Selective Derivatization of N-Terminal Cysteines Using Cyclopentenediones

Article abstract of DOI:10.1021/acs.orglett.6b02301

The outcome of the Michael-type reaction between thiols and 2,2-disubstituted cyclopentenediones varies depending on the thiol. Stable compounds with two fused rings were formed upon reaction with 1,2-aminothiols (such as N-terminal cysteines in peptides). Other thiols gave reversibly Michael-type adducts that were in equilibrium with the starting materials. This differential reactivity allows differently placed cysteines to be distinguished and has been exploited to prepare bioconjugates incorporating two or three different moieties.

Full text of DOI:10.1021/acs.orglett.6b02301

Letter
pubs.acs.org/OrgLett
Selective Derivatization of N‑Terminal Cysteines Using
Cyclopentenediones
Omar Brun,† Jordi Agramunt,† Lluis Raich,†,‡ Carme Rovira,†,‡,∥ Enrique Pedroso,†,§
and Anna Grandas*,†,§
‡
§
†Departament de Química Inorgan
̀
ica i Organ
̀
ica, seccio
1-11, 08028 Barcelona, Spain
∥Institucio
́
Catalana de Recerca i Estudis Avanca̧ ts (ICREA), Passeig Lluis Companys 23, 08010 Barcelona, Spain
́
Q. Organ
̀
ica, IQTCUB, and seccio
́
Q. Organ
̀
ica, IBUB), Facultat de
Química, Universitat de Barcelona, Martí i Franques
̀
S
* Supporting Information
ABSTRACT: The outcome of the Michael-type reaction between thiols and 2,2-disubstituted cyclopentenediones varies
depending on the thiol. Stable compounds with two fused rings were formed upon reaction with 1,2-aminothiols (such as N-
terminal cysteines in peptides). Other thiols gave reversibly Michael-type adducts that were in equilibrium with the starting
materials. This differential reactivity allows differently placed cysteines to be distinguished and has been exploited to prepare
bioconjugates incorporating two or three different moieties.
Four CPDs differing in one of the C2 substituents (1−4,
Figure 1) were prepared (see the Supporting Information,
section 2).
he Michael-type addition of a thiol to a maleimide is the
Tthiol-involving click reaction most frequently exploited for
bioconjugation.1 It is water compatible, quick, and clean and
takes place in high yield without the need of any catalyst.
However, it has recently been found that the resulting Michael-
type adducts (MTAs) may undergo thiol exchange and
hydrolysis to an extent depending on both the nature of the
maleimide and the environment.2 Thiol exchange may cause
premature loss of payload from therapeutically relevant
compounds such as antibody−drug conjugates,3 and as a result
of hydrolysis, conjugates may lose their structural integrity and
become a mixture of products.
Since thiols are natural components of peptides and proteins,
several groups are actively searching for new thiol-involving
conjugation methods providing stable linkages.4
Figure 1. Synthesized CPDs.
First experiments aimed at examining the effect of the
cysteine position within peptide chains on the Cys−CPD
reaction. Therefore, peptides H-WGRGC-NH2 (5), H-
KYAYCG-NH2 (6), and H-CYG-NH2 (7) were reacted with
CPDs 1 or 2 (water, 37 °C, Scheme 1 and Supporting
Information, sections 3 and 4), and the reaction progress was
monitored by HPLC.
In reactions involving 5 and 6, it was observed that peptides
were not fully consumed in spite of the presence of an excess of
CPD (5 equiv) in the mixture and that the amount of the
MTAs in the crude decreased as the unreacted peptide
underwent oxidation to the disulfide dimer. Isolated MTAs
2,2-Disubstituted cyclopent-4-ene-1,3-diones (CPDs), which
formally differ from N-substituted maleimides in that the N-
alkyl group is replaced by a dialkylated carbon, hold promise for
conjugation purposes because the MTAs resulting from
reaction with thiols cannot be hydrolyzed. Although the
homologue of acid 2 (see below) and N-acetylcysteine reacted
to give a stable MTA as a mixture of four stereoisomers,5
2,2,4,5-tetramethyl-cyclopent-4-ene-1,3-dione failed to form
thiol adducts with N-acetylcysteamine.6
Here, we investigate the reaction of CPDs with cysteine-
containing compounds and their potential in the preparation of
bioconjugates. We have found that the outcome of the Cys−
CPD reaction varies depending on the position of the cysteine
residue within peptide (or, in general, polyamide) chains.
Received: August 3, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.6b02301
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4,7,7-trisubstituted 2-thia-5-azabicyclo[4.3.0]nona-1(9),5-
diene-8-one (see the Supporting Information, section 9.3, for
an assessment of the stability of these adducts).
Scheme 1. Reaction of CPDs with Different Thiols
Methyl cysteinate was also reacted with CPDs 2−4
(Supporting Information, section 5). The evolution of the
reaction was the same as with CPD 1. Initially formed M−18
Da adducts (14, 16, and 18) evolved into the M−20 Da ones
(15, 17, and 19, respectively), which proves that the reaction
outcome is independent of the CPD substituents at the 2
position.
