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sultant crude product was purified by preparative RP-HPLC (see
the Supporting Information for details) and converted into the tet-
rasodium salt by ion exchange chromatography (Dowex 50WX 50–
100) to give 3 as a greenish solid (65 mg, 25% over 3 steps from
To a solution of the thus obtained Park’s nucleotide 1 (2.5 mg,
2.2 mmol) in acetone (190 mL), dansyl chloride (5.9 mg, 22 mmol)
and NaHCO3 (0.25m in water, 190 mL, 48 mmol) were added. The re-
action mixture was stirred at 248C for 3 h. After centrifugation, re-
suspension of the pellet in water (270 mL) and repeated centrifuga-
tion, the supernatants were combined and evaporated under re-
duced pressure. The resultant residue was resuspended in water
(500 mL) and centrifuged again. The supernatant was purified by
semi-preparative RP-HPLC (see the Supporting Information for de-
tails) and converted into the tetrasodium salt by ion exchange
chromatography (Dowex 50WX 50–100) to give 3 as a slightly
1
28). [a]2D0 = +22.4 (c=1.6, MeOH). H NMR (300 MHz, D2O): d=8.48
(d, J=8.7 Hz, 1H, dansyl-H-2), 8.27 (d, J=8.7 Hz, 1H, dansyl-H-8),
8.23 (d, J=7.4 Hz, 1H, dansyl-H-4), 7.90 (d, J=8.1 Hz, 1H, uracil-H-
6), 7.70–7.65 (m, 2H, dansyl-H-3, dansyl-H-7), 7.38 (d, J=7.6 Hz, 1H,
dansyl-H-6), 5.95–5.91 (m, 2H, ribose-H-1, uracil-H-5), 5.46 (dd, J=
7.2, 3.2 Hz, 1H, Glc-H-1), 4.34–4.32 (m, 2H, ribose-H-2, Glc-H-4),
4.29–4.23 (m, 4H, Ala-H-2, Glc-H-3, ribose-H-4, propionyl-H-2),
4.23–4.10 (m, 5H, Lys-H-2, Glu-H-2, Glc-H-2, ribose-H-5), 4.10–4.06
(m, 1H, Ala-H-2), 3.96–3.93 (m, 1H, Glc-H-6a), 3.85–3.59 (m, 6H, 2
x Ala-H-2, Glc-H-4, Glc-H-5, Glc-H-6b, ribose-H-3), 2.90 (t, J=6.5 Hz,
2H, Lys-H-6), 2.85 (s, 6H, N(CH3)2), 2.27–2.21 (m, 2H, Glu-H-4), 2.14–
2.05 (m, 1H, Glu-H-3a), 1.99 (s, 3H, N-Ac-CH3), 1.88–1.81 (m, 1H,
Glu-H-3b), 1.41 (d, J=7.3 Hz, 3H, CH3), 1.38 (d, J=6.8 Hz, 3H, CH3),
1.42–1.39 (m, 2H, Lys-H-4), 1.30 (d, J=7.2 Hz, 6H, 2 x CH3), 1.26–
1.15 (m, 2H, Lys-H-5), 1.13–1.03 (m, 1H, Lys-H-3a), 1.03–0.94 ppm
(m, 1H, Lys-H-3b); 13C NMR (75 MHz, D2O): d=179.8 (C=O), 177.6
(C=O), 175.8 (C=O), 175.4 (C=O), 174.2 (C=O), 174.0 (C=O), 173.5
(C=O), 152.2 (uracil-C-4), 150.9 (uracil-C-2, dansyl-C-5), 141.6 (uracil-
C-6), 134.1 (dansyl-C-1), 130.2 (dansyl-C-2), 129.8 (dansyl-C-4), 129.0
(dansyl-C-8a), 129.0 (dansyl-C-4a), 128.8 (dansyl-C-7), 124.1 (dansyl-
C-3), 119.1 (dansyl-C-8), 116.0 (dansyl-C-6), 102.7 (uracil-C-5), 94.7
(d, JCP =5.5 Hz, Glc-C-1), 88.6 (ribose-C-1), 83.2 (d, JCP =9.2 Hz,
ribose-C-4), 79.9 (propionyl-C-2), 78.0 (Glc-C-3), 73.8 (ribose-C-2),
73.0 (ribose-C-3), 69.7 (Glc-C-4), 68.1 (Glc-C-5), 65.1 (d, JCP =4.2 Hz,
ribose-C-5), 60.4 (Glc-C-6), 54.4 (Ala-C-2), 54.2 (Ala-C-2), 53.5 (d,
1
greenish solid (1.2 mg, 38%). H NMR spectra and MS data of this
material were identical to the corresponding data for synthetically
obtained 3.
Cloning of the mraY gene from S. aureus
The mraY gene of S. aureus subsp. aureus MRSA252 (BX571856.1)
was purchased from Mr. Gene (see the Supporting Information for
the gene sequence) and provided in a pMA vector. This mraY gene
was cut with the restriction enzymes NcoI and XhoI (New England
Biolabs) and ligated into a pET28a-(+) vector (Novagen), which
had been cut by using the same enzymes. The resultant plasmid
thus featured a gene encoding the MraY enzyme with a C-terminal
His6 tag. The plasmid was amplified with the E. coli strain XL-1 blue
based on the kanamycin resistance provided by the pET28a vector,
and its sequence was confirmed by DNA sequencing (Eurofins
MWG Operon).
