1. 50 µm
b
)
c
)
Identification of Highly Catalytic Polymers
Protocol
Graduate Students
Shazia Baig
Trinh Dinh
Pratyusha Mogalisetti
Stephanie Schubert
Soyoon Sarah Hwang
Prarthana Khanna
Payel Ghatak
Nicolas Iverson
Limor Cohen
Post Doctoral Associates
Dr. Barrett Duan
Dr. Candice Etson
Dr. Danlu Wu
Dr. Stephanie Walter
Dr. Mark Hartman
Dr. Shudan Bian
Dr. Bishnu Regmi
Dr. Liangxia Xie
Dr. Yael Simakov
Group Members
Principal Investigator
Dr. David R. Walt
University Professor & Robinson Professor of Chemistry & Howard Hughes Medical Institute Professor
Undergraduate Students
Tabitha Amondi
Peter Cavanagh
Douglas Davis
Derek DuPont
Thomas Moody
Kevin Ngan
Alex Sakers
Jason Shnipes
Petar Todorov
Bridget Yang
Bruce 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 Well
1.4×109 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 or
oligomer 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
Amide
10-10 ~ 10-11 sec-1
Phosphate ester
10-20 ~ 10-16 sec-1
Glycoside
10-14 ~ 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): 7.041
Standard Deviation: 7.506
Control 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): 5.44
Standard Deviation: 9.01
Control 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 sequences
46fL×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 sequences
46fL×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