# Structural Biochemistry/Proteins/Synthesis

The most popular method to synthesize peptides of more than 50 amino acids in length is automated solid-phase peptide synthesis. R. Bruce Merrifield first developed this method, and it can be used for both DNA and RNA. To begin the process, the carboxyl-terminal amino acid of the desired sequence is anchored to polystyrene beads, and the peptide is synthesized backwards from the C-terminal end to the N-terminal end (contrary to the usual sequence from the N-terminal end to the C-terminal end). The t-Boc protected group of this amino acid is then removed by a wash with trifluoroacetic acid (CF3COOH) and methylene chloride (CH2Cl2), which does not break covalent bonds. The next amino acid with t-boc (di-tri-butyl dicarbonate), a protected N-terminal, and a DCC (dicyclohexylcarbodiimide)-activated C-terminal is added to the reaction column. After the formation of the peptide bond, the excess reagents and dicyclohexylurea are washed away with an appropriate solvent. For the elongation of the peptides, the next amino acids continue to be added in the same manner. At the end of the synthesis, the peptide is released from the polystyrene beads by adding hydrofluoric acid (HF), which cleaves the ester bond without destroying the peptide bonds. Protected groups on the reactive side chains, such as lysine or histamine, also are removed at this time. The huge advantage of this method, besides the fact it is automated, lies in the purification step. Because the impurities are not bound to the reaction column, they can be washed away without losing the synthesized product. In the laboratories, this technique is used to synthesize drugs, such as insulin.

## Processes
### Transcription

It starts in the nucleus. It is very similar to the DNA replication process in which the DNA is "unzipped" by helicase, producing one nucleotide chain ready to be replicated.
Transcription 3 Steps summary –> Producing an RNA message from DNA
(A) Binding and Initiation
DNA transcription unit divided into TATA Box and Enhencer region. TBP is bind to TATA region, other transcription factors (a protein has bound to the region) such as TFIIA and TFIIB are bonded to TATA regions as well. The RNA polymerase cannot bind to the DNA directly unless a transcription factor is bind first. Transcription begins when RNA polymerases bind to the enhancer region( or called the initiation site), separate it into two strands by requiring ATP energy Initiation initiate the location of the DNA strand to begin transcription.
(B) Elongation
RNA polymerase moves along the DNA promoter region by performs two elongate steps:
1) it untwists (unwind) the double helix DNA about 10 bases at a time at 3.4 A.
2) adds nucleotides to the 3’ end of the growing RNA.
As the RNA polymerase moves along, the growing mRNA molecule was replicated base on base. Transcription goes about 60 nucleotides per second. DNA’s nucleotides Adenine will be complimentary to RNA’s Uracil base. DNA’s nucleotides Guanine will pair with Cytosine.
(C) Termination
Transcription proceeds until the RNA polymerase reaches a termination site. No more RNA nucleotides will be added and the mRNA is released. So, mRNA will move out of the nucleus into the cytoplasm for the further use in protein synthesis.

### Translation

The mRNA codons translates to amino acid polypeptide chains in three steps.
3 steps general guidance of translation
Initiation 2. Small subunits ribosomal attaches to mRNA. Large Subunit of ribosome is bind to small subunit with A site (entry for tRNA.)and P site ( leaving door for tRNA.) first attach to a tRNA. anticodon( nucleotide triplet in tRNA) is attaching to A site (entry site) to paired with 3 nucleotide codons from mRNA. tRNA carries an amino acid. As shown by the graph below, tRNA. carry an amino acid on the top
Elongation 3. Initiator tRNA. then moved to P site and A site is opened for the second triplet coded tRNA. to enter along with another amino acid. After the second tRNA. is bind to A site. The amino acid is then bonded together by peptide bonds. Afterwards the third tRNA comes in right after the second tRNA. move to P site. (Moving along from 3’’ to 5’’) 4. ribosomal enzymes link the amino acid into a chain. The process will continue until the stop codon (UAA) is reached.
Termination
5. a stop codon is reached (UAA, UAG, or UGA). A protein called a release factor binds in the A-site to the termination codon. The ribosomes adds a water molecule to the end of the polypeptide chain. 6. ribosome dissociates into its component parts

## Advantages

Good yield and high purity. All reactions are carried out in the single vessel, eliminating losses caused by the repeated transfer of products. This method is good for synthesizing long chain of peptide (50 residues and above).

