DUAL AUTO- ROBOTIC PEPTIDE LIBRARY & ORGANIC
& BIO-ORGANIC SYNTHESIZER FOR RESEARCHING
"AIDS" CANCER AND OTHER FATAL DISEASES
Department of Robotic and Automation British Columbia Institute of Technology Vanc. B.C. Canada
Department of Bio-Chemistry University of British Columbia Vancouver B.C. Canada
Durgo's Scientific "AIDS" Research Surrey B.C. Canada.
By
Jozsef A Durgo PhD,.DSc
(International Business Communications Conference)
Combinatorial Libraries for molecular Diversity
Stanford CH, San Francisco, CA, USA. Aug. 11-12 1994.
The construction of the Dual-Auto Robotic Peptide Library Synthesizer is so effective that it can synthesize millions to billions of peptides in a few days by the use of my newly developed Dual Auto-Robotic station.
Dual means A&B channels, where the system has parallel synthesis running under identical conditions and the computer is continuously making comparison and recording all the information between a standard and the unknown or between a duplicated peptide synthesis
Theoretical Schematic Diagram of the Dual Auto-Robotic peptide
Library & Organic & Bio-Organic Synthesizer Station For Researching
"Aids" Cancer and Other Fatal Diseases.
The robotic station has four major units; #1. The Dual mixing and dividing apparatus, #2. The Dual teflon reaction syringes, #3.Industrial robot, #4. The I.B.M. computer which controls the whole robotic station.
Dual Auto-Robotic peptide Library Synthesizer.
The Dual system possesses a number of outstanding features. One example is the adaptability of the Dual robotic station to any other existing chemistry and computer programs. Another example is that the Dual vacuum block reaction system is superior and outstanding because the resin will not stick on the walls of the teflon reaction syringes. Another major advantage of the reaction syringe is the capability of vacuuming in the reagent and solvent with the robot's assistance. My superiority over any other system is that it does not require the use of solenoid valves and pressurized bottles. Further more, the mixing and dividing Dual chamber is made from teflon which prevents the resin from sticking to the chamber walls. In conclusion, the design and development of my Dual Auto-Robotics Peptide Library Synthesizer is "state of the art" and due to the demand for this synthesizer, developing it into a commercial model will be a breakthrough and a welcome advantage for all scientist that are highly involved with peptide library synthesis.
The Dual Vacuum Block Reaction Syringe System.
Shown above is a picture that illustrates the vacuum gage, the argon gas pressure gage and robot light sensor. On each side, A and B, are 19 reaction syringes with frits at the bottom. The solvent and reagent teflon delivery lines and built-in, 2-way reversible valves are controlled by the vacuum and the argon pressure. The background shows the SRS-M1 industrial robot.
Close-up photo of the Dual reaction syringes & Robot Arm
The Robot pulls the syringe piston up and vacuums the reagents and solvents. Then the robot pushes the syringe piston down to remove the reagents and solvents from the resins. This is an enormous advantage because it does not require the use of solenoid valves and pressurized bottles.
Top view photograph of the Dual Mixing/Dividing Chamber
The dual mixing chamber apparatus is a complex unit in itself. There are enormous advantages in having the capability of comparing identical synthesis on channels A & B, or between a control and a manipulated synthesis.
Dual Mixing/Dividing Apparatus
The photograph shown above is the injection port for injecting markers or already marked resins. Also shown are auto-robotic sensor switches, manual valves for single or dual operation, and the microscope for viewing resins in the mixing chamber.
Theoretical Schematic Diagram of the
Dual Mixing/Dividing Apparatus (Radioactive Section)
Radioactive operation is one of the most important features for identifying labelled resins or labelled amino-acids, az a special marker
.
This is a description of the dual mixing/dividing synthesis of support bound peptide libraries with the CR-M1 industrial robot system and IBM PC Host interface. All the synthesizer functions, the robot and the programs are run by the IBM Computer.
