A Tetrazine-Labile Vinyl Ether Benzyloxycarbonyl Protecting Group (VeZ): An Orthogonal Tool for Solid-Phase Peptide Chemistry

Article abstract of DOI:10.1021/acs.orglett.8b00898

The vinyl ether benzyloxycarbonyl (VeZ) protecting group is selectively cleaved by treatment with tetrazines via an inverse electron-demand Diels-Alder reaction. This represents a new orthogonal protecting group for solid-phase peptide synthesis, with Fmoc-Lys(VeZ)-OH as a versatile alternative to Fmoc-Lys(Alloc)-OH and Fmoc-Lys(Dde)-OH, as demonstrated by the synthesis of two biologically relevant cyclic peptides.

Full text of DOI:10.1021/acs.orglett.8b00898

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
A Tetrazine-Labile Vinyl Ether Benzyloxycarbonyl Protecting Group
(VeZ): An Orthogonal Tool for Solid-Phase Peptide Chemistry
Matteo Staderini,† Alessia Gambardella,† Annamaria Lilienkampf, and Mark Bradley*
EaStCHEM School of Chemistry, University of Edinburgh, Joseph Black Building, David Brewster Road, Edinburgh, EH9 3FJ, U.K.
S
* Supporting Information
ABSTRACT: The vinyl ether benzyloxycarbonyl (VeZ)
protecting group is selectively cleaved by treatment with
tetrazines via an inverse electron-demand Diels−Alder reaction.
This represents a new orthogonal protecting group for solid-
phase peptide synthesis, with Fmoc-Lys(VeZ)-OH as a versatile
alternative to Fmoc-Lys(Alloc)-OH and Fmoc-Lys(Dde)-OH,
as demonstrated by the synthesis of two biologically relevant
cyclic peptides.
eptides are valuable scaffolds due to their intrinsic
making the target protein inactive until uncaged by a
tetrazine.24 Our group reported a polymeric drug delivery
system, which featured a vinyl ether as a dienophile that, upon
activation, uncaged a phenol(ate) that underwent subsequent
1,6-elimination and cargo liberation.20 Herein, we introduce the
vinyl ether benzyloxycarbonyl (VeZ) group as a tetrazine-
cleavable protecting group that was used to mask the ε-amino
group on lysine, with Fmoc-Lys(VeZ)-OH used as a Fmoc/tBu-
orthogonal protected building block in the solid-phase synthesis
of cyclic peptides.
Three VeZ derivatives were synthesized with zero, one, or
two methoxy groups incorporated into the phenyl ring, as
increasing electron density was envisioned to promote the
invDA reaction by reducing the highest occupied molecular
orbital (HOMO) and lowest unoccupied molecular orbital
(LUMO) gap with the diene.19,25 The VeZ moiety was
prepared via vinylation of the phenolic hydroxy group in the
corresponding aldehydes 1a−c using the vinyl boronic
anhydride pyridine complex and copper(II) acetate,26 followed
by reduction of the aldehydes 2a−c with NaBH4 to give the
benzylic alcohols 3a−c, which were subsequently transformed
into the corresponding carbonates 4a−c by treatment with 4-
nitrophenyl chloroformate (Scheme 1). Carbonates 4a−c were
coupled onto Fmoc-Lys-OH and Cbz-Lys-OH, with transient
protection of the carboxyl group with MSTFA,27 allowing
incorporation of the VeZ protecting group on the side chain
through a carbamate linkage to give 5a−d (24−55% over four
steps) (Scheme 1).
