2-(Dibenzylamino)butane-1,4-dithiol (DABDT), a Friendly Disulfide-Reducing Reagent Compatible with a Broad Range of Solvents

Article abstract of DOI:10.1021/acs.orglett.9b04106

Disulfide-reducing agents play a crucial role in bioconjugate chemistry. Herein, we describe the synthesis of a new disulfide-reducing agent [2-(dibenzylamino)butane-1,4-dithiol (DABDT)] that is conveniently prepared from inexpensive aspartic acid. Being

Full text of DOI:10.1021/acs.orglett.9b04106

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
2‑(Dibenzylamino)butane-1,4-dithiol (DABDT), a Friendly Disulfide-
Reducing Reagent Compatible with a Broad Range of Solvents
Sinenhlanhla N. Mthembu,†,∥ Anamika Sharma,†,∥ Fernando Albericio,*,†,‡,§
and Beatriz G. de la Torre*,∥
†Peptide Science Laboratory, School of Chemistry and Physics, University of KwaZulu-Natal, Durban 4001, South Africa
‡
́
Department of Organic Chemistry, University of Barcelona, Martí i Franques 1-11, Barcelona 08028, Spain
§CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, and IQAC, CSIC, Jordi Girona, Barcelona
08028, Spain
∥KRISP, College of Health Sciences, University of KwaZulu-Natal, Westville, Durban 4001, South Africa
S
* Supporting Information
ABSTRACT: Disulfide-reducing agents play a crucial role in
bioconjugate chemistry. Herein, we describe the synthesis of a
new disulfide-reducing agent [2-(dibenzylamino)butane-1,4-
dithiol (DABDT)] that is conveniently prepared from
inexpensive aspartic acid. Being nonmalodorous and highly
soluble in a broad range of solvents, DABDT is user-friendly.
We have demonstrated its performance both in solution and in
the solid phase.
iven their unique chemical and physical properties, free
Gthiols and disulfide bonds are principal actors in many
biological processes. The major representative of thiol-
containing molecules in nature is cysteine (Cys), one of the
20 genetically encoded amino acids present in proteins.
Although the amino acid composition of different proteins is
variable and the protein composition varies between
organisms, the presence of Cys is generally very low.
Nevertheless, this amino acid plays a key role in nature as its
thiol side chain is often involved in enzymatic reactions, and
oxidation of two Cys residues leads to the formation of
disulfide bridges, which are essential for stabilizing the tertiary
structure of proteins and peptides.1 Disulfide bridges can be
Figure 1. Most used disulfide-reducing agents in their reduced and
intra- and intermolecular.2
oxidized forms.
Given the relevance of Cys and disulfide bridges, disulfide-
reducing agents are widely used in many molecular biology
protocols to stabilize free sulfhydryl groups and reduce
disulfide bonds in peptides and proteins.3,4 The most common
DTT is that its reactivity is lower at physiological pH due to its
high pKa, thereby hindering the thiolate form. In this regard,
TCEP is a considerable improvement. It is active over a wide
pH range (5.0−9.0), stable to air oxidation, and odorless. Its
oxidized state is irreversible, unlike the previous reducers, but it
is soluble only in aqueous solvents, thereby preventing its
broad application (Figure 1).
dithiol reducers are β-mercaptoethanol (β-ME), dithiothreitol
(DTT),5 and tris(2-carboxyethyl)phosphine (TCEP),6 each
with a series of advantages and disadvantages. For example, β-
ME is an inexpensive and user-friendly reducing agent.
However, it has a strong unpleasant smell. It shows poor
reducing capacity and kinetically slow performance compared
to those of DTT but greater stability.7,8 The great reducing
capacity and low stability of DTT are caused by the stability of
the cycle in its oxidized form. Nevertheless, the limitation of
Received: November 17, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.9b04106
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
An ideal reducing agent should be almost odor free, have a
low pKa, and show good solubility in a broad range of solvents,
including organic ones. In this regard, it is important to
consider that the synthesis of cystine/cysteine-containing
peptides often involves the concourse of more than one kind
of thiol-protecting group, which can be removed by different
chemical mechanisms.
Such protecting groups are those where the thiol of the Cys
is masked through a disulfide bridge, like S-tert-butyl (S-tBu)
and S-2,6-dimethoxybenzyl (S-DMP),9 which is more labile
than S-tBu and commercially available. In peptide chemistry, it
is important to have a reducing agent that is soluble in non-
aqueous solvents, because very often the disulfide-based
protecting group is removed when the peptide is fully
protected and is even anchored to the solid support where
the peptide was elongated using the solid-phase peptide
synthesis strategy (SPPS).
