poster for walt lab
TRANSCRIPT
50 µm
b)
c)
Identification of Highly Catalytic Polymers
Protocol
Graduate StudentsShazia BaigTrinh Dinh
Pratyusha MogalisettiStephanie SchubertSoyoon Sarah HwangPrarthana KhannaPayel GhatakNicolas IversonLimor Cohen
Post Doctoral Associates Dr. Barrett DuanDr. Candice EtsonDr. Danlu WuDr. Stephanie WalterDr. Mark Hartman Dr. Shudan BianDr. Bishnu RegmiDr. Liangxia XieDr. Yael Simakov
Group MembersPrincipal Investigator
Dr. David R. WaltUniversity Professor & Robinson Professor of Chemistry & Howard Hughes Medical Institute Professor
Undergraduate StudentsTabitha AmondiPeter CavanaghDouglas DavisDerek DuPontThomas MoodyKevin NganAlex SakersJason ShnipesPetar TodorovBridget YangBruce Bausk
Walt Laboratory: Synthetic Polymers as Catalysts for Difficult Reactions
Results: Proof of Catalysis?
a)
b)
c)
Light reflected down the fiber core
Light refracted into the cladding material
Fiber-optic arrays of 50,000 individual 4.5 m fibers are bundled into a 2 mm hexagonal array.
This optical fiber schematic illustrates how light is guided through the core material via total internal reflection. The core material possesses a higher refractive index than the cladding material, so as long as the angle of incidence is greater than the critical angle of the fiber, the light entering the core material can be internally reflected at the boundary between the two materials.
Fiber Optic Bundles
Peptide Catalysts: Innovation
a)
Effect of PEG Functionalization
Step 1: Oligomer Synthesis (Gellman Lab)
Step 2: Pool Screening, Mechanical Sealing (Walt Lab)
8 Image Fiber
50,000 wells (46 fL)
28,000 Oligomers per Well1.4×109 oligomers (50,000 wells)
*An appropriate low enzyme concentration ensures that the wells contain a maximum of only a single enzyme molecule while the rest are empty.
Big Goal: Discover a given polymer oroligomer pool containing rare members with significant catalytic activity.
What we need to do?
Identify a reaction with rationally variable “degree of difficulty”
Femtoliter-well array screening method
Our Preliminary observations of those pools will give us a general idea about the length and residues of oligomers required to achieve high catalytic activity
• Better throughput: can Image 8 fibers
• More Reproducible
Amide10-10 ~ 10-11 sec-1
Phosphate ester10-20 ~ 10-16 sec-1
Glycoside10-14 ~ 10-15 sec-1
Transamidation/Hydrolysis of amide, phosphate ester or glycoside
Advantages of Peptide Oligomers
-Building blocks with inherent chirality and functional diversity-Easy to synthesize-High degree of stability
Challenges
-Difficult rational design -Few polymers with unnatural bones used for reactivity study-Very little effort to identify oligomeric catalysts with unnatural backbones
• Establish a reliable experimental protocol to identify rare highly active catalysts for fluorogenic versions of difficult reactions; any fluorogenic reaction can then be tested
• Deliver catalysts with unprecedented activity levels among non-biological systems.
• Provide new insight on fundamental requirements for achieving significant catalytic activity for "difficult" reactions
• Enable such studies to be conducted in many different laboratories
Innovation
PEG MW 700
PEG MW 2000
Mean (PercentageChange): 7.041Standard Deviation: 7.506Control Threshold Value = Mean(Percentagechange)+3*(Standard Deviation) = 29.559
Fluorescent Wells with Percentage Increase More than 29.559 (# Wells: 18)
Fluorescent Wells with Percentage Increase More than 32.47 (# Wells: 13)
Mean (PercentageChange): 5.44Standard Deviation: 9.01Control Threshold Value = Mean(Percentagechange)+3*(Standard Deviation) = 32.47
5 μM Pro-Pro-Rhodamine Control Experiment (7 Fiber Experiments/PEG-functionalized Fiber/57 Min)
5 μM Pro-Pro-Rhodamine Control Experiment (3 Fiber Experiments/PEG-functionalized Fiber/57min)
Min)
5 μM Pro-Pro-Rhodamine with 2.5 μM MM-153-A(4 Fiber Experiments/PEG-functionalized Fiber/57 Min)Lys-XXXX-Phe-XXXX-Phe-XXXX-Phe-XXXX-Lys (MM153 A) X = 1:1:1 Tyrosine/Histidine/Glutamic
Diversity:3^16 = 4.3 x 107 sequences46fL×2.5 μM ×6.02×1023×50000×4=1.38×1010
1.38×1010 / 4.3 x 107 = 322
(~11 active polymers per fiber)(~42 active polymers per fiber, without PEG-Functionalization)
5 μM Pro-Pro-Rhodamine with 2.5 μM MM-153-B(3 Fiber Experiments/PEG-functionalized Fiber/57 Min)
Lys-XXXX-Phe-XXXX-Phe-XXXX-Phe-XXXX-Lys(MM153 B) X = 4:1:1 Tyrosine/Histidine/Glutamic
Fluorescent Wells with Percentage Increase More than 29.559 (# Wells: 16294)
Diversity:3^16 = 4.3 x 107 sequences46fL×2.5 μM ×6.02×1023×50000×3=1.03×1010
1.03×1010 / 4.3 x 107 = 239
(~17 active polymers per fiber)(~93 active polymers per fiber, without PEG-Functionalization)
Bulk measurement with respect to control:MM153B = 1.7 ± 0.2
Fluorescent Wells with Percentage Increase More than 29.559 (# Wells: 15022)
Bulk measurement with respect to control:MM153A = 1.3 ± 0.4