Replacement of the disulfide bridge in a KLK3-stimulating peptide using orthogonally protected building blocks

Article abstract of DOI:10.1021/ml400419g

Peptide B-2 , which is one of the most potent kallikrein-related peptidase 3 (KLK3)-stimulating compounds, consists of 12 amino acids and is cyclized by a disulfide bridge between the N- and C-terminal cysteines. Orthogonally protected building blocks were used in the peptide synthesis to introduce a disulfide bridge mimetic consisting of four carbon atoms. The resulting pseudopeptides with alkane and E-alkene linkers doubled the proteolytic activity of KLK3 at a concentration of 14 μM. They were almost as potent as the parent B-2 peptide, which gives a 3.6-fold increase in the proteolytic activity of KLK3 at the same concentration.

Full text of DOI:10.1021/ml400419g

Letter
pubs.acs.org/acsmedchemlett
Replacement of the Disulfide Bridge in a KLK3-Stimulating Peptide
Using Orthogonally Protected Building Blocks
Kristian Meinander,*,† Miikka Pakkala,‡,∥ Janne Weisell,‡ Ulf-Hakan Stenman,§ Hannu Koistinen,§
̊
Ale Narvanen,‡ and Erik A. A. Wallen†
́
̈
̈
†Division of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, FIN-00014 Helsinki, Finland
‡School of Pharmacy, University of Eastern Finland, P.O. Box 1627, FIN-70211 Kuopio, Finland
§Department of Clinical Chemistry, Haartman Institute, University of Helsinki and Helsinki University Central Hospital,
Haartmaninkatu 8, FIN-00290 Helsinki, Finland
S
* Supporting Information
ABSTRACT: Peptide “B-2”, which is one of the most potent kallikrein-related peptidase 3 (KLK3)-stimulating compounds,
consists of 12 amino acids and is cyclized by a disulfide bridge between the N- and C-terminal cysteines. Orthogonally protected
building blocks were used in the peptide synthesis to introduce a disulfide bridge mimetic consisting of four carbon atoms. The
resulting pseudopeptides with alkane and E-alkene linkers doubled the proteolytic activity of KLK3 at a concentration of 14 μM.
They were almost as potent as the parent “B-2” peptide, which gives a 3.6-fold increase in the proteolytic activity of KLK3 at the
same concentration.
KEYWORDS: Disulfide bridge mimetic, human kallikrein-related peptidase 3, prostate specific antigen, proteolytic activity,
pseudopeptide, peptide synthesis
allikrein-related peptidase 3 [KLK3, also known as
prostate specific antigen (PSA)] is currently the most
substrates and to have a higher affinity for KLK3 (J. M. Mattson
and H. Koistinen, unpublished results). These peptides
represent the most potent KLK3-stimulating compounds, and
the search for low-molecular weight compounds with the same
biological activity has so far been unsuccessful, although one
low-molecular weight compound possessing both a very weak
stimulating effect on its own and a weakly enhancing effect on
the stimulation by 1a has been identified.12
K
widely used serum marker for prostate cancer.1−3 KLK3 has
been shown to inhibit angiogenesis.4−6 This anti-angiogenic
activity is associated with the proteolytic activity of KLK3 and
can be enhanced by peptides that stimulate KLK3.5,6
Furthermore, a high level of KLK3 expression in tumors is
associated with a low microvessel density, and the level of
expression of KLK3 decreases in poorly differentiated tumors,
suggesting a tumor suppressing role for KLK3.7−9
In an earlier study of 1a,13 it was shown that most of the
amino acid side chains are essential for the KLK3 stimulating
activity. To improve the in vivo stability of the KLK3-
stimulating peptides, alternatives to the disulfide bridge for
cyclizing the peptide have been studied.14 These alternatives are
Peptide 1a (B-2) and its equipotent amide analogue, 1b,
stimulate the proteolytic activity of KLK3.10 Peptide 1a along
with two other peptides with similar biological activity was
identified using phage-display technology.10,11 The two most
potent peptides are 1a/1b and C-4 (structure not shown).10
Although previously published results indicate that peptide C-4
has a stronger stimulating effect on KLK3 with small peptide
substrates, 1a has proven to be more potent with larger protein
Received: October 18, 2013
Accepted: December 15, 2013
Published: December 16, 2013
© 2013 American Chemical Society
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based on cyclizing the peptide in a head-to-tail fashion using an
amide bond. The only potent replacement for the terminal
disulfide bridge consisted of γ-aminobutyric acid at the N-
terminus and aspartic acid at the C-terminus connected via an
amide bond (peptide 2). The analogue had improved stability
in plasma compared to that of 1a.14 The same study also
showed that the terminal NH2 group could be removed from
the peptide without a significant loss of biological activity.
