Tan, ElaineTorrelavega, Leda DaleVillahermosa, 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 PeptidesTARGET 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? Solid supports in which reactions can

occur Insoluble polymer Must be a well-solvated system 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

Chlorotrityl Polystyrene Resin Highly-solvated Hydrophobic beads are solvated by

nonpolar solvents such as dichloromethane

Fmoc: 9-fluorenylmethyloxycarbamoyl chlorideServes as protecting group for amines.

Cl- serves as leaving group.Lone pair of Nitrogen amine attacks

carbonyl C, displacing Cl-.

(the protected amino acid)

Protection of Amino Acid Unit

At this point, the Fmoc-protected unit is acid-stable.

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

Coupling of Protected Glu Unit to Resin

Dichloromethane Hydrophobic solvent

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

and dimethylamine.

How Deprotection Occurs

Dibenzofulvene AdductCarbon Dioxide

Deprotected Glu Unit

Progress of deprotection can be followed by quantifying the adduct.

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 of the next amino acid.

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.

Peptide Coupling

Where the solvent used is DMF.

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

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.

Cyclohexane

• Unreactive

• Non-polar

• Hydrophobic

• Serves as a solvent

Acetic Acid

• Serves as a Lewis acid

• activates C=C bond (???)

• upon activation, N nucleophile can attack

• trichloroacetimidate serves as a leaving 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

Tetrakis Palladium (0)

• Serves as a catalyst

• Process begins by oxidative addition to Pd(0) center

1,3-dimethylbarbituric acid

•Cleaves allyl and deprotects, giving –OH

Why use 1,3-dimethylbarbituric acid instead of TFA?

• 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 SequencesTARGET 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.

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

The yields obtained for the two target peptides were not given.

The description given was “good yield and purity”.

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!