Chemical generation and modification of peptides containing multiple dehydroalanines

Article abstract of DOI:10.1039/c5cc05469a

Chemical formation of dehydroalanine has been widely used for the post-translational modification of proteins and peptides, however methods to incorporate multiple dehydroalanine residues into a single peptide have not been defined. We report the use of methyl 2,5-dibromovalerate which can be used to cleanly carry out this transformation.

Full text of DOI:10.1039/c5cc05469a

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Chemical generation and modification of peptides containing
multiple dehydroalanines
Received 00th January 20xx,
Accepted 00th January 20xx
P. M. Morrison,a P. J. Foley,a S. L. Warrinera and M. E. Webba*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Chemical formation of dehydroalanine has been widely used for achieved utilising dehydratase enzymes from lanthipeptide
the post-translational modification of protein and peptides, pathways.18-23 All of the chemical methods of Dha generation were
however methods to incorporate multiple dehydroalanine reviewed extensively by Davis and coworkers,24 who have
residues into a single peptide have not been defined. We report pioneered the double-alkylation elimination reagents to convert
the use of methyl 2,5-dibromovalerate which can be used to cysteine to Dha. This approach is selective for Cys residues, utilises
cleanly carry out this transformation.
mild reaction conditions and avoids the incorporation of non-
canonical amino acids however it had not been applied to the
simultaneous generation of multiple dehydroalanine residues in a
single peptide, as has previously only been achieved using
unnatural amino acids.16, 25
Incorporation of dehydroalanine (Dha) into proteins and peptides
can be used to generate a diverse range of chemical modifications.
The electrophilic nature of the Dha residue means that it is readily
modified in a site-selective fashion using thiols; this has been
Our initial aim was to create constrained peptides in a two-step
utilised to label proteins with dyes1 and sugars2, 3, incorporate strategy via the creation of multiple Dha residues and their
unnatural side chains4-6
,
and install post-translational
subsequent cyclisation with
a small molecule polythiol core
modifications7, 8. A particular advantage of using Dha is that a single
modified protein can be reacted with an array of thiols to generate
a range of protein conjugates.5, 6 Metal-catalysed chemistry has also
been used to create unnatural amino acids from Dha9, such as those
with boron- and silicon-containing side chains10 and fluorescent
alanine derivatives.11 Dha itself is an integral constituent of
modified ribosomal peptides such as the lanthipeptides and
thiopeptides, being used to generate lanthionine and pyridine
cross-links as well as remaining unmodified in many lantibiotic
structures. Finally, incorporation of Dha has been used as a
chemical biology tool for example as a electrophilic probe in the
investigation of diubiquitinase activity and selectivity.12, 13
(Scheme 1). Similar molecules have been shown to bind to proteins
with nanomolar affinities and large surface areas – making them
potential modulators of protein-protein interactions.26-28 The
addition of a thiol to Dha scrambles the stereochemistry at the
Chemical formation
SH
of multiple
dehydroalanine
residues
N
H
N
H
O
O
A1
3
3
SH
Br
A2
Both its application for site-selective protein modification and
the involvement of Dha in lanthipeptide biosynthesis have driven
the development of methods to chemically generate Dha in peptide
structures. Dehydroamino-acids cannot be incorporated using
standard peptide synthesis strategies and a masked amino acid is
commonly incorporated and subsequently converted to Dha.14-16
Biosynthetic methods rely on incorporation of selenocysteine
derivatives which are then unmasked using peroxide oxidation2, 17
whereas enzymatic transformation of serine residues can be
B
SH
SH
O
2
Br
Br
1
S
S
HN
O
*
*
NH
S
A: 8 stereoisomers *=mix of L/D
B: 1 stereoisomer *=all L
O
*
NH
Scheme 1. Routes to bicyclic peptide structures involving cysteine modification. A: A
two-step modification in which cysteine is first converted to dehydroalanine followed
by cyclisation with a trithiol, ultimately generating 8 stereoisomers of the peptide
produced in route B. B: alkylation of 3 cysteine residues with a central mesitylene core.
