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Introduction and Methods

Introduction

Many new biomolecules are created by conjugating proteins to synthetic polymers. One important method of biomolecule conjugation is disulfide exchange between a thiol on a biomolecule and an activated disulfide present on a polymer. From previous studies in our lab, when these disulfide polymers were included in the network of a hydrogel, incorporation of proteins by disulfide exchange was sterically hindered. We were therefore interested in the relative steric effect of the different components in disulfide exchange. We investigated the differences in both the exchange rate and maximum extent of disulfide exchange depending on three variables: pH, thiol size, and polymer size.

Steric Effects in Peptide and Protein Exchange with Activated Disulfides Jason Kerr, Jessica L. Schlosser, Donald R. Griffin, Darice Y. Wong and Andrea M. Kasko

Department of Bioengineering , University of California Los Angeles

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Kinetic Rate Constants

Figure 1: Kinetic rate constants for each variable averaged over all other factors.

Figure 2: Kinetic rate constants for individual experimental conditions, grouped by thiol.

Figure 3: Maximum extent of exchange for each variable averaged over all other factors.

Figure 4: Maximum extent of exchange for individual experimental conditions, grouped by thiol. Figure 5: Illustrations of protein-polymer pairings representative of exchange behavior

Maximum Disulfide Exchange Overall Comparison

Scheme 1: Chemical synthesis of PDG-SS-Pyr with different macromer sizes for comparison of the effect of PEG chain lengths on disulfide exchange kinetics.

Synthesis and Exchange Conditions

pH and Polymer have mild influence on exchange

Protein S-Protein SH

Table 1: Relative influence of each factor on the kinetic rate constants and maximum extent of disulfide exchange. (✓= influence, ✗= no influence)

The relative influence of 3 factors on the kinetics of disulfide exchange were investigated: 1) thiol size and steric accessibility, 2) macromer size, and 3) pH. Kinetic rate constants increase with increasing pH, but maximum exchange is not affected by pH in the range tested. Small molecule disulfides react faster and more completely than disulfides with polymer chains, but increasing sizes of the chains has no effect. Larger proteins, and particularly proteins with additional local steric barriers react more slowly and incompletely. With decreasing pH and increasing protein size, the disulfide size becomes less important in determining the kinetics of disulfide exchange. Overall, the sterics of the proteins, at any given pH, are more influential than macromer size and pH in determining the rate and extent of disulfide exchange.

Factors

Influence on Kinetics Influence on Max Exchange

pH ✓✓ ✗

Protein ✓✓✓ ✓✓✓

Macromer ✓ ✗

Thiol access dominates exchange

S-S

S-S

= pyridine= o-NB

SH

SH

SUCC-SS-Pyr

PEG-PDG-SS-Pyr

GSH

βLG

+

+

kexch ~ 0.035 s-1

kexch ~ 0.010 s-1

max exch. ~100%fastest, most completesterics negligible

max exch. ~ 75%moderate, variablethiol and disulfide sterics interact

S-S

PEG-PDG-SS-Pyr

+ SH

BSA

kexch ~ 0.001 s-1

max exch. ~ 30%slowest, least completethiol sterics dominate

pH = 8.0

pH = 8.0

all pH

This material is based upon work supported by: the National Institutes of Health (NIH) and the Milton Gottlieb Scholarship Fund.

! pH = ! k

! Thiol access = ! k

! PEG length ≠ ! k

! pH ≠ ! max exchange

! Thiol access = ! max exchange

! PEG length ≠ ! max exchange

! pH = ! k but ! pH ≠ ! max exchange Higher pH deprotonates the thiol, increasing the ratio of thiolate anion, thus rate of disulfide exchange, but not maximum disulfide exchange.

! PEG length ≠ ! k and ! PEG length ≠ ! max exchange PEG chain has a moderate affect on k and max exchange vs. no PEG chain, but their flexibility and distance from the disulfide bond means differences in chain length are relatively unimportant.

! Thiol access = ! k and ! max exchange Local and global steric conditions affect thiol accessibility. SN2 reactions depend on access and positioning. This factor is most influential.

PEGProtein SS SS Protein

Results

Conclusion Acknowledgements

O

OO

OOH

NO2

O

OO

OO

NO2EDC/DMAPDCM24hr, RT O O

OO

O

O2N

n

HO

OO

OO

NO2

O OO

OO

O2NOH

n

NaBH4

N S S OO

OOH

O

OO

OO

NO2

O OO

OO

O2NO

n

N S S OO

O

NSSOO

O

DCM24hr, RT

EDC/DMAPDCM24hr, RT

O

n= 45, 91, 227

Compound A Compound B

OHO OHn

O

OO

OO

NO2

O OO

OO

O2NO

n

N S S OO

O

NSSOO

O

n= 44, 90, 226

N S S OO

OOH

R-SHpH6.07.07.48.0

O

OO

OO

NO2

O OO

OO

O2NO

n

R S S OO

ORSSO

O

O

n= 44, 90, 226

R S S OO

OOH

NHS

Monitored at 340nm

or

or

R=GSH, βLG ,BSA

Scheme 2: Experimental disulfide exchange conditions. Reaction rate constants were determined from change in absorbance of pyridine-2-thione measured at 340nm when released from the macromers. !

References

[1] PDB ID: 2Q2M, Vijayalakshmi, L., Krisha, R., Sankarayarayananan, R., Vijayan, M. Proteins 71: 214-249 (2007). [2] PDB ID: 3NPO, Loch, J., Lewinski, K. J. Mol Recognit. 24: 341-349 (2011). [3] PDB ID: 4F5S, Bujacz, A. Acta Crystalogr. Sect. D. 68: 1278-1289 (2012). [4] PDB ID: 3V03, Majorek. K.A., Porebski, P.J., Chruszcs, M., Almo, S.C., Minor, W. Mol. Immunol. 52: 174-182 (2012).

Glutathione

~50%!dimer!!!!!!!!!!!!!!!!!!!!!!~50%!monomer!

Bovine Serum Albumin β-Lactoglobulin

100%!dimer!!!!!!!!!!!!!!!!!!!!!!100%!monomer!

DTT!

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