Synthesis of constrained tetracyclic peptides by consecutive CEPS, CLIPS, and oxime ligation

Article abstract of DOI:10.1021/acs.orglett.9b00378

In Nature, multicyclic peptides constitute a versatile molecule class with various biological functions. For their pharmaceutical exploitation, chemical methodologies that enable selective consecutive macrocyclizations are required. We disclose a combination of enzymatic macrocyclization, CLIPS alkylation, and oxime ligation to prepare tetracyclic peptides. Five new small molecular scaffolds and differently sized model peptides featuring noncanonical amino acids were synthesized. Enzymatic macrocyclization, followed by one-pot scaffold-assisted cyclizations, yielded 21 tetracyclic peptides in a facile and robust manner.

Full text of DOI:10.1021/acs.orglett.9b00378

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Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Synthesis of Constrained Tetracyclic Peptides by Consecutive CEPS,
CLIPS, and Oxime Ligation
Dieuwertje E. Streefkerk,†,∇ Marcel Schmidt,†,‡,∇ Johannes H. Ippel,§ Tilman M. Hackeng,§
Timo Nuijens,‡ Peter Timmerman,†,∥ and Jan H. van Maarseveen*,†
†Van ‘t Hoff Institute for Molecular Sciences (HIMS), Science Park 904, 1098 XH Amsterdam, The Netherlands
‡EnzyPep B.V., Urmonderbaan 22, 6167 RD Geleen, The Netherlands
§Department of Biochemistry (CARIM), University of Maastricht, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands
∥Pepscan Therapeutics, Zuidersluisweg 2, 8243 RC Lelystad, The Netherlands
S
* Supporting Information
ABSTRACT: In Nature, multicyclic peptides constitute a
versatile molecule class with various biological functions. For
their pharmaceutical exploitation, chemical methodologies that
enable selective consecutive macrocyclizations are required. We
disclose a combination of enzymatic macrocyclization, CLIPS
alkylation, and oxime ligation to prepare tetracyclic peptides.
Five new small molecular scaffolds and differently sized model
peptides featuring noncanonical amino acids were synthesized.
Enzymatic macrocyclization, followed by one-pot scaffold-
assisted cyclizations, yielded 21 tetracyclic peptides in a facile
and robust manner.
However, despite its ease, the applicability is limited to the
eptides are Nature’s most diverse toolkit and fulfill a
Pplethora of functions, ranging from hormonal to preparation of monocyclic and bicyclic peptides.
antimicrobial activities.1 In the past decade, especially cyclic
Recently, our group introduced a novel concept to expand
the CLIPS technology to furnish tricyclic and tetracyclic
peptides via a one-pot procedure, by combining it with two
orthogonal ligation methods: Cu(I)-catalyzed alkyne−azide
cycloaddition (CuAAC) and enzymatic head-to-tail cyclization
using omniligase-1 (chemo-enzymatic peptide synthesis,
CEPS).11,21 Here, we present a successful expansion of the
set, utilizing oxime ligation. This well-established orthogonal
ligation method involves the condensation reaction between an
aminooxy group and a carbonyl electrophile.22−25 In contrast
to CuAAC, the formed oxime bond shows E/Z isomerism,
which is influenced by their substituents.26
To explore the combination of CEPS, CLIPS, and oxime
ligation, a novel type of small-molecule scaffold was developed,
comprising two reactive primary bromides (CLIPS) in
combination with either (a) an aldehyde or (b) an aminooxy.
Both the aminooxy and aldehyde were chemically protected
(with Boc and diethyl-acetal groups) and are only liberated
after the initial CLIPS reaction to ensure a controlled,
regioselective cyclization. CEPS cyclization followed by
CLIPS and subsequently oxime ligation was envisaged the
most straightforward approach, based on previous experi-
ence.21 For oxime ligation, the peptide contains either (a) the
aminooxy or (b) the ketone moiety. Therefore, we set out to
peptides have attracted increased attention as a highly
promising class of therapeutics.2−4 Key features of macrocylic
peptides include increased metabolic stability and improved
binding affinity, compared to linear molecules.5 The set of
macrocycles in nature ranges from small monocyclic to highly
constrained multicyclic peptides such as cyclotides6,7 or the
“last-resort” antibiotic vancomycin, which is a prime example
of a multicyclic peptide drug.8,9 While Nature produces these
complex, highly constrained, multicyclic compounds in a
relatively straightforward manner using cascades of enzyme-
catalyzed reactions, their chemical synthesis is often elaborate
resulting in low overall yields.10 Clearly, there is a need to
overcome these challenges by the development of novel
synthetic methodologies. Thanks to their inherent properties,
such as excellent regioselectivity and stereoselectivity, the use
of enzymes has recently gained increased attention as a tool for
peptide head-to-tail cyclization. Enzymes such as omniligase-1,
butelase, or sortase11−13 have been successfully employed for
this purpose.14 For example, omniligase-1 efficiently catalyzes
head-to-tail cyclization of linear C-terminal glycolate-ester
peptides in aqueous solution.11,15 In addition, peptide
cyclization using small molecule organic scaffolds has been
widely explored. For example, CLIPS technology (Chemical
Linkage of Peptides onto Scaffolds)16 is used for peptide
cyclization in vitro and for the generation of large phage-
displayed libraries of thioether-bridged bicyclic peptides.16−20
Received: January 29, 2019
© XXXX American Chemical Society
A
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Letter
Figure 1. Schematic representation of the CEPS/CLIPS/oxime cyclizations and the evaluated scaffold and peptides: (a) aminooxy-bearing peptides
with scaffolds T4-2(CO)/T4-3(CO) (Strategy I); (b) peptides comprising ketone-functionalized amino acids in combination with scaffolds
T4-1(ONH2)/T4-2(ONH2)/T4-3(ONH2) (Strategy II), for the synthesis of tetracyclic peptides; and (c) peptide codes and the corresponding
sequences.
