Download Structural Biochemistry/Proteins/Synthesis

Structural Biochemistry/Proteins/Synthesis
Structural Biochemistry/Proteins/Synthesis
Introduction
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.
Translation
The mRNA codons translates to amino acid polypeptide chains in three steps:
1) Initiation
2) Elongation
3) Termination
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
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Structural Biochemistry/Proteins/Synthesis
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
Dicyclohexylcarbodiimide (DCC)
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.
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.
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Structural Biochemistry/Proteins/Synthesis
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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.
Using a dipeptide as an example, the solid-phase synthesis of peptide
on chloromethylated polystyrene proceeds as follows.
Polystyrene
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.
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.
Structural Biochemistry/Proteins/Synthesis
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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 NH2 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),
CF3COOH. 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 choloroformate react with the N-terminus of alanine,
forming benzyloxycarbonyl alanine (alanine with the N-ternimus
protected by Z-group). Typically, triethylamine is used as catalyst for
this reaction.
Boc-group, synthesized and removed
Second Step: The protected alanine is treated with ethyl
choloroformate. 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.
Trifluoroacetic acid used to remove t-Boc group
Final Step: The Z-protected group was removed by hydrogenolysis
under mild condtion 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.
Synthesis of Ala-Valine, using solution-phase
synthesis
Structural Biochemistry/Proteins/Synthesis
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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.
Z-group protecting group
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.
Fmoc protecting group
Piperidine. Used to remove Fmoc group
Article Sources and Contributors
Article Sources and Contributors
Structural Biochemistry/Proteins/Synthesis Source: http://en.wikibooks.org/w/index.php?oldid=1957598 Contributors: Alenalee, B1huynh, Beijingwu, Dklok, Jsl008, Kttran, MariaAlvarado,
Panic2k4, Pwtse, Thenub314, Tinojasontran, Vilau, Willie liang
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Image:piperidine.jpg Source: http://en.wikibooks.org/w/index.php?title=File:Piperidine.jpg License: Public Domain Contributors: Tinojasontran
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