Sulfo-click reaction via in situ generated thioacids and its application in kinetic target-guided synthesis

Article abstract of DOI:10.1039/c1cc14724b

Herein, we describe a practical, one-pot variant of the sulfo-click reaction, in which 9-fluorenylmethyl-protected thioesters are rapidly deprotected and reacted further with sulfonylazides to give N-acyl sulfonamides.

Full text of DOI:10.1039/c1cc14724b

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COMMUNICATION
Sulfo-click reaction via in situ generated thioacids and its application in
kinetic target-guided synthesiswz
Niranjan Kumar Namelikonda and Roman Manetsch*
Received 31st July 2011, Accepted 15th August 2011
DOI: 10.1039/c1cc14724b
Herein, we describe a practical, one-pot variant of the sulfo-click
reaction, in which 9-fluorenylmethyl-protected thioesters are
rapidly deprotected and reacted further with sulfonylazides to
give N-acyl sulfonamides.
straightforward although various synthetic protocols are
available.9 Reliable protocols have been reported, in which
protected thioacids are deprotected prior to the amidation
step. For example, 2,4,6-trimethoxybenzyl (Tmob) thiol and
trityl thiol were used to prepare the corresponding thioesters
from carboxylic acids.10 These Tmob or trityl thioesters were
then selectively deprotected and reacted with sulfonylazides in
the presence of 2,6-lutidine to arrive at the N-acyl sulfonamides.10
The generation of the thioacids from these thioesters, however,
requires prolonged exposure to an excess of trifluoroacetic
acid for complete deprotection and an aqueous workup to
remove the residual acid, since the sulfo-click reaction is
optimally conducted under slightly basic conditions.
In the last decade, various click chemistry approaches mani-
fested themselves as simple yet reliable tools for material and
life sciences.1 Other bioorthogonal transformations known as
the thiol-click reaction or the sulfo-click reaction emerged as
alternatives though they are slightly limited in their reactivity
profile and substrate scope.2,3 Herein, we report a rapid, one-
pot sulfo-click reaction taking advantage of an in situ activation
of thioesters into thioacids followed by the amidation step.
Adoption of these reaction conditions furthermore eliminated
the major limitation of our developed sulfo-click-based kinetic
target-guided synthesis approach4 improving it to fully
complement the established in situ click chemistry approach.5
Though N-acyl sulfonamides are known for more than a
century, in recent years they gained great attention in medicinal
chemistry.6 Commonly, N-acyl sulfonamides are synthesized
via reactions between sulfonamides and carboxylic acids with
carbodiimide reagents or between acid halides and sulfo-
namides under basic conditions. Recently there have been
reports on the preparation of N-acyl sulfonamides by a copper
catalyzed reaction between sulfonylazides and alkynes.7 An-
other modern approach entails the reaction between sulfonyl-
azides and thioacids3 or sulfonylazides and selenocarboxylates8
(Scheme 1). Especially, the amidation with thioacids stands
out by its ease and bioorthogonality and thus was later named
sulfo-click reaction by Liskamp.3c
Crich and coworkers recently developed an amidation
reaction between 2,4-dinitrobenzenesulfonamides and thioacids,
in which the thioacids have been prepared starting from
corresponding 9-fluorenylmethyl (Fm) thioesters with 20%
piperidine in DMF.11 Analogously, we envisioned a strategy
in which corresponding thioacids are rapidly generated and
directly used without any workup and purification in a sulfo-
click amidation. However, Williams et al. reported that the
sulfo-click reaction with piperidine generates piperidine
amides of the corresponding thioacids, sulfonamides and only
small amounts of targeted N-acyl sulfonamides.3b Because the
nucleophilic piperidine attacks the carbonyl carbon and out-
competes the intramolecular S-acyl transfer, the unwanted
piperidine amide is generated as one of the two main products.
To start the optimization of a one-pot deprotection/amidation
reaction, various thioesters were prepared from corresponding
carboxylic acids and FmSH following the reported procedures.12
In search of a non-nucleophilic base which would rapidly
deprotect the Fm thioester and promote the amidation,
1,8-diazabicycloundec-7-ene (DBU) has served the purpose.
A convenient procedure was attained, which performs well
with diverse thioacids and sulfonylazides after screening the
different proportions of DBU from 5% to 3% DBU in DMF.
Optimal deprotection was achieved in one minute at room
temperature by a mixture of 3.5% DBU/DMF and subsequent
Though the sulfo-click reaction has been proven to be
reliable in various applications, it is currently underutilized
due to limitations with respect to the preparation and handling
of thioacids. In general, thioacids are nucleophilic, readily
dimerize, and suffer from storage and stability issues. The
synthesis and purification of thioacids are also not always
Department of Chemistry, University of South Florida, 4202 E Fowler
Ave., Tampa, FL 33620, USA. E-mail: manetsch@usf.edu;
Tel: +1 8139747306
w This article is part of the ChemComm ‘Emerging Investigators 2012’
themed issue
z Electronic supplementary information (ESI) available: Experimental
section and spectral data of the compounds. See DOI: 10.1039/
c1cc14724b
Scheme 1 Reaction between sulfonylazides and thioacids or sulfonyl-
azides and selenocarboxylates to yield N-acyl sulfonamides.
