Science / Solid Phase Peptide Synthesis (Spps)

Solid Phase Peptide Synthesis (Spps)

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Autor:  anton  23 November 2010
Tags:  Peptide,  Synthesis
Words: 1792   |   Pages: 8
Views: 528

Solid Phase Peptide Synthesis (SPPS)


Solid phase synthesis is a process in which synthetic reactions are carried out on a solid support. The idea was developed by R .B. Merrifield to synthesise polypeptides and this work earned him the Nobel Prize in 1984.

Solid phase synthesis can be used in many ways for example to create carbohydrates, peptides and oligonucleotides.

I will be looking at solid phase peptide synthesis.

A peptide is when two amino acids are joined together resulting in the loss of a water (H2O) molecule. The resulting C-N bond between the two amino acids is referred to as a peptide bond. When a large number of amino acids are joined together by peptide bonds (polypeptide chains) they can form proteins. Proteins are present in every living cell and they have a variety of uses as they can be enzymes, hormones, antibiotics and receptors.

In this experiment I will be forming a cyclic dipeptide, where the ends of the peptide are joined together. Dipeptides are used in everyday living. An example of a dipeptide that may be used in daily life is Aspartame which is an artificial sweetener.

There are many areas of importance for synthetic peptides. A few examples are stated below:

To chemists as the ultimate proof of the structure of natural products, To biochemists as models for studying the specificity and mechanism of action enzymes, To physical chemists as models for the investigation of protein conformation, To pharmacologists as sources of products with modified or selective hormonal activity, To immunologists as tools for defining and understanding immunological specificity.

There are also many high profile uses of peptides in drug design, chemotherapy and immunology e.g.

• In diagnostics with the preparation on mono and polyclonal antibodies.

• As hormones for therapeutic agents.

• In receptor characterisations and isolation

• For clarification in enzyme-peptide substrate interactions

• As inhibitors of proteolytic enzymes.

And the major objectives in the field on peptide synthesis are

1. To verify the structure of naturally occurring peptides as determined by degradation techniques.

2. To study the relationship between structure and activity of biologically active protein and peptides and establish their molecular mechanisms.

3. To synthesise peptides that are of medical importance such as hormones and vaccines.

4. To develop new peptide-based immunogens.

R. B. Merrifield pioneered Solid phase peptide synthesis as a way of simplifying and accelerating the process of peptide synthesis so that the synthesis of long peptides would be more practical and that so as development continues it may be automated.

The revolutionary premise of solid phase peptide synthesis is that a peptide can be formed with any desired amino acid sequence but the peptide is anchored to an insoluble support at one end of the peptide chain. Once the required peptide has been formed the insoluble support can be cleaved away with a reagent that will liberate the peptide into solution. All the reactions involved are brought to 100% completion and this is done with the use of excess reagents, which allows the reactions to proceed to completion in the minimum time and results in faster synthesis and so a homogeneous product can be obtained. One of the advantages of this method of solid phase peptide synthesis is that the arduous purification steps that are involved in the synthesis of the peptide are eliminated and are replaced with simple washing and filtration of the solid. The entire process of solid phase peptide synthesis can be carried out in only one suitably designed vessel so that the transfer of materials from one container to another is cut out of the process.

Basic outline

There are synthetic polymers that have a reactive (X) group. These groups are made to react with the carboxyl group of an amino acid so the amino acid is covalently bonded to the polymer. During this step the amine group of the amino acid must be covered with a protecting group (Y) so that the amine does not react with the polymer. The protecting group must be such that it can be selectively removed without damage to the bond holding the amino acid to the polymer. A second N-protected amino acid then acylates the exposed amine group of the first amino acid forming the first peptide bond. By repeating the deprotection and coupling steps and by using the proper N-protected amino acid, the peptide of desired sequence can be assembled on the polymer. At the end of the synthesis a different reagent is applied to cleave specifically the bond joining the first amino acid to the polymer and the free peptide is liberated into solution.




In my project I will come across two different protecting groups. Protecting groups are attached to the amine of the amino acid and it stops the amine group from taking part in unwanted side reactions. When the amine is required to form a peptide bond the protecting group can be easily removed. The first protecting group that I will come across is 9-fluorenylmethoxycarbonyl (Fmoc). The Fmoc group protects the amine group from reacting with anything when the amino acid is being attached to the resin. The Fmoc group can be removed by adding a 20% piperidine solution in DMF. The reaction mechanism for this reaction can be seen in the diagram below.

The second protecting group that is involved in my project is the Butyloxycarbonyl (Boc) protecting group. This was the original protecting group that was used in the early days of solid phase peptide synthesis. Fmoc was only introduced by Carpino in 1972. The Boc group also protects the amine group on the amino acid but deprotection of Boc requires trifluoroacetic acid followed by washing and neutralisation. The reaction mechanism for the deprotection of Boc is shown in the diagram below.