Dual Auto Robotic Peptide synthesizer, and the Peptide Libraries Synthesizer Program, Based on use of Portioning-Mixing Method.
In order to Vacillate the discovery of new bioactive Peptides developed and a new Synthetic procedure introduced First in l988, and 1993" Simple Devise"developed by J.A.Durgo G.Dibo A.Furka published in Edmonton, based on Dr. Merrifield's solid phase method. The coupling cycles normally used in the solid phase Synthesis are REPLACED by the following three operations:
l.
Dividing the solid support into equal portions.2.
Coupling a different amino acid to each portion.3.
Mixing the portionsThere are 19 dual teflon reaction syringes (each are of 10 ml. capacity) with frits at the bottom. The starting solid support in each teflon syringe is 100 mg. (10 umol capacity) aminomethylated polystyrene resin acylated by BOC-GABA (200-400 mesh) .BOC protected amino-acid derivatives are used in the coupling stepsOperation 1 to 7, refer to each of the 19 "dual" samples.
"DUAL" AUTO-ROBOTICS PEPTIDE SYNTHESIZER PROGRAM
Description of the"Portioning-Mixing" synthesis of support bound peptide libraries with CSR-M1 Industrial Robot System, IBM-PC Host Interface, All the synthesizer function, the robot and the programs running by the IBM.PC. Computer.
There are 19 reaction "DUAL" Teflon Syringes (ca.10 ml each) having frits at the bottom. The starting solid support in each Teflon Syringes is 100 mg (ca 10 umol) aminomethylated polystyrene resin acylated by Boc-GABA (200-400 mesh). Boc-protected amino acid derivatives are used in coupling steps. Operations 1 to 7 refer to each of the 19 "Dual" samples.
T
he "Dual" IBM, PC. Computer program steps are as follow:A.B. = 0. Dual operation Start.
A.B. 1. Swelling
A.B. 1.1. Add 3 ml dichloromethane (DCM)
A.B. 1.2. Argon gas mix for 5 min.
A.B. 1.3. Remove the solvent by vacuum
A.B. 1.4. Repeat 1.1. to 1.3.
A.B. 2. Deprotection
A.B. 2.1 Add 3 ml of 2:1 (v/v) mixture of DCM and
trifluoroacetic acid (TFA)
A.B. 2.2. Argon gas mix for 2 min.
A.B. 2.3. Remove the liquid by vacuum
A.B. 2.4. Repeat 2.1
A.B. 2.5. Argon gas mix for 30 min.
A.B. 2.6. Repeat 2.3.
A.B. 3. Washing No. 1
A.B. 3.1. Add 3 ml DCM
A.B. 3.2. Argon gas mix for 5 min.
A.B. 3.3. Remove the liquid by vacuum
A.B. 3.4. Add 3 ml methanol and repeat 3.2. then A.B. 3.3 3.5. Repeat 3.1.to 3.3.
A.B. 3.6. Repeat 3.4.
A.B. 3.7. Repeat 3.1. to 3.3. twice
A.B. 4. Deprotonation
A.B. 4.1. Add 3 ml of 9:1 (v/v) mixture of DCM and diisopropyl-ethyl amine
A.B. 4.2. Argon gas mix for 2 min.
A.B. 4.3 Remove the liquid by vacuum
A.B. 4.4 Repeat 4.1.
A.B. 4.5. Argon gas mix for 9 min.
A.B. 4.6. Repeat 4.3.
A.B. 5. Washing No. 2.
A.B. 5.1. Add 3 ml DCM
A.B. 5.2. Argo gas mix for 9 min
A.B. 5.3. Remove the liquid by vacuum
A.B. 5.4. Repeat 5.1. to 5.3. twice
A.B. 6. Coupling
A.B. 6.1 Add 400 umol Boc-amino acid dissolved in 2.5ml solvent The solvent in the case of derivatives of His, Asn, Gln, Arg and trp, is dimethylformamide (DMF).In all other cases is DCM.