investigate two different strategies (Figure 1). For Strategy I,
the peptide bears the aminooxy functionality, with the scaffold
(T4-2(CO)/T4-3(CO)) bearing the aldehyde, (Figure
1a), while Strategy II features a reversed orientation of the
aminooxy and ketone moieties (T4-1(ONH2)/T4-2(ONH2)/
T4-3(ONH2)) (Figure 1b). The set of scaffolds we studied
comprises the rigid T4-1(ONH2)27 and the flexible scaffolds
T4-2(ONH2)/T4-2(CO) and T4-3(CO)/T4-3(ONH2),
B
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all containing rotatable amide bonds in combination with a
bromoacetamide.28 [For the synthesis of the scaffolds, see the
Supporting Information (SI).]
bicycle, by the addition of trifluoroacetic acid in dichloro-
methane (2:1, v/v). The peptide was dried in vacuo before
oxime ligation was instigated in aqueous dimethylsulfoxide
(DMSO) (0.5 mM, 16 h at 40 °C). Reactions were monitored
via UPLC analysis (column temperature = 50 °C).
In Strategy I, the aminooxy residue is introduced in the form
of aminooxy-homoserine (hS(ONH2)),26 whereas in Strategy
II, two different keto-amino acids, namely para-acetyl phenyl-
alanine (F(CO)), being well-studied29−33 and a tert-butyl
ketone derivative of aspartic acid (D(CO)), were incorpo-
rated into the peptide, respectively. In Strategy I (Figure 1a),
scaffold aldehydes are used, presumably resulting in E-
configured oximes only. In contrast, Strategy II (Figure 1b)
starts from the keto-containing amino acids F(CO) and
D(CO); hence, a mixture of E/Z-oximes may be expected,
with an increased preference for the E-isomer (D(CO) >
Following these generalized procedures, we first investigated
the reaction of hS(ONH2)-containing monocyclic hexadeca-
peptides with scaffolds T4-2(CO) and T4-3(CO)
(Strategy I, Figure 1a). The CLIPS reactions, yielding the
bicyclic peptides, went smoothly (see the SI). For the oxime
ligation, these peptide−scaffold combinations are the least
hindered as it contains the aldehyde, which, in principle,
should solely yield the E-isomer upon oxime ligation. Both
tetracycles c13333-hS(ONH2)·T4-2(CO)c/o and c13333
-
hS(ONH2)·T4-3(CO)c/o were formed as a mixture of two
different products (tR = 0.82/0.83 min and tR = 0.70/0.72 min)
with identical molecular weights. This can likely be attributed
to hindered rotation around either the aryl-C(O) or the
HN-CO bond in the scaffolds, as a result of the relatively
small peptide ring size (Figures 2a and 2d), matching earlier
t
F(CO)), because of the steric hindrance of the Bu group.