The first step of this project was to make 9-Fluorenylmethoxycarbonylproline (Fmoc-Pro) from Fmoc-Cl. The method that was used was an experimental method and was devised from the formation of Fmoc-Gly. The product formed was to be used in the second step of the project which was to make a cyclic dipeptide by solid phase peptide synthesis.

Formation of Fmoc-Pro.

Glycine (0.57g) was dissolved in 10% Na2CO3 (20.2 ml) then Fmoc-Cl (1.96g) was added while stirring and cooling in an ice bath until it had dissolved. The mixture was then stirred at room temperature for two hours. The mixture was then poured into H2O (400 ml) and extracted twice with ether in a separating funnel. This was done to remove small amounts of 9-fluorenylmethanol and the high-melting polymer dibenzofulvene. The aqueous layer was then be cooled in an ice bath and acidified with HCl. The acidity was checked with blue litmus paper, when the litmus paper turned red the solution was sufficiently acidic. The white precipitate that had been formed was then extracted with ethyl acetate. The ethyl acetate layer was then extracted with water and dried with MgSO4. The liquid was evaporated with a rotary evaporator and recrystallised with ether. The weight of the product was recorded.

Solid phase peptide synthesis of a cyclic dipeptide

There were five steps involved in this method.

Step 1- attachment of the first amino acids to the resin

Hydoxymethyl resin (100mg or 0.1g) was directly weighed into the plastic reactor vessel. CH2Cl2 (2 ml) was added and the mixture was stirred for 1 min on a magnetic stirrer. Fmoc-proline (146mg or 0.146g) was added to the reactor vessel and diisopropyl carbodiimide (DIC) (100µl) was dropped in to the reactor vessel using a microsyringe. Dimethylaminopyridine (DMAP) (approximately 5mg (a spatula tip)) was added and the reactor vessel sealed and left stirring overnight.

The next day the reaction mixture was filtered using a sintered filter. The resin that was filtered on the sinter contained the compound that is required. The resin was then washed with the following reagents in the following order:-

4 x 2ml CH2Cl2

4 x 2ml MeOH

4 x 2ml Et2O

The resin was dried under high vacuum for 30 minutes.

Step 2- Removal of the Fmoc protecting group

20% piperidine solution in DMF (3ml) was added to the reactor vessel and stirred for 20 minutes. The liquid was then filtered off and a further 3 ml of the 20% piperdine solution in DMF was added and stirred for another 20 minutes. The Fmoc protecting group should now have been removed.

The resin was filtered and washed with the following reagents in the following order:-

4 x 2ml DMF

4 x 2ml CH2Cl2

4 x 2ml MeOH

4 x 2ml Et2O

The resin was dried under high vacuum for 30 minutes.

Step 3- Attachment of the second amino acid

DMF (3 ml) was added to the reactor vessel and Boc-Glycine (76mg (0.076g)) was added. DIC (100µl) was dropped into the reactor vessel using a microsyringe and then hydroxybenzotriazole (67mg (0.067g)) was added. The reactor vessel was sealed and stirred overnight.

The next day the resin was filtered and washed with the following reagents in the following order:-

4 x 2ml DMF

4 x 2ml CH2Cl2

4 x 2ml MeOH

4 x 2ml Et2O

The resin was dried under high vacuum for 30 minutes.

Step 4- Removal of the Boc protecting group

CH2Cl2 (2ml) was added to the reactor vessel along with trifluoroacetic acid (1ml). The reactor vessel was sealed and stirred for 30 minutes.

The solvent was filtered off and trifluoroacetic acid (1 ml) added and stirred for 30 minutes.

The solvent was filtered and the resin washed with:-

4 x 2ml CH2Cl2

The Boc protecting group has now been removed but the bound peptide exists as a salt and not as a free amine so neutralisation was necessary.

The resin was washed with the following reagents in the following order:-

3 x 3 ml 10% Et3N in CH2Cl2

4 x 2ml CH2Cl2

4 x 2ml MeOH

4 x 2ml Et2O

The resin was dried under high vacuum for 30 minutes.

Step 5- Cyclysation and cleavage from the resin

The resin was transferred to a 5ml round bottom flask that had a magnetic stirrer bar. The transfer was done by tapping the resin from the reactor vessel onto a weighing boat and then the resin was poured into the 5 ml round bottom flask.

5% acetic acid in toluene (3 ml) was added to the resin.

The reactor vessel was stirred and heated overnight at 80єC on an oil bath. A condenser was connected to the round bottom flask so that none of the product was lost due to evaporation.

The next day the reaction mixture was filtered using a small filter funnel into a 5 ml round bottom flask that had been dried and weighed carefully.

The toluene was removed using a rotary evaporator followed by drying under high vacuum.

The remaining solid cyclic dipeptide was washed with Et2O twice. This was done by adding small amounts of the Et2O with a pasteur pipette while swirling the flask and then removing the Et2O using the same pipette taking care not to remove any of the solid.

The product was dried under high vacuum and the amount of dipeptide obtained was calculated and an NMR was prepared using all the cyclic dipeptide formed.

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