General and Facile Route to Isomerically Pure Tricyclic Peptides Based on Templated Tandem CLIPS/CuAAC Cyclizations

Article abstract of DOI:10.1002/anie.201709127

We report a one-pot ligation/cyclization technology for the rapid and clean conversion of linear peptides into tricyclic peptides that is based on using tetravalent scaffolds containing two benzyl bromide and two alkyne moieties. These react via CLIPS/CuAAC reactions with cysteines and azides in the peptide. Flexibility in the scaffolds is key to the formation of isomerically pure products as the flexible scaffolds T4₁ and T4₂ mostly promote the formation of single isomeric tricycles while the rigid scaffolds T4₃ and T4₄ do not yield clean products. There seems to be no limitation to the number and types of amino acids present as 18 canonical amino acids were successfully implemented. We also observed that azides at the peptide termini and cysteine residues in the center gave better results than compounds with the functional groups placed the other way round.

Full text of DOI:10.1002/anie.201709127

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Accepted Article
Title: General and Facile Route to Isomerically Pure Tricyclic Peptides
using Templated Tandem CLIPS/CuAAC Cyclizations
Authors: Jan Herman Van Maarseveen, Peter Timmerman, Gaston
Richelle, Sumeet Ori, and Henk Hiemstra
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201709127
Angew. Chem. 10.1002/ange.201709127
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10.1002/anie.201709127
Angewandte Chemie International Edition
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General and Facile Route to Isomerically Pure Tricyclic Peptides
using Templated Tandem CLIPS/CuAAC Cyclizations
Gaston J. J. Richelle, Sumeet Ori, Henk Hiemstra, Jan H. van Maarseveen,* and Peter Timmerman*
symmetrical scaffold to assemble linear peptides into tricycles,
while Suga,[19] Nuijens[20] and our group[21] used various types of
backbone cyclization in combination with T3-CLIPS. These and
Abstract: We report a one-pot ligation-cyclization technology for
the rapid and clean conversion of linear peptides into tricyclic
peptides using tetravalent scaffolds containing two benzyl
bromides and two alkyne functionalities that react via
CLIPS/CuAAC reactions with cysteines and azides in the peptide.
Flexibility in the scaffolds is key to the formation of isomerically
pure products, since flexible T41 and T42-scaffolds mostly
promote formation of single isomeric tricycles, while rigid T43 and
T44-scaffolds resist formation of clean products. There seems no
limitation to the number and type of amino acids present, since 18
canonical amino acids were successfully implemented. We also
observed that azides at the peptide termini and cysteines in the
center gave better results than vice versa.
Cyclic peptides have emerged as
a promising class of
therapeutics[1–4] showing a wide therapeutic window that ranges
from antifertility[5] to anticancer[6–8] and antiviral[9–11] applications.
As the number of cyclic peptides entering clinical trials has
drastically increased over the last years,[6] the search for novel
synthetic routes to multicyclic peptides has gained serious
Figure 1. Structure of vancomycin and a tricyclic peptide format inspired by its
multicyclic structure.
interest.[12–23]
In 2005, our group introduced “CLIPS-technology”
other examples emphasize the interest in multicyclic peptides,
despite the fact that formation of multiple isomers, laborious
reaction protocols as well as difficulties in generating structural
(Chemical LInkage of Peptides onto Scaffolds), involving tandem
ligation-cyclization of tris(cysteine)-containing linear peptides
withα,α’,α’’-tribromomesitylene (T3) to give the corresponding
bicyclic peptides.[12] Heinis et al. applied this technology in phage
display libraries, reaching diversity levels as high as 109 – 1013
different bicycles.[13] Biological evaluation against various
proteins[13–16] identified potent bicyclic peptides with Ki values in
the nM-range. In some cases, the best inhibitors displayed only
(sub)µM activities,[24] suggesting that higher-order structures, like
tricycles, may be needed to reach improved activity levels.
