Tan, Elaine

Torrelavega, Leda Dale

Villahermosa, Mheira

Dr. Voltaire Organo

Tubulins Make Up Microtubules

 Microtubules perform
several functions in
biological systems:
 Cell division
 Nerve cell
differentiation
 Transport inside the
cells
Tubulin undergoes Posttranslational
Modifications

Since posttranslational modifications take place, we can

differentiate between different populations of
microtubules.
 Choice of Tubulin Subunit as “Differentiator”

To do so, we need to investigate on the number of Glu-units

which can vary from 1 to 20.
 The Need to Know the Oligo-Glu Length

 Monoclonal antibodies:
 Can detect the presence or absence of Glu-units
 But insensitive to length of modification

Clearly, the need to synthesise antibodies that are sensitive

to length modification of tubulins is a must.

THIS STUDY ADDRESSES THE PROBLEM.

Objective

General Objective:
To generate specific antibodies that can
discriminate between the different lengths of
oligo-Glu modifications

Specific Objective:
To synthesise peptides with corresponding
defined side chains
Strategy # 1

1. Incorporate main chain Glu with selectively

cleavable allyl side-chain protection
2. Selectively deprotect
3. Activate carboxylic group on the resin
4. Couple H-Glu(tBu)-Oallyl with further side-chain
elongation after allyl cleavage

Not reliable. Only one to two side-chain Glu residues

could be introduced.
Strategy # 2

1. Preassemble suitably protected building

blocks with required side-chain length
2. Utilize their assembly in the main chain
The Target Peptides
TARGET PEPTIDE # 1:
CYEEVGVDSVEGEG-E(Ex)-EEGEEY

TARGET PEPTIDE # 2:
CQDATADEQG-E(Ex)-FEEEGEDEA

These target peptides are model peptide sequences.

Target 1: Mammalian alpha-1-tubulin
Target 2: Mammalian beta-1-tubulin
Step 1. Resin Loading

But first, what is a resin? Chlorotrityl Polystyrene Resin

 Solid supports in which reactions can  Highly-solvated
occur  Hydrophobic beads are solvated by
 Insoluble polymer nonpolar solvents such as
 Must be a well-solvated system dichloromethane
 Physically stable, permit rapid filtration
 Inert to reagents and solvents
 Must swell just right
 Too little swelling: reagents will not
penetrate
 Too much swelling: may not fit wells of
automated synthesizers
 Protection of Amino Acid Unit
(the protected amino acid)
Fmoc: 9-fluorenylmethyloxycarbamoyl chloride
Serves as protecting group for amines.

At this point, the Fmoc-protected unit is

Cl- serves as leaving group. acid-stable.
Lone pair of Nitrogen amine attacks
carbonyl C, displacing Cl-.
Coupling of Protected Glu Unit to Resin

Dichloromethane
 Hydrophobic solvent

Diisopropylethylamine
 Tertiary amine
 Used as a base
 N atom: shielded by 2 isopropyl groups & ethyl group, only 1 proton can
easily fit
 Readily attacks unprotected –OH group

At this point, the Fmoc-protected unit has been coupled

to the resin.
Deprotection of The Fmoc-protected Unit

Piperidine
 Heterocyclic amine
 Serves as a base that cleaves off the Fmoc group
 Deprotects N-terminus, freeing it for another coupling reaction

(Reaction mechanism shown on next slide.)

Dimethylformamide
 Hydrophilic
 Common solvent
 Washes away the coupling reagents previously used

Must be kept in the dark. May undergo photolysis to form CO and

dimethylamine.
 How Deprotection Occurs
The deprotected amino
group of the Glu unit is
neutral.
Minimizes the use of
other reagents.
Prevents aggregation of
peptides during
neutralization of the
amino group and coupling
Progress of of the next amino acid.
deprotection can
be followed by
quantifying the Deprotected Glu Unit
adduct.

Carbon Dioxide
Dibenzofulvene Adduct
Addition of Glu-unit was performed (x-
1) more times.

Oligo – Glu unit

Step 2. Assembly of Precursors

• Has 2 protecting groups

- Fmoc
- Allyl
-COOH Activation
• The carboxyl groups must first be activated in order for coupling to occur.
• There are usually two types of activators – carbodiimides (too reactive,
cause racemization) and triazolols (reactivity just right).

TBTU converts –COOH group into a more reactive -COOR group.

 Why do we need to activate?
• C-terminus: site where amino acid monomers are added
• increase electrophilicity of carboxylate group
• make oxygen a better leaving group

Peptide Coupling

Where the solvent used is DMF.

At this point, the peptide has been coupled. Cleavage from the resin
can now take place.
 Cleavage from Resin

Trifluoroacetic Acid
• Strong carboxylic acid
• Volatile
• Electronegative trifluoromethyl group
• Cleaves peptide from resin

At this point, the peptide has been cleaved

from the resin.
Step 3. Formation of t-Butyl Esters

Upon cleavage using TFA, you get a free

carboxylic function.

Free –OH can be esterified.

Why do we need to esterify?

We need to esterify to get a good leaving group for the incorporation of
the Oligo-Glu building block into the peptide sequences.
 Acetic Acid
Cyclohexane • Serves as a Lewis acid
• Unreactive • activates C=C bond (???)
• Non-polar • upon activation, N nucleophile can attack
• Hydrophobic • trichloroacetimidate serves as a leaving
• Serves as a solvent group

The problem with the use of acetic acid:

• Acetic acid can react with t-butyl
trichloroacetimidate (How???)
The solution:
• Have a tenfold-excess of t-butyl
trichloroacetimidate
At this point, we have formed the t-butyl ester. The next step is to deprotect
by cleaving the allyl group.
 Step 4. Cleavage of the Allyl Group

1,3-dimethylbarbituric acid
Tetrakis Palladium (0) •Cleaves allyl and deprotects,
• Serves as a catalyst giving –OH

• Process begins by Why use 1,3-dimethylbarbituric

oxidative addition to Pd(0) acid instead of TFA?
center • TFA is too strong, it can cause
degradation.
At this point, we have finally formed the oligo-Glu building block.
Let’s look at the yields of what we’ve
done so far.
 Assembly of Precursors: 95% - 98%
 Formation of t-Butyl Esters: 80% - 90%
 Cleavage of the Allyl Group: 80% - 90%
Step 5. Incorporation of the Oligo-Glu
Building Blocks into the Peptide Sequences
TARGET PEPTIDE # 1:
CYEEVGVDSVEGEG-E(Ex)-EEGEEY
At this point, what’s left Is to remove the protecting groups and cleave the
peptide from the resin.
 Final Removal of Protecting Groups
Triisopropylsilane (TIPS)
• Serves as a scavenger
• Removes the tBU protecting groups
• (Look for mechanism.)

Cleavage from Resin

This is accomplished by the use of TFA.

Finally, we have formed the first target peptide.

 TARGET PEPTIDE # 2:
CQDATADEQG-E(Ex)-FEEEGEDEA
The assembling of the target peptide # 2 is similar to that of target peptide # 1.

The yields obtained for the two target peptides were not given.
The description given was “good yield and purity”.

Orthogonality of the Fmoc method proves to be an advantage.

An orthogonal process means that a final product occurs by 2 independent
mechanisms.
• Deprotection of alpha-amino group: piperidine/DMF, TFA
• Final cleavage from resin: TIPS and TFA
Conclusion

 The Fmoc-method successfully synthesized

the target peptides to be used as an antibody
in investigating on oligoglutamylation of
tubulin upon undergoing posttranslational
modifications.
Thank you!