## Synthetic Peptides

Peptides can be made synthetically by linking an amino group of one amino acid to the carboxyl group of another; this being an example of a condensation reaction. A condensation reaction is the reaction when two molecules come together, releasing water, to form one molecule.
Peptide synthesis can be specific; meaning specific/desired products can be formed. To make unique products and to prevent side reactions, protecting groups such as tert-butyloxycarbonyl (t-Boc) are used. T-Boc is used in the first step of the formation of simple peptides. This protecting group, in order to block the alpha-amino group, reacts with the alpha-amino group forming a complex [[Image:known as t-butyloxycarbonyl amino acid. The blocking of the amino group is followed by the activation of the carboxyl group of the same amino acid. The carboxyl group is activated by dicyclohexylcarbodiimide (DCC).
Now, with the alterations being done to the amino group and the carboxyl group of the first amino acid, a second amino acid can be linked to the first amino acid. The second amino acid has a free amino group, meaning not blocked, and it links to the activated carboxyl group of the first; forming a rigid peptide bond and releasing dicyclohexylurea. The carboxyl group of the newly formed dipeptide is activated with DCC and ready to react with a third amino acid which has a free amino group. Again, a new peptide bond is formed and dicyclohexylurea is released. This process can be performed continuously until the desired peptide is synthesized. To end the synthesis, dilute acid, which removes the t-Boc and leaves the peptide undisturbed, is added.
Solid-phase method is used to form synthetic peptides that contain more than 50 amino acids. It involves binding the last amino acid's carboxyl group to polystyrene beads. The anchored amino acids t-Boc is removed, and the next amino acid with t-Boc protected amino group and DCC activated carboxyl group is added to the amino acid with polystyrene beads. The peptide bond forms, and the peptide with polystyrene beads is filtered and washed, so the peptide is pure before the synthesis is continued. The following amino acids are linked with the same process until the desired peptide is synthesized. Finally, the finished peptide is removed from the beads by using hydrofluoric acid(HF).
Peptide ligation is used to synthesize peptides with more than 100 amino acids. The long peptide is formed from two or more smaller sized peptides with no protecting groups on them. Native thiol ligation is the most powerful and widely used peptide ligation. The long peptide is formed from peptides with thioester on C-terminal carboxyl group and the other peptides with cysteine on N-terminal. The thioester on C-terminal carboxyl group of one peptide reacts with the cysteine on N-terminal of another peptide to form a thioester-linked intermediate. The intermediate is then rearranged(S->N acyl shift) to form a peptide bond. The small sized unprotected peptides are linked by this process to synthesize the long peptide.

## Utilization

Synthetic peptides are made for many purposes. These peptides can act as antigens, which will stimulate the immune system of the body to produce antibodies that target such peptide. These antibodies can then be used to isolate a protein. Peptides can also isolate receptors for hormones.
Synthetic peptides can also be used as drugs. Such example is the synthetic analog of Vasopressin, also known as 1-Desamino-8-D-arginine vassopressin. This synthetic peptide is used to treat patients with diabetes insipidus who lacks the peptide hormone vasopressin, which cause them to urinate excess liquid from their body. By using the analog of vasopressin to substitute for the natural vasopressin, such patients can be treated.
File:Vasopressin.jpg
Lastly, synthetic peptides can be used to gain a greater understanding of the 3D structure of proteins. Using synthetic proteins to study the 3D structure of proteins is extremely helpful because such peptides can include many amino acids that are not found in normal proteins; meaning these peptides are not limited to just the 20 standard amino acids. This result in a much greater variety of structures.

## Solid-Phase Peptide Synthesis

Polypeptide synthesis can be automated, known as the Merrifield solid-phase peptide synthesis, which uses a solid support of polystyrene to support a peptide chain. Polystyrene is a polymer whose subunits are derived from ethenylbenzene.
The beads of polystyrene are insoluble and rigid when they are dry; however, they swell in certain organic solvents, dichloromethane for example. Therefore, reagents are able to move in and out of the polymer matrix easily. The phenyl groups on polystyrene are functionalized by electrophilic aromatic substitution.
File:Electrophilic Chloromethylation of Polystyrene.jpg
Using a dipeptide as an example, the solid-phase synthesis of peptide on chloromethylated polystyrene proceeds as follows.
1. Attach protected amino acid
2. Deprotect amino terminal
3. Coupling to the second protected amino acid
4. Deprotect amino terminal
5. Disconnect dipeptide from polystyrene