Vancomycin, a last-resort antibiotic glycopeptide wrapped around
an aromatic core provides an illustrative example of such a
complex architecture (Figure 1).[25] Currently, complex syntheses
impede the large-scale production of vancomycin-like drugs, and
complicates structural diversification processes aimed at
optimizing their biological activities.[26]
diversity remain
tetrakis(cysteine)-containing
a
delicate issue. CLIPS-reactions of
peptides with 1,2,4,5-
tetrabromodurene (T4) would provide a straightforward route to
manufacturing tricycles. However, this reaction yields a mixture of
six different regioisomers,[18] which renders this strategy not
suitable for a therapeutic development trajectory (Figure 2a).
Here we describe a novel ligation-cyclization strategy that
combines CLIPS with copper-catalyzed azide-alkyne cyclization
(CuAAC). We observed that CuAAC-chemistry is fully compatible
with both peptide[27,28] and CLIPS[22,23] chemistry and does not
require protection of any of the amino acid side chains (Figure 2b).
Four different T4-scaffolds were synthesized containing two
CLIPS- and two CuAAC-functionalities (Figure 2c), whereby for
the latter we choose for alkynes at the scaffold (circumvents
undesired risk of explosion with multiple azides on low-molecular-
weight compounds) and azides in the peptides (Fmoc azido
amino acids are readily available[29]). All four T4x-scaffolds display
very different numbers of rotatable bonds connecting the alkynes
to the core (4/2/1/0 for T41/2/3/4, respectively). This allowed us to
evaluate how this ’’flexibility’’ governs the outcome of the
reactions. Moreover, T41 and T42 have a freely rotatable bond
(aryl-aryl bond for T41, aryl-amide bond for T42), which should
prevent formation of regioisomeric products, while such rotation
is not possible for the rigid T43 and T44. Synthesis routes for T41
and T42 are depicted in Scheme 1, for details see SI sections 1
and 2.
Examples of synthetic tricyclic peptides have been reported,
as both Ruchala[17] and Wu[18] used either a Th- or a D2h-
[*]
Gaston J. J. Richelle, MSc, Sumeet Ori, MSc, Prof. dr. Henk
Hiemstra, Prof. dr. Jan H. van Maarseveen, Prof. dr. Peter
Timmerman
Bioinspired Organic Synthesis
Van ‘t Hoff Institute for Molecular Sciences (HIMS)
Science Park 904, 1098 XH Amsterdam (The Netherlands)
E-mail: J.H.vanMaarseveen@uva.nl
Prof. dr. Peter Timmerman
Pepscan Therapeutics
Zuidersluisweg 2, 8243 RC Lelystad (The Netherlands)
E-mail: P.Timmerman@pepscan.com
Supporting information for this article is given via a link at the end of
the document
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10.1002/anie.201709127
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Figure 2. a) Reaction of linear peptides containing four cysteines (red circles) with T4 give a mixture of six isomers (o = ortho, m = meta, p = para); b) Schematic
representation of the CLIPS/CuAAC ligation-cyclization of linear peptides containing two thiols (red open circles) and two azides (green open circles) that react with
either two bromomethyl groups (red hazy circles) and two alkynes (green hazy circles) on a T4x-scaffold (x = 1-4) to first generate a monocyclic CLIPS-peptide (e.g.
[1555-T4x]) and subsequently a tricyclic peptide (e.g. [I555-T4x]) with two thioether linkages (red filled circles) and two triazole linkages (green filled circles); c) Chemical
structures of the T4x-scaffolds, either with free (T41 and T42) or blocked internal rotation (T43 and T44).
After optimization of the reaction conditions (SI sections 4 and 5),
the linear peptide (1333, Figure 3a; for peptide overview, see Table
1), with 3x3 amino acids separating the two reactive cysteines (C)
scaffolds T41 and T42, which resulted in complete conversion into
the CLIPS-products within 30 min (Figure 3b). Then, a pre-
incubated mixture of CuSO4/THPTA/NaAsc in H2O was added to
trigger the CuAAC cyclizations, which led to complete conversion
into the expected tricyclic peptides within minutes (Figure 3c).
