Organic & Biomolecular Chemistry
Paper
conjugation reaction had gone to completion (or almost). The 16.8 Hz, 1H), 2.63–2.56 (m, 2H), 2.37–2.17 (m, 2H), 2.06–1.94
conjugate was then purified by HPLC (semi-preparative con- (m, 2H), 1.92 (s, 3H), 1.19 (s, 3H) ppm.
ditions), lyophilized, redissolved for UV quantitation and final
purity assessment by HPLC, and finally characterized by ESI 159.3, 151.5, 137.6, 111.4, 100.76, 84.9, 83.7, 60.5, 53.1, 50.0,
13C NMR (D2O, 101 MHz): δ 205.2, 203.8, 177.0, 166.5,
(HPLC-MS, positive mode) or MALDI-TOF MS (positive mode).
35.8, 29.3, 29.1, 18.8, 11.5 ppm.
ESI-HRMS (negative mode): m/z: 420.1414 [M − H]−,
M calcd for C19H22N3O8 420.1412.
CuAAC reactions
Aqueous solutions of CuSO4 and sodium ascorbate, and a
solution of TBTA in either MeOH or 3 : 2 (v/v) MeOH/H2O were
added to an aqueous solution of the alkyne-containing conju-
gate. An aqueous solution of the azide-containing moiety was
then added. The resulting mixture was reacted in a MW oven
for 1 h at 90 °C (60 °C in the case of the 17 + 9 reaction).
Relative molar ratios of the reagents: Alkyne-containing conju-
gate/CuSO4/sodium ascorbate/TBTA/azide-containing moiety
1 : 4 : 24 : 1 : 1 (1 : 1 : 24 : 1 : 1 for the 18 + 10 reaction). Molar
concentration of the reagents: Alkyne-containing conjugate,
0.5 mM; CuSO4, 0.5 mM; sodium ascorbate, 12 mM; TBTA,
0.5 mM; azide-containing moiety, 2 mM (0.6 mM for the 18 +
10 reaction).
After the MW reaction, the crude was analyzed by HPLC
and the target double conjugate purified (semi-preparative
HPLC), lyophilized, redissolved for UV quantitation and final
purity assessment by HPLC and/or HPLC-MS, and finally
characterized by MALDI-TOF MS (positive mode).
Mass spectrometric characterization data of isolated conju-
gates, conjugates structures and HPLC traces of the conju-
gation crudes can be found in Section 4 of the ESI (Table S2†).
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was supported by funds from the Ministerio de
Economía y Competitividad (grants CTQ2014-52658-R and
CTQ2017-84779-R). The Generalitat de Catalunya (2014SGR187
and 2017SGR391) is also acknowledged. The authors wish to
thank Mr Guillem Morató-Aragonés for synthesizing com-
pound 1.
Notes and references
1 E. P. Diamandis and T. K. Christopoulos, Clin. Chem., 1991,
37, 625.
2 R. M. Lösel, R. Philipp, T. Kálai, K. Hideg and
W. E. Trommer, Bioconjugate Chem., 1999, 10, 578.
3 M. J. Davies, A. Shah and I. J. Bruce, Chem. Soc. Rev., 2000,
29, 97.
CPD + azide reactions
Equimolar amounts of CPD-acid (20) and AZT (9) were dis-
solved (water was generally the solvent) and heated (90–100 °C)
in a MW oven (or eventually a metal block), sometimes in the
presence of salts (LiCl or MgCl2), or SDS. Reaction times were
often 1.5–2 h, but were sometimes extended to 2–3 days. The
progress of the reaction was monitored by HPLC-MS analysis.
Details on reaction conditions as well as HPLC profiles are
shown in Table S3 (Section 5 of the ESI†).
4 I. L. Medintz, H. T. Uyeda, E. R. Goldmann and
H. Mattoussi, Nat. Mater., 2005, 4, 435.
5 L. M. De León-Rodríguez and Z. Kovacs, Bioconjugate
Chem., 2008, 19, 391.
6 M. S. T. Gonçalves, Chem. Rev., 2009, 109, 190.
7 L. Ducry and B. Stump, Bioconjugate Chem., 2010, 21, 5.
8 L. Q. Ying and B. P. Branchaud, Chem. Commun., 2011, 47,
8593.
Compounds 23 and 27 were isolated by HPLC from the
experiments described in entries 5 and 3, respectively, of
9 J. M. Niers, J. W. Chen, R. Weissleder and B. A. Tannous,
Anal. Chem., 2011, 83, 994.
Table S3.† After lyophilization they were characterized by NMR 10 K. S. Yang, G. Budin, C. Tassa, O. Kister and R. Weissleder,
(see ESI, Section 5†) and HR-ESI MS:
Angew. Chem., Int. Ed., 2013, 52, 10593.
23: 1H NMR (D2O, 400 MHz): δ 7.68 (s, 1H), 6.47 (t, J = 11 R. L. Juliano, Nucleic Acids Res., 2016, 44, 6518.
6.7 Hz, 1H), 5.50–5.43 (m, 1H), 5.21 (s, 1H), 4.54–4.48 (m, 1H), 12 A. Beck, L. Goetsch, C. Dumontet and N. Corvaïa, Nat. Rev.
3.93–3.75 (m, 2H), 3.08–2.98 (m, 1H), 2.88–2.77 (m, 1H),
Drug Discovery, 2017, 16, 315.
2.47–2.25 (m, 2H), 2.00–1.89 (m, 2H), 1.84 (s, 3H), 1.15 (s, 3H) 13 A. Maruani, M. E. B. Smith, E. Miranda, K. A. Chester,
ppm.
13C NMR (D2O, 101 MHz): δ 195.5, 177.5, 166.5, 157.4,
V. Chudasama and S. Caddick, Nat. Commun., 2015, 6,
6645.
151.6, 149.6, 137.8, 111.5, 85.8, 83.9, 66.8, 62.8, 60.7, 59.5, 14 A.-C. Knall, M. Hollauf, R. Saf and C. Slugovc, Org. Biomol.
36.7, 31.1, 29.0, 17.6, 11.5 ppm. Chem., 2016, 14, 10576.
ESI-HRMS (negative mode): m/z: 448.1475 [M − H]−, 15 S. Ariyasu, H. Hayashi, B. Xing and S. Chiba, Bioconjugate
M calcd for C19H23N5O8 448.1474. Chem., 2017, 28, 897.
27: 1H NMR (D2O, 400 MHz): δ 7.70 (s, 1H), 6.29 (t, J = 16 D. Zewge, G. Butora, E. C. Shere, D. M. Tellers, R. D. Sidler,
5.6 Hz, 1H), 6.06 (s, 1H), 4.31 (dt, J = 8.0, 17.2 Hz, 1H),
4.17–4.13 (m, 1H), 3.92 (d, J = 13.6 Hz, 1H), 3.76 (d, J =
J. Gouker, G. Copeland, V. Jadhav, Z. Li, J. Armstrong and
I. W. Davies, Bioconjugate Chem., 2018, 29, 1859.
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