Selective catalytic hydrogenation of the N-acyl and uridyl double bonds in the tunicamycin family of protein N-glycosylation inhibitors

Article abstract of DOI:10.1038/ja.2017.141

Tunicamycin is a Streptomyces-derived inhibitor of eukaryotic protein N-glycosylation and bacterial cell wall biosynthesis, and is a potent and general toxin by these biological mechanisms. The antibacterial activity is dependent in part upon a π-π stacking interaction between the tunicamycin uridyl group and a specific Phe residue within MraY, a tunicamycin-binding protein in bacteria. We have previously shown that reducing the tunicamycin uridyl group to 5,6-dihydrouridyl (DHU) significantly lowers its eukaryotic toxicity, potentially by disrupting the π-stacking with the active site Phe. The present report compares the catalytic hydrogenation of tunicamycin and uridine with various precious metal catalysts, and describe optimum conditions for the selective production of N-acyl reduced tunicamycin or for tunicamycins reduced in both the N-acyl and uridyl double bonds. At room temperature, Pd-based catalysts are selective for the N-acyl reduction, whereas Rh-based catalysts favor the double reduction to provide access to fully reduced tunicamycin. The reduced DHU is highly base-sensitive, leading to amide ring opening under mild alkaline conditions.

Full text of DOI:10.1038/ja.2017.141

The Journal of Antibiotics (2017), 1–7
2017 Japan Antibiotics Research Association All rights reserved 0021-8820/17
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&
ORIGINAL ARTICLE
Selective catalytic hydrogenation of the N-acyl and
uridyl double bonds in the tunicamycin family
of protein N-glycosylation inhibitors
Neil PJ Price1, Michael A Jackson1, Karl E Vermillion1, Judith A Blackburn1, Jiakun Li2 and Biao Yu2
Tunicamycin is a Streptomyces-derived inhibitor of eukaryotic protein N-glycosylation and bacterial cell wall biosynthesis, and is
a potent and general toxin by these biological mechanisms. The antibacterial activity is dependent in part upon a π-π stacking
interaction between the tunicamycin uridyl group and a specific Phe residue within MraY, a tunicamycin-binding protein in
bacteria. We have previously shown that reducing the tunicamycin uridyl group to 5,6-dihydrouridyl (DHU) significantly lowers its
eukaryotic toxicity, potentially by disrupting the π-stacking with the active site Phe. The present report compares the catalytic
hydrogenation of tunicamycin and uridine with various precious metal catalysts, and describe optimum conditions for the
selective production of N-acyl reduced tunicamycin or for tunicamycins reduced in both the N-acyl and uridyl double bonds.
At room temperature, Pd-based catalysts are selective for the N-acyl reduction, whereas Rh-based catalysts favor the double
reduction to provide access to fully reduced tunicamycin. The reduced DHU is highly base-sensitive, leading to amide ring
opening under mild alkaline conditions.
The Journal of Antibiotics advance online publication, 1 November 2017; doi:10.1038/ja.2017.141
INTRODUCTION
Recent work by Hakulinen et al.15 has shown that tunicamycin
Tunicamycin is a potent and selective inhibitor of the UDP-N-acetyl-
binds at a cytoplasmic cavity of MraY, a bacterial PNPT transmem-
D-hexosamine:isoprenol-1-phosphate UDP-HexNAc-1-P translocase brane protein. Significantly, the tunicamycin uracil ring is shown to
bind to a uridyl binding pocket within the MraY protein, composed of
Gly-176, Asn-221, and Phe-228. Other essential residues, Asp-175,
Glu-300, and Lys-74 are adjacent to the 2-carbonyl of the uracil ring.15
This tunicamycin uracil binding pocket (UBP) shares a partial
overlapping binding mode also found for the uracil group of
muraymycin D, a related nucleotide inhibitor of MraY.16,17 Compar-
able π-π stacking of Phe-228 occurs with both tunicamycin and
muraymycin, and also with a truncated MraY substrate, UDP-
MurNAc-L-Ala, providing a mechanism for the binding of the
tunicaminyl uracil motif.15,17
The present work investigates the catalytic hydrogenation of
tunicamycin, in an attempt to selectively reduce the N-acyl and/or
uridyl double bonds on a preparative scale (Scheme 1). We report that
hydrogenation at ambient temperature and pressure with Pd/C catalyst
results in selective reduction of the conjugated 2′′′, 3′′′-N-acyl double
bond to give Tun-R1. Substituting Rh/Al2O3 catalyst results in the
reduction of both double bonds to give Tun-R2. Moreover, the
reduced uridyl ring is shown to be is highly sensitive to base-catalyzed
ring opening. We have previously shown that Tun-R1 and Tun-R2
both exhibit considerably reduced eukaryotic toxicity when compared
to the tunicamycin natural products.18
(PNPT) family of enzymes, and is widely used to block the first step
in eukaryotic protein N-glycosylation or of the assembly of bacterial
cell walls.1–4 It consists of an 11-carbon dialdose sugar, tunicamine,
which is N-glycosidically linked to uracil, and αβ-1,11-O-glycosidically
linked to N-acetylglucosamine (Figure 1). Several Streptomyces species
produce tunicamycins, notably S. chartreusis and S. lysosuperificus,5,6
although other strains are known to produce related compounds, such
as streptovirudins, corynetoxins, and quinovosamycins.7–10 Tunica-
mycin biosynthesis has been studied in S. chartreusis NRRL B-3882
and showed that the 11-carbon tunicamine dialdose is derived from
ligation of 6-carbon pyranose and 5-carbon furanose units, the latter
derived from uridine or ribose.11–14 This suggested that there may be
an active uridine salvage pathway in the tunicamycin-producing S.