Information and Figures S8−S14). At this point, we tested
several synthetic approaches to prepare the final dithiol.
Initially, we attempted direct conversion using Lawesson’s
reagent.14,15 Although this reagent is applied mainly for the
conversion of carbonyl compounds to thiocarbonyls, Nishio et
al.16,17 reported the direct conversion of alcohols into thiols.
However, this procedure did not work in our hands. Then, the
next option was to convert the hydroxyl groups in a leaving
group,18 which would allow posterior conversion to iso-
thiouronium salt19 or thioesters.20 We chose chloride as the
leaving group because the reaction using SOCl2 is clean and
the byproducts are easily removed under vacuum. The
conversion of the diol to dichloride was monitored by TLC.
When the reaction was completed and the mixture evaporated
to dryness, the compound obtained was used directly for the
next step. First, we attempted the formation of iso-thiouronium
salts using thiourea, which affords sodium salt of thiolate upon
reaction with NaOH. However, the dithiane along with the
thiolane was obtained mainly as products.21 Second, we
attempted thioester formation using potassium thioacetate. In
this case, S,S′-[2-(dibenzylamino)butane-1,4-diyl] diethane-
thioate (4) was obtained in high yield and purity (see the
Supporting Information and Figures S15−S21).
With this in mind, and on the basis of the previous water-
soluble reducing agent developed by Raines and co-workers,10
we developed a new reducing agent, 2-(dibenzylamino)butane-
1,4-dithiol (1, DABDT), suitable for non-aqueous media
(Figure 2).
Finally, the thioester hydrolysis was attempted under both
acidic (aqueous HCl in MeOH)22 and basic (LiOH in MeOH)
conditions, but the results were unsatisfactory. We then
proceeded to reduce the thioester to thiol by means of LAH.23
In this case, the reaction was fast and total conversion was
observed. Nevertheless, DABDT obtained through this
reaction was quickly oxidized to DABDTox in different steps
of the workup. Although, to the best of our knowledge, there is
no precedent in the literature about the use of NaBH4 to
reduce thioesters, we tested this reagent. Thus, NaBH4 (10
equiv) was added in two portions to the dithioester dissolved
in MeOH, and the reaction mixture was left for 15 min. After
workup, DABDT (>97% pure) was obtained in 96% yield (see
the Supporting Information and Figures S22−S28). In this
case, DABDT was fully stable through the workup process.
The oxidation using LAH may have been catalyzed by the
presence of traces of aluminum.
Figure 2. Chemical structure of 2-(dibenzylamino)butane-1,4-dithiol
(DABDT).
a
Scheme 1. Synthetic Protocol for Synthesizing DABDT
The solubility of DABDT was tested in several organic
solvents, such as N,N-dimethylformamide (DMF), dichloro-
methane (DCM), tetrahydrofuran (THF), ethyl acetate
(EtOAc), acetonitrile (ACN), and methanol (MeOH), the
concentration in all cases being >1 g/mL. Furthermore,
DABDT showed stability in the solid form at room
temperature under nitrogen, and even a solution of 5%
DABDT in ACN under atmospheric conditions kept >70% in a
reduced form for >10 days.
To explore the reducing capacity of DABDT, the reduction
of a side chain-protected Cys residue in the form of a SDMP
[Fmoc-Cys(SDMP)-OH] was attempted in solution. DABDT
rapidly reduced Fmoc-Cys(SDMP)-OH in a 1:5 ratio in the
presence of 3% DIEA at room temperature. HPLC analysis
immediately after mixture showed that 80% of the protecting
group had been removed, as indicated by the formation of
Fmoc-Cys-OH, the release of the protecting group HSDMP in
the form of free thiol, and the oxidized (cyclic) form of
DABDT (see the Supporting Information and Figures S29−
S35). Nevertheless, by following the time course reaction, we
observed the reversibility of the reaction with the concomitant
diminution of the peaks corresponding to HSDMP and Fmoc-
Cys-OH (Figure S37). To stabilize the thiol groups, H2O (3%)
a
(i) Benzyl chloride (4 equiv)/K2CO3 (4 equiv) in water, 80 °C,
microwave for 40 min; (ii) LAH, rt, 4 h; (iii) SOCl2/DCM, 60 °C, 3
h; (iv) potassium thioacetate in ACN/DMF with 0.1 equiv of
triethylamine, 60 °C, 4 h; (v) NaBH4 (10-fold excess), rt, 30 min.