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Scheme 2 Sulfo-click reaction between sulfonylazides and thioacids
generated from 9-fluorenylmethyl-protected thioesters.
amidation with 1 equivalent of sulfonylazide was completed in
another minute to obtain N-acyl sulfonamide along with DBF 4
(Scheme 2). All products were purified and isolated in good to
excellent yields. Representative examples of this rapid depro-
tection/amidation reaction are shown in Table 1. Reactions
between aromatic or aliphatic azides and aromatic or aliphatic
thioacids performed well in the one-pot two-reaction sequence.
Functional groups like an acetamide 3i, a hydroxyl 3f and an
ether 3e and 3f were tolerated in the reaction. Heterocycles like
caffeine 3h, thiazole 3b and 3i, piperazine 3g, and tetrahydroi-
soquinoline 3c were also compatible with the reaction conditions.
This reaction was also examined in high dilutions of thioacids
and sulfonylazides. At 20 mM concentration of thioester and
sulfonylazide, the reaction attained completion at room
temperature in 12 hours. It is noteworthy that the deprotection/
amidation reaction worked similarly well with Cs2CO3 with
good yields. First the deprotection completed with 5 equivalents
of Cs2CO3 in DMF in 90 minutes and after the addition of the
sulfonylazide, the N-acyl sulfonamide was formed cleanly in
30 minutes at room temperature.
Scheme 3 Preparation of a semi-synthetic derivative of the natural
product artemisinin.
confirmed by the doublet at d 4.8 ppm with a vicinal equatorial-
axial coupling constant of H-10 with H-9 and only one
singlet at d 5.38 ppm, which corresponds to H-12.
As a second example, a peptidic N-acyl sulfonamide has
been synthesized. After Boc deprotection of Fm thioester 9,
the resulting amine was reacted with Boc-Ala-OH under
standard peptide coupling conditions providing the dipeptidic
Fm thioester 10 (Scheme 4). The sulfo-click reaction between
the Fm thioester 10 and p-chlorobenzenesulfonyl azide
with 3.5% DBU/DMF resulted in two minutes in the
N-acyl sulfonamide 11 in good yields without detectable
epimerization.
The use of Fm thioesters in the sulfo-click reaction is also
compatible with applications requiring bioorthogonal conditions.
Previously, we developed similar to in situ click chemistry a
kinetic target-guided synthesis (TGS) approach, in which
the sulfo-click reaction between thioacids and sulfonyl-
azides was used for the screening of compounds with biological
activity.4 In kinetic TGS, the biological target is engaged in the
assembly of its own inhibitory bidentate ligand from a pool of
complementary reacting fragments. In proof-of-concept
studies, we demonstrated that incubation of the protein target
Bcl-XL with a library of thioacids and sulfonylazides leads to
the selective formation of four hit N-acyl sulfonamides
displaying inhibitory activity towards Bcl-XL.4b Although
the initial proof-of-concept study underlines the potential of
kinetic TGS, the issues related to the preparation and handling
of the thioacid fragments restrict the kinetic TGS approach to
a minor degree.
The good yields and the high chemoselectivity of the
amidation reaction are especially useful for the preparation
of highly complex biomolecules. First, artelinic acid, a semi-
synthetic derivative of the natural product artemisinin, has
been successfully derivatized with a fluorescent dansyl moiety
using the new amidation reaction conditions. Commercially
available dihydroartemisinin 5 was reacted under known
Lewis acid conditions with the thioester of the p-hydroxy-
methyl benzoic acid 1j resulting in the thioester of artelinic
acid (Scheme 3).13 The major product obtained was the
10b isomer 6 along with trace amounts of the 10a isomer 7.
Next, the sulfo-click reaction of the isomeric mixture contain-
ing 6 and 7 was deprotected with 3.5% DBU/DMF and
reacted with dansylazide to provide the dansyl analogue 8 of
artelinic acid in excellent yields after the trace amounts of the
10a isomer has been removed by preparative HPLC. By
1H NMR spectroscopy, the structure of the 10b isomer 8 was
Inspired from the success of the rapid one-pot in situ
deprotection/amidation reaction sequence, known thioacid
fragments TA1–TA3 were prepared as Fm thioesters
TA10–TA30 and then applied in a kinetic TGS approach to
sulfonylazides SZ1–SZ6 in the presence of Bcl-XL after the
thioesters have been deprotected.4 Not surprisingly, deprotection
of the three Fm thioesters TA10–TA30 with 3.5% DBU/DMF
cleanly furnished thioacids TA1–TA3 in one minute. The
resulting thioacids TA1–TA3 were immediately diluted with
methanol to 2 mM stock solutions (about 85 fold dilution) and
then directly used for the kinetic TGS screening of Bcl-XL.