chartreusis. Most of the known tunicamycin-related compounds
contain an N-glycosidically linked uridyl group that acts as a substrate
analog of the nucleotide on the UDP-HexNAc donor substrate of
PNPT. However, several of the streptovirudins isolated from S.
griseoflavus subsp. thuringiensis have related structures to the tunica-
mycins except that the uridyl group is replaced by 5,6-dihydrouracyl
(Figure 1).7
1Agricultural Research Service, US Department of Agriculture, National Center for Agricultural Utilization Research, Peoria, IL, USA and 2State Key Laboratory of Bioorganic and
Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China
Correspondence: Dr NPJ Price, Agricultural Research Service, US Department of Agriculture USDA-ARS, National Center for Agricultural Utilization Research, 1815 N University
Street, Peoria, IL, 61604, USA.
E-mail: neil.price@ars.usda.gov
Received 9 August 2017; revised 29 September 2017; accepted 2 October 2017

Selective hydrogenation of tunicamycins
NJ Price et al
2
O
N
R1
MeN
O
O
HN
HN
O
O
N
O
H
N
H
N
H
N
HOOC
HO
O
O
OH
O
O
O
HO
OH OH
HO
HO
OH
OH
OH OH
O
NH
SMe
O
O
HN
or
R1
=
NH2
NH
Tunicamycin
O
HO
HO
b
a
Napsamycin C and Mureidomycin B
O
HN
O
O
N
O
HN
HO
OH
O
O
O
N
O
O
O
O
O
O
HO
HO
OH
OH OH
N
H
O
NH
O
O
OH OH
OH
HN
Streptovirudin
Jawsamycin
O
Figure 1 Chemical structures of tunicamycin and various dihydrouridyl-based natural products. Napsamycin C, R1= a; Mureidomycin B, R1= b. The
chemically-reducible double bonds in tunicamycin are denoted by black arrows.
Scheme 1 Selective catalytic hydrogenations of the tunicamycin antibiotics. A full colour version of this figure is available at the Journal of Antibiotics journal online.
RESULTS
undesirable uridyl ring opening.19–21 Kochetor et al.22 have described
catalytic hydrogenation of uridine over Rh/Al2O3 catalyst in aqueous
solution requiring the use of lithium acetate buffer, which requires
Selective catalytic hydrogenation of the tunicamycin uridyl and
N-acyl double bonds
Methods have been investigated for the selective chemical reduction of subsequent removal. Using uridine as a model for the tunicamycin
the tunicamycins. Reduction of the tunicaminyl double bonds with uridyl motif we obtained complete reduction to 5,6-dihydrouridine in
sodium borohydride in the presence of Pd/C catalyst, although methanol using Rh/Al2O3-catalyzed hydrogenation at 1 atm. pressure
successful on a small scale, was complicated by the post reaction and room temperature. The reaction progress was monitored by
removal of borate and acetate co-products.18 Moreover, unless the pH MALDI-TOF MS by following the conversion of the [M+H]+ and
is strictly controlled the borohydride treatment can lead to an [M+Na]+ ions at m/z 245.2 and 267.2 (for uridine) to m/z 247.2 and
The Journal of Antibiotics

Selective hydrogenation of tunicamycins
NJ Price et al
3
Figure 2 Hydrogenation of uridine with Pd/C (a, c) or Rh/Al2O3 (b, d) catalyst. (a, b) MALDI-TOF MS showing [M+H]+ and [M+Na]+ ions for uridine (a) and
5,6-dihydrouridine (b), respectively. m/z 273.2 is a MALDI-MS matrix ion. (c, d) 1H-NMR shows quantitative reduction of the uridyl 5, 6-double bond after
hydrogenation with the Rh/Al2O3 catalyst (d).