DABDT was conveniently prepared from aspartic acid (Asp)
in five synthetic steps (Scheme 1). First, Asp was reacted with
benzyl bromide to achieve the perbenzoylated molecule. This
reaction was carried out in aqueous media under microwave
(MW) radiation,11,12 yielding the desired product, dibenzyl
N,N-dibenzylaspartate (2), in high yield and purity (see the
Supporting Information and Figures S1−S7). The second step
involved the reduction of the two benzyl esters to alcohol using
LiAlH4 (LAH), as described in the literature.13 In this reaction,
the product, 2-(dibenzylamino)butane-1,4-diol (3), was also
obtained in high yield and purity (see the Supporting
B
DOI: 10.1021/acs.orglett.9b04106
Org. Lett. XXXX, XXX, XXX−XXX

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Letter
was added to the reaction medium.24,25 Under these
conditions, the back reaction was not observed, and total
reduction of Fmoc-Cys(SDMP)-OH was achieved instanta-
neously.
In view of this successful result, we tested lower ratios of the
reducing agent, 1:3 and 1:1. In both cases, the reaction was
instantaneous (Figure 3), but in the 1:1 case, Fmoc-cystine
formation was observed because DABDT was totally
consumed. Therefore, an excess of DABDT is required
under these conditions.
Figure 4. HPLC chromatogram of disulfide bridge reduction using
DABDT in a peptide anchored on a solid support.
Figure 3. HPLC chromatogram of disulfide bridge reduction in
Fmoc-Cys(SDMP)-OH using DABDT at different ratios.
The use of N-methylmorpholine (NMM), a weaker base
than DIEA, led to full reduction in 40 min (slower compared
to DIEA). Similar results were obtained when the reaction was
attempted with DTT, thereby confirming the similar behavior
of DABDT and DTT. The removal of SDMP was also
attempted using DCM as the solvent. However, due to the
insolubility of H2O in DCM, the former was replaced by
MeOH. In this case, the removal of SDMP was completed in
20 min, which is slower compared to ACN (see the Supporting
Information and Figures S38 and S39).
Figure 5. HPLC chromatogram of disulfide bridge reduction using
DABDT in oxytocin anchored on a solid support.
Once the reducing capacity of DABDT had been
demonstrated in solution, it was tested in the solid phase.
For this purpose, the following three peptidyl resins were
synthesized: (i) Cys at the N-terminal position [Ac-Cys-
(SDMP)-Gly-Phe-Leu-Rink amide resin], (ii) Cys in an
intermediate position flanked by amino acids without a side
chain protecting group [Fmoc-Ala-Cys(SDMP)-Leu-Rink
amide resin], and (iii) Cys in an intermediate position flanked
by amino acids with a bulky side chain protecting group
[Fmoc-Asn(Trt)-Cys(SDMP)-Asn(Trt)-Rink amide resin].
The peptidyl resins were treated with 3 equiv of DABDT in
a DMF/DIEA/H2O mixture (94:3:3) twice for 5 min each.
After cleavage of the peptide from the resin, analysis of the
peptides revealed the absence of protected Cys (Figure 4).
To check the reduction of other kinds of disulfide bonds, a
peptide model carrying dithiodipropionic acid [HOOC-CH2-
CH2-S-S-CH2-CH2-COOH (DTDPA)] at the N-terminus was
prepared. Thus, DTDP-Gly-Phe-Leu-NH-Rink-resin was trea-
ted as described above. Again, the cleaved peptide showed the
full reduction of the disulfide bond (Figure 4).
HPLC and compared with the product obtained before
reduction (Figure 5). The peak at 12.6 min corresponded to
the dimer formed during the synthesis, because SDMP was not
fully stable. The disappearance of this peak after reduction
indicates that DABDT is able to reduce that dimer.
In conclusion, here we prepared DABDT from inexpensive
commercially available Asp using a highly convenient synthetic
pathway. A key step was the reduction of thioesters with
NaBH4. Unexpectedly, the use of the strongest LAH as a
reducing agent was accompanied by spontaneous formation of
the oxidized form of DABDT.
