C-terminal acetylene derivatized peptides via silyl-based alkyne immobilization

Article abstract of DOI:10.1021/ol401332r

A new Silyl-based Alkyne Modifying (SAM)-linker for the synthesis of C-terminal acetylene-derivatized peptides is reported. The broad scope of this SAM2-linker is illustrated by manual synthesis of peptides that are side-chain protected, fully deprotected, and disulfide-bridged. Synthesis of a 14-meric (KLAKLAK)₂ derivative by microwave-assisted automated SPPS and a one-pot cleavage click procedure yielding protected 1,2,3-triazole peptide conjugates are also described.

Full text of DOI:10.1021/ol401332r

ORGANIC
LETTERS
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Vol. XX, No. XX
000–000
C‑Terminal Acetylene Derivatized
Peptides via Silyl-Based Alkyne
Immobilization
Martin Strack, Nils Metzler-Nolte, and H. Bauke Albada*
Inorganic Chemistry I, Bioinorganic Chemistry, Faculty of Chemistry and Biochemistry,
€
Ruhr-Universitat Bochum, Universitatsstrasse 150, 44801 Bochum, Germany
€
h.b.albada@gmail.com
Received May 13, 2013
ABSTRACT
A new Silyl-based Alkyne Modifying (SAM)-linker for the synthesis of C-terminal acetylene-derivatized peptides is reported. The broad scope of
this SAM2-linker is illustrated by manual synthesis of peptides that are side-chain protected, fully deprotected, and disulfide-bridged. Synthesis of
a 14-meric (KLAKLAK)2 derivative by microwave-assisted automated SPPS and a one-pot cleavage click procedure yielding protected 1,2,3-
triazole peptide conjugates are also described.
From the dawnofresin-basedpeptide synthesis in 1963,1
the field has advanced to the current level that allows
the preparation of polypeptides, and their use in various
ligation strategies to afford even entire proteins.2,3 As a
result of these advancements, peptides are being used
in biomedical research,4 in catalysis,5 and in advanced
materials,6 to name a few. In light of the many applications
of peptides, new synthetic methods for a more con-
venient preparation of diverse peptides are desired.
One of the most prominent diversification methods re-
cently utilized is the solid-phase compatible CuI/DiPEA
1,3-dipolar alkyneÀazide cycloaddition reaction, as initi-
ally described by Meldal and co-workers.7 This reaction is
used not only in peptide conjugation8 but also across the
entire chemical landscape.9
In view of the widespread applications of this so-called
click reaction and the availability of silyl-based protecting
groups for acetylene moieties,10 we pursued the prepara-
tion of a linker that is based on a silyl-protected alkyne and
that can be used in standard solid-phase peptide synthesis
(SPPS). The first generation of this class of silyl-based
alkyne modifying (SAM)-linkers was recently described by
our group; after fully automated microwave-assisted
SPPS, fluoride-mediated cleavage yielded a C-terminal
arylacetylene derivatized peptide.11 Notably, SAM-linkers
allow orthogonal click modifications: side-chain posi-
tioned acetylene moieties can be clicked during the pep-
tide-chain assemblyand orthogonally tothe silyl-protected
acetylene moiety that is used for immobilization; this can
(1) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149.
(2) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H.
Science 1994, 266, 776.
(3) Kochendoerfer, G. G.; Chen, S.-Y.; Mao, F.; Cressman, S.;
Traviglia, S.; Shao, H.; Hunter, C. L.; Low, D. W.; Cagle, E. N.;
Carnevali, M.; Gueriguian, V.; Keogh, P. J.; Porter, H.; Stratton,
S. M.; Wiedeke, M. C.; Wilken, J.; Tang, J.; Levy, J. J.; Miranda,
L. P.; Crnogorac, M. M.; Kalbag, S.; Botti, P.; Schindler-Horvat, J.;
Savatski, L.; Adamson, J. W.; Kung, A.; Kent, S. B. H.; Bradburne, J. A.
Science 2003, 299, 884.
(8) Meldal, M.; Tornøe, C. W. Chem. Rev. 2008, 108, 2952.
(9) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596.
(4) Kastin, A. J. Handbook of Biologically Active Peptides, 1st ed.;
Elsevier Inc.: Burlington, MA, 2006.
(5) Miller, S. J. Acc. Chem. Res. 2004, 37, 601.
(6) Gazit, E. Chem. Soc. Rev. 2007, 36, 1263.
(7) Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002,
67, 3057.
(10) Diederich, F.; Stang, P.; Tykwinski, R. Acetylene Chemistry:
Chemistry, Biology and Material Science; Wiley-VCH: Weinheim, 2004.
(11) Strack, M.; Langklotz, S.; Bandow, J. E.; Metzler-Nolte, N.;
Albada, H. B. J. Org. Chem. 2012, 77, 9954.
r
10.1021/ol401332r
XXXX American Chemical Society