Double triazole formation was confirmed by the fact that TCEP
incubation, which reduces azides to amines did not cause any
change, while addition of TCEP prior to the CuAAC-reaction
showed complete double reduction (MW-52 Da) in ~12 h. UPLC-
analysis of the CuAAC reactions showed a slightly better profile
for T41 as compared to T42, as judged from the elevated baseline
for T42. This is likely due to increased polymerization of the mono-
and bicyclic intermediates as a result of restricted flexibility of T42
compared to T41. Purification by RP-HPLC yielded the tricyclic
peptides [I333-T41] (28%) and [I333-T42] (18%) based on crude
linear precursor. CLIPS-reaction of 1333 and T43 resulted in
formation of the desired monocycle, however, CuAAC reaction
afforded the tricyclic peptide as a mixture of two isomers, along
with severe side product formation. This supports our view of
flexibility in the linkers being essential for obtaining clean and
isomerically pure products. Successful conversion of other 333-
peptides (2333 to 9333, Table 1) using both T41 and T42 proved that
the technology does tolerate a wide range of different canonical
Scheme 1. a) Synthesis of T41 scaffold; b) Synthesis of T42 scaffold; dba =
dibenzylidene acetone, DIPEA
= N,N-diisopropylethylamine, DMAP = 4-
dimethylaminopyridine, DPEPhos = bis[(2-diphenylphosphine)phenyl] ether,
amino
acids
in
the
peptides
(18
in
total:
dppf = (diphenylphosphino)ferrocene, PPTS = pyridinium p-toluenesulfonate.
A/D/E/F/G/H/I/K/L/N/Q/P/R/S/T/V/W/Y, SI 8.7). Changing the
order to a CuAAC/CLIPS process was not successful due to
and azides (Aha = azidohomoalanine), was reacted with flexible
Table 1. Overview of all peptides evaluated in this study; for UPLC-MS data of the CLIPS/CuAAC reactions, see SI section 8.7.
Code
Sequence
Code
Sequence
Code
Sequence
Code
Sequence
1333
2333
3333
4333
5333
CQWG[Aha]KAS[Aha]FSEC
CNSN[Aha]SKE[Aha]TWNC
CQYR[Aha]KIL[Aha]KGRC
CAIP[Aha]RYR[Aha]NVTC
CTHW[Aha]QEK[Aha]SGNC
6333
7333
8333
9333
10222
CHPY[Aha]RQV[Aha]TVDC
CDHV[Aha]KFY[Aha]RHDC
CNEG[Aha]SHN[Aha]GIKC
CQLQ[Aha]GSY[Aha]RFIC
CQW[Aha]KA[Aha]FSC
11222
12222
13222
14111
15111
CES[Aha]FA[Aha]KKC
ACGS[Aha]FE[Aha]KNCG
NACEE[Aha]FK[Aha]KSC
CQ[Aha]K[Aha]FC
16444
17555
18333
19111
CQWGA[Aha]KASE[Aha]FSEKC
CQWGAS[Aha]KASEV[Aha]FSEKGC
[Aha]QWGCKASCFSE[Aha]
[Aha]QCKCF[Aha]
CE[Aha]F[Aha]KC
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10.1002/anie.201709127
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Figure 3. a) UPLC-MS of crude linear peptide 1333; CLIPS (b) and CuAAC (c) UPLC reaction analysis of 1333 with T41 , T42 and T43; MW of T43 measured minus
Br-; • = deletion products present in crude linear peptide; # = excess scaffold; ¤ = mono-CuAAC intermediate; £ = S-S oxidized monocycle; */† = two isomers.
unwanted copper-induced cysteine S-S oxidation during CuAAC.
We then studied 222-peptides, where for peptide 10222 only
[X222-T41] was formed as a single tricycle (Figure 4a), while for
[X222-T42] a second isomer was observed in an otherwise clean
chromatogram (Figure 4b). We believe that the presence of the
second isomer originates from restricted rotation caused by the
tertiary amide bond in [X222-T42], for which the energy barrier is
significantly higher than for rotation around the aryl-aryl bond in
[X222-T41].[30,31] With T43 reactions, again two isomeric products
were observed along with severe side product formation (Figure
4c). For peptides 11222 to 13222 (SI 8.7), we observed the same
pattern, i.e. clean formation of the monocyclic CLIPS-products
[#222-T4x] with all four scaffolds. While tricycles on T41/2 were
formed in a clean manner, severe side products/polymer
formation for T43 and no detectable products for T44 were
observed. This reveals that it is mainly the loop size and choice
of scaffold rather than the nature of the amino acids in the loops
that governs the outcome of the reactions.
