Synthesis of photoactive analogues of a cystine knot trypsin inhibitor protein

Article abstract of DOI:10.1021/ol702461z

We describe the preparation of a recently described photoactive amino acid analogue (photoMethionine) by two novel synthetic routes, one of which is flexible and enantiospecific, and the site-specific chemical incorporation of photoMethionine into a defined and functionally active protein using a combination of solid-phase peptide synthesis and modern chemical ligation methodology. Site-specific labeling of proteins with this amino acid analogue through chemical synthesis provides valuable probes for photoaffinity cross-linking studies.

Full text of DOI:10.1021/ol702461z

ORGANIC
LETTERS
2
007
Vol. 9, No. 26
497-5500
Synthesis of Photoactive Analogues of
a Cystine Knot Trypsin Inhibitor Protein
5
Thomas Durek,†,‡ Junliang Zhang, Chuan He, and Stephen B.H. Kent*
§
§
,†,‡,§
Institute for Biophysical Dynamics, Department of Chemistry, and Department of
Biochemistry and Molecular Biology, The UniVersity of Chicago,
Chicago, Illinois 60637
skent@uchicago.edu
Received October 9, 2007
ABSTRACT
We describe the preparation of a recently described photoactive amino acid analogue (photoMethionine) by two novel synthetic routes, one
of which is flexible and enantiospecific, and the site-specific chemical incorporation of photoMethionine into a defined and functionally active
protein using a combination of solid-phase peptide synthesis and modern chemical ligation methodology. Site-specific labeling of proteins
with this amino acid analogue through chemical synthesis provides valuable probes for photoaffinity cross-linking studies.
1
3
Chemical cross-linking, i.e., the formation of a covalent
molecule metabolites. However, in the case of peptides and
bond between spatially interacting protein molecules, has
emerged as an important biochemical tool in the proteomics
era. The cross-linking approach enables identification of
interacting proteins under in vivo conditions and in conjunc-
tion with state of the art mass spectrometry is a useful way
to elucidate the complex interaction networks that proteins
are known to participate in. In contrast to the classical
intrinsically reactive cross-linkers such as N-hydroxysuccin-
imide esters or maleimides, photoactivatable cross-linkers
require UV illumination to initiate cross-link formation
and offer the unique advantage of permitting temporal and
spatial control over reactivity. Most of the photoactivatable
reagents reported to date are based on arylazides, aryldiaz-
irines, and arylketones, which upon irradiation generate
highly reactive nitrene, carbene, or triplet biradical
proteins, their bulky aryl-structure and limited relatedness
to most naturally occurring amino acids can be disadvanta-
geous, especially when sterically demanding protein-protein
interaction interfaces are the subject of investigation.
Recently, novel alkyldiazirine amino acid analogues that
closely resemble the aliphatic amino acids Leu, Ile, and Met
4
have been described. These amino acid analogues were
shown to be incorporated into proteins by the biosynthetic
machinery of mammalian cells, which underscores their
structural similarity to the naturally occurring amino acids.
In this paper we report on the efficient site-specific incor-
poration of one of these analogues (“photoMethionine”
(phMet), Figure 1A) into a small protein using modern
chemical synthesis methods.
2
species, respectively. These functional groups have been
incorporated in the past into a number of biologically relevant
molecules including peptides, DNA, lipids, and small
(2) (a) Sinz, A. Mass Spectrom. ReV. 2006, 25, 663. (b) Sadakane, Y.;
Hatanaka, Y. Anal. Sci. 2006, 22, 209. (c) Chan, E. W. S.; Chattopadhaya,
S.; Panicker, R. C.; Huang, X.; Yao, S. Q. J. Am. Chem. Soc. 2004, 126,
14435. (d) Sieber, S. A.; Niessen, S.; Hoover, H. S.; Cravatt, B. F. Nat.
Chem. Biol. 2006, 2, 274.
(
3) (a) Nassal, M. J. Am. Chem. Soc. 1984, 106, 7540. (b) Kauer, J. C.;
†
Institute for Biophysical Dynamics.
Department of Biochemistry and Molecular Biology.
Department of Chemistry.
Ericksonviitanen, S.; Wolfe, H. R.; Degrado, W. F. J. Biol. Chem. 1986,
261, 695.
(4) Suchanek, M.; Radzikowska, A.; Thiele, C. Nat. Methods 2005, 2,
‡
§
(1) Brunner, J. Annu. ReV. Biochem. 1993, 62, 483.
261.
1
0.1021/ol702461z CCC: $37.00
© 2007 American Chemical Society
Published on Web 11/28/2007