P
biological and chemical properties.1 Cyclic peptides often
show good in vivo stability, while demonstrating enhanced
biological activities compared to their linear counterparts.2
Given the growing interest in these constrained molecules as
therapeutic and biomedical tools, there is a demand for efficient
synthetic approaches.3 In solid-phase peptide synthesis (SPPS),
protecting groups orthogonal to Fmoc are typically used to
temporarily mask side-chain functionalities to enable controlled
synthesis via the so-called Fmoc/tBu approach.4−6 For the
generation of cyclic or complex peptides, such as those
decorated on their side chains,7 the demands are more complex
with the need for another tier of orthogonality, typically
provided by Alloc8 or Dde (or ivDde),9 both which have their
limitations. The cleavage of the Alloc group requires the use of
palladium salts, potentially complicating the purification of
peptides aimed at biological and medical applications, while the
standard conditions used for the removal of the Dde (2−5%
hydrazine in DMF) are not compatible with the Fmoc group
and can reduce the Alloc group, furthermore, the Dde moiety
can undergo migration.10,11 Therefore, efforts have been made
to develop a variety of other orthogonal cleavage strategies that
offer mild cleavage conditions for the selective deprotection of
side-chain groups.12−17
Inverse electron-demand Diels−Alder (invDA) reactions
between tetrazines and electron-rich dienophiles have been
widely used in bioconjugation chemistry.18,19 Recently, they
have found applications in drug delivery/prodrug activa-
tion20−23 and protein activation chemistries,24 with the
tetrazine acting as a trigger, which upon the invDA reaction
starts a cascade process resulting in the liberation of the cargo
and formation of pyridazine as a side product. For example,
Robillard developed a biorthogonal system using a trans-
cyclooctene as a strained dienophile that allowed drug release
through an elimination reaction mediated by a tetrazine.23 Li
used an invDA-mediated deprotection strategy for bioorthog-
onal protein activation where the ε-amino group of a catalytic
lysine residue was likewise modified with a trans-cyclooctene
Treatment of the VeZ protecting group with a tetrazine was
envisioned to trigger a cascade process starting with the
removal of the vinyl ether group to liberate the phenol(ate),
followed by amino liberation through a 1,6-elimination reaction
(Figure 1). Initially, 5a (5.6 mM) was treated with the widely
used tetrazine, 3,6-di-2-pyridyl-1,2,4,5-tetrazine20 (17 mM), in
Received: March 19, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b00898
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Scheme 1. Synthesis of the VeZ Protected Lysines 5a−d
Table 1. Tetrazine-Mediated Deprotection of Fmoc-
Lys(VeZ)-OH 5a
a
temp
(°C)
conv
(%)
ratio
d
entry
solvent
time (h)
6:Lys
1
rt
DMF
DMF
DMF
24
24
24
18
18
18
18
18
18
2
0
0
−
−
−
2
40
60
rt
3
0
4
10% H2O/DMF
10% H2O/DMF
10% H2O/DMF
25% H2O/DMF
50% H2O/DMF
2
1:0
1:0
1:0
1:1
1:1.4
0:1
2:1
2:1
1:1
1:3
1:10
5
40
60
60
60
60
58
80
92
100
100
33
43
48
58
71
6
7
8
b
9
50% buffer /DMF
c
10
11
12
13
14
40
50% buffer/DMF
50% buffer/DMF
50% buffer/DMF
50% buffer/DMF
50% buffer/DMF
c
40
4
c
60
1
c
60
2
c
60
4
a
Reaction conditions: Fmoc-Lys(VeZ)-OH 5a (5.6 mmol), 3,6-di-2-
b
pyridyl-1,2,4,5-tetrazine (17 mmol). Sodium citrate buffer (100 mM,
pH 5). The reaction was carried out under μW irradiation. Fmoc-
c
d
Lys-OH.
increased electron deficiency is known to enhance reaction
rates in invDA reaction chemistry,19 while a lower pH would
also promote protonation of the carbamate, thereby enhancing
the 1,6-elimination reaction. Thus, when the reaction with the
tetrazine was carried out at 60 °C for 18 h using a 1:1 (v/v)
mixture of pH 5 sodium citrate buffer and DMF, full conversion
of compound 5a into the corresponding ε-amino free lysine
was observed (Table 1, entry 9). This reaction could be
accelerated by performing the deprotection reaction under
microwave (μW) irradiation conditions (60 °C for 4 h) giving
the VeZ deprotected lysine as the main product (longer
irradiation times did not improve conversion due to rapid
degradation of the tetrazine).
These optimized VeZ deprotection conditions were directly
transferred onto the solid support using the water compatible
resin ChemMatrix (loading 1 mmol/g, 35−100 mesh, loaded
with an Fmoc-Rink-amide linker) that was subsequently
functionalized with Cbz-Lys(VeZ)-OH (5d) (Scheme 2).