Scheme 1. Synthesis of SAM2-Linker 1
Figure 1. Click modifications possible by Rink Amide (left) and
SAM (right) derivatized solid supports.
was reduced with DiBAL-H followed by acidic hydrolysis
to yield corresponding aldehyde 1in good yields (Scheme 1).
After this, SAM2-linker 1 was immobilized on a TentaGel
HL NH2 solid support (loading: 0.54 mmol/g) via reduc-
tive amination using 1 equiv of aldehyde 1 and 1.4 equiv of
NaB(OAc)3H. An IR spectrum of linker-loaded resin 7
showed a faint peak at 2172 cmÀ1, which can be assigned to
the carbonÀcarbon triple bond (see Supporting Information).
Unprotected amino groups were acylated with acetic
anhydride, followed by Tr-removal using a mixture of
DCM/TFA/TIS (94:4:2)15 and coupling of Fmoc-Leu-
OH and Fmoc-Phe-OH using standard SPPS procedures
at ambient conditions, resulting in a loading of 0.33 mmol/
g as determined by Fmoc-determination. Thus, an overall
yield of 61% was achieved with respect to the amount of
used linker 1 in seven steps. As peptide elongation steps
usually proceed quantitatively, the determined yield most
likely correlates to the immobilization of linker 1.
be conjugated in a final cleavage-click procedure12 (Figure1).
Generally speaking, SAM-linkers are particularly useful in
those cases where N-terminal or side-chain click modifica-
tions of peptides are detrimental for their activity, or when
an additional final click-conjugation reaction is required
thathastobe orthogonaltoside-chainorN-terminalbased
labeling reactions.
In principle, our first SAM-linker already contained
some important properties: it was conveniently prepared
on gram scale and showed general SPPS compatibility.
However, the presence of an arylacetylene makes the linker
incompatible with acids that are required to remove pro-
tecting groups in Fmoc/t-Bu peptide chemistry. Acid-
catalyzed hydration of the carbonÀcarbon triple bond
generates an acetophenone derivative, which can be used
in other ligation strategies, but not in the desired click
reaction with an azide anymore. Thus, we prepared an
improved version of the SAM-linker that is stable toward
acid treatments, especially applicable for peptides that
need a free N-terminus for their activity.13
Table 1. Amino Acid Composition of the Peptide Library
Herein, this versatile and economic second member of
the family of SAM-linkers is presented. In order to assess
the compatibility of silyl-based alkyne immobilization of
the SAM2-linker with SPPS, five nonapeptides containing
18 proteinogenic amino acid derivatives were synthesized
using manual Fmoc-based SPPS. In this focused library,
acid-stable (Acm for Cys), highly acid-labile (Mtt or Tr for
Lys or His and Cys, respectively), and standard (Pbf, Boc,
t-Bu) protecting groups were used to investigate a wide
spectrum of side-chain protection (Table 1). This library
was used to assess various cleavage conditions. Lastly,
compatibility of the SAM2-linker with on-resin disulfide-
bond formation and with microwave-assisted automated
SPPS was investigated.
Synthesis of SAM2-linker 1 was performed in three steps
with an overall yield of 62%. Propargylamine 2 was
protected using triphenylmethylchloride 3, affording Tr-
protected propargylamine 4 in high yield.14 Subsequently,
4 was treated with n-butyllithium in the presence of
(3-cyanopropyl)diisopropylchlorosilane (Cl-CPDIS) 5 to
yield bis-protected propargylamine 6 with a yield of 90%.
Finally, the cyano-group of trityl-protected silyl-alkyne 6
Parallel synthesis of the focused peptide library was
performed on five equal batches of Fmoc-Leu-Phe-
SAM2-TG using 20% piperidine/NMP for Fmoc-removal
and a mixture of Fmoc-AA(PG)-OH/HOBt/TBTU/Di-
PEA for coupling reactions (see Supporting Information).
Each coupling was analyzed with a Kaiser test16 to determine
completion of the acylation reaction. Fmoc-determination
of the completed peptide chains confirmed the stability of
SAM2-linker 1 during SPPS (see Supporting Information).
After final Fmoc-deprotection, fluoride-mediated cleavage
with 0.1 M TMAF 4H2O in DMF/MeOH (9:1) yielded
77À89% of crude materials (70À94% purity). Subsequent
3
(12) Friscourt, F.; Boons, G.-J. Org. Lett. 2010, 12, 4936.
(13) Albada, H. B.; Chiriac, A.-I.; Wenzel, M.; Penkova, M.; Bandow,
J. E.; Sahl, H.-G.; Metzler-Nolte, N. Beilstein J. Org. Chem. 2012, 8, 1753.
(14) Bhandari, M. R.; Yousufuddin, M.; Lovely, C. J. Org. Lett.
2011, 13, 1382.
(15) For the list of abbreviations, see Supporting Information.
(16) Schwetlich, K. Organikum, 4th ed.; Wiley-VCH: Weinheim, 2001.
B
Org. Lett., Vol. XX, No. XX, XXXX