loop because its side chain appears to be in close proximity
to a number of functional groups near the active site of
trypsin. The amino acid sequence of EETI-II is given in
Figure 1B. Synthesis of EETI-II wild-type and phMet
modified analogues was achieved by native chemical ligation8a
of three unprotected peptide segments, starting at the
C-terminus and extending the polypeptide chain toward the
8
b
N-terminus (Figure 1C). Although polypeptide chains of
this size can be made by optimized stepwise SPPS, we
believe that the modular synthesis demonstrated in this work
is particularly useful for the efficient preparation of ana-
logues. The ligation-based synthetic strategy also serves as
a prototype for the preparation of larger protein molecules
containing photoactive moieties.
4
Racemic Boc-phMet was synthesized in good yield by a
novel route involving alkylation of a glycine Schiff base with
1-iodo-3,3′-azibutane; details can be found in the Supporting
Information.
The racemic phMet was incorporated into the EETI-II
1
8 R
6
segment Gly -Arg - thioester in place of Leu by optimized
9
stepwise Boc SPPS chemistry. Side chain deprotection and
concomitant cleavage from the resin by treatment with
anhydrous HF gave the two diastereomeric peptide-
R
thioesters, which were separated by RP-HPLC and isolated.
Stereospecific synthesis of Boc-L-phMet was realized ac-
cording to Sch o¨ llkopf’s bislactim ether method, as described
in the Supporting Information (Scheme 1). This enabled us
Scheme 1. Synthesis of Boc-L-phMet
Figure 1. (A) PhotoMethionine and (B) amino acid sequence of
EETI-II. Cysteines used as strategic sites for native chemical ligation
of unprotected peptide segments are labeled in bold. The loop
interacting in a substrate-like manner with the active site of trypsin
is underlined. The site of incorporation of photoMethionine is
6
marked with an asterisk (Leu ). (C) Synthetic scheme for the
assembly of EETI-II from three unprotected peptide segments by
sequential native chemical ligation.
As a target for incorporation of phMet we chose the small
squash protein EETI-II (Ecballium elaterium trypsin inhibitor
type II), which interacts with trypsin and other related serine
5
proteases with high affinity (Kd in the range of pM-nM).
to determine the absolute configuration of phMet in both
diastereomeric peptides (see the Supporting Information). We
found the diazirine-modified amino acid to be stable toward
the repeated cycles of TFA treatment and HBTU/DIEA
activation used in chain assembly by Boc chemistry SPPS;
however, final HF treatment resulted in partial (∼30%) loss
6
EETI-II is a so-called “microprotein”, comprising a 30
amino acid residue polypeptide chain that contains six
cysteines that form three disulfides in the native protein
molecule. The inhibitor binds its target enzyme in a substrate-
like manner via an N-terminal loop comprising the amino
3
7
acids -Pro -Arg-Ile-Leu-Met - forming a tight and highly
complementary interaction interface (see the Supporting
(7) (a) Le Nguyen, D.; Heitz, A.; Chiche, L.; Castro, B.; Boigegrain, R.
A.; Favel, A.; Coletti-Previero, M. A. Biochimie 1990, 72, 431. (b) Kratzner,
R.; Debreczeni, J. E.; Pape, T.; Schneider, T. R.; Wentzel, A.; Kolmar, H.;
Sheldrick, G. M.; Uson, I. Acta Crystallogr. D: Biol. Crystallogr. 2005,
61, 1255.
7
Information). A crystal X-ray structure of the EETI-II:
7b
trypsin complex has been determined recently. We envi-
6
sioned incorporation of phMet in place of Leu in the binding
(
8) (a) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. Science
994, 266, 776. (b) Bang, D.; Kent, S. B. Angew. Chem., Int. Ed. 2004, 43,
2534.
(9) (a) Schnoelzer, M.; Alewood, P.; Jones, A.; Alewood, D.; Kent, S.
1
(
5) Le Nguyen, D.; Nalis, D.; Castro, B. Int. J. Pept. Protein Res. 1989,
3
1
4, 492.
(
6) Craik, D. J.; Clark, R. J.; Daly, N. L. Exp. Op. InVestig. Drugs 2007,
B. Int. J. Pept. Protein Res. 1992, 40, 180. (b) Hackeng, T. M.; Griffin, J.
H.; Dawson, P. E. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10068.
6, 595.
5498
Org. Lett., Vol. 9, No. 26, 2007