Table 1 Relevant NMR assignments for tunicamycin, uridine, and hydrogenated analogs, DHU, TunR1, and TunR2
Tunicamycin
13C
Uridine
DHU
Tun-R1
Tun-R2
Residue
1H
13C
1H
13C
1H
13C
1H
13C
1H
Uridyl/DHU
2
4
5
6
152.6
166.1
103.1
142.8
—
—
151.9
166.4
102.6
142.2
—
—
154.1
171.8
30.1
—
—
150.8
165.5
102.4
87.2
—
—
154.1
171.6
30.7
—
—
5.89
8.05
5.79
7.77
2.71
5.78
7.93
2.69
36.4
3.55/3.50
36.4
3.58/3.48
Tunicaminyl/ribosyl
1'
89.9
6.05
89.7
5.80
87.6
5.80
87.2
5.94
87.3
5.87
N-acyl chain
174.9
36.7
25.6
29.5
—
175.8
36.3
25.5
29.2
—
C = O
2'''
169.7
124.9
146.5
33.1
—
2.21
1.63
1.37
2.21
1.61
1.35
6.07
6.94
2.32
3'''
4'''
Abbreviation: DHU, 5,6-dihydrouridyl.
All spectra were acquired in d4-methanol. Chemical shifts are given in p.p.m.
269.2 (for dihydrouridine) (Figures 2a and b). Following a straightforward occurred (Figure 3c). This suggested a selective reduction scheme for
recovery by centrifugation or filtration as needed to remove spent catalyst, tunicamycin, whereby Pd/C-catalyzed hydrogenation is used to
the product was characterized by NMR (Figures 2c and d and selectively reduce the N-acyl double bond, and Rh/Al2O3-catalyzed
Supplementary Data). The uridyl H5 and H6 methine protons (at 5.8 hydrogenation for double reduction of both N-acyl and uridyl double
and 7.8 p.p.m., respectively) are entirely absent after 3 h reaction, with bonds (see Figure 1 for the tunicamycin structure). This was
concomitant generation of new methylene signals at 3.5 and 3.7 p.p.m. investigated by monitoring the hydrogenation of tunicamycin under
(Figure 2d and Table 1). These latter two signals and the adjacent ribosyl these selective conditions by MALDI-TOF MS (Figure 3), and by
H1′ integrate to 2:2:1, indicating a clean reduction of the double bond proton NMR analysis of the double bond region (5–8 p.p.m.;
without degradation or racemization of the uridyl motif. The dihydrour- Figure 4).
idine product was further characterized by 13C-NMR, DEPT, COSY,
HSQC, HMBC, and NOESY NMR (Table 1; Supplementary Data). The
HMBC experiment was crucial in showing that the carbonyl 13C signals
(154.6 and 173.8 p.p.m.) are not reduced during the hydrogenation.
MALDI-TOF MS peaks at m/z 853.8, 867.9, and 881.9 correspond
to the [M+Na]+ adduct ions for Tun-15:1, Tun16:1, and Tun17:1,
tunicamycin components with N-pentadecenoate, N-hexadecenoate
and N-heptadecenoate N-acyl groups, respectively (Figure 3a). The
Similar results were obtained on uridine using Rh/C catalyst in place single reduction of these molecules is evident from ions at m/z 855.8,
of the Rh/Al2O3. However, with Pd/C no reduction of the uridyl ring 869.9 and 883.9, and the double reduction by m/z 857.7, 871.7 and
The Journal of Antibiotics

Selective hydrogenation of tunicamycins
NJ Price et al
4
by 30 min with the Rh/Al2O3 catalyst (Figure 4 (i)). After 1 h the
reduction of both double bonds is apparently completed, and with no
evidence of further modification after 3 h. This double-reduced
tunicamycin product was readily recovered by filtering off the catalyst,
and was fully characterized by COSY, HSQC, TOCSY, 13C, and
HMBC NMR (Supplementary Data). The HMBC spectra of the high
field region indicated that the four carbonyls (154.1, 171.6, 172.2, and
175.8 p.p.m.) in the dihydrouridyl-tunicamycin are still intact (Figure
4 (ii)) as observed previously with the uridine model compound. The
HMBC signal at 154.1 p.p.m. is assigned as the dihydrouridyl ring C2
carbonyl, and is seen to correlate with the pseudoribosyl αH1′ signal
(5.90 p.p.m.) and the reduced H6a and H6b signals at 3.5 – 3.6 p.p.m.