DABDT is soluble in a broad range of solvents, such as
DMF, ACN, THF, DCM, EtOAc, and MeOH. As a solid, it is
stable and even dissolved in ACN for at least a reasonable
period of time. DABDT has demonstrated to remove the
SDMP protecting group of the thiol of the Cys cleanly in
solution and in the solid phase. Furthermore, DABDT reduced
the N-terminal dithio-dipropionyl moieties present in the N-
terminus of protected peptides anchored on a resin. The
complete reduction of SDMP at room temperature in all of the
cases can be summarized in Table 1.
Given the inexpensive and convenient preparation of
DABDT, its absence of odor, stability, and excellent perform-
ance, we envisage that it will be rapidly adopted by the
scientific community as the disulfide-reducing agent of choice
in substitution of DTT.
Finally, as proof of concept, the linear Fmoc-oxytocin
(Fmoc-CYIQNCPLG-NH2), a nonapeptide containing two
Cys residues, was synthesized in SPPS using Fmoc-Cys-
(SDMP)-OH as a building block. At the end of the chain
elongation, the peptidyl resin was treated three times with 5
equiv of DABDT and a DIEA/H2O/ACN mixture (3:3:94) for
5 min each. After cleavage, the crude peptide was analyzed by
C
DOI: 10.1021/acs.orglett.9b04106
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Table 1. Reduction by DABDT of Disulfide-Containing Compounds
solvent/base
additive
time (min)
% Fmoc-Cys-OH
Solution Phase
Fmoc-Cys(SDMP)-OH:DABDT
a
b
1:5
1:5
1:3
1:3
1:1
1:5
1:3
1:1
ACN/DIEA
ACN/DIEA
ACN/DIEA
ACN/NMM
ACN/DIEA
DCM/DIEA
DCM/DIEA
DCM/DIEA
−
H2O
H2O
H2O
1
1
1
80
a
100
100
100
100
100
100
100
a
40
a
H2O
1
1
1
a
MeOH
MeOH
MeOH
a
20
Solid Phase
Fmoc-C(SDMP)-GFL-R (1:3)
Fmoc-DTDP-GFL-R (1:3)
Fmoc-AC(SDMP)L-R (1:3)
Fmoc-NC(SDMP)N-R (1:3)
Fmoc-Oxytocin-Resin (SDMP) (1:5)
DMF/DIEA
DMF/DIEA
DMF/DIEA
DMF/DIEA
DMF/DIEA
H2O
H2O
H2O
H2O
2 × 5
2 × 5
2 × 5
2 × 5
3 × 5
100
100
100
100
100
H2O
b
a
Immediate HPLC analysis after mixing of the reagents showed total conversion. Reversibility of the reaction was observed with the concomitant
diminution of the peaks corresponding to HSDMP and Fmoc-Cys-OH.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.9b04106.
■
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Procedure for the synthesis of DABDT, H and 13C
NMR spectra, and HPLC data of intermediates and
peptides (PDF)
1
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AUTHOR INFORMATION
Corresponding Authors
■
(9) Postma, T. M.; Giraud, M.; Albericio, F. Org. Lett. 2012, 14 (21),
5468−5471.
*E-mail: albericio@ukzn.ac.za.
*E-mail: garciadelatorreb@ukzn.ac.za.
ORCID
(10) Lukesh, J. C., III; Palte, M. J.; Raines, R. T. J. Am. Chem. Soc.
2012, 134 (9), 4057−4059.
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Fernando Albericio: 0000-0002-8946-0462
Beatriz G. de la Torre: 0000-0001-8521-9172
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Notes
The authors declare no competing financial interest.
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Chem. 2007, 72 (17), 6382−6389.
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ACKNOWLEDGMENTS
■
This work was funded in part by the National Research
Foundation (NRF) and the University of KwaZulu-Natal
(South Africa), the Spanish Ministry of Economy, Industry,
and Competitiveness (MINECO) (CTQ2015-67870-P),
CIBER-BBN, and the Generalitat de Catalunya (2017 SGR
1439). The authors thank Yoav Luxembourg (Luxembourg Bio
Technologies, Ness Ziona, Israel) for encouraging this work
and for the gifts of coupling reagent.
(21) Seebach, D.; Beck, A. K.; Bichsel, H. U.; Pichota, A.; Sparr, C.;
Wunsch, R.; Schweizer, W. B. Helv. Chim. Acta 2012, 95 (8), 1303−
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DOI: 10.1021/acs.orglett.9b04106
Org. Lett. XXXX, XXX, XXX−XXX