9
18
R
of the diazirine functionality by addition of HF. These
byproducts were readily removed by HPLC purification.
The peptides EETI-II(Thz -Gly )- thioester and EETI-II-
1
9
30
(Cys -Pro ) were covalently joined by native chemical
ligation in 6 M guanidine hydrochloride, 0.2 M sodium
phosphate, 30 mM mercaptophenylacetic acid, 20 mM tris-
(2-carboxyethyl)phosphine hydrochloride, pH 6.8 (see the
Supporting Information for details), after which Thz was
converted to Cys by treatment with methoxylamine hydro-
chloride at pH 4.0. Peptide EETI-II(Cys -Pro ) was isolated
in good yield (65%). Full-length wild-type and photoacti-
vatable analogues of EETI-II were finally obtained by
reacting unmodified and modified EETI-II(Gly -Arg )-
thioester peptides with EETI-II(Cys -Pro ) in separate
9
18 R
Peptide segments EETI-II(Thz -Gly )- thioester and EETI-
19
30
II(Cys -Pro ) were synthesized by Boc SPPS according to
9
published procedures. 1,3-Thiazolidine-4-carboxylic acid
9
9
9
30
1
8
R
9
30
native chemical ligation reactions using the conditions
described above. Native chemical ligation of the diazirine-
modified peptides proceeded smoothly, without accumulation
of major side products (Supporting Information). We con-
clude that the diazirine functionality is fully compatible with
native chemical ligation reaction conditions, including a
reducing environment as well as the presence of thiol
nucleophiles. Folding of the full-length polypeptides with
concomitant disulfide formation was initiated in all cases
simply by diluting the crude reaction mixture after the final
ligation into buffer containing a redox system consisting of
10 mM oxidized glutathione and 2 mM reduced glutathione.
Folding was complete within 2 h as determined in LC
analysis by the appearance of a dominant product eluting at
earlier retention time. Mass spectrometric analysis revealed
the loss of 6 Da upon folding, which is in agreement with
the formation of the three disulfides that are an integral part
5
of the cystine-knot fold of EETI-II. Three synthetic proteins,
the two diastereomeric analogues ([Leu6L-phMet]EETI-II
and [Leu6D-phMet]EETI-II) and the wild-type EETI-II, were
isolated in good yield by reverse-phase HPLC (Figure 2).
We next determined the ability of the synthesized protein
analogues to bind trypsin and inhibit its proteolytic activity.
R
To this end, trypsin was assayed using N -benzoyl-Arg-(4-
nitro)anilide as a chromogenic substrate in the presence of
11
increasing concentrations of inhibitors (Figure 3). [Leu6L-
phMet]EETI-II displayed strong inhibitory activity against
trypsin that was essentially indistinguishable from wild-type
EETI-II (Figure 3). Although precise determination of the
d
(very low) corresponding dissociation constants (K ) describ-
ing this interaction is generally difficult under these condi-
tions, inspection of the fitted data seems to suggest that they
are in the lower nanomolar range (data fitting revealed a K
d
of 0.7 ( 1.7 nM for both wild-type protein and the Leu6L-
phMet analogue). In contrast [Leu6D-phMet]EETI-II showed
Figure 2. LC-MS analysis of the purified EETI-II proteins. The
wild-type protein (A) and the Leu6L-phMet analogue (B) were of
high purity [insets: ESI-MS of the major peaks; the observed mass
for WT EETI-II was 3082.7 ( 0.1 Da; calculated mass 3082.6 Da
only weak activity against trypsin, with a determined K
d
of
about 800 ( 120 nM. These results indicate that substitution
6
6
of Leu by L-phMet in EETI-II does not significantly impair
its ability to interact with and inhibit trypsin. Replacement
(
average isotopes); the observed mass for [Leu6L-phMet]EETI-II
was 3108.4 ( 0.2 Da; calculated mass 3108.7 Da (average
isotopes)].
6
6
of Leu by D-phMet, on the other hand, is much less
tolerated, which underscores the high selectivity constraints
imposed by trypsin’s active site on EETI-II binding.
We next examined photo-cross-linking of [Leu6L-phMet]-
EETI-II to trypsin upon illumination with near-UV light. The
9
(
Thz) was used as a protected form of Cys in order to
8
b,10
prevent side reactions during the first ligation reaction.
(
11) Erlanger, B. F.; Cohen, W.; Kokowsky, N. Arch. Biochem. Biophys.
(10) Villain, M.; Vizzavona, J.; Rose, K. Chem. Biol. 2001, 8, 673.
1961, 95, 271.
Org. Lett., Vol. 9, No. 26, 2007
5499