The C4 carbonyl in the dihydrouridyl ring is assigned at 171.6 p.p.m.,
and also correlates to the H6a and H6b protons, plus the reduced H5
CH2 at 2.70 p.p.m. The N-acetyl and N-acyl carbonyls are assigned at
172.2 p.p.m. and 175.8 p.p.m., respectively, and show correlations to
the N-acetyl methyl signal at 2.03 p.p.m., or the N-acyl α-CH2 signals
at 2.21 p.p.m. Moreover, a comparison of the HSQC, 1H, and 13C
NMR spectra of the Rh/Al2O3-catalyzed hydrogenation product with
dihydrouridyl-tunicamycin (Tun R2) produced by 50 h at reflux with
NaBH4/Pd/C showed them to be identical (Supplementary Data),
although the catalytic hydrogenation is achieved in 3 h and without
the required post-reaction cleanup of borate salts.
Figure 3 MALDI-TOF MS analysis of tunicamycins (a), and after catalytic
hydrogenation with Pd/C (b) or Rh/Al2O3 (c) catalysts. (a). Tun-14:1, m/z
839.8; Tun-15:1, m/z 853.8; Tun-16:1, m/z 867.9; Tun-17:1, m/z 881.9.
(b). Tun-14:0, m/z 841.8; Tun-15:0, m/z 855.8; Tun-16:0, m/z 869.9;
Tun-17:0, m/z 883.9. (c) DHU-Tun-14:0, m/z 843.7; DHU-Tun-15:0, m/z
857.7; DHU-Tun-16:0, m/z 871.7; DHU-Tun-17:0, m/z 885.8. The MS
spectra were acquired in reflectron mode using 2,5-dihydroxybenzoic acid as
matrix.
Photoreduction and alkaline lability of the dihydrouridyl group
Cerutti et al.23 reported in 1965 that photoexcitation of certain
heterocyclic compounds enhances their reduction by sodium borohy-
dride, and used this to selectively photoreduce uridine in RNA
polynucleotides. As part of this work they also reported a preparative
reaction for the selective photoreduction of uridine to 5,6-
885.8, mass increases of two or four protons, respectively (Figures 3b dihydrouridine.24 More recently, the photoreduction of carbonyls
and c). However, these data are not selective for the position of the with borohydride has been reported,25 and others found that UV
reductions. This required a quantitative analysis of the uridyl H5 and irradiation of polyunsaturated allylic benzoates results in a selective
H6 double bond protons at 7.91 p.p.m. and 5.77 p.p.m. for the redox E/Z interconversion of the allylic double bond.26 This suggested a
state of the uridyl group, and at 6.83 and 5.95 p.p.m. for the N-acyl possible selective photoreduction of the uridyl group of tunicamycins
double bond (Figure 4 (i); Table 1). The integrals of these signals without the attendant reduction of the N-acyl double bond which, if
indicate that after 30 min hydrogenation with Pd/C catalyst that the successful, might be an expedient route for the chemical conversion of
uridyl H5/H6 double bond is still intact, and that the H5/H6 uridyl tunicamycins into streptovirudins (Figure 1). However, our attempts
protons and the tunicaminyl pseudoribosyl αH1′ signal (5.90 p.p.m.) to replicate the photoreduction of uridine under Cerutti conditions
integrate to 1:1:1 (Figure 4 (i)). Noticeably, the N-acyl double bond is were unsuccessful. House and Miller found that the dihydrouridine
completely reduced by this time point, with the loss of the H2′′′ and residue found in tRNA is stable at 25 °C, but with a short half-life at
H3′′′ methine signals at 6.85 and 5.95 p.p.m. At 3 h there is also a 70% higher temperatures and pH leading to a ring opening of the
attenuation of the uridyl H5/H6 protons relative to the pseudoribosyl dihydrouridine to ureidopropanol riboside.19 Cleavage of the dihy-
αH1′, indicating about a 70% conversion to dihydrouridyl (Figure 4 drouridine ring has also been reported at mild alkaline conditions and
(i)). This is also evident from the chemical shift of the pseudoribosyl upon reduction by sodium borohydride. In both cases, cleavage of the
αH1′ on the reduced dihydrouridyl-tunicamycin, which now appears dihydrouridine ring is followed by cleavage of the RNA chain (Behm-
at 5.88 p.p.m. (Figure 4 (i)). These data show that the Pd/C catalyst is Ansmant et al,27).