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alpha carbon, often limiting the yield due to formation of the preliminary experiments with a model peptide containing two
-Cys derivatives. We aimed to take advantage of the cysteines (H2N-ACGDDACG-CO2H) the use of the most common
stereochemical scrambling and increase the diversity of reagent, dibromoadipamide led to formation of an
peptide loop structures accessible upon cyclisation while undesired stapled by-product (Scheme 2a). While the
incorporating -amino acids which have previously been proportion of the stapled by-product could be decreased by
shown to improve protease resistance in the bicyclic peptide increasing the equivalents of
D
3,
D
3
used in the reaction, even at
structures.29 Successful application of this strategy was high ratios of reagent to cysteine (50:1) residual stapled
however dependent upon developing a strategy to incorporate peptide was still observed. Corresponding experiments with
the multiple dehydroalanine residues.
peptides containing three cysteines gave complex mixtures
We planned to utilise cysteine double alkylation for Dha which included a yet higher proportion of stapled peptides.
We hypothesised that the stapled by-product forms either
by double alkylation of the dibromoadipamide across two
cysteine residues (Scheme 2a, Route A), or cysteine
interception of the cyclic sulfonium ion (Route B) in a manner
similar to elongated thiol modification previously observed by
Nathani et al.30 An unsymmetrical reagent containing both an
conversion due to the mild reaction conditions and wide
tolerance of other amino acids. cyclic sulfonium
A
intermediate, formed through double cysteine alkylation, is
eliminated to generate the dehydroalanine residue.7, 24 In
a)
HS
HS
α
-bromoacyl group and an alkyl bromide would mitigate the
observed stapling; the first alkylation step at the -position
would be rapid, whereas a second alkylation at the -alkyl
α
N
N
H
O
ω
H
O
bromide would be slow. By changing the relative rates of the
two steps we anticipated that all cysteine residues would be
monoalkylated before the intramolecular alkylation or
sulfonium ion formation, preventing either potential
competing reaction pathway (Scheme 2a, Route C).
R1
R2
3 R1=R2=CONH2
4 R1=CO2Me, R2=H
Br
R1
Br
R2
S
S
3/4
We therefore generated methyl dibromovalerate
PBr3-mediated bromination and ring-opening of valerolactone
(Scheme 2b). We used to promote alkene formation in the
4 via
N
O
Br
N
H
5
4
H
O
R2
same set of model peptides and observed clean conversion to
dehydroalanine residues. Similar conversion was observed for
peptides containing three cysteine residues and we were
readily-able to purify these peptides by HPLC. To investigate
the scope of the reagent, we investigated the reaction of the
R1
undesired stapled byꢀproduct
A
HS
S
B2
N
H
N
O
R2
peptide H2N-LTFCEYWAQLCSAA-CO2H
and (Figure 1a) in a variety of solvent conditions. We
monitored by LCMS the formation of both the product peptide
containing two Dha residues and the undesired stapled
by-products and wherein the two cysteine residues have
reacted with a single molecule of and respectively. At low
ratios of to , we still observed some formation of the
stapled by-product however this was significantly reduced
relative to formation of when was used. At higher ratios of
to (greater than 5:1), little to no stapling could be
observed. In DMSO/H2O mixtures, no stapling was observed
even at low ratios of reagent to peptide . Additionally, the
reaction could be conducted in acetonitrile (SI Figure S1),
which is not compatible with since it is not soluble in this
6 using both reagents 3
H
B1
O
S
R1
4
HS
N
H
7
C
N
O
8
9
H
O
3
4
Br
4
6
Br
R2
9
R2
8
3
R1
R1
4
6
S
S
N
N
4
6
O
H
H
O
N
desired product
N
3
O
H
O
H
solvent. All subsequent conversions were carried out in
DMSO/H2O followed by HPLC purification.