purification by preparative HPLC provided side-chain
protected, acetylene derivatized peptides 9aÀe in 24À32%
yield (Scheme 2). Importantly, except for Asp(Ot-Bu), all
side-chain protecting groups;e.g. Trp(Boc), Lys(Mtt), or
Cys(Tr);remained intact during cleavage and purification.
Since Glu(Ot-Bu) did not release its protecting group upon
fluoride treatment, we assume aspartimide formation follo-
wed by hydrolysis to be the route to tert-butyl ester cleavage in
Asp(Ot-Bu).
After this successful synthesis of protected acetylene-
derivatized peptides, compatibility of the SAM2-linker
with CuAAC was investigated by applying a one-pot
cleavage click procedure for the direct synthesis of peptide
conjugates. For this, azidomethylferrocene 12, an air- and
water-stable, redox-active metallocene, was used as a sub-
strate.17 In short, peptide-loaded resin was combined with
washing the side-chain deprotected resin-bound peptides
with DiPEA, fluoride cleavage yielded the corresponding
deprotected, acetylene-derivatized peptides in 29À37%
yield after purification. Methionine oxidation in peptide
11e was avoidedusing1,2-ethylenedithiolinstead of 2-mer-
captoethanol in the deprotection cocktail. Whereas Cys
containing peptide 11b did not show acceptable purity after
fluoride cleavage, Cys(Acm) containing peptide 11a was
cleanly obtained. Therefore, the use of this amino acid is
recommended for the synthesis of free thiol functions.19
Scheme 2. Solid-Phase Synthesis of the Peptides and Chemical
Diversificationa
a methanolic solution of TMAF 4H2O, 12, DiPEA, and
3
CuI and was heated under microwave irradiation to 60 °C
for 30 min. The click-cleaved products were concentrated
to dryness and directly subjected to HPLC purification
affording side-chain protected, ferrocene-labeled peptide
conjugates 10aÀe in 24À53% yield (Table 2). Again, all
side-chain protecting groups, excluding Asp(Ot-Bu), were
stable toward these reaction conditions. Thus, the one-pot
cleavage click reaction is a highly promising route for the
synthesis of C-terminally modified peptide conjugates.
After this, the stability of the SAM2-linker toward
treatment with TFA/scavengers was investigated. For this,
all resin-bound library members were treated with a mix-
ture of TFA/H2O/TIS/TIA/2-ME (82.5:10:2.5:2.5:2.5,
% v/v) at ambient conditions for 1.5 h. Since no peptide
material was isolated from the TFA solutions, linker 4 is
shown to be stable against treatment with a concentrated
TFA/scavenger cocktail, making this SAM2-linker a
highly acid-stable alkyne-modifying linker. For compar-
ison, commonly used di-/trialkoxy BAL linkers release
peptides upon treatment with 1À30% TFA.18 After
a Yields of the purified peptides are given.
Lastly, SAM2 solid-support 7 was applied in the synth-
esis of two biologically relevant peptides: the disulfide-
bridged somatostatin analogue octreotate and the antimi-
crobial 14-meric (KLAKLAK)2 sequence. The peptide
chain of octreotate analogue 13 was assembled manually
Table 2. Experimental Data of C-Terminally Modified Peptides
a Reported yields are after HPLC-purification, crude yields were typically in the range of 77-89% (only for 9a-e, see Supporting Information).
Org. Lett., Vol. XX, No. XX, XXXX
C

