reSeArCH Letter
Methods
azides with this method and then included in the azide library (some azides in Plate
Other diazotransfer reagents reported in the literature. Sulfonyl azide deriv- 8 were made from insoluble primary amines; see Supplementary Information 1).
atives28–32 and a 2-azido-1,3-dimethylimidazolium salt33 have previously been Procedure for the CuAAC reaction with the azide library. The acceleration
developed as diazotransfer reagents.
of the CuAAC reaction by use of an appropriate ligand was found to be cru-
Preparation of FSO2N3. A 100-ml cylindrical plastic bottle was charged with cial, because low conversion into triazoles was encountered without any ligand.
aqueous NaN3 solution (0.50 M in 40 ml H2O, containing 1.3 g (20 mmol) NaN3) Tris-hydroxypropyltriazolylmethylamine (THPTA) was chosen as the ligand for
and methyl tert-butyl ether (MTBE, 40 ml). Compound 1 (7.9 g, 24 mmol) was the copper, to achieve a balance between accessibility and reactivity.
dissolved in MeCN (2 ml), and the resultant viscous solution was added rapidly to
An aqueous acidic buffer (pH ≈ 5) was prepared with sodium ascorbate (2.48 g,
the stirred NaN3/H2O/MTBE mixture in an ice-water bath. This was followed by a 12.5 mmol), Na2HPO4 (7.0 g, 49.3 mmol), citric acid (4.87 g, 25.4 mmol) and water
rinse of the vial used for preparing the solution of 1 with additional MeCN (2 ml), (to a total volume of 100 ml). From the azide library, prepared as described above,
which was also added to the reaction mixture. The reaction mixture was stirred the 96 azide solutions (100 µl from each well, DMSO as major solvent; around
vigorously (approximately 600 r.p.m.) in an ice-water bath for 10 min in the loosely 50 mM, containing 5 µmol azide) were transferred to an empty 96-well microplate.
sealed plastic bottle, then the mixture was poured into a glass separating funnel. To each well of this newly loaded plate was added the sodium ascorbate/Na2HPO4/
One of the byproducts, 1,2-dimethylimidazole, remains in the aqueous phase and citric acid buffer (40 µl). The plate was sealed and swirled at 800 r.p.m. at 30°C
buffers it at a weakly basic pH (around 8) at the end of the reaction, and release of for 15 min. A solution of N-(3-ethynylphenyl)acetamide (4a, 100 mM in DMSO;
hydrazoic acid (HN3) is prevented. The mixture was kept in the funnel at room 47.5 µl, containing 4.75 µmol 4a) was added to each well, followed by an aqueous
temperature for 30 min for phase separation. The organic phase was separated from solution of CuSO4/THPTA (20 mM of CuSO4 and 20 mM THPTA in H2O; 12.5
the aqueous phase, and this organic phase—containing FSO2N3—was kept in a µl, 0.25 µmol). The plate was sealed and swirled at 800 r.p.m. at 40°C for 6 h, to
loosely sealed plastic bottle at room temperature for at least 12 h. The orange-red afford the corresponding triazole product in each well. For the detailed procedure
residual aqueous phase (approximately 1 ml in volume), which developed during see Supplementary Information 1, and for the labelled UPLC chromatograms of
the 12-hour resting period, was removed with a plastic pipette. The colourless the CuAAC reaction on the 1,224-azide library see Supplementary Information 2.
organic phase could be used as a solution of FSO2N3 in MTBE without further
Besides its role in the CuAAC reaction, we discovered that sodium ascorbate
purification. The concentration and yield of the FSO2N3 solution was measured quickly quenches any remaining FSO2N3 in cases involving electron-deficient ani-
by 19F NMR with a known amount of TsF or PhCF3 added as an internal standard. lines or low-purity amines. The addition of sodium ascorbate before the alkyne
We have repeated this procedure more than 50 times, with a consistent yield of ensures any residual FSO2N3 does not interfere with the CuAAC catalyst system.
FSO2N3 in the range of 86%–93%, and concentration in the range of 420–470 mM. General patterns in the UPLC results of the 1,224 tandem diazotransfer-CuAAC
Supplementary Information 1 contains detailed safety precautions and procedures. reactions. Among the 1,224 primary amine substrates undergoing tandem
General procedure for the preparation of the isolated azide compounds. To diazotransfer–CuAAC reaction, the following general patterns were noticed in
a 50-ml glass round-bottom flask was added sequentially the primary amine 2 UPLC analysis: first, the majority of the ester functional groups were partially hydro-
(1.0 mmol; aliphatic, aromatic or heteroaromatic; as free naked amine or as HCl, lysed into carboxylates, or partially transesterified with methanol (methanol was
MsOH, TsOH or tartrate salt), FSO2N3 solution (containing 1.0 mmol FSO2N3, used as a solvent for HPLC analysis) during the HPLC run; second, most aliphatic
approximately 200 mM in DMF/MTBE 1:1, v/v, approximately 5 ml, volume primary amines and electron-rich anilines gave high conversion into 1,2,3-triazoles,
adjusted according to the concentration; prepared according to the above pro- but some electron-deficient anilines and heteroaromatic amines showed partial
cedure and diluted with equal volume of DMF) and aqueous KHCO3 solution conversion into triazoles with remaining substrate; third, some amines with a low
(3.0 M, 1.33 ml, containing 4.0 mmol KHCO3). The reaction mixture was stirred molecular mass (mostly those less than 100 Da) were converted into the triazole with
for 5 min at room temperature, while monitoring by LC–MS. After completion, a substantial amount of alkyne remaining, probably due to the volatility of the azide.