A.B. 6.2. Add 520 umol (80mg) 1-hydroxybenztriasole dissolved in 0.5 ml 2:1 (v/v mixture ofDCM and DMF.
A.B. 6.3. Add 400 umol (62 Uldiisopropyl carbodiimide.
A.B. 6.4. Argon gas mix for 3 h
A.B. 6.5. Remove the liquid by vacuum
A.B. 7. Washing No. 3.
A.B. 7.1. Add 3 ml DMF
A.B. 7.2. Argon gas mix for 5 min.
A.B. 7.3. Remove the liquid by vacuum
A.B. 7.4. Repeat 7.1. to 7.3. twice
A.B. 7.5. Add 3 ml DMF
A.B. 7.6. Argon gas mix for 5 min.
A.B. 7.7. Remove the liquid by vacuum
A.B. 7.8. Repeat 7.5. to 7.7. twice
A.B. 8. Argon gas Mixing
A.B. 8.1. Combine the resins of the 19 "Dual" reaction Syringes by rinsing them into a Portioning-Mixing chamber A. & B. with a 2:1 (v/v) mixture of DCM and DMF
A.B. 8.2. Complete the volume of the combined suspension to 95 ml with DCM-DMF 2:1mixture.
A.B. 8.3. Argon gas mix the suspension thoroughly for 20 min.
A.B. 9. Portioning
A.B. 9.1. Put 5 ml of suspension (of 8.3.) into each of the "Dual" 19 reaction vessels
A.B. 9.2. Remove the solvent from the resin samples by vacuum
A.B. 10. Repetition of cycles
A.B. 10.1. The operations 1.1. to 9.2. are executed as many times as the number of residues in Peptides.In the last cycle Portioning is omitted so the process ends up with the suspension describe under 8.3.
A.B. 11. Final removal of all protecting groups
A.B. 11.1. Remove the liquid by vacuum (From mix)
A.B. 11.2. Add 5 ml DCM
A.B. 11.3. Argon gas mix for 5 min.
A.B. 11.4. Remove the solvent by vacuum
A.B. 11.5. Repeat 11.2. to 11.4.
A.B. 11.6. Add 10 ml TFA and argon gas mix for 10' A.B. 11.7. Add 25 ml 10:1 (v/v) mixture of TFA and trifluoromethanesulfonic acid and Argon gas mix for 1 hr.
A.B. 11.8. Remove the acidic liquid by vacuum
A.B. 11.9. Add 5 ml dry ether
A.B. 11.10. Argon gas mix for 2 min.
A.B. 11.11. Remove the liquid by vacuum
A.B. 11.12. Repeat 11.9. to 11.11.
A.B. 11.13. Add 5 ml methanol
A.B. 11.14. Argon gas mix for 2 min.
A.B. 11.15. Remove the liquid
A.B. 11.16. Repeat 11.12. and 11.13.
A.B. 11.17. Dry the resin in vacuum. 4.hrs.
A.B. 11.18. END.
FINAL CONCLUDING RESULTS OF THE "DUAL" AUTO-ROBOTICS PEPTIDE LIBRARIES SYNTHESIZER TRIAL.
The main operation is based on the use of the "Simple Device Method" was applied to the Dual Auto Robotic Peptide Library Synthesizer along with a program that executes the whole robotic station. Using the "Dual" operation, a parallel synthesis was applied under identical conditions on channel A and B. The IBM PC computer made non-stop comparisons between the two channels to supervise a high quality synthesis. As a PhD,.DSc. I would like to actively pursue further advancement in Bio-Medical Engineering, Biochemistry and Biology with concentration in peptide libraries synthesis and affiliated research. As a result of the positive findings and achievements in my research, my next major goal is to develop new treatment oriented scientific and medical equipment which will provide hope for the millions of people around the world who are suffering and dying from a variety of diseases.