Using 3 × 1 h deprotection cycles with fresh tetrazine solution,
with subsequent coupling of Fmoc-Gly-OH and cleavage off the
resin, gave the dipeptide 7 in quantitative yield, showing the
efficient cleavage of the vinyl ether and the subsequent 1,6-
elimination reaction.
Figure 1. Mechanism of tetrazine-mediated vinyl ether decaging and
amine liberation.
DMF at various temperatures (up to 60 °C); however, no
reaction of the vinyl ether was observed after 24 h (Table 1).
Since water is known to dramatically accelerate Diels−Alder
reactions, perhaps by promoting hydrophobic interactions
between the dienophile and tetrazine or acting as a general
acid,19 5a was treated with tetrazine in 10% H2O/DMF (v/v)
at 60 °C. Under these conditions, 80% conversion of the vinyl
compound into the phenol derivative 6 was observed (as
determined by HPLC-MS), with 6 isolated in 65% yield after
column chromatography. The introduction of two methoxy
groups (compound 5b) resulted in slower conversion into the
corresponding phenolate, whereas 5a (one methoxy group) and
5c (no methoxy groups) showed comparable reactivity in
1
reactions monitored by H NMR (Supporting Information
(SI), Figures S1−S3).
It was somewhat surprising that the phenol intermediate 6
was isolable. The subsequent 1,6-elimination from 6 was
promoted by increasing the water content in the reaction
mixture; however, even 50% H2O/DMF (v/v) only resulted in
a 1:1.4 ratio of the phenol 6 and the desired Fmoc-Lys-OH
(Table 1). Protonation of the tetrazine and its consequent
To explore possible epimerization under these deprotection
conditions, two diastereoisomeric peptides Ac-Phe-Lys(Ac)-
NH2 8a and Ac-phe-Lys(Ac)-NH2 8b were synthesized on a
ChemMatrix resin that had been functionalized with a Rink
linker, viat coupling of Fmoc-Lys(VeZ)-OH. Upon depro-
tection of the Fmoc group, either Fmoc-Phe-OH or Fmoc-phe-
B
DOI: 10.1021/acs.orglett.8b00898
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
examples of side-chain to side-chain lactam-bridge peptides
(Scheme 3). MTII 9 (Ac-Nle-c[Asp-His-phe-Arg-Trp-Lys]-
NH2) is a potent agonist of melanocortin receptors,28 while 10
(Ac-1Nal-Arg-c[Lys-Nle-Ala-Asp-Asp]-Phe-NH2) is a potential
anticancer agent that exerts its activity by inhibiting Bcl-2.29
The fully protected linear peptides were synthesized on a Rink-
amide functionalized ChemMatrix resin using a Fmoc-based
solid-phase chemistry with DIC and Oxyma as the coupling
combination and Fmoc-Lys(VeZ)-OH 5a as a building block.
After the selective cleavage of the 2-phenylisopropyl group (O-
2-PhiPr) from the side chain of aspartic acid using 3% TFA in
CH2Cl2, the VeZ group was cleaved using the tetrazine-
mediated protocol (3 × 1 h of dipyridyl tetrazine (30 mM) in
1:1 (v/v) sodium citrate buffer and DMF at 60 °C, μW). On-
resin cyclization was achieved using PyBOP/HOBt/DIPEA in
DMF and was followed by removal of the side chains protecting
groups and cleavage from the solid support by TFA, followed
by purification by preparative HPLC, giving the cyclic peptides
9 and 10 in 47% and 39% isolated yields, respectively.
In conclusion, the vinyl ether benzyloxycarbonyl (VeZ)
protecting group has been introduced as a temporary masking
group for amino functionalities during solid-phase peptide
synthesis. It can be removed by treatment with a tetrazine via
devinylation and subsequent 1,6-elimination chemistries. The
VeZ protecting group was stable under standard Fmoc-based
SPPS conditions and could be selectively removed under
relatively mild conditions without affecting other common side-
chain protecting groups or the Rink-amide linker. The
applicability of the approach was demonstrated by the solid-
phase synthesis of two biologically relevant cyclic peptides. It is
interesting to note that the tetrazine-mediated inverse Diels−
Alder reaction was hugely accelerated by the presence of even
10% water and that the 1,6-elimination reaction of the “safety-
catch linker”30 was not spontaneous, being promoted by acidic
conditions.