Scheme 1. Synthesis of Building Blocks 8a, 8b, 9a, and 9b
spectra). This was further confirmed by the reduction of the
double bond in 6b that yielded only one product (7b).
Interestingly, the hydrolysis of the methyl ester in 6b was
found to enrich the E-form of building block 8b, yielding the E-
isomer in pure form. The cause for this difference in reactivity
between the two isomers is unclear, but the observation is
confirmed by NMR spectroscopy of both the isolated product
and unreacted starting material.
Figure 1. Structures of synthesized pseudopeptides and the linkers
used.
The synthesized mimetics 8a, 8b, 9a, and 9b were introduced
into the peptide following a previously published protocol.14
The peptide was attached to the resin (Rink Amide AM) from
the side chain of Asn6, followed by coupling of amino acids and
mimetics using Fmoc chemistry, and then finally cyclizing the
pseudopeptide between residues Asn6 and Tyr7 (Scheme 2).
Longer reaction times (4 h) were used for coupling the
orthogonally protected building blocks to ensure a high yield in
these steps. The yield-limiting step in the syntheses of the
pseudopeptides was expected to be the head-to-tail cyclization.
The main cause of this is the length of the peptide and the
bulky tyrosine at the terminal site of the cyclization point that
has previously resulted in low yields.14 One common impurity
according to MS studies of pseudopeptides 3a and 3b was a
linear peptide resulting from incomplete cleavage of the allylic
ester used for protecting the main chain carboxylic acid of the
aspartic acid, thus preventing the head-to-tail cyclization.
To verify the stereochemistry of the double bond in the
purified pseudopeptide 3c, we also analyzed it by NMR using a
method we have utilized in a previous study.18 This showed
that the peptide exclusively contained the E-isomer. After
purification of the pseudopeptides to >90% purity, the yields of
peptides were 0.6−5%.
We were interested in replacing the disulfide bridge in
peptide 1a with some of the orthogonally protected disulfide
bridge mimetics based on hydrocarbon linkers that we had
developed previously,15 to afford pseudopeptides 3a and 3b. As
the terminal NH2 group does not seem to be critical for
biological activity, analogues 3c and 3d were also included in
the series. Replacing the disulfide bridge with a hydrocarbon
linker has been reported to considerably increase the in vivo
stability and bioavailability of peptides.16,17 A similar alkene
linker has already been shown to be a successful disulfide bridge
replacement in the other KLK3-stimulating peptide, C-4.18
However, in the C-4 peptide, the alkene linker was introduced
after the peptide synthesis using a ring-closing metathesis
reaction that gave an inseparable mixture of the E- and Z-
alkenes.
The synthetic route to pseudopeptides 3a and 3b started
with the synthesis of the mimetics containing orthogonal
protecting groups compatible with the Fmoc protocol for solid
phase peptide synthesis (SPPS). The synthesis of building
blocks 8a and 9a needed for synthesis of pseudopeptides 3a
and 3b, respectively, is presented in Scheme 1 and is based on
our previously published procedure.15
Mimetics 8b and 9b lacking the terminal NH2 group were
synthesized in a manner analogous to that of 8a and 9a
(Scheme 1). However, the obtained cross metathesis product
(6b) was in this case an inseparable 3:1 mixture of the E- and Z-
The effect of the pseudopeptides on the proteolytic activity
of KLK3 was determined in an assay using a chromogenic
chymotrypsin substrate.13,14,19 The ability of pseudopeptides
3a−3d to stimulate the activity of KLK3 is summarized in
Table 1. All four pseudopeptides stimulated the activity of
KLK3; however, the effects were weaker than that of 1b, and
1
isomers (as determined by H and 13C NMR spectroscopy,
assignment of peaks using gCOSY, gHSQC, and gHMBC
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ACS Medicinal Chemistry Letters
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a
more potent than the E-alkene linker. This can possibly be
attributed to the higher flexibility of the saturated linker that
allows the peptide to adopt a conformation closer to that of the
biologically active conformation of the parent peptide.