b)
O
O
i) Br2, PBr3
100 °C, 2 h
Having defined robust chemistry to generate multiple
dehydroalanine residues, we turned to the kallikrein inhibitor
PK1528 as a model system to exemplify the increased diversity
generated in our two-step strategy for bicyclic peptide
O
MeO
Br
ii) MeOH, TsOH
75 °C, 3 h
Br
5
4
77%
Scheme 2. Conversion of multiple cysteine residues to dehydroalanine. a) The
conversion of two cysteine residues with 3 is complicated by formation of a stapled by-
product via routes A or B. Stapling is avoided when methyl 2,5-dibromovalerate 4 is
used as the lower reactivity of the alkyl bromide slows steps A and B1, leading to
conversion to dehydroalanine-containing peptides via Route C. b) The synthesis of 4
from δ-valerate.
formation.
The
linear
PK15-derived
peptide
10
(H2N-ACSDRFRNCPADEALCG-CO2H) was converted to its
dehydroalanine-containing analogue 12 in good yield. To
replicate the mesitylene core of PK15 11 we synthesised the
trithiol
trismercaptomethylbenzene
2
from
2 | J. Name., 2012, 00, 1-3
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conversion of peptide 10 to the stereochemically-scrambled mixture 13 via
dehydroalanine-containing peptide 12
tris(bromomethyl)benzene (TBMB) 1. Addition of 2 to 12 gave
.
a cyclised product as a mixture of stereoisomers (SI Figure S2).
Figure 2. The inhibition of human plasma kallikrein by the mixture of stereoisomers 13.
PK15 LLL Ligand 8 (IC50 2.7 ± 0.10 nM) Purified mixture of isomers 13 (IC50 144 ± 31
nM) Crude reaction mixture in the conversion of 12 to 13. (IC50 238 ± 21 nM)
▲
■
●
Table 1. IC50 data for the 8 stereoisomers of PK15 11 present in mixture 13
Figure 1. Optimisation of dehydroalanine conversion by LCMS. a) Peptide 6 was
converted to 7 using either 3 or 4. The level of by-products 8 or 9 were monitored by
LCMS when using 3 or 4 respectively. b) The ratio of stapled by-product 8/9 produced
in the conversion of cysteine to dehydroalanine in peptide 6 using either 3 or 4 in a
variety of base and solvent systems. At low ratios of 3 to 6, high levels of stapled by-
product 8 are observed. This is not entirely mitigated by increasing the equivalents of 3
used, with a persistent residual fraction of stapled peptide observed. The use of 4
demonstrated much reduced staple character, increasing the production of peptide 7
containing multiple dehydroalanine residues. ■ K2CO3, 1:1 H2O/DMF; ♦ K2CO3, 1:1
H2O/DMSO; red/black = use of 3/4 respectively
Isomer
IC50 /nM
Isomer
IC50 /nM
a
LLL
2.7 ± 0.10
3740 ± 480
240 ± 14
DDD
DDL
DLD
DLL
> 1 × 107
> 1 × 107
1700 ± 50
311 ± 15
LDD
LDL
LLD
> 1 × 107
[a] Naming refers to stereochemical configuration at Cys2, Cys9 and Cys16 in sequence
We evaluated the biological activity of the mixture of
stereoisomers 13 obtained via cyclisation using a kallikrein
inhibition assay. The mixture retained high potency (IC50 144 ±
We were conscious that the bicyclic product has the same
mass as monocyclic and uncyclised single-addition products.
To confirm the presence of only bicyclic peptide structures, the
purified product mixture was individually reacted with either
31 n
21 n
M
), even as a crude mixture before purification (IC50 238 ±
). To further investigate the effect of this stereochemical
M
excess N-methyl maleimide or β-mercaptoethanol. No reaction
of -mercaptoethanol was observed, and a single addition of
scrambling on the PK15 sequence we resynthesized the
individual stereoisomers using solid phase peptide synthesis
β
N-methylmaleimide to the free amino-terminus of the peptide
was observed. The same addition was observed on the bicyclic
peptide PK15 11 which was synthesised independently via
and cyclisation with TBMB 1. Following purification by HPLC,
they were then tested in the kallikrein inhibition assay (Table 1
and SI Figure S3). The range of observed activity spans more
direct cyclisation of peptide 10 with TBMB 1 28
.