J
CP =8.2 Hz, Glc-C-2), 51.0 (Ala-C-2), 49.8 (Glu-C-2), 52.0 (Lys-C-2),
Overexpression of MraY from S. aureus in E. coli
45.0 (N(CH3)2), 42.2 (Lys-C-6), 31.9 (Glu-C-4), 30.3 (Lys-C-4), 28.2
(Glu-C-3), 27.8 (Lys-C-5), 22.2 (N-Ac-CH3), 22.0 (Lys-C-3), 18.8 (CH3),
17.6 (CH3), 17.0 (CH3), 16.6 ppm (CH3); 31P NMR (121 MHz, D2O): d=
À11.11 (d, J=20.6 Hz, 1P), À12.92 ppm (d, J=20.6 Hz, 1P); IR (ATR):
The mraY-containing plasmid was transformed into E. coli Lemo21
cells (New England Biolabs), which were plated on lysogeny broth
(LB) agar containing kanamycin (50 mgmLÀ1) and chloramphenicol
(30 mgmLÀ1). A single colony was picked to induce an overnight
n˜ =3294, 2943, 1741, 1695, 1634, 1541, 1455, 1145, 791, 628 cmÀ1
.
culture (10 mL) of LB media containing kanamycin (50 mgmLÀ1
)
HRMS (ESI): calcd for C52H75N10O28P2S 1381.3954, found 1381.3944
[MÀH]À.
and chloramphenicol (30 mgmLÀ1), which was incubated at 378C
and 180 rpm for 16 h. A culture of LB media (500 mL) containing
kanamycin (50 mgmLÀ1), chloramphenicol (30 mgmLÀ1) and l-rham-
nose (1 mm) was inoculated with the overnight culture (500 mL)
and then grown at 378C and 180 rpm to OD600 0.6. This was fol-
lowed by induction of MraY expression with IPTG (1 mm) and incu-
bation at 378C and 180 rpm for 4 h. Cells were centrifuged (4600g,
20 min, 48C) and the pellet was resuspended in buffer A (50 mm
Tris-HCl buffer pH 7.5, 1 mm MgCl2, 2 mm b-mercaptoethanol;
15 mL total). Egg white lysozyme (spatula tip), DNAse I (spatula tip)
and a tablet of cOmpleteTM EDTA-free protease inhibitor cocktail
(Merck) were added. Cells were lysed by using sonication (30%,
30/100 pulse, 15 min on ice) and then incubated at 48C for 30 min.
The lysate was centrifuged (17000g, 45 min, 48C) and the superna-
tant was centrifuged again (180000g, 1 h, 48C). The resultant
pellet was resuspended in buffer A (1.7 mL), flash frozen in liquid
nitrogen and stored at À808C (aliquots of 20 mL). This MraY-con-
taining crude membrane preparation (overall protein concentration
ꢀ20.5 mgmLÀ1 as determined by OD280, Nanodrop UV spectrome-
ter) was diluted with water (final overall protein concentration
1 mgmLÀ1) and then directly used for MraY activity assays without
further purification.[9c,10]
Semi-synthesis of dansylated Park’s nucleotide 3
The isolation of naturally occurring Park’s nucleotide 1 was carried
out according to a modified protocol from Kohlrausch and Hçlt-
je.[18a] S. aureus SA113 was grown in Antibiotic Medium 3 (Difco, 3ꢁ
100 mL) at 378C and 140 rpm for 24 h as precultures (OD578 =1.1–
1.2). From each preculture, approximately 70 mL were transferred
into Antibiotic Medium 3 (2 L each, starting OD578 ꢀ0.06) to give
the main cultures. These were incubated at 378C and 140 rpm
until OD578 0.8–0.9 was reached. Penicillin G (Sigma Aldrich,
75 mgmLÀ1 cell culture) was added and the mixtures were incubat-
ed at 378C for 1 h under stirring. Cells were collected by centrifu-
gation (12000g, 48C, 15 min, at this point cells from all cultures
were combined), resuspended in water (0.1 g wet weight mLÀ1
water) and slowly stirred into boiling water (double volume). Boil-
ing was continued for 30 min, the suspension was allowed to cool
to RT and finally stirred at 48C overnight. It was then centrifuged
(200000g, 48C, 1 h) and the supernatant was lyophilized. The re-
sultant residue was dissolved in water and applied to gel permea-
tion chromatography (GPC, column Toyopearl TSK-40F (Tosoh Bio-
science, 2.5ꢁ120 cm), eluent 8:20:2000 pyridine-AcOH-water) to
yield crude 1. Further purification was achieved by preparative RP-
HPLC and final desalting by GPC as described before for UDP-mur-
amyl tripeptide (DAP-type)[18b] to give 1 (ꢀ10 mg from the 6 L of
bacterial culture).
Fluorescence-based MraY assay
Based on Bugg’s method,[9c,10,12] fluorescence intensity over time
was measured at lex =355 nm and lem =520 nm (BMG Labtech PO-
LARstar Omega, 384-well plate format). To start the MraY-catalyzed
&
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Chem. Eur. J. 2016, 22, 1 – 8
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ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!