In this work, we have presented the replacement of a
terminal disulfide bridge using an orthogonally protected
building block. Together with the pseudopeptides of the
KLK3-stimulating peptide C-4, which we have published
previously,18 we have now successfully introduced similar
hydrocarbon-based disulfide bridge replacements in two
structurally different KLK3-stimulating pseudopeptides. Nota-
bly, pseudopeptides 3a−3d have been synthesized employing a
synthetic strategy different from that used for pseudopeptides
based on C-4. We thereby conclude that the presented disulfide
bridge mimetics and the synthetic strategies for introducing
them could also be applied to other biologically relevant
peptides.
Scheme 2. Synthesis of Pseudopeptides 3a−3d
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures for the synthesis and purification of
peptides 3a−3d and compounds 6a and 7−9 and information
about the biological assay. This material is available free of
charge via the Internet at http://pubs.acs.org.
a
The X in intermediate 10 refers to a bridge mimetic corresponding to
building block 8a, 8b, 9a, or 9b.
AUTHOR INFORMATION
■
Corresponding Author
Table 1. Stimulation of KLK3 Activity by the Synthesized
Pseudopeptides at 14 μM (N = 2)
*Telephone: +358-9-191 59172. Fax: +358-9-191 59556. E-
mail: kristian.meinander@helsinki.fi.
compound
activity standard error (%)
Present Address
KLK3 alone
100
∥M.P.: Nesteoil Corp., P.O. Box 310. FIN-06101 Porvoo,
Finland.
1b
3a
3b
3c
3d
360 5.4
160 8.1
250 10
160 0.5
170 15
Funding
This work was supported by the Academy of Finland (Grants
126969, 135937, and 140929), the Graduate School of Organic
Chemistry and Chemical Biology, Research Funds of the
University of Helsinki, the Helsinki University Central
Hospital, the Finnish Cancer Foundation, the Sigrid Juselius
the activity of the best one (3b) was equal to that of the
previously published analogue with a linker consisting of
aspartic acid and γ-aminobutyric acid as the bridge mimetic
(peptide 2, 250% stimulation compared to that with KLK3
alone).14 Pseudopeptides 3a and 3b containing the disulfide
bridge mimetics increased the enzymatic activity to 160 and
250%, respectively, of that of KLK3 alone. Similarly,
pseudopeptides 3c and 3d stimulated KLK3 activity to 160
and 170%, respectively.
In this work, we have shown that it is possible to replace the
disulfide bridge in KLK3-stimulating peptide 1a with a
hydrocarbon linker without a significant loss of biological
activity. Interestingly, the four-carbon linkers containing an
alkane or E-alkene seem to be able to mimic the disulfide bridge
well despite the carbon atoms in the bridge being smaller in size
than the corresponding sulfur atoms in the natural disulfide
bridge. Furthermore, we have also confirmed that the terminal
NH2 group next to the disulfide bridge is not critical for the
biological activity of the pseudopeptides, although removing it
slightly decreases the stimulating effect of the pseudopeptide.
Even though the stimulating effect of the synthesized
pseudopeptides is weaker than that of the parent peptide,
they still significantly stimulate KLK3. In the pseudopeptides
both with and without the terminal NH2 group, the fully
saturated linker seems to result in a KLK3 stimulator slightly
Foundation, Finska Lakaresallskapet, and the Magnus Ehrn-
̈
̈
rooth foundation.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully thank Prof. Jouko Vepsalainen and Dr. Pasi
Soininen for help with the NMR analysis of peptide 3c, Ms.
̈
̈
Marianne Niemela for the biological testing, and Ms. Tiina
̈
Ahonen for help with the peptide synthesis.
ABBREVIATIONS
■
KLK3, human kallikrein-related peptidase 3; DCE, 1,2-
dichloroethane; HOAT, 1-hydroxy-7-azabenzotriazole; HATU,
2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hex-
afluorophosphate; HBTU, N,N,N′,N′-tetramethyl-O-(1H-ben-
zotriazol-1-yl)uronium hexafluorophosphate; TBTU,
N,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl)uronium tetra-
fluoroborate; DCM, dichloromethane; DIPEA, N,N-diisopro-
pylethylamine; Fmoc, 9-fluorenylmethoxycarbonyl; Boc, N-tert-
butoxycarbonyl; tBu, tertiary butyl; SPPS, solid phase peptide
synthesis
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kallikrein-related peptidase 3 activity. Med. Chem. Commun. 2013,
549−553.
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NOTE ADDED AFTER ASAP PUBLICATION
■
This paper published ASAP on December 19, 2013. Scheme 1
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