Sulfonylazides SZ1–SZ6 and thioacids TA1–TA3 derived
Table 1 N-Acyl sulfonamides prepared from sulfonylazides and
thioacids generated from 9-fluorenylmethyl-protected thioesters with
3.5% DBU/DMF. The yields are given in parentheses
Scheme 4 Preparation of a peptidic N-acyl sulfonamide.
Chem. Commun., 2012, 48, 1526–1528 1527
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equimolar concentrations were analyzed by LC/MS-SIM. In
this comparison, the peak area of the piperidine amide was
significantly larger than the peak area of the N-acyl sulfon-
amide SZ4TA2, which clearly indicates that the electrospray
ionization of the piperidine amide is better than that of
SZ4TA2. Thus, it can be concluded that though the piperidine
amide can be identified by LC/MS-SIM, it has been formed in
the incubation sample only to minor amounts in comparison
to the N-acyl sulfonamide SZ4TA2. These results suggest that
the unwanted byproduct piperidine amide did not have an
unfavorable impact on the kinetic TGS incubations and the hit
compound SZ4TA2 is easily detectable by LC/MS-SIM
analysis.
Scheme 5 Kinetic TGS incubations with TA2 generated from TA20.
from the corresponding thioesters TA10–TA30 were incubated
as binary mixtures at 20 mM concentration each in the
presence and absence of Bcl-XL in the appropriate phosphate
buffer at 37 1C for 6 hours. At the same time as a control
experiment, all binary building block combinations with
sulfonylazides SZ1–SZ6 and the thioacids TA1–TA3, which
were synthesized and purified as thioacids, were incubated
with and without Bcl-XL. Contrary to the previous results,4
the amplification factor for hit combination SZ4TA2, the ratio
between SZ4TA2 in the Bcl-XL-templated reaction and
SZ4TA2 in the non-templated reaction, was not detected in
the incubation samples containing the fragments SZ4 and
TA2, which derived from the thioester TA20. The strong
basicity of DBU was considered to change the pH of the
Bcl-XL-containing incubation sample and thereby possibly
affect the protein conformation or even denaturing it.
In conclusion, we have developed a practical, one-pot
variant of the sulfo-click reaction, in which Fm thioesters
are rapidly deprotected and further reacted with sulfonylazides
to give N-acyl sulfonamides in high yields. This variant of the
sulfo-click reaction has also been shown to be applicable for
kinetic TGS screening. We are currently using this kinetic TGS
approach for the screening of various protein–protein inter-
action targets.
We thank Dr. Hong-Gang Wang for the help and guidance
to express and purify Bcl-XL and Sameer Kulkarni for fruitful
discussions. We are also grateful to the James and Esther King
Biomedical Research Program (NIR grant 07KN-08) for
financial support.
Therefore, the kinetic TGS experiments were repeated with
a weaker base (Scheme 5). Piperidine was considered to be a
good candidate, even though it has been proven to undergo a
side reaction yielding piperidine amides. After the deprotection
of the Fm thioesters, it was assumed that the dilution and the
buffering effect by the phosphate buffer of the incubation
samples may significantly decrease the nucleophilicity of
piperidine and thus suppress the formation of unwanted
piperidine amides. The thioacids TA1–TA3 were rapidly
generated by the treatment of thioesters TA10–TA30 with a
mixture of 5% piperidine/DMF and immediately diluted with
methanol to 2 mM stock solutions. Kinetic TGS incubation
samples were prepared with and without Bcl-XL in all possible
fragment combinations and then analyzed by LC/MS-SIM.
Gratifyingly, an amplification factor for hit combination
SZ4TA2 in the incubation samples containing the thioacid
TA2 derived from the thioester TA20 (see Fig. S1, traces C and
D in ESIz) was calculated to be in the same range as the
amplification for the Bcl-XL-templated incubations containing
the purified thioacid TA2 (see Fig. S1, traces A and B in ESIz).
Next, studies were conducted to check whether the
unwanted piperidine amide was formed during the kinetic
TGS incubations with thioacid TA2 derived from thioester
TA20. LC/MS-SIM analysis set to simultaneously detect ions
with masses corresponding to SZ4TA2 and piperidine amide
identified the formation of both N-acyl sulfonamide SZ4TA2
and piperidine amide in both the non-templated reaction and
the Bcl-XL-templated reactions (Fig. S2 in ESIz). As expected,
the amount of SZ4TA2 was increased in the Bcl-XL-containing
incubation sample, while the piperidine amide was detected at
approximately the same amounts in the Bcl-XL-templated and
non-templated incubations. Further attempts were made to
estimate the amounts of SZ4TA2 and piperidine amide
formed during the kinetic TGS. Both compounds were synthesized
and mixtures containing SZ4TA2 and piperidine amide at
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