Journal of Medicinal Chemistry
Brief Article
Synthesis of Erythromycin Alkyne (11). To a solution of
erythromycin A and 9-O-oxime (100 mg, 0.133 mmol) in MeCN (1.2
mL) were added propargyl bromide (80% solution in toluene, 18.4 μL,
0.165 mmol) and KOH (13 mg, 0.21 mmol). The mixture was stirred
at ambient temperature for 3 h followed by the addition of CHCl3 (2
mL). The resulting mixture was filtered, concentrated in vacuo, and
redissolved in 1:1 MeCN/H2O (3 mL). The product was purified by
HPLC: Gilson preparative system, Phenomenex C18 column, flow rate
5 mL/min, detected at 220 nm. Gradient: starting from 10% MeCN
(0.04% AcOH) and 90% water (0.04% AcOH), ramping up to 40%
MeCN over 10 min, then ramping up to 95% MeCN over 1 min,
staying at 95% MeCN for 2 min, ramping down to 20% MeCN over 1
min, then staying at 20% MeCN for 2 min. Product containing
fractions were pooled, frozen, and lyophilized to yield the product as a
white fluffy powder (31 mg, 30%). Retention time (analytical) = 12.1
Author Contributions
S.A.C. performed all chemical synthesis and in vitro testing and
wrote the manuscript. X.L., S.H., and M.Y. performed in vivo
testing. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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We thank Dr. Angie Morales, Dr. Randy Whitall, Jing Zheng,
and Bela Reiz for their assistance with mass spectrometry.
These investigations were supported by the Natural Sciences
and Engineering Research Council of Canada (NSERC),
Alberta Innovates Health Solutions, the Flight Attendant
Medical Research Institute (FAMRI, Grant 103007), National
Institutes of Health Grant AI109317-01A1, and NIH Grant
AI101973-01 to M.W.
1
min; H NMR (DMSO-d6, 500 MHz) δ 5.14 (dd, 1H, J = 11.1, 1.8
Hz), 4.75 (d, 1H, J = 4.8 Hz), 4.52−4.49 (m, 2H), 4.13 (br s, 1H),
4.06, (m, 1H) 4.00−3.95 (m, 1H), 3.84−3.76 (m, 2H), 3.65 (app td,
1H, J = 11.2, 3.8 Hz), 3.56−3.53 (m, 2H), 3.49 (d, 1H, J = 6.5 Hz),
3.24 (s, 3H), 3.15 (s, 3H), 3.09 (s, 3H), 2.93 (d, 1H, J = 9.4 Hz), 2.80
(app dt, 1H, J = 16.3, 7.2 Hz), 2.64 (app q, 1H, J = 7.0 Hz), 2.28 (d,
1H, J = 14.9 Hz), 2.07−2.02 (m, 1H), 1.99−1.94 (m, 1H), 1.86−1.78
(m, 1H), 1.65 (s, 1H), 1.60−1.48 (m, 3H), 1.41−1.23 (m, 5H), 1.19−
0.99 (m, 18H), 0.75 (t, 3H, J = 7.4 Hz); HRMS (ES) calcd for
C40H70N2O13 [M + H]+ 786.4951, found 786.4941.
ABBREVIATIONS USED
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ATCC, American Type Culture Collection; CFU, colony
forming units; CuAAC, copper-catalyzed azide alkyne cyclo-
addition; DIPEA, diisopropylethylamine; DMF, dimethylfor-
mamide; DMSO, dimethylsulfoxide; Eryc, erythromycin A;
Fmoc, fluorenylmethyloxycarbonyl; HATU, O-(7-azabenzotria-
zol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate;
HBTU, N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)-
uronium hexafluorophosphate; HPLC, high-performance liquid
chromatography; LCMS, liquid chromatography−mass spec-
trometry; MALDI, matrix assisted laser desorption ionization;
MDR, multidrug resistant; MIC, minimum inhibitory concen-
tration; Oct, octanoyl; OM, outer-membrane; PEG, poly-
ethylene glycol; Rif, rifampicin; SPPS, solid-phase peptide
synthesis; TFA, trifluoroacetic acid; THF, tetrahydrofuran; Tri,
tridecaptin; Van, vancomycin; XDR, extremely drug resistant
Synthesis of Rifampicin Alkyne (14). Rifaldehyde (100 mg,
0.138 mmol) was suspended in dry THF (0.5 mL). 1-Amino-4-
propargylpiperazine (12) (19 mg, 0.138 mmol) was added and the
mixture stirred vigorously for 15 min. The mixture was then diluted
with CH2Cl2 (7 mL) and washed with 5.5 mL of a solution of ascorbic
acid (2.0 g) in 3:1 H2O/brine (40 mL). The aqueous phase was then
extracted with CH2Cl2 (7 mL) and the combined CH2Cl2 extracts
were dried over anhydrous Na2SO4 and concentrated in vacuo to yield
rifampicin alkyne (14) as a red solid (110 mg, 94%). Retention time
(analytical) = 19.7 min; 1H NMR (CDCl3, 500 MHz) δ 12.02 (s, 1H),
8.29 (s, 1H), 6.58 (dd, 1H, J = 15.5, 12.3 Hz), 6.39 (d, 1H, J = 11.1
Hz), 6.21 (d, 1H, J = 13.6 Hz), 5.94 (dd, 1H J = 15.4, 5.0 Hz), 5.11
(dd, 1H, J = 12.7, 6.8 Hz), 4.95 (d, 1H, J = 10.7 Hz), 3.78−3.73 (m,
1H), 3.60 (d, 1H, J = 4.7 Hz), 3.48 (d, 1H, J = 6.8 Hz), 3.43 (s, 1H),
3.38 (s, 1H), 3.22−3.19 (m, 1H), 3.11−3.09 (m, 1H), 3.05−3.01 (m,
4H), 2.76−2.69 (m, 2H), 2.41−2.36 (m, 1H), 2.28 (s, 1H), 2.23 (s,
3H), 2.08−2.06 (m, 6H), 1.80 (s, 3H), 1.73−1.70 (m, 1H), 1.57−1.52
(m, 1H), 1.39−1.34 (m, 1H), 1.02 (d, 3H, J = 7.0 Hz), 0.90 (d, 3H, J =
7.1 Hz), 0.61 (d, 3H, J = 76.9 Hz), −0.30 (d, 3H, J = 6.9 Hz). HRMS
(ES) calcd for C45H57N4O12 [M − H]− 845.3978, found 845.3977.
General Procedure for Conjugate Synthesis. The peptide
azide (2.5 μmol) and alkyne (7.5 μmol) were dissolved in 1:1
H2O/tBuOH (125 μL). A 100 mM solution of CuSO4 (15 μL, 1.5
μmol) and freshly prepared 500 mM solution of ascorbic acid (12 μL,
6.0 μmol) were added, and the mixture was stirred at 50 °C until
complete consumption of the azide starting material observed by using
MALDI. The product was then purified by HPLC.
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ASSOCIATED CONTENT
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* Supporting Information
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Synthetic procedures, compound characterization, and
details on in vitro and in vivo testing (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
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Corresponding Author
*Phone: 780-492-5475. Fax: 780-492-8231. E-mail: john.
F
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