selective toward the tunicamycin N-acyl group in methanol at room
To investigate the pH stability of the dihydrouridine group we
temperature.
acquired in situ NMR spectra of the dihydrouridine prepared by Rh/
An analogous series of reactions was done on tunicamycin with Rh/ Al2O3-catalyzed hydrogenation of uridine in the presence of either
Al2O3 catalyst using methanol as the solvent. After 30 min reaction deuterated NaOD or CF3COOD (Figure 5). The DHU is stable under
1
time the N-acyl double bond H-NMR signals are still present, but acid condition, and the NMR data were unchanged over a period of
reduced by ~ 90%, indicating that the reduction of the N-acyl group is 18 h (Figure 5b). However, in the presence of the NaOD the
not completed by this time (Figure 4 (i)). This is in contrast to that dihydrouridine was quantitatively hydrolyzed to ureidopropanol ribo-
observed with the Pd/C catalyst, which gave complete reduction of the side, without any cleavage of the adjacent ribosyl N-glycosidic bond
N-acyl double bond by 30 min. However, unlike on Pd/C, the (Figure 5c). This result was confirmed when dihydrouridine was
tunicamycin uridyl group is also partially hydrogenated by 30 min treated with 0.25M sodium hydroxide for 18 h at ambient temperature
with the Rh/Al2O3 catalyst (Figure 4 (i)). This is evident from the and analyzed by MALDI TOF mass spectrometry (data not shown).
attenuated integrals of H5/H6 relative to αH1′, and from the de novo The molecular ions for DHU ([M+H]+, m/z 247.2; [M+Na]+, m/z
αH1′ of the dihydrouridyl-pseudoribosyl at 5.88 p.p.m. The relative 269.2) were increased by 18 Da to m/z 265.2 and m/z 287.2 by the
integrals of these signals show that the uridyl group is ~ 30% reduced hydrolysis of the cyclic peptide bond.
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Selective hydrogenation of tunicamycins
NJ Price et al
5
Figure 4 (i) Proton NMR spectra (5.6 – 8.2 p.p.m. region) of tunicamycin after hydrogenation with either Pd/C (a–c) or Rh/Al2O3 catalysts (d–f). Timed
reactions with H2 gas (1 atm. in methanol) were stopped at 30 min (a, d), 1 h (b, e) or 3 h (c, f). Signals arise from the starting tunicamycin (uridyl ring;
H5, 5.77 p.p.m.; H6, 7.91 p.p.m.; pseudoribosyl anomeric H1′(U), 5.90 p.p.m.; N-acyl H2′′′, 5.95 p.p.m.; H3′′′, 6.83 p.p.m.); the N-acyl-reduced
tunicamycin, TunR1 (uridyl ring; H5, 5.77 p.p.m.; H6, 7.91 p.p.m.; pseudoribosyl anomeric H1′(U), 5.90 p.p.m.); and the fully reduced dihydrotunicamycin,
TunR2 (pseudoribosyl anomeric H1′(d), 5.88 p.p.m.). (ii) HMBC NMR spectrum of the four carbonyl correlation for the dihydrouridyl-tunicamycin produced
by Rh/Al2O3-catalyzed hydrogenation. The 13C carbonyl signals are (A). uridyl-2; (B). uridyl-4; (C). N-acetyl; (D). N-acyl. The associated 1H signals are
assigned in Table 1.
