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Himabindu Lanka

Professor John Taylor’s lab

The approach is to design, synthesize and study peptide analogues. The synthesis used in this approach is solid-phase peptide synthesis. One of the projects this lab concentrates on research of design in peptide-based antiviral drugs against HIV-1. [4]

Human Immunodeficiency Virus (HIV) is a disease that affects more than 40 million people. Many efforts have been and still are being made to inhibit HIV’s fusion into the host cell. The efforts include the clinical use of T-20. T-20 is a 36 residue long peptide. Before understanding the mechanism of T-20 in inhibiting HIV, first it is important to understand the mechanism of HIV-fusion into the host cell. HIV infects any cells with CD4 and its co receptors (chemokine receptors). Once HIV enters the body, its glycoprotein gp120 binds to CD4 (T-cell receptor) and its co-receptor. Also, at this point gp41, a trimeric mushroom-shaped glycoprotein, gets activated and reorganized.  Then it dissociates from gp120 and folds into a six-helix bundle making the fusion between the viral membrane and host cell membrane. [2]

T-20 is thought to bind to gp41 and either prevents folding or does an untimely folding of it meaning the folding of six helix bundle might happen before such as when gp120 interacts with CD4 and its co receptor. [3]

It is currently used only in extreme situations such as when retroviral drugs are ineffective because it costs about $30000 per year.[1] Reasons for its expensiveness include the large quantities of the Fmoc amino acid residues used to synthesis, multiple steps (72+ steps), and the purification techniques used. Before the purification technique used was reverse phase HPLC which is a very expensive purification method. The goal of the project is to make the production of T-20 cost effective. How to make it cost-effective? Reduce the amount of starting material utilized, and synthesize Co(III) complexes. In doing so, we are able to use cheaper purification techniques such as Ion exchange chromatography with low performance reverse phase. On the whole, we want to avoid HPLC for purification which is a really expensive technique.

The general procedure of this project includes

1. Solid-phase peptide synthesis of each segment using 2-chlorotrityl chloride, an insoluble, resin as a reaction vessel
2. HPLC and Mass Spectrometry to check for the presence and purity of the prepared segment
3. Synthesis of Cis-[CoIII(en)2OAcA.A.OtBut)]2+  (N-terminal Protecting group)
4. Coupling peptide to the Cobalt Complex
5. Segment Condensation to bring the peptide together

 Applying Co (III) Complexes to the N-terminus of the protected peptide segment has many advantages for the purposes of this project. One of the many advantages includes easy solubilization of protected-peptide segments in aqueous organic solvents such as water-methanol, DMF-water, acetonitrile-water. As the peptide gets larger the solubility decreases and becomes hard to assemble the segment together. We believe that by applying these complexes solves this problem. 

My goal was to synthesize one segment of the T-20 peptide: Acetyl-YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF-Amide using solid phase peptide synthesis. Solid phase peptide synthesis involves coupling of the amino acid residues to a resin in a single reaction vessel. Advantage to this synthesis includes it is an automated approach as well easy filtration of the solvents. The segment that I worked on was: LIHSLIEESQ. The sequence of coupling for the segment synthesis is as follows:

1a. Coupling of first amino acid with resin

1b. UV-Visible Spectroscopy analysis

1c. Deprotection of the Fmoc

1d. Coupling of the next amino acid

1e. Kaiser Test to check for coupling efficiency

1f. Repeat steps b-e until segment is completed

In order to check for purity and presence of product, HPLC and Mass spectrometry were performed. My segment did not show the presence of product there. Then, UV-Vis Spectroscopy was performed. This technique showed negative result as well. Finally, I performed a Kaiser test in which a purplish blue color shows acknowledging the presence of amines. So far three of the five segments have been synthesized. In the future, the lab is going to complete the synthesis of the remaining two segments, synthesize the Cobalt complex, couple segments with this complex and assemble segment via segment condensation to give T-20

References

1. DeNoon, Daniel. HIV Fusion Inhibitor Gets FDA Nod, Medscape Medical News, 2003.
2. Shibo Jiang and Asim K. Debnath, “Development of HIV entry inhibitors targeted to the coiled coil regions of gp41”, Biochemical and Biophysical Research Communications, 269, 2000.
3. http://www.roche.com/pages/facets/16/fusinh.htm
4. http://rutchem.rutgers.edu/content_dynamic/faculty/john_taylor.shtml
5. Proposal by Professor John W. Taylor: Advantageous Application of Co (III) - Protected Amino acids to the synthesis and purification of peptides by segment condensation methods.

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