than
4 orders of magnitude, albeit with none of the
a)
i) thiourea, THF
75 °C, 4 h
ii) KOH, H2O
105 °C, 16 h
stereoisomers being more potent inhibitors of kallikrein than
the literature PK15 LLL-sequence. This differential activity
demonstrates that the peptide loops adopt significantly
different conformations in response to changes in
stereochemistry at a single centre, validating our initial
hypothesis that stereochemical scrambling of a peptide library
will effectively increase its diversity. Intriguingly, analysis of
inhibition by the stereochemically-scrambled peptides
revealed an unexpected interaction between the two peptide
loops; while the LLD-stereoisomer was inactive in the
concentration range tested, a further stereocentre inversion to
generate a DLD-variant rescues activity slightly, suggesting that
the two loops must interact with each other. The wide range
of activities means that we envisage that a deconvolution-by-
resynthesis approach could be readily used to identify the
Br
HS
Br
Br
HS
SH
58%
1
2
b)
1
ACSDRFRNCPADEALCG
mesitylene core
10
NH4HCO3
MeCN/H2O (3:7)
r.t., 1 h
ACSDRFRNCPADEALCG
K2CO3
11
4
H2O:DMSO (1:1)
37 °C, 3 h
mesitylene core
2
AXSDRFRNXPADEALXG
ACSDRFRNCPADEALCG
H2O/DMF(1:1)
37 °C, 3 h
*
*
*
12
13
* = mix of L/DꢀCys
X = Dha
Scheme 3. a) The synthesis of 2 to be used as the central core. b) The two-step
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16 S. Burrage, T. Raynham, G. Williams, J. W. Essex, C. Allen, M.
Cardno, V. Swali and M. Bradley, Chem.-Eur. J., 2000, 6,
1455-1466.
17 F. P. Seebeck and J. W. Szostak, J. Am. Chem. Soc., 2006, 128,
7150-7151.
most active stereoisomer, if the chemistry were to be adapted
to a screening context.
In summary, we have reported an effective strategy to
generate multiple dehydroalanine residues in peptides via mild
chemical
2,5-dibromovalerate
stapled by-products observed with other reagents.
Additionally, is more readily soluble and has a wider solvent
conversion
of
cysteines
with
methyl
18 C. Chatterjee, G. C. Patton, L. Cooper, M. Paul and W. A. van
der Donk, Chemistry & Biology, 2006, 13, 1109-1117.
19 N. Garg, L. M. Salazar-Ocampo and W. A. van der Donk, Proc
Natl Acad Sci U S A, 2013, 110, 7258-7263.
20 J. A. Majchrzykiewicz, J. Lubelski, G. N. Moll, A. Kuipers, J. J.
Bijlsma, O. P. Kuipers and R. Rink, Antimicrob. Agents Ch.,
2010, 54, 1498-1505.
21 C. Chatterjee, M. Paul, L. Xie and W. A. van der Donk, Chem.
Rev., 2005, 105, 633-684.
22 P. J. Knerr and W. A. van der Donk, Annu. Rev. Biochem.,
2012, 81, 479-505.
23 Y. O. You, M. R. Levengood, L. A. Ihnken, A. K. Knowlton and
W. A. van der Donk, ACS Chem. Biol., 2009, 4, 379-385.
24 J. M. Chalker, S. B. Gunnoo, O. Boutureira, S. C. Gerstberger,
M. Fernández-González, G. J. L. Bernardes, L. Griffin, H. Hailu,
C. J. Schofield and B. G. Davis, Chem. Sci., 2011, 2, 1666.
25 Y. Zhu, M. D. Gieselman, H. Zhou, O. Averin and W. A. van
der Donk, Organic & Biomolecular Chemistry, 2003, 1, 3304-
3315.
26 A. Angelini, L. Cendron, S. Chen, J. Touati, G. Winter, G.
Zanotti and C. Heinis, ACS Chem. Biol., 2012, 7, 817-821.