## Purpose of dicyclohexylcarbodiimide (DCC)

Dicyclohexylcarbodiimide (DCC) is used specifically in peptide synthesis in order to activate the electrophilicity of the carboxylate group. This allows the C-terminus to be more favorable as an attachment site for other amino acids. Then the negatively charged oxygen will act as a nucleophile which attacks the center carbon in DCC. This intermediate will eventually be converted into urea, a stable end product that is relatively unreactive throughout the remaining peptide synthesis process. In addition, DCC's activation ability may sometimes racemize peptide bonds if not monitored correctly, therefore sometimes triazoles may be used instead which do not racemize the stereochemistry of peptides.
File:Solid-Phase Synthesis of Peptide.jpg

## Advantage of solid-phase synthesis

The advantage of solid-phase synthesis is that the products can be isolated easily since all the intermediates are immobilized on polystyrene. Thus, the products can be purified by filtration and washing. Repetition of the deprotection-coupling process will be able to synthesize larger peptides. A machine, designed by Merrifield, is able to carry out the series of manipulations automatically.

## Protecting Groups

Peptide bond can be formed from the carboxyl group and amino group on the main chains of amino acids. It also can be formed from the side chains to synthesize an undesired peptide. In order to synthesize a desired peptide, protecting groups are used to prevent the formation of undesired products. They also prevent the polymerization from the excess amino acids used in the reaction. Protecting groups also aid in ensuring that the stereochemistry of certain amino acids remain unchanged. Configurations of amino acids may have their stereoisomers changed or racemized if not properly protected as well.

## t-butyloxycarbonyl(t-Boc) protecting group

It is used to protect the N-terminal amino groups as well as the side chains of lysine, arginine, asparagine, and glutamine. Di-t-butyldicarbonate reacts with the NH$_2$ of amino acid to form a t-Boc-amino acid. t-Boc group can be removed under acidic condition. Typically, they are treated with strong acid or Trifluoroacetic acid(TFA), CF$_3$COOH. In the lab, Boc-amino acids are also available to buy since it can be synthesized easily in large quantity. People who synthesize peptides do not have to make Boc-amino acid on their own. Solid phase synthesis is effective because it allows the protein to remain in a primary structured configuration rather than being complicated by secondary or tertiary intermolecular interactions.

## Solution-Phase Peptide Synthesis (Using Benzyloxycarbonyl(Z) as protecting group)

Benzyloxycarbonyl is used to protect the N-terminal amino groups as well as the side chains of lysine, arginine, asparagine, and glutamine. The synthesis starts at the N-terminus and ends at C-terminus. For example, here are steps to synthesize a simple peptide such as Ala-Val:
First Step: Benzyl chloroformate react with the N-terminus of alanine, forming benzyloxycarbonyl alanine (alanine with the N-terminus protected by Z-group). Typically, triethylamine is used as catalyst for this reaction.
Second Step: The protected alanine is treated with ethyl chloroformate. Carboxyl group of the alanine was activated by forming anhydride. It is sensitive to any nucleophilic attack from the N-terminus of Valine.
Third step: Valine is added to the protected, activated alanine. This forms peptide bond, connecting Valine and Alanine. We'll have the product of Z-Ala-Valine. Notice that the N-terminus is still being protected after this step.
Final Step: The Z-protected group was removed by hydrogenolysis under mild condition with metal such as Pd acting as catalyst. (check the image for detailed reactions in each step)
In order to synthesize a larger protein, we have the repeat the second and third step. Activating the C-terminus and then, coupling the next amino acid. The advantages of this synthesis are it works very fast, and have a good percentage yield of the product. However, it can only be used for small protein chain. The yields become smaller with larger protein. Therefore, solid-phase is more preferred with large protein.

## 9Fluoronylmethyoxycarbonyl(Fmoc) protecting group

It is used to protect the N-terminal amino groups as well as the side chains of lysine, arginine, asparagine, and glutamine. Fmoc can be removed by piperidine/DMF.

## t-butyl and benzyl protecting groups

They are used to protect the C-terminal carboxyl groups as well as the side chains of serine, threonine, tyrosine, glutamate, and aspartate. t-butanol or benzenol reacts with the hydroxyl groups or carboxyl groups of amino acids to form t-butyl or benzyl amino acid. t-butyl or benzyl can be removed by strong acid and catalytic hydrogenation.