In order to thoroughly investigate the combination of CEPS,
CLIPS, and oxime ligation for the preparation of tetracyclic
peptides, a library of eight peptides containing different
numbers of amino acids (n) between the CLIPS and oxime
junction points (n = 3, 4, 5) was designed (Figure 1c). The
synthesis of peptides containing both the C-terminal glycolate-
type ester, as required for the CEPS macrolactamization,15 and
the protected aminooxy/ketone amino acids, was performed
using classical automated SPPS (see the SI). Generally, the
ketone-containing peptides were easier to obtain (little to no
side reactions) than peptides containing amino acid
hS(ONH2), of which the latter often resulted in a low-yielding
synthesis and troublesome purifications. Common side-
reactions included isopropylidene formation of the aminooxy
functionality with traces of acetone, incomplete SPPS-coupling
of Fmoc-hS(ONHBoc)−OH, and elimination of the aminooxy
moiety.34 Peptides containing D(CO) exhibited low
solubility in aqueous solutions, potentially requiring the
addition of solubilizing agents (e.g., urea) for efficient
enzymatic cyclization. Nevertheless, all linear peptide Cam-
esters (n = 3, 4, 5) were efficiently head-to-tail cyclized using
omniligase-1 (>85% average efficiency; see the SI). Initially, we
explicitly avoided placing one of the oxime-reactive non-
canonical amino acids, hS(ONH2), F(CO), D(CO), in
the enzymatic recognition sequence of the peptide (N-terminal
P1′ and P2′ and C-terminal P4−P1)35 in order to ensure a
high cyclization efficiency. However, it turned out that all three
amino acids were well-tolerated at the majority of positions
(F(CO) = all pockets; hS(ONH2) = P3, P2, P1′, P2′,
D(CO) = P3, P2; see the SI), despite distinct differences in
reaction rate that could, in turn, be compensated for by adding
an increased amount of biocatalyst. Generally, these results
demonstrate the broad applicability and compatibility of the
CEPS cyclization, even when using noncanonical amino acids.
Next, we explored the CLIPS and oxime ligation reactions
using the scaffolds given in Figure 1. Generally, CLIPS
reactions with the monocyclic peptides proceeded cleanly
under standard reaction conditions (0.5 mM peptide solution,
∼0.9 equiv of scaffold), aqueous NH4HCO3 solution (pH
>8.0), within 20 min at room temperature (rt)) to give the
corresponding bicyclic products (see the SI). This was
followed by deprotection of the scaffolds for oxime ligation.
For Strategy I, the acetals in T4-2(CO)/T4-3(CO) were
hydrolyzed by the addition of a 15% (v/v) aqueous TFA
solution. Under these conditions, oxime ligation occurs
instantaneously. For Strategy II, Boc-removal to liberate the
aminooxy moiety of T4-1(ONH2)/T4-2(ONH2)/T4-
3(ONH2) was performed after lyophilization of the CLIPSed
Figure 2. UPLC traces of Strategy I oxime ligations with hS(ONH2)-
containing bicyclic peptides (n = 3, 4, 5) with scaffolds (a−c) T4-
3(CO) and (d−f) T4-2(CO). Peak masses: M1 = first
encountered peak (tetracycle); M2, etc. = peaks with longer tR.
observations.21 Separation of the products was not attempted
and was deemed impossible, because the isomerism is
considered conformational, rather than configurational, assum-
ing both products are in thermodynamic equilibrium. This
assumption was confirmed by the fact that, for c13333
-
hS(ONH2)·T4-3(CO)c/o, a third isomer was initially
observed at tR= 0.73 min, that disappeared overnight at rt.
Interestingly, an increased loopsize (n = 4) in the cyclic 20-
peptide led to the formation of two broad product peaks for
the tetracyclic constructs c44444hS(ONH2)·T4-2(CO)c/o
and c44444hS(ONH2)·T4-3(CO)c/o (see Figures 2b and
2e). We attribute this peak broadening to slow equilibration
between several conformers on UPLC time scale, en route to
coalescence, while being comparatively faster than the
c13333hS(ONH2) tetracycles. Finally, for the largest 24-
membered peptides c65555-hS(ONH2)·T4-2(CO)c/o and
c65555-hS(ONH2)·T4-3(CO)c/o, the UPLC clearly shows a
single product (see Figures 2c and 2f), confirming that
C
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Figure 3. UPLC chromatograms of Strategy II oxime ligations with F(CO) peptides and scaffolds T4-1(ONH2)/T4-2(ONH2)/T4-3(ONH2)
reacted for 16 h at 40 °C. Peak masses: M1= first encountered peak (tetracycle); M2, etc.= peaks with longer tR.
Figure 4. UPLC chromatograms of Strategy II oxime ligations with D(CO) peptides and scaffolds T4-1(ONH2)/T4-2(ONH2)/T4-3(ONH2)
reacted for 16 h at 40 °C. Peak masses: M1= first encountered peak (tetracycle); M2, etc. = peaks with longer tR. [Legend: (★) mono-oxime still
●
present; ( ) disulfide starting material not reacted during CLIPS.]
conformational isomers in these macrocycles are in rapid
equilibrium at the UPLC-time scale. This is consistent with
previous findings that the number of product isomers obtained
is linearly correlated to the difference in peptide lengths and
attributable to conformational/rotational rather than config-
urational/structural isomers.21
Because oxime formation within the systems studied for
Strategy II (Figure 1b) starts from ketones, E/Z mixtures may
arise. Although intermolecular oxime formation between linear
peptides containing two F(CO) residues with methoxy-
amine or benzylhydroxylamine gave only single products, the
prediction of the precise E/Z pattern of more-constrained
constructs remains challenging. Interestingly, in contrast to
Strategy I, not the peptide length appeared to be critical for the
isomer distribution, but the type of scaffold, either T4-
1(ONH2), T4-2(ONH2), or T4-3(ONH2), did seem to have
an effect (Figure 3).