27 S. Chen, D. Bertoldo, A. Angelini, F. Pojer and C. Heinis,
Angew. Chem. Int. Ed. Engl., 2014, 53, 1602-1606.
28 C. Heinis, T. Rutherford, S. Freund and G. Winter, Nat. Chem.
Biol., 2009, 5, 502-507.
4
. This strategy avoids the formation of
4
tolerance. This reagent has multiple potential applications,
including in the synthesis of modified peptides and modified
peptide precursors for lanthipeptides. In this report, we have
utilised the reagent in a two-step approach to generate
stereochemically diverse bicyclic peptide structures from a
single peptide precursor. Generation of such mixtures in a
peptide or phage-display context and their subsequent
deconvolution has the potential to increase the diversity of
peptide libraries while simultaneous incorporating in-built
resistance to cellular and plasma
L-amino- and
L-carboxypeptidases as well as increasing the potential range
of core molecules which can be used in bicyclic peptide library
generation.
We acknowledge support from EPSRC (EP/K03135X/1) for
equipment. Personal funding to PMM was provided by the
Wellcome Trust (grant 096687/Z/11/Z).
29 S. Chen, D. Gfeller, S. A. Buth, O. Michielin, P. G. Leiman and
C. Heinis, ChemBioChem, 2013, 14, 1316-1322.
30 R. Nathani, P. Moody, M. E. Smith, R. J. Fitzmaurice and S.
Caddick, ChemBioChem, 2012, 13, 1283-1285.
References
1
2
3
E. Branigan, C. Pliotas, G. Hagelueken and J. H. Naismith, Nat.
Protoc., 2013, 8, 2090-2097.
J. Wang, S. M. Schiller and P. G. Schultz, Angew. Chem.,
2007, 119, 6973-6975.
C. Aydillo, I. Compañón, A. Avenoza, J. H. Busto, F. Corzana, J.
M. Peregrina and M. M. Zurbano, J. Am. Chem. Soc., 2014,
136, 789-800.
4
5
Y. A. Lin, O. Boutureira, L. Lercher, B. Bhushan, R. S. Paton
and B. G. Davis, J. Am. Chem. Soc., 2013, 135, 12156-12159.
N. Timms, C. L. Windle, A. Polyakova, J. R. Ault, C. H. Trinh, A.
R. Pearson, A. Nelson and A. Berry, ChemBioChem, 2013, 14,
474-481.
6
7
8
F. Rowan, M. Richards, M. Widya, R. Bayliss and J. Blagg,
PLoS One, 2014, 9, e103935.
J. M. Chalker, L. Lercher, N. R. Rose, C. J. Schofield and B. G.
Davis, Angew. Chem. Int. Edit., 2012, 51, 1835-1839.
K. P. Chooi, S. R. G. Galan, R. Raj, J. McCullagh, S.
Mohammed, L. H. Jones and B. G. Davis, J. Am. Chem. Soc.,
2014, 136, 1698-1701.
9
C. J. Chapman, A. Matsuno, C. G. Frost and M. C. Willis,
Chem. Commun., 2007, 3903-3905.
10 F. Bartoccini, S. Bartolucci, S. Lucarini and G. Piersanti, Eur. J.
Org. Chem., 2015, 2015, 3352-3360.
11 P. M. T. Ferreira, L. S. Monteiro, G. Pereira, E. M. S.
Castanheira and C. G. Frost, Eur. J. Org. Chem., 2013, 2013,
550-556.
12 N. Haj-Yahya, H. P. Hemantha, R. Meledin, S. Bondalapati, M.
Seenaiah and A. Brik, Org. Lett., 2014, 16, 540-543.
13 M. P. C. Mulder, F. El Oualid, J. ter Beek and H. Ovaa,
ChemBioChem, 2014, 15, 946-949.
14 N. M. Okeley, Y. Zhu and W. A. van Der Donk, Org. Lett.,
2000, 2, 3603-3606.
15 Y. Zhu and W. A. van der Donk, Org. Lett., 2001, 3, 1189-
1192.
4 | J. Name., 2012, 00, 1-3
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