Biosynthesis of 4-aminoheptose 2-epimers, core structural components of the septacidins and spicamycins

Article abstract of DOI:10.1038/ja.2014.15

Septacidins and spicamycins are acylated 4-aminoheptosyl-β-N- glycosides produced by Streptomyces fimbriatus and S. alanosinicus, respectively. Their structures are highly conserved, but differ in the stereochemistry of the 4-aminoheptosyl residues. The o

Full text of DOI:10.1038/ja.2014.15

The Journal of Antibiotics (2014) 67, 405–414
2014 Japan Antibiotics Research Association All rights reserved 0021-8820/14
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ORIGINAL ARTICLE
Biosynthesis of 4-aminoheptose 2-epimers, core
structural components of the septacidins and
spicamycins
Neil PJ Price1, Takayuki Furukawa2, Fang Cheng3, Jianzhao Qi3, Wenqing Chen3 and David Crich2
Septacidins and spicamycins are acylated 4-aminoheptosyl-b-N-glycosides produced by Streptomyces fimbriatus and
S. alanosinicus, respectively. Their structures are highly conserved, but differ in the stereochemistry of the 4-aminoheptosyl
residues. The origin of this stereochemistry is unknown, but is presumably because of the difference in their biosynthetic
pathways. We have synthesized the septacidin 4-aminoheptose to verify the difference between septacidin and spicamycin.
Isotopic enrichment studies were undertaken using S. fimbriatus, and show that the septacidin heptose is derived from the
pentose phosphate pathway. This indicates conserved pathways leading to the biosynthesis of 4-amino-4-deoxy-L-gluco-
heptose or 4-amino-4-deoxy-L-manno-heptose.
The Journal of Antibiotics (2014) 67, 405–414; doi:10.1038/ja.2014.15; published online 19 March 2014
Keywords: 4-amino-4-deoxyheptose; biosynthetic pathway; septacidin; spicamycin; synthesis
INTRODUCTION
With these questions in mind, we first sought to confirm the
The septacidins (1) and spicamycins (2) are unique but structurally reported stereochemistry for the carbohydrate motifs. The absolute
related natural products produced by fermentation of diverse configuration of septacidin has been tentatively assigned by Agahigian
actinomyces species.1,2 Both are differentiation inducers of leukemia et al.6 Septacidin was acid hydrolyzed to the aminoheptose, which was
cells, and a modified spicamycin (KRN5500) is in clinical trials subsequently converted to an aminohexose by cleavage of the C6–C7
for neuropathic pain suppression.3,4 In 2011, the US Food and bond by periodate oxidation. A proton NMR study of the methyl
Drug Administration designated KRN5500 a Fast Track Drug glycoside tetraacetate derivative of this aminohexose was used to
Development Program.5 Both series of compounds are comprised assign it as 4-amino-4-deoxy-L-gluco-pyranose, but unfortunately this
of four structural motifs: (1) a 4-deoxy-4-amino-L-heptose sugar; (2) was only undertaken at low resolution with a low field (60 MHz)
a 6-N-glycosidically linked adenine group; (3) an amide-linked NMR instrument.6
glycine; and (4) a variety of N-glycyl-linked acyl groups (Figure 1).
On the basis of this work, the absolute configuration of spicamycin
To date, very little is known about their biosynthesis, although it is was inferred to be 6-(4-amino-4-deoxy-L-glycero-b-L-manno-
interesting for two reasons. First, the heptosyl N-glycosidic linkage is heptopyranosylamino)-9H-purine.7 More recently, anicemycin, an
reported to be a to primary amine group, an uncommon structural analog of spicamycin (Figure 1), has been isolated from an unrelated
feature that is found in only few natural products, and which implies Streptomyces strain. The relative configuration has been shown by
the presence of potentially unusual N-glycosyltransferase biosynthetic MS and NMR to be the same as spicamycin,8 but unfortunately
enzymes. Second, the 4-deoxy-4-amino-L-heptosyl sugar motifs are the absolute configuration was not determined. The absolute
reported to be gluco-heptose for septacidin, but manno-heptose for configuration of spicamycin has also been determined by X-ray
spicamycin.1,2,6–8 This crucial difference in the stereochemistry at the crystallography,7 but this has not yet been completed for the
20-position has marked implications on the potential biosynthetic septacidins or anicemycins.
pathways for septacidins and spicamycins. Either the presence of a
The spicamycin/septacidin series of aminosugar antibiotics have
unique 2-epimerase functionality is implied or the two different only been addressed by total synthesis on one previous occasion by
aminoheptoses may be biosynthesized by entirely diverse pathways. Chida’s group. These workers accessed spicamycin from myo-inositol
Moreover, this simple change in the stereochemistry at one position by a lengthy route involving resolution of a non-symmetrical
has implications on the relative biological activities of septacidins and derivative, introduction of nitrogen with inversion of configuration,
spicamycins.
oxidative ring opening, two-carbon homologation, one-carbon
1USDA-ARS, National Center for Agricultural Utilization Research, Peoria, IL, USA; 2Department of Chemistry, Wayne State University, Detroit, MI, USA and 3Key Laboratory of
Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan, China
Correspondence: Dr NPJ Price, USDA-ARS, National Center for Agricultural Utilization Research, 1815 North University Street, Peoria, IL 61604, USA.
E-mail: neil.price@ars.usda.gov
Received 19 September 2013; revised 3 January 2014; accepted 28 January 2014; published online 19 March 2014