Using scaffold T4-1(ONH2) resulted in the formation of
four products for all peptide lengths (Figures 3a−c). This was
innate to the scaffold, since the quaternary ammonium center
is prochiral. Upon peptide ligation in an unfavorable manner,
there is no rotational relaxation of the system, yielding two
isomers, which do not exchange within a time frame of one
month. When using scaffold T4-2(ONH2), we did not observe
thermodynamic equilibration, but obtained the product with
2−4 different isomers, depending on the specific peptide used
(see Figures 3d−f). For example, in the case of c54444-F(C
O)·T4-2(ONH2)c/o (Figure 3e), two distinct products were
formed, that proved thermodynamic stability and did not
interconvert upon separation of both peaks. In contrast, at
elevated temperatures (40 °C), c23333‑F(CO)·T4-
3(ONH2)c/o (Figure 3g) forms one thermodynamic product
from initially four isomers. For c54444-F(CO)·T4-
3(ONH2)c/o, however, very close running products were
obtained and could only be separated using a slow eluting
UPLC gradient (Figure 3h). Lastly, c75555-F(CO)·T4-
3(ONH2)c/o instantaneously yields a single product (Figure
3i), suggesting that larger cycles might also result in the
formation of a single product.
Since the n = 4 peptides were chosen based on the sequence
of a previously published tetracyclic peptide,21 we opted to
compare the structure of c54444-F(CO)·T4-2(ONH2)c/o
with the NMR structure of the identical peptide fused with a
T42 scaffold.21,36 However, NMR studies revealed that the
tetracyclic structure of c54444-F(CO)·T4-2(ONH2)c/o is
present in many different conformations, because of hindered
rotation around the scaffold’s amide bond, as well as a slow
D
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Author Contributions
∇These authors contributed equally.
Notes
equilibrium of the E/Z oxime bond. This prevented a detailed
structure determination of the scaffold (see the SI).
Similar to F(CO), D(CO) also contains an asymmetric
ketone. However, because of the bulky tert-butyl group, it is
expected that oxime ligation occurs selectively at one face of
the ketone only, thus yielding single-isomeric products. Initial
experiments with methoxyamine revealed that the free amino
acid is fairly unreactive and intermolecular reactions took
weeks to complete. On the other hand, intramolecular
reactions are much faster, yet slower, compared to F(CO).
Since D(CO) is very apolar and poorly reactive, only the
The authors declare the following competing financial
interest(s): PT is employee of Pepscan Therapeutics at
which part of the work has been carried out. The methodology
described may be commercialized in the future.
ACKNOWLEDGMENTS
■
This project was financed by The Netherlands Organization
for Scientific Research (NWO-TTW, Project No. 12557). The
authors wish to thank Dr. Rodney Lax (EnzyPep B.V.) for
useful discussions and providing corrections to this manuscript.
Ed Zuidinga (UvA) and Dennis Suylen (Maastricht) are kindly
acknowledged for exact mass analysis.
most divisive loop lengths (c33333-D(CO) and c85555
-
D(CO)) were investigated (Figure 4). The reactivity of
both peptides is quite similar and for all scaffolds (T4−
1(ONH2) to T4−3(ONH2)), a single main product was
obtained, which was especially surprising in the case of scaffold
T4-1(ONH2) (see Figures 4a and 4b). However, all reactions
did not go to completion and mono-oximed products were still
present after several weeks. It seems that only thermodynami-
cally favorable tetracycles are formed, and unfavorable
conformations of the mono-oxime do not react further.
Clearly, the gain of selectivity comes at the cost of the
reaction rate, but is the only system with consistent single-peak
results of the tetracyclic peptide for all peptide/scaffold
combinations.
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AUTHOR INFORMATION
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Corresponding Author
*E-mail: j.h.vanmaarseveen@uva.nl.
ORCID
Marcel Schmidt: 0000-0001-9618-1376
Peter Timmerman: 0000-0001-6687-5297
Jan H. van Maarseveen: 0000-0002-1483-436X
E
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DOI: 10.1021/acs.orglett.9b00378
Org. Lett. XXXX, XXX, XXX−XXX