Figure 3. Inhibition of trypsin by wild-type EETI-II (b) and
photoaffinity labeled analogues (] for Leu6L-phMet; 0 for Leu6D-
phMet). Data were fitted to the solution of a quadratic equation:
(s) WT; (‚‚‚) Leu6D-phMet; and (---) Leu6L-phMet.
two proteins were mixed in a 1:1 ratio at a concentration of
about 100 µM in aqueous buffer and the sample was
illuminated with a 450 W mercury lamp for 5 min. MALDI-
TOF analysis of the cross-linking reaction before and after
UV illumination (Figure 4) revealed the appearance of a
product with a mass 3.1 kDa higher than the calculated
molecular mass of trypsin (23.2 kDa), reflecting formation
of a chemical cross-link between trypsin and EETI-II (3.1
kDa).
Figure 4. Photo-cross-linking of [Leu6L-phMet]EETI-II and
trypsin. The two proteins were mixed in a 1:1 ratio at a concentra-
tion of ∼100 µM. (A) MALDI-TOF MS spectrum before illumation.
(
B) MALDI-TOF MS spectrum after illumination of the sample
with a 450 W mercury lamp for 5 min (λ > 300 nm). [Inset (upper
panel): SDS-PAGE analysis of the cross-linking reaction.]
experiments to help dissect protein-protein and peptide-
1
2
protein interactions in living cells.
We were unable to detect any species with higher
molecular weight, which further underscores the high
specificity of the cross-linking reaction. Photo-cross-linking
efficiency was about 40% judged by SDS-PAGE analysis
Acknowledgment. This research was supported by the
Office of Science (BER), U.S. Department of Energy, Grant
No. DE-FG02-07ER64501 (to S.B.H.K.).
(Figure 4 (inset)); the efficiency of cross-linking was not
increased by illumination times of up to 30 min.
Supporting Information Available: Experimental pro-
cedures and spectroscopic data for described compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
In summary we have reported the efficient synthesis of
Boc-protected analogues of photoMethionine by two new
synthetic routes giving access to both D and L stereoisomers.
The amino acid analogue is compatible with Boc chemistry
solid-phase peptide synthesis and with the native chemical
ligation of unprotected peptide segments. These methods will
enable the preparation of photoactivatable variants of peptides
and proteins and their use in photochemical “fishing”
OL702461Z
(12) During preparation of this manuscript, Muir and co-workers reported
on the semisynthesis of a phMet-containing analogue of the MH2 domain
of the SMAD2 protein by expressed protein ligation: Vila-Perello, M.; Pratt,
M. R.; Tulin, F.; Muir, T. W. J. Am. Chem. Soc. 2007, 129, 8068.
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