resin was done by treatment with excess TMAF 4H2O in
3
DMF/MeOH (9:1, v/v) using microwave irradiation
(Tmax = 50 °C) for 30 min. After HPLC purification,
side-chain protected acetylene-modified (KLAKLAK)2
peptide 14 was obtained in 37% yield. A small amount
of peptide 14 (1 mg) was treated with TFA/H2O/TIS
(95:2.5:2.5), and HPLC analysis of this deprotected pro-
duct 15 showed high purity; MS analyses of peptides 14
and 15 confirmed formation of both the desired protected
and deprotected products.
In summary, a second generation silyl-based alkyne-
modifying linker is described which is generally applicable
in Fmoc/t-Bu-based solid-phase peptide synthesis. This
SAM2-linker yields C-terminal prop-2-yn derivatized peptide
carboxamides upon cleavage with tetramethylammonium
fluoride (TMAF). Except for Asp(Ot-Bu), compatibility
of protected peptides with fluoride cleavage is shown in the
synthesis of a focused peptide-library containing a broad
variety of proteinogenic, side-chain protected amino acid
derivatives. A cleavage click procedure allows a one-step
synthesis of protected 1,2,3-triazole peptide conjugates in
moderate to good yields after purification. In addition, this
SAM2-linker is the first generally applicable, highly acid-
stable silyl-based alkyne modifying linker yielding depro-
tected, acetylene-modified peptides after fluoride cleavage.
Furthermore, the linker is compatible with Tl(CF3COO)3
mediated disulfide-bond formation and applicable in the
synthesis of peptides of intermediate length as shown for
the 14-meric (KLAKLAK)2 sequence. We expect that the
SAM2-linker will find widespread applications in the syn-
thesis of C-terminally functionalized peptide bioconjugates.
Figure 2. Structure of octreotate analogue 13 and 14-meric
(KLAKLAK)2 sequence 14.
as described above. Fmoc-Cys(Tr)-OH was chosen in order to
follow disulfide-bond formation qualitatively with Ellman’s
reagent.20 Having removed the trityl groups, the disulfide-
bridgewasformedontheresinusingTl(CF3COO)3 at ambient
conditions.21 Subsequent cleavage with TMAF 4H2O and
3
purification by preparative HPLC yielded side-chain pro-
tected, disulfide-bridged octreotate 13 in 36% yield (Figure 2).
In addition, we assessed the compatibility of SAM2 resin 7
with microwave-assisted automated synthesis in the pre-
paration of the 14-meric (KLAKLAK)2 sequence, an
antimicrobial apoptosis inducing peptide.22 Using Fmoc-
Lys(Boc)-SAM2-TentaGel (loading: 0.30 mmol/g), auto-
mated SPPS was performed. After completion of the
reaction sequence a loading of 0.18 mmol/g was deter-
mined by Fmoc quantification after the 27 reaction steps
that were needed to complete the synthesis of the peptide.
Cleavage of the side-chain protected peptide from the
Acknowledgment. This research was supported by the
“Fonds der Chemischen Industrie” (fellowship to M.S.),
the State of North-Rhine Westphalia (NRW), Germany,
and the European Regional Development Fund, “Invest-
ing in your Future”.
(17) van Staveren, D. R.; Metzler-Nolte, N. Chem. Rev. 2004, 104,
5931. Sehnert, J.; Hess, A.; Metzler-Nolte, N. J. Organomet. Chem. 2001,
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637À639, 349. Jios, J. L.; Kirin, S. I.; Buceta, N. N.; Weyhermuller, T.;
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Vedova, C. O.; Metzler-Nolte, N. J. Organomet. Chem. 2007, 692, 4209.
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Husken, N.; Gasser, G.; Koster, S. D.; Metzler-Nolte, N. Bioconjugate
Chem. 2009, 20, 1578. Gasser, G.; Ott, I.; Metzler-Nolte, N. J. Med.
Chem. 2011, 54, 3.
(18) Boas, U.; Brask, J.; Jensen, K. J. Chem. Rev. 2009, 109, 2092.
(19) Fujii, N.; Otaka, A.; Watanabe, T.; Okamachi, A.; Tamamura,
H.; Yajima, H.; Inagaki, Y.; Nomizu, M.; Asano, K. J. Chem. Soc.,
Chem. Commun. 1989, 283.
(20) Riddles, P. W.; Blakeley, R. L.; Zerner, B. Reassessment of
Ellman’s reagent. In Methods in Enzymology; Hirs, C. H. W., Timasheff,
S. N., Eds.; Academic Press: 1983.
(21) Yajima, H.; Fujii, N.; Funakoshi, S.; Watanabe, T.; Murayama,
E.; Otaka, A. Tetrahedron 1988, 44, 805.
(22) Javadpour, M. M.; Juban, M. M.; Lo, J. L.; Bishop, S. M.;
Alberty, J. B.; Cowell, S. M.; Becker, C. L.; McLaughlin, M. L. J. Med.
Chem. 1996, 39, 3107.
Supporting Information Available. Detailed experimen-
tal procedures for on-resin preparations, NMR spectra of
compounds 1 and 6, IR spectrum of SAM2-loaded resin
7, and HPLC chromatograms as well as ESI mass spectra
of all peptides presented in this study are provided. This
material is available free of charge via the Internet at
http://pubs.acs.org.
The authors declare no competing financial interest.
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Org. Lett., Vol. XX, No. XX, XXXX