Dynamic combinatorial chemistry with diselenides and disulfides in water

Article abstract of DOI:10.1039/c4cc00523f

Diselenide exchange is introduced as a reversible reaction in dynamic combinatorial chemistry in water. At neutral pH, diselenides are found to mix with disulfides and form dynamic combinatorial libraries of diselenides, disulfides, and selenenylsulfides.

Full text of DOI:10.1039/c4cc00523f

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COMMUNICATION
Dynamic combinatorial chemistry with diselenides
and disulfides in water†
Cite this: Chem. Commun., 2014,
50, 3716
Brian Rasmussen, Anne Sørensen, Henrik Gotfredsen and Michael Pittelkow*
Received 21st January 2014,
Accepted 12th February 2014
DOI: 10.1039/c4cc00523f
www.rsc.org/chemcomm
Diselenide exchange is introduced as a reversible reaction in
dynamic combinatorial chemistry in water. At neutral pH, disele-
nides are found to mix with disulfides and form dynamic combina-
torial libraries of diselenides, disulfides, and selenenylsulfides.
Dynamic combinatorial chemistry is a methodology that har-
nesses the power of reversible chemical reactions to form
dynamic combinatorial libraries (DCLs).1 At the heart of a
DCL is the reversible reaction that connects the building
blocks, and thus allows the DCLs to form and to equilibrate.
A particularly important challenge in dynamic combinatorial
chemistry is to identify reversible reactions that equilibrate in
water at physiological pH. Of the many reactions introduced for
dynamic combinatorial chemistry only a few have found prac-
Fig. 1 Three exchange reactions for dynamic combinatorial chemistry in
water that work simultaneously: diselenide exchange (top), disulfide
exchange (middle), and selenenylsulfide exchange (bottom).
tical use in DCLs that enable recognition of biologically impor- diselenide exchange can be combined with disulfide exchange
tant molecules in water. These include boronic ester exchange,2 to give DCLs where disulfides, diselenides and mixed selenenyl-
imine exchange,3 hydrazone exchange,4 and disulfide exchange,5 sulfides coexist (Fig. 1), and moreover that simple diselenides
which is the most widely used. Disulfide exchange comes closest to can be used as catalysts for formation of disulfide based DCLs
being of practical use at physiological pH, but even this exchange at neutral pH with only 1 mol% diselenide catalyst.
has some drawbacks; one of them being that the disulfide
exchange reaction is most effective at slightly alkaline pH.
Disulfide based DCLs are set up by dissolving appropriate
thiol-containing building blocks in buffered aqueous solutions.
Selenols are more acidic than their corresponding thiols Air oxidation of the thiols to disulfides and simultaneous
(pKa (Cys) = 8.18, pKa (Sec) = 5.24)6 and higher concentrations of disulfide equilibration take place during DCL formation. This
highly nucleophilic selenolates are present at lower pH than convenient protocol is not directly applicable to diselenide
their thiolate counterparts.7 At neutral pH, kinetic studies based DCLs because selenols are oxidised to diselenides too
using cysteamine and selenocysteamine have shown that the quickly to be isolated. Instead the starting point for diselenide
rate constants for diselenide exchange are up to seven orders of based DCLs are mixtures of diselenides.
magnitude higher than the corresponding rate constants for
the disulfide exchange.8
To study how to conveniently initiate the exchange process and
reach equilibrium, we prepared DCLs from the diselenides (1)2,
Herein we introduce diselenide exchange as a reversible (2)2, and (3)2. The diselenides were mixed in different orders in
reaction for dynamic combinatorial chemistry in water that the presence of 20 mol% exchange initiator 4-mercaptobenzoic
works at physiological pH. Additionally, we show how acid (Fig. 2 and Fig. S16–S19, ESI†). Within a day the three
libraries reached the same composition of exchanged diselenides.
These reactions were carried out both in ammonium acetate
buffer at pH 7.6 and in phosphate buffer at pH 7.9 and in both
buffer systems the libraries remained stable for at least 40 days.
Department of Chemistry, University of Copenhagen, Universitetsparken 5,
DK-2100 Copenhagen, Denmark. E-mail: pittel@kiku.dk; Web: www.pittelkow.kiku.dk
† Electronic supplementary information (ESI) available: Experimental proce-
Next, the efficiency of the exchange reaction at different pH
values was studied by carrying out the same type of exchange
dures, spectral characterisation data, description of the library preparations,
and data from these. See DOI: 10.1039/c4cc00523f
3716 | Chem. Commun., 2014, 50, 3716--3718
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Fig. 3 Structures of the building blocks (top). The combined diselenide,
disulfide, and selenenylsulfide based DCL from bis-diselenide macrocycle
(4)2 and bis-disulfide macrocycle (5)2 (middle). The partial ESI mass
spectrum showing the presence of different sized macrocycles in the