DISCUSSION
streptovirudins have dihydrouracil analogs (Figure 1), and these
The uridyl structural motif is a fairly widespread chemophore for a compounds all target the bacterial MraY members of the PNPT
number of natural products.28,29 However, the 5,6-dihydrouridyl enzyme family. For the napsamycins a biosynthetic gene, npsU, has
group is less common. Mureidomycins, napsamycins, and been identified that encodes a putative reductase that is responsible for
The Journal of Antibiotics

Selective hydrogenation of tunicamycins
NJ Price et al
6
O
O
N
O
OH
H2N
HN
HN
O
O
N
N
O
O
O
O
HO
HO
HO
OH OH
OH OH
OH OH
Figure 5 Base-catalyzed quantitative 5,6-dihydrouridyl ring opening to give ureidopropanol riboside (a). 1H-NMR spectra of 5,6-dihydrouridine in D2O after
treatment with CF3COOD (b) or NaOD (c) at ambient temperature for 18 h.
the reduction of the uridyl group to dihydrouridyl.30 A related group 150 μJ maximum output, and 3000 shots are accumulated. Samples under
analysis were dissolved in methanol:water (50:50 v/v, 5 μl) prior to co-
crystallization with the matrix. The matrix used was 2,5-dihydrobenzoic acid.
NMR spectra were obtained on a Bruker Avance III instrument (Bruker
BioSpin, Billerica, MA, USA) operating at 500.11 Mhz using a 5 mm z-gradient
BBI probe at 27 ºC. The samples under analysis were dissolved in CD3OD
(Sigma-Aldrich Inc.). Chemical shifts are reported as p.p.m. from TMS
calculated from the lock solvent.
of 5,6-dihydrouridyl-containing nucleotides includes jawsamycin (FR-
-900848) from Streptoverticillium fervens (Figure 1),31 the sesquiterpe-
noid nucleotide esters farnesides A and B from Streptomyces sp. CNT-
372,32 and an anti-influenza virus compound called JBIR-68 from
Streptomyces sp. RI18.33 Moreover, a putative reductase gene. jaw1, has
been identified in Streptoverticillium fervens that is responsible for the
reduction of dehydrojawsamycin to jawsamycin.34 Noticeably,
although jaw1 and npsU are both classified as PPOX class F-420-
dependent reductases, and they are both involved in the reduction of
uridyl double bonds, there is very low homology between these two
Hydrogenation of uridine
Uridine (500 mg) was dissolved in 32 ml methanol to which 100 mg Rh/Al2O3
were added. This suspension was placed in
a Parr Instruments (Parr
genes,34 and their use for the prediction of genes involved in the Instruments, Inc., Moline, IL, USA) stirred reactor using a glass liner. The
reactor was purged with H2 to remove air and charged to 6.6 bar H2 pressure.
The reaction was allowed to proceed at 21 °C and gave complete conversion to
DHU in 6 h.
potential reduction of tunicamycins is therefore not viable. Catalytic
hydrogenation and photoreduction methods were therefore investi-
gated for the potentially selective chemical reduction of tunicamycins.
Photoinduced reductions were unsuccessful, but the use of selected
hydrogenation catalyst (either Pd or Rh) are shown to selectively
reduce the double bonds of tunicamycins. Hence, this provides a
straightforward way to scale up the preparation of reduced toxicity
tunicamycin analogs, Tun-R1 and Tun-R2, for further biological
testing.
Synthesis of N-acyl reduced tunicamycin (Tun-R1)
Twenty milligrams of 5 wt% Pd/C were suspended in 3 ml methanol contain-
ing 30 mg tunicamycin. This suspension was stirred at room temperature and
purged briefly with H2. The septum-capped vial was charged at 1 atm. H2 until
the reaction was complete. The product was isolated by removing the catalyst
by centrifugation.
MATERIALS AND METHODS
Materials
Tunicamycin was purchased from Alfa Aesar, Ward Hill, MA. 5 wt% Pd/C was
from the Calsicat division of Mallinkrodt Chemical, Erie, PA and the 5 wt%
Rh/Al2O3 was from BASF Engelhard, Iselin, NJ. All other chemicals, including
the MALDI-TOF MS matrix, were obtained from Sigma-Aldrich Inc., St Louis,
MO, USA.
Synthesis of doubly reduced tunicamycin (Tun-R2)
As described for the singly reduced tunicamycin except that 50 mg 5 wt% Rh/
Al2O3 were suspended in 3 ml methanol containing 30 mg tunicamycin. The
product was isolated using the same procedure as for isolation of the N-acyl
reduced tunicamycin.