Scheme 2. Tetrazine-mediated deprotection on the solid-
phase
OH was coupled to the lysine followed by the cleavage of the
VeZ group using the tetrazine-mediated protocol described
above. Acetylation of the free amino groups and cleavage from
the resin gave the diastereoisomers 8a and 8b that were clearly
resolvable by HPLC, with analysis showing no evidence of
epimerization (Figure S5).
In order to evaluate the orthogonality and compatibility
between the VeZ group and other protecting groups commonly
used in peptide chemistry (e.g., Fmoc, Dde, Allyl, Alloc, Boc),
as well as standard cleavage protocols, stability studies were
carried out in solution alongside 5a and 5d. The VeZ group was
readily cleaved from the lysine side chain under strongly acidic
conditions (95% TFA), but was stable in 2% TFA and 20%
HFIP for more than 3 h (Table S1). The VeZ group was also
compatible with standard Fmoc-based SPPS conditions
(carbodiimide-actived couplings and 20% piperidine in
DMF), Alloc removal (Pd(PPh3)4, PhSiH3 in DMF), and
Dde removal (2% hydrazine in DMF and NH2OH·HCl/
imidazole in NMP/DMF), confirming its suitability for peptide
synthesis. However, the Alloc group was not stable upon
treatment with tetrazine (4 h μW irradiation at 60 °C), whereas
the S-trityl group was observed to be robust under these
conditions (Table S2).
To test its versatility, Fmoc-Lys(VeZ)-OH 5a was applied to
the synthesis of two cyclic peptides, melanotan II (9) (MTII)28
and a constrained BAD BH3 peptide analogue 10,29 both
Scheme 3. Side-Chain to Side-Chain Cyclization of Linear Peptides Using the Tetrazine-Cleavable VeZ as an Orthogonal
SideChain Protecting Group
C
DOI: 10.1021/acs.orglett.8b00898
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
(21) Neumann, K.; Jain, S.; Geng, J.; Bradley, M. Chem. Commun.
2016, 52, 11223−11226.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.8b00898.
■
S
́
(22) Jimenez-Moreno, E.; Guo, Z.; Oliveira, B. L.; Albuquerque, I. S.;
Kitowski, A.; Guerreiro, A.; Boutureira, O.; Rodrigues, T.; Jimen
́
ez-
Oses, G.; Bernardes, G. J. L. Angew. Chem., Int. Ed. 2017, 56, 243−247.
́
(23) Versteegen, R. M.; Rossin, R.; ten Hoeve, W.; Janssen, H. M.;
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(24) Li, J.; Jia, S.; Chen, P. R. Nat. Chem. Biol. 2014, 10, 1003−1005.
(25) Blencowe, C. A.; Russell, A. T.; Greco, F.; Hayes, W.;
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(26) McKinley, N. F.; O’Shea, D. F. J. Org. Chem. 2004, 69, 5087−
5092.
Supporting figures/tables, experimental procedures,
compound characterization, and NMR spectra (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: mark.bradley@ed.ac.uk.
ORCID
(27) Todorovski, T.; Fedorova, M.; Hennig, L.; Hoffmann, R. J. Pept.
Sci. 2011, 17, 256−262.
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MacNeil, T.; Van der Ploeg, L. H. T.; Hruby, V. J. J. Pept. Res.
2003, 62, 199−206.
Mark Bradley: 0000-0001-7893-1575
Author Contributions
†M.S. and A.G. contributed equally.
(29) Shepherd, N. E.; Harrison, R. S.; Ruiz-Gomez, G.; Abbenante,
G.; Mason, J. M.; Fairlie, D. P. Org. Biomol. Chem. 2016, 14, 10939−
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the European Research Council
(Advanced Grant ADREEM ERC-2013-340469) and the
EPSRC funded Implantable Microsystems for Personalised
Anti-Cancer Therapy (IMPACT) Programme (Grant re. EP/
K034510/1).
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DOI: 10.1021/acs.orglett.8b00898
Org. Lett. XXXX, XXX, XXX−XXX