DCL (bottom). The intensities of the high mass signals are scaled with a
factor three relative to the low mass signals.
Fig. 2 (a) Setup of diselenide DCLs from different starting points with 20
mol% 4-mercaptobenzoic acid as an initiator in water at pH 7.6. (b) HPLC
chromatograms (monitored at 290 nm) of the DCLs after equilibration for
four days.
studies as above at pH 7.0, 6.1, 5.1, 4.0, and 2.9. It was found
that the exchange reaction stayed efficient down to pH 5.0
The high efficiency of the above diselenide based DCLs
whereas no exchange was observed at pH 4.0 and 2.9. Collec- combined with their interplay with sulfur systems inspired us
tively, these findings show that diselenide based libraries (i) to study how diselenides could affect disulfide based DCLs.
can be conveniently set up from diselenides, (ii) are efficiently Previous studies have shown that selenols catalyse the disulfide
exchanging over a broad pH range including physiological pH, exchange reaction,9 and that diselenides catalyse the oxidation
(iii) are stable for extended time, and (iv) are easily tameable by of thiols.10 Consequently, selenium additives have found use as
lowering the pH.
catalysts in protein folding studies,11 and substitution of Cys
After these proof-of-concept studies, more complex DCLs with Sec in proteins has led to an improved control of the
were prepared. For this, oxidised dimeric structure (4)2, folding process.12 In the search for a similar effect on the rate of
equipped with two diselenide functionalities, was used giving library formation, we examined a simple DCL of macrocyclic
the possibility of DCLs consisting of oligo-diselenide macro- disulfides formed from 3,5-dimercaptobenzoic acid, 6. When 6
cycles. When the dimer was dissolved in ammonium acetate buffer was dissolved in ammonium acetate buffer a disulfide based
together with diselenide (1)2 and 5 mol% 4-mercaptobenzoic acid DCL consisting of a cyclic trimer and a cyclic tetramer was
as an initiator, diselenide exchange was observed. In the formed formed (Fig. 4a). The DCL was studied at pH 7.0, 7.6, 8.3, 9.4,
DCL, significant quantities of exchange products between (1)2 and and 10.0. The libraries were formed efficiently in a day at pH 9
(4)2 were identified together with mass peaks corresponding to and 10, whereas one week was required for reactions to
higher diselenide macrocycles (Fig. S20 and S21, ESI†).
complete at the lower pH values. The rates of library formation
To form DCLs with even higher diversity, (4)2 was mixed at pH 7.0, 7.6, 8.3 in the presence of 10 mol% diselenide (1)2
with the bis-disulfide macrocycle (5)2 in phosphate buffer were monitored over time, and it was observed that the catalyst
(pH 7.8) in the presence of 5-mercaptoisophthalic acid caused accelerated library formation (Fig. 4b). This catalytic
(Fig. 3). The resulting DCL consisted of two homotetramers, effect was observed to be general since at pH 7.0 the time
(4)4 and (5)4, and hexamers, (4)6 and (5)6, along with a mixed required for library formation was reduced from 7 to 2 days, at
selenenyl based tetramer, (4)2(5)2, and two mixed hexamers, pH 7.6 from 7 to 2 days, and at pH 8.3 from 7 to 1 day. The
(4)4(5)2 and (4)2(5)4. The amount of the different library mem- acceleration of the DCLs was still pronounced down to 1 mol%
bers varied over time showing how the diselenide exchange catalyst loading (Fig. S29–S34, ESI†), thereby providing this as a
took place alongside both disulfide and selenenylsulfide new approach to catalyse disulfide based DCL formation in
exchanges on the way to equilibrium. Analysis of this complex water.13
dynamic mixture was aided by the characteristic isotope dis-
Analysis of the LC/MS data gave indications of the origin
tribution in selenium containing molecules to unambiguously of the catalytic effect. During the formation of the DCLs, a
determine the structure of the formed library members small peak corresponding to linear selenenylsulfide (6)2(1)1
(Fig. S23–S27, ESI†).
was observed and after complete library formation this peak
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To conclude, we have demonstrated for the first time that
diselenide based DCLs equilibrate under thermodynamic con-
trol at neutral pH and that combinations of diselenide and
disulfide based DCLs give mixtures where disulfides, disele-
nides and selenenylsulfides coexist. We have also shown that
diselenides catalyse the formation of disulfide based DCLs at
physiological pH. These discoveries pave the way to form DCLs
with diselenides and disulfides at truly physiological pH, and
currently we are studying how addition of templates affects the
distribution of library members under such conditions.
This work was supported by the Lundbeck Foundation. We
thank Assoc. Prof. Lars Henriksen and Dr Sophie R. Beeren for
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3718 | Chem. Commun., 2014, 50, 3716--3718
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