Photochemical reactions and base-catalyzed ring opening
Photoinduced reductions were undertaken essentially as described by Cerutti
et al.24 Uridine (3 mg) and sodium borohydride (5 mg) were dissolved in 3 ml
of aqueous methanol (2:1 v/v). This was allowed to spread as a thin film in a
5 ml glass Petri dish, and was irradiated open with short wavelength UV light
for 2 h at a distance of 2 cm. The solution was transferred to a tube and treated
Analytical techniques
Matrix-assisted laser desorption/ionization time-of-flight mass spectra
(MALDI-TOF MS) were acquired in a Bruker-Daltonics Microflex instrument
(Bruker-Daltonics, Billerica, MA, USA) running in reflectron mode. Ion source
1 and 2 were set to 19.0 and 14.0 kV, respectively, with lens and reflector with Dowex 50W H+ to destroy the excess borohydride, and analyzed by
voltages of 9.20 and 20.00 kV. The laser (337.1 nm) was typically at 60% of MALDI-TOF MS. For the base-catalyzed ring opening reactions, DHU was
The Journal of Antibiotics

Selective hydrogenation of tunicamycins
NJ Price et al
7
prepared by catalytic hydrogenation with Rh/Al2O3 as described, and subse-
quently treated for 18 h at room temperature using 0.25 M aqueous sodium
hydroxide. The products were analyzed directly by MALDI TOF MS. In
addition, a small scale hydrolysis was undertaken in situ in an NMR tube using
10 mg of DHU in 0.25 M sodium deuteride dissolved in deuterated water. The
reaction was monitored by 1H-NMR. Control reactions were undertaken by
replacing the NaOD with deuterated trifluoroacetic acid or under neutral
conditions in D2O.
12 Tsvetanova, B. C., Keimle, D. J. & Price, N. P. J. Biosynthesis of tunicamycin
andmetabolic origin of the 11-carbon dialdose sugar, tunicamine. J. Biol. Chem. 277,
35289–35296 (2002).
13 Wyszynski, F. J., Hesketh, A. R., Bibb, M. J. & Davis, B. G. Dissecting tunicamycin
biosynthesis by genome mining: cloning and heterologous expression of a minimal gene
cluster. Chem. Sci 1, 581–589 (2010).
14 Chen, W. et al. Characterization of the tunicamycin gene cluster unveiling unique steps
involved in its biosynthesis. Protein Cell 1, 1093–1105 (2010).
15 Hakulinen, J. K. et al. MraY-antibiotic complex reveals details of tunicamycin mode
of action. Nat. Chem. Biol. 13, 265–267 (2017).
16 Chung, B. C. et al. Crystal structure of MraY, an essential membrane enzyme
for bacterial cell wall synthesis. Science 341, 1012–1016 (2013).
doi:10.1038/ja.2017.101. [Epub ahead of print].
17 Chung, B. C. et al. Structural insights into inhibition of lipid I production in bacterial
cell wall synthesis. Nature 533, 557–560 (2016).
CONFLICT OF INTEREST
The authors declare no conflict of interest.
18 Price, N. P. J. et al. Modified tunicamycins with reduced eukaryotic toxicity that
enhance the antibacterial activity of β-lactams. J. Antibiot (e-pub ahead of print 27
September 2017; doi:10.1038/ja.2017.101).
19 House, C. H. & Miller, S. L. Hydrolysis of dihydrouridine and related compounds.
Biochemistry 35, 315–320 (1996).
ACKNOWLEDGEMENTS
We thank Trina Hartman for technical assistance, and Dr Joseph O Rich for pre-
review of the manuscript. A provisional patent application (patent no. 62/450,760)
has been filed. BY acknowledges the support of the National Natural Science
Foundation of China (21372253 and 21432012). Mention of any trade names or
commercial products is solely for the purpose of providing specific information
and does not imply recommendation or endorsement by the US Department of
Agriculture. USDA is an equal opportunity provider and employer.
20 Igo-Kemenes, T.
& Zachau, H. G. On the specificity of the reduction of
transfer ribonucleic acids with sodium borohydride. Eur. J. Biochem. 10,
549–556 (1969).
21 Cerutti, P. & Miller, N. Selective reduction of yeast transfer ribonucleic acid with
sodium borohydride. J. Mol. Biol. 26, 55–66 (1967).
22 Kochetkov, N. K., Budovskii, E. I., Shibaev, V. N.
& Eliseeva, G. I. The
synthesis of dihydrouridine diphosphate glucose. Russ. Chem. Bull. 14,
884–885 (1965).
23 Cerutti, P., Ikeda, K.
& Witkop, B. The selective photoreduction of uridine in
polynucleotides. J. Am. Chem. Soc. 87, 2505–2507 (1965).
24 Cerutti, P., Kondo, Y., Landis, W. R. & Witkop, B. Photoreduction of uridine and
reduction of dihydrouridine with sodium borohydride. J. Am. Chem. Soc. 90,
771–775 (1968).
