Site Specific Introduction of Unnatural Amino Acids

1
Site Specific Introduction of Unnatural Amino
Acids at Sites Critical to Insulin Receptor
Recognition Biological Activity
B.Quan, D.Smiley, V.Gelfanov, and R.DiMarchi
Department of Chemistry Indiana University,
Bloomington Indiana 47405 U.S.A
Experimental Design Synthesis of B-chain analogs
B chain analogs with unnatural amino acids at
B24 and B25 were successfully synthesized by
solid phase methodology using Fmoc chemistry.
Peptides were cleaved from the support in strong
anhydrous acid and purified under acidic
conditions by preparative high performance
reverse-phase chromatography in the free
sulfhydryl form. Each peptide was characterized
by MALDI-MS and HPLC analysis to be at least 90
pure prior to chain combination. Chain
Combination Reaction Each B-chain analog was
mixed with a molar equivalent of native A-chain
S-sulfonate (gift from Eli Lilly) in a 0.05 M
glycine buffer, pH 10.5. A molar equivalent of
DTT relative to each S-sulfonate was used to
facilitate disulfide interchange. The reaction
was conducted at 4ºC, overnight. The product was
purified by preparative high performance reverse
phase chromatography in a slightly alkaline
NH4HCO3 buffer with CH3CN elution. The insulin
analogs were obtained in step yield that varied
from 5-30, and characterized by MALDI-MS and
HPLC analysis to be greater than 95
pure. Receptor Binding The binding affinity of
each insulin analog for the insulin receptor was
measured in a competition binding assay utilizing
scintillation proximity assay technology. Serial
3-fold dilutions of the peptides were made in
scintillation proximity assay buffer (0.05 M
Tris-HCl, pH 7.5, 0.15 M NaCl, 0.1 w/v bovine
serum albumin) in a 96 well plate (Corning Inc.,
Acton, MA) with 0.05 nM (3-125I-iodotyrosyl)
TyrA14 Insulin. An aliquot of 1-6 micrograms of
plasma membrane fragments prepared from cells
over-expressing the human insulin receptor and 1
mg/well polyethyleneimine-treated wheat germ
agglutinin type A scintillation proximity assay
beads (Amersham Biosciences, Piscataway, NJ) were
added to each well. The plate was incubated for
twelve hours at room temperature and measurements
made with MicroBeta1450 liquid scintillation
counter (Perkin-Elmer, Wellesley, MA).
Non-specifically bound (NSB) radioactivity was
measured in the wells with fourfold greater
concentration of cold native ligand than the
highest concentration in test samples and total
bound radioactivity was detected in the wells
with no competitor. Percent specific binding was
calculated as following Specific Binding
Bound-NSB / Total bound- NSB x 100. IC50 values
were determined by using Origin software
(OriginLab, Northampton, MA).
Abstract
Insulin constitutes a hormone of central
importance in physiology and a vital element in
glucose management. Its use in diabetes care has
been of seminal significance for nearly a
century. The advent of rDNA biosynthesis provided
human insulin in virtually unlimited quantity.
More importantly, it provided a mechanism by
which improved biosynthetic insulin analogs could
be synthesized, evaluated and registered as new
medicines. The advent of chemical biotechnology
(biosynthesis with unnatural amino acids)
provides a new venue for optimizing insulin
pharmacology through the use of synthetic
chemistries that otherwise would be prohibitively
expense for commercialization. Our work has
focused on two amino acids in the C-terminus of
the B-chain that are central to insulin activity,
specifically positions B24 and 25. The selective
introduction of a series of Phe-related
derivatives at each position has yielded an
insightful perspective on insulin receptor
recognition. Position 24 has proven highly
restrictive to changes that can be introduced and
the results emphasize the importance of backbone
conformation to full potency. In comparison,
position B25 is quite accommodating to sizable
changes in side-chain structure but surprisingly
highly demanding in that the phenyl ring must be
positioned at the beta-carbon. These collective
observations establish a foundation for
application of unnatural amino acids as a route
to insulin pharmacology that may not be
obtainable with natural amino acids alone.
Fig 3. Binding Affinity of Insulin Analogs with
Comparable Amino Acid Substitution at B24 B25
B24 IC50 nM
C
H
C
H
18.25
22.54
2
2
C
H
3
N
Results Conclusion Nineteen different amino
acids (17 non-natural) were substituted for the
native phenylalanine residues at positions B24
and B25 in insulin. The analogs were successfully
prepared by SPPS from individual A and B chains,
in total yields that varied between 1 and 10.
The relative binding affinity of this group of
insulin analogs for the insulin receptor was
determined to differ by more than thousand-fold
from the most potent to the least potent peptide
studied. Consistent with previous observations
predominantly employing native amino acids, we
observed that insulin activity was highly
dependent upon aromatic character at these two
residues (1). Position B24 was extremely
restrictive to structural modification while B25
was extremely permissive. In Figure 3 we report
the extreme difference between the same
substitutions at B24 and B25. Each of the B25
insulin analogs were of high affinity while only
one of the B24 analogs did not exhibit a sizable
reduction in binding affinity. The highly
homologous B24 Phe(4-F) analog is a rare example
of an l-amino acid with comparable potency to the
native hormone, and represents an opportunity for
structural study in the receptor recognition site
with isotopically enriched amino acid. Further
study at B24 (Figure 4) illustrates that even a
single methyl group added to the phenyl ring is
deactivating and most notably when ortho to the
ring. Phenylalanine heterocycle-based mimetics
were studied. An appreciable difference was
observed among two commonly used derivatives. The
5-membered thiophene ring provided nearly native
insulin affinity but the more alkaline 6-membered
pyridine ring was sizably reduced in
potency. Additional study at position B25 (Figure
5) demonstrated that movement of the phenyl ring
closer or further from the peptide backbone had a
significant deactivating impact upon binding
affinity (2). Additional modifications that
selectively introduced negative charge (4-COOH)
and size (4-Br) relative to the native Phe were
largely without effect. Although, the alkaline
amino-methyl substitution (4-CH2NH2) reduced
binding affinity to a significant degree. This
observation is consistent with the more subtle
reduction noted for the less alkaline 4-NH2
modification (Figure 3).
C
H
C
H
6.78
0.63
2
2
S
C
H
3
C
H
2
5.25
C
H
3
Fig 4. Binding Affinity of Insulin Analogs
Substituted at Position B24
Fig 1. Primary Structure of Human Proinsulin
Insulin B25 Phe(4-CF3)
0.46 ? 15cm Vydac C18 1ml/min, 45C, 214nm,
Linear gradient of CH3CN in 0.1 TFA(aq)
Theoretical MW 5875
Ubiquitin (MH) average
Ubiquitin (M2H) 2average
References
1. Mirmira, R.G., Nakagawa S.H., and Tager H.S.
(1991) J. Biol. Chem. 266, 1428-1436 2.
Nakagawa, S. H. And Tager, H.S. (1986) J. Biol.
Chem. 261, 7332-7341
Fig 2. Purified Insulin Analog Chromatographic
Mass Spectral Analysis
Acknowledgements Beili Quan is financially
supported by an Indiana University Gill Center
Fellowship.
Fig 5. Binding Affinity of Insulin Analogs
Substituted at Position B25