other causing additional intermolecular CuAAC reactions to
overtake. CuAAC of [15111-T41] gave slightly better results, but
also here two isomers were obtained, illustrating that restricted
aryl-aryl bond rotation in [#111-T41] tricycles seems a general
phenomenon. To further investigate this, we studied the
cyclization of single azide peptides CE[Aha]FAKC and
CEAF[Aha]KC with scaffold T41, which showed complete
conversion into single isomeric peptides (see SI section 7a),
showing the low rotation barrier around the aryl-aryl bond in the
bicyclic intermediate becoming more hindered only in the last
cyclization step when bis(azido)peptides are cyclized.
We also investigated ligation-cyclization of peptides 16444
and 17555. Conversion into isomerically pure tricycles [XVI444-T4x]
and [XVII 555-T4x] was successful for both T41 and T42 (SI 8.7),
albeit that several deletion variants present in the linear peptides
were also detected in the tricyclic products.
Finally, we studied two sequence variants of peptides 1333
and 14111, in which the positions of the Cys- and Aha-residues
were interchanged, while the rest of the sequence was kept the
same. Reaction of peptide 18333 with either scaffold T41/2 cleanly
gave both the tricyclic products [XVIII333-T41/2] as a single isomer
(Figure 5a). The product from reaction with the rigid scaffold T43
showed still the formation of two different isomers as expected.
Cyclizations of monocycles like [18333-T41/2] are apparently far
Subsequently, we studied the cyclization of peptide 14111 (SI
8.7). Even though the CLIPS-product [14111-T41] was cleanly
formed, tricycle [XIV111-T41] now also gave a mixture of two
isomers (ratio 2:3), likely due to restricted rotation around the aryl-
aryl bond, which was also the case for scaffold T42. It seems that
the azides and alkynes in [14111-T41/2] cannot easily reach each
Figure 4. a) UPLC CuAAC reaction analysis of 10222 on T41 (a), T42 (b) and T43 (c); */† = two isomers.
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Figure 5. UPLC CuAAC analysis of peptide 18333 (a) and 19111 (b) with T41 (i), T42 (ii) or T43 (iii); • = tricycle thioether oxidation; */† = two isomers.
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or T42 (Figure 5b) giving fairly clean formation of the
corresponding tricyclic peptides [XIX111-T41/2], implying that Cys-
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that are otherwise difficult to manufacture.
In summary, we developed a widely applicable one-pot
technology for the locking of linear peptides into isomerically pure
tricycles. No restrictions were observed as to the nature and
number of amino acids in the loops (except for additional Cys and
Met which were not investigated). The technology is currently
under investigation for high-diversity generation to enable
screening and identification of therapeutically relevant tricyclic
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Acknowledgements
These investigations were supported by The Netherlands
Research Council for Chemical Sciences (CW) with financial aid
from NWO Domain Applied and Engineering Sciences (TTW).
[24]
[25]
[26]
Conflict of Interest
The authors declare no conflict of interest.
[27]
[28]
Keywords: peptide • cyclization • tricycle • CLIPS • CuAAC •
single • isomer • one-pot
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10.1002/anie.201709127
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COMMUNICATION
Entry for the Table of Contents (Please choose one layout)
COMMUNICATION
Accessing single isomeric
Gaston J. J. Richelle, Sumeet
Ori, Henk Hiemstra, Jan H.
van Maarseveen, and Peter
Timmerman
tricyclic
peptides:
Rotatable
tetravalent
scaffolds containing both two
bromomethyl- and two
Page No. – Page No.
alkyne-functionalities were
reported that enable locking
of linear peptides containing
two Cys- and two Aha-
General and Facile Route to
Isomerically Pure Tricyclic
Peptides using Templated
Tandem CLIPS/CuAAC
Cyclizations
residues via
a
one-pot
ligation-cyclization
procedure
topologies,
limitations
into
with
observed
tricyclic
no
in
number and type of amino
acids displayed in the loops.
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