25 Choi, J. H., Kim, D. W. & Shim, S. C. Photo-enhanced reduction of carbonyl compounds
by sodium borohydride. Tetrahedron Lett. 27, 1157–1160 (1986).
26 Hu, T. & Corey, E. J. A novel and selective photoisomerization of allylic benzoates. Org.
Lett. 3, 3547–3548 (2001).
1
2
Tamura, G Tunicamycin, (Japan Scientific Societies Press, Tokyo, Japan, 1982).
Heifetz, A., Keenan, R. W. & Elbein, A. D. Mechanism of action of tunicamycin on
theUDP-GlcNAc: dolichyl phosphate GlcNAc-1-phosphate transferase. Biochemistry
18, 2186–2192 (1979).
Price, N. P. & Momany, F. A. Modeling bacterial UDP-HexNAc: polyprenol-P HexNAc-1-
P transferases. Glycobiology 15, 29–42 (2005).
Lehrman, M. A. A family of UDP-GlcNAc/MurNAc: polyisoprenol-P GlcNAc/MurNAc-1-
Ptransferases. Glycobiology 4, 768–771 (1994).
Eckardt, K Tunicamycins, streptovirudins, and corynetoxins, a special subclass of Q8
3
4
5
6
27 Behm-Ansmant, I., Helm, M.
& Motorin, Y. Use of specific chemical reagents
for detection of modified nucleotides in RNA. J. Nucleic Acids 2011, 1–17
(2011).
28 Kimura, K.-I. & Bugg, T. D. H. Recent advances in antimicrobial nucleoside antibiotics
targeting cell wall biosynthesis. Nat. Prod. Rep. 20, 252–273 (2003).
29 Chen, W. et al. Natural and engineered biosynthesis of nucleoside antibiotics in
Actinomycetes. J. Ind. Microbiol. Biotechnol. 43, 401–417 (2016).
nucleoside antibiotics. J. Nat. Prod. 46, 544–550 (1983).
Doroghazi, J. R. et al. Genome sequences of three tunicamycin-producing streptomyces
strains, S. chartreusis NRRL 12338, S. chartreusis NRRL 3882, and S. lysosuperificus
ATCC 31396. J. Bacteriol. 193, 7021–7022 (2011).
Eckardt, K., Ihn, W., Tresselt, D. & Krebs, D. The chemical structures of streptovirudins.
J. Antibiot 34, 1631–1632 (1981).
Vogel, P., Stynes, B. A., Coackley, W., Yeoh, G. T. & Petterson, D. S. Glycolipid toxins
from parasitised annual ryegrass: a comparison with tunicamycin. Biochem. Biophys.
Res. Commun. 105, 835–840 (1982).
Edgar, J. A. et al. Corynetoxins, causative agents of annual ryegrass toxicity; their
identification as tunicamycin group antibiotics. J. Chem. Soc. Commun. 1982,
222–224 (1982).
7
8
30 Kaysser, L. et al. Identification of
a napsamycin biosynthesis gene cluster by
genome mining. Chembiochem. 12, 477–487 (2011).
31 Yoshida, M. et al. A novel antifungal antibiotic, FR-900848. I. Production, isolation,
physico-chemical and biological properties. J. Antibiot. 43, 748–754 (1990).
32 Zafrir Ilan, E. et al. Farnesides A and B, sesquiterpenoid nucleoside ethers from a
marine-derived Streptomyces sp., strain CNT-372 from Fiji. J. Nat. Prod. 76,
1815–1818 (2013).
9
10 Price, N. P. et al. Quinovosamycins: new tunicamycin-type antibiotics in which the α, β-
1″,11'-linked N-acetylglucosamine residue is replaced by N-acetylquinovosamine. J.
Antibiot. 69, 637–646 (2016).
11 Price, N. P. J. & Tsvetanova, B. C. Biosynthesis of the tunicamycins: a review. J.
Antibiot 60, 485–491 (2007).
33 Takagi, M. et al. Anti-influenza virus compound from Streptomyces sp. RI18. Org. Lett.
12, 4664–4666 (2010).
34 Hiratsuka, T. et al. Biosynthesis of the structurally unique polycyclopropanated
polyketide-nucleoside hybrid jawsamycin (FR-900848). Angew. Chem. Int. Ed. Engl.
53, 5423–5426 (2014).
Supplementary Information accompanies the paper on The Journal of Antibiotics website (http://www.nature.com/ja)
The Journal of Antibiotics