM), for example. The next amino acid in the sequence is coupled to the resin-bound peptide with a coupling agent such as dicyclohexylcarbodiimide (DCC) in a solution of DCM and dimethyl formamide (DMF), for example. An activating agent such as 1-hydroxybenzotrazole (HOBt) may be used to improve the rate and selectivity of the coupling reaction and to decrease racemization. The unreacted amino acid, reagents, and by-products are removed from the resin by washing and filtration. The washing and filtration process is then repeated. The N-terminus of the peptide is then de-blocked, another peptide is added to the chain, the system is then washed and filtered, etc. The process is repeated until all the desired amino acids have been added to the peptide chain in the desired order. The remaining blocking groups are then removed from the peptide, the peptide is cleaved from the resin, and the peptide is collected.
SPPS originally was performed, and often still is performed, in shaken or stirred flasks in which the resin is dispersed. To suspend and mix the resin in a fluid phase, several times the amount of liquid which the resin will absorb or hold is required for the system. Thus, if the resin will hold 10 ml of liquid, 50-100 ml of liquid will be required to disperse and suspend the resin. The increased amount of liquid leads to the use of dilute solutions (typically a 150 mmol amino acid/liter of solution) to minimize costs, since the amino acids are expensive. With dilute solutions, it is difficult to obtain high concentrations of amino acids and hence fast chemical reactions between the amino acids and the growing chain. Further, when the resin is washed and filtered (in a flask) via batch dilution, it is virtually impossible, in a limited number of batch washes, to remove from the resin all the DCM and TFA used. As a result, the contact time of the peptide chain with the DCM and TFA cannot be accurately controlled.
A number of design issues in the reactor development are unique to peptide synthesis. The resin used in solid-phase peptide synthesis is usually a gel resin with a low degree of cross-linking. These resins swell in certain solvents (such as DCM), and shrink in other solvents (such as methanol). The resin volu
REFERENCES:
patent: 4192798 (1980-03-01), Verlander et al.
patent: 5186824 (1993-02-01), Anderson et al.
Stapleton Michael C.
Stepaniuk Nicholas
Tomazi Keith
Mallinckrodt Inc.
Mohamed Abdel A.
Woodward Michael P.
LandOfFree
Reactor and method for solid phase peptide synthesis does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Reactor and method for solid phase peptide synthesis, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Reactor and method for solid phase peptide synthesis will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-521467