EtOAc (40 ml) was added and the mixture was washed sequentially with brine The only functional group other than the primary amino group that can react under
(60 ml × 6), water (60 ml × 2) and brine (60 ml), dried over Na2SO4, concentrated the diazotization conditions was found to be thiol (R–SH), which dimerized to form
by rotary evaporation and dried under vacuum to afford the azide product 3. For disulfide (R–S–S–R) but did not interfere with the subsequent CuAAC step (for an
products containing acidic functional groups, this extraction process was modified example of disulfide formation, see Supplementary Information 2).
with acidified aqueous phase (acidified with aqueous HCl). Detailed procedures
and the various modifications, as well as the characterization data for each com-
pound, can be found in Supplementary Information 1.
Data availability
The UPLC chromatograms (UV absorption) of the reaction mixtures for the
preparations of the azide compounds in Fig. 2 are available in Supplementary
Information 2. The structures of the 1,224 compounds in the azide library, and the
UPLC chromatograms of the 1,224 CuAAC reaction mixtures, are also shown in
Supplementary Information 2. Information on the source of each amine substrate
used is available upon reasonable request from the corresponding authors.
Preparation of the amine library in microplates. Because most free amines—
primary or otherwise—are unstable upon long-term storage in air, the majority
of the primary aliphatic amines were purchased as their acid salts (hydrochloride,
tosylate, tartrate, mesylate and hydrobromide salts worked equally well in our
library); the aliphatic primary amines purchased with naked –NH2 groups were
treated with methanesulfonic acid to form mesylate salts. All the primary amines
in this library were stored as solutions in DMSO in microplates. Each well of the
microplate contained the solution of a single amine compound, with the molar
concentration of reactive –NH2 groups at 100 mM. For details of this procedure
see Supplementary Information 1.
DMSO was chosen as the solvent for all the amines to balance the three require-
ments of solubility, reactivity and application: first, DMSO can dissolve the majority
of the primary amines (including the acid salts); second, DMSO was found to be
one of the few solvents (along with DMF) that gave rapid reaction rates in our
diazotransfer reaction; and third, DMSO is the major component of the solvent
in the resultant azide library, and later the triazole library if CuAAC is to follow
after diazotransfer—owing to the relatively low toxicity of DMSO, this allows the
completed triazole library solutions to pass directly into the biological screens.
Procedure for the preparation of one of the microplates of the azide library. To a
96-well deepwell microplate (in which each well has a volume of 1,200 µl), 96 amine
solutions (100 mM in DMSO, 200 µl each, containing 20 µmol of each amine) were
transferred. Aqueous KHCO3 solution (3.0 M, 26.7 µl each well, containing 80 µmol
KHCO3) was added, followed by the FSO2N3 solution (200 mM in DMSO/MTBE
1:1, 100 µl each well, containing 20 µmol FSO2N3). Additional DMSO (73 µl) was
added so that the total volume of mixture in each well was approximately 400 µl. The
microplate was then sealed and incubated in microplate shaker at 800 r.p.m. and 30°C
for 1 h. The microplate—containing the corresponding 96 azides—was stored at 4°C,
and has been found to be stable without substantial degradation for 6 months; for
more details see Supplementary Information 1. This method was most convenient
with, but is not limited to, soluble primary amines. Most insoluble primary amines,
including most amino acids, could also be transformed quantitatively to soluble
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synthesis of azides from primary amines. Adv. Synth. Catal. 352, 2515–2520
(2010).
29. Katritzky, A. R., Khatib, M. E., Shakov, O. B., Khelashvili, L. & Steel, P. J.
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30. Fischer, N. et al. Sensitivities of some imidazole-1-sulfonyl azide salts. J. Org.
Chem. 77, 1760–1764 (2012).
31. Ye, H. et al. A safe and facile route to imidazole-1-sulfonyl azide as a
diazotransfer reagent. Org. Lett. 15, 18–21 (2013).
32. Stevens, M. Y., Sawant, R. T. & Odell, L. R. Synthesis of sulfonyl azides via
diazotransfer using an imidazole-1- sulfonyl azide salt: scope and 15N NMR
labeling experiments. J. Org. Chem. 79, 4826–4831 (2014).
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Acknowledgements We acknowledge the National Natural Science Foundation
of China (NSFC 21672240, NSFC 21421002), the Strategic Priority Research
Program of the Chinese Academy of Sciences (XDB20020300), the Key Research
Program of Frontier Sciences (CAS, grant no. QYZDB-SSW-SLH028) and
Shanghai Sciences and Technology Committee (18JC1415500, 18401933502)
for financial support. We thank J. Yang for help with the thermal and mechanical
stability tests; K. Ding for support; and P. Gao, T. Wang, Y. Liang, X. Zhan and
J. Chen for assistance with the management of the amine compounds.
Author contributions G.M. developed the procedure for the preparation of
fluorosulfuryl azide. T.G., G.M., Y.S. and T.M. prepared the 1,224-amine library.
G.M. and T.G. developed the procedure for the diazotransfer reaction. T.G., T.M.,