In all previous experiments, we had perceived that the M−18
Da to M−20 Da adduct oxidation was the rate-limiting step.
Upon reacting methyl cysteinate and 2 under different
conditions (Supporting Information, section 6), it was found
that the 14 to 15 conversion rate increased with temperature
and decreased when water was replaced by 1:1 mixtures of
water and an organic cosolvent (the conversion rate was higher
using dimethyl sulfoxide than using methanol or acetonitrile). It
was also found that use of an excess of CPD increased the 14 to
15 conversion rate, but not the use of an excess of the thiol
component.
In the case of the 1 + cysteine reaction, the M−20 Da adduct
(21) underwent reversible hydrolysis of the imine to yield an
M−2 Da zwitterionic compound (22, see Supporting
Information, section 7).
Homocysteine (Hcy) was also included in this study because
of its biological relevance in cardiovascular and neurological
pathologies.10 It has been reported that Cys and Hcy can react
with the same compound, generally at different rates, to
generate homologous structures differing in only one
methylene.4a,11 However, as in reactions involving N-acylated
1,2-aminothiols, the M−20 Da and M−18 Da adducts were not
detected, and only the MTA (23) was formed (Supporting
Information, section 8). The isolated MTA reverted to the
parent compounds. DFT calculations (Supporting Information,
section 12) confirmed that the energy barriers of the first two
steps leading to imine formation (7-membered ring) are higher
for Hcy than for H-Cys-OH or H-Cys-OMe. This discrim-
ination provides a basis on which separation and/or detection
systems could be devised.
At this point, it was time to assess whether the Cys−CPD
reaction could be of use to prepare conjugates. It was first
verified that CPD 2 can be attached to resin-linked peptide and
peptide nucleic acid (PNA) chains and the corresponding
CPD-polyamides 24a−c be obtained after the trifluoroacetic
acid mediated deprotection reaction (Scheme 2). Then, peptide
24a was reacted with the cysteine-derivatized PNA 25d and
PNA 24c with peptide 25e. Peptide−oligonucleotide con-
jugates were also synthesized by reacting peptides 24a and 24b
with cysteine-derivatized oligonucleotides (25f−h) prepared
following described procedures.13 In all cases, the target
conjugates 26 were satisfactorily obtained (2−5 h reaction
times).
(8 and 9, respectively) were found to revert to the parent CPD
and peptide after 60 min incubation in water. These results
differ from previously described ones5 and from formation of
the MTA upon reacting N-acetylcysteine and 2,4,5-tetrame-
thylcyclopent-4-ene-1,3-dione (though in 15% yield6). In
contrast to their maleimide counterparts,7 Michael additions
to cyclopent-4-ene-1,3-diones may be reversible.
DFT calculations showed that maleimide−thiol MTAs have a
greater stability than CPD−thiol MTAs (Supporting Informa-
tion, sections 12 and 13), which is in agreement with the
equilibria observed in the experiments reported here.
The mass corresponding to the Michael-type adduct of 7 + 1
(M Da, Scheme 1b) was not observed. Instead, when the
reaction was monitored by HPLC/MS it was observed that two
new products with masses of 18 and 20 Da lower were formed
and that the one with the M−18 Da mass (10) evolved into the
final, stable compound whose mass was M−20 Da (11). We
thus surmised that the Michael-type adduct very quickly
underwent intramolecular imine formation to generate a 6-
membered ring, which would account for the presence of the
M−18 Da compound 10 in the reaction mixture. Subsequent
loss of two H atoms would render the oxidized M−20 Da
adduct 11.8
Intramolecular cyclization to form an imine-containing six-
membered ring is not unknown and occurs when cysteine
reacts with α-bromo ketones.9 In our case, the presence of the
carbonyl group in the five-membered ring likely favors the
unprecedented subsequent oxidation, which yields the CO-
CC−CN conjugated system. The UV−vis spectrum of the
M−20 Da adducts exhibits a maximum around 330 nm, which
is consistent with such a conjugated structure.
Since the outcome of the reaction between CPDs and
cysteines differs depending on whether the amine is free or
acylated, we decided to investigate the possibility of selectively
labeling N-terminal cysteines in the presence of differently
placed cysteines, which is not straightforward. Aldehydes14 and
2-cyanobenzothiazoles4a are among the few reagents that react
selectively with N-terminal cysteines. In the other cases, even
though it has been shown that local environment may affect
cysteine reactivity and render them chemically distinguish-
To confirm the structure of the M−20 Da adduct, CPD 1
was reacted with methyl cysteinate (which mimicked a peptide
containing an N-terminal cysteine), and the final adduct (13,
Scheme 1b) was isolated and characterized by NMR (1H, 13C,
gHSQC) and electrospray HRMS. This proved that 13 is a
B
DOI: 10.1021/acs.orglett.6b02301
Org. Lett. XXXX, XXX, XXX−XXX

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Letter
Scheme 2. Synthesis of Cys−CPD Conjugates12
incubated with 2 (1.1 equiv). After 90 min reaction time, 3-
maleimidopropanoic acid (5 equiv) was added. HPLC/MS
analysis of the crude 1 h later showed the presence of two new
peaks (see Figure S38), whose mass fit with that of the doubly
derivatized peptide (29a). A similar procedure was used to
prepare conjugates 29b and 29c (Scheme 3), assembling three
different moieties, namely a peptide (H-CWGRGC-NH2, 30), a
PNA (24c or 31), and a tagging agent (either biotin or a
fluorophore). These results, as well as those summarized in
Scheme 2, prove that formation of the M−20 Da adduct takes
place irrespective of the moiety linked to the cysteine carboxyl
group.
To sum up, this work has revealed that CPDs react
differently from maleimides, even though CPDs can be formally
considered maleimide mimics. Strikingly, the outcome of the
reaction between CPDs and cysteines whose amine is free (1,2-
aminothiols) differs from that of the reactions involving N-
acylated cysteines and Hcy. Only the former react irreversibly
with CPDs to give water-stable bicyclic nonadienone adducts
that can be exploited for bioconjugation. In the other cases, the
Michael-type adducts (MTAs) revert to the starting materials.
The different behavior of maleimides and cyclopentenediones is
reproduced by DFT calculations.
CPDs also distinguish N-terminal cysteines from cysteines
placed at different positions, thus pointing to new alternatives
for the regioselective derivatization of peptides and proteins, to
which N-terminal cysteines can be added for conjugation
purposes.4a,16 The reaction between CPDs and cysteines with a
free amine has been successfully exploited to prepare a variety
of conjugates, both incorporating two and three different
components.
able,15 regioselectivity is typically difficult to achieve unless
cysteines are orthogonally protected.
In a preliminary experiment, CPD 2 was incubated with a 1:1
mixture of peptides 6 (H-KYAYCG-NH2) and H-CSYAKYG-
NH2 (25e) (Supporting Information, section 10). As expected
from previous experiments, HPLC/MS analysis of the reaction
mixture showed that 25e had reacted completely (yielding M−
20 Da adducts 27), whereas 6 had not at all. This result
confirmed that N-terminal cysteines could be selectively labeled
in the presence of peptides with cysteines at internal positions.
Next, peptide H-CSYACKYG-NH2 (28) was reacted with
two model alkylating agents, namely CPD 2 and 3-
maleimidopropanoic acid (Scheme 3). The peptide was first
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.6b02301.
Experimental procedures, compound characterization
data, spectra, HPLC profiles, quantum chemical
calculations and orbital analysis, and Cartesian coor-
dinates (PDF)
Scheme 3. Double-Derivatization Experiments
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: anna.grandas@ub.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by funds from the Ministerio de
■
́
Economıa y Competitividad (Grant Nos. CTQ2014-52658-R
and CTQ2014-55174-P and the project RNAREG, Grant No.
CSD2009-00080, funded under the CONSOLIDER INGENIO
2010 program) and AGAUR (Grant No. SGR2014-987). O.B.
and L.R. were recipient fellows of the MINECO (FPI) and the
Universitat de Barcelona (APIF), respectively.
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