Septacidin and spicamycin glycone 2-epimers
NPJ Price et al
406
Figure 1 Structures of the septacidins (top left) and spicamycins (top right). Note the difference in the core 4-aminoheptose stereochemistry at C-2, in the
identity of N-linked fatty acids. Anicemycin (lower) is a spicamycin analog with a specific unsaturated N-acyl group. The numbering system is as described
previously.8
dehomologation and eventual introduction of the N-glycoside.9 The complex in dimethylsulfoxide was most satisfactory and gave ketone 7
coupling constants (JH1,H2o1 Hz, JH2,H3 ¼ 3.2 Hz, JH3,H4 ¼ 9.4 Hz, in 95% yield. Stereoselective reduction of 7 was best achieved with the
JH4,H5 ¼ 9.4 Hz) observed in the 1H NMR spectrum of this Luche reagent23 in methanol at 0 1C, enabling, after benzoylation,
synthetic compound supported the b-manno-heptose configuration. isolation of the 7-epi-sialoside 8 in 66% yield with a selectivity of 17:1.
Model D-series, b-D-manno-heptose and b-D-gluco-heptose analogs, of Removal of the acetonide under standard conditions followed by
spicamycin and septacidin have also been reported by Chida et al.10 treatment with sodium metaperiodate then afforded the aldehyde 9 in
The synthesis of the first lower homologs of both spicamycin and 84% yield. Reduction with sodium borohydride, saponification of the
septacidin has been described in the literature by two groups who benzoate groups and acetylation gave the octulosonic acid derivative 10
adopted largely analogous routes. Thus, Acton and co-workers11 in 84% yield. Application of the Barton decarboxylation procedure17
prepared 7-nor-septacidin analogs from L-fucose by incorporation using tert-dodecanethiol24,25 as the hydrogen atom source lead to the
of nitrogen at the 4-position, with inversion of configuration and isolation of the 2-deoxy-b-thioglycoside 11 in 38% yield and a minor
installation of the N-glycoside, whereas Mons and Fleet13 obtained 7- amount of the a-isomer. Oxidation of 11 with meta-
nor-spicamycin analogs from L-rhamnose through azide displacement chloroperoxybenzoic acid at ꢀ78 1C gave a glycosyl sulfoxide, which
of an activated hydroxyl group, with inversion of configuration and on heating to 80 1C in toluene underwent elimination to provide the
eventual installation of the N-glycoside.12
glycal 12, albeit in only 25% yield for the two steps. Finally, reaction of
To confirm the relative stereochemistries, an authentic sample of the the glycal 12 with catalytic osmium tetroxide in the presence of N-
septacidin glycone was prepared, starting from a neuramic acid methylmorpholine N-oxide followed by acetylation gave the protected
thioglycoside. In principle, the enantiomer of septacidin should be septacidin glycone 13 in 64% yield, with excellent selectivity for the
available from 4-amino-4-deoxy-D-glucose, which can be obtained desired gluco-isomer consistent with the precedent (Scheme 2).
by degradation of the aminoglycoside antibiotic apramycin or by
The naturally occurring aminoheptoses were obtained by acid
substitution at the 4-position of D-galactose, followed by one-carbon hydrolysis of native septacidin and spicamycin. This required a two-
homologation at the 6-position. Alternatively, functionalization of step lysis treatment, the first a methanolysis step to de-N-acylate the
L-fucose at the 6-position by a C–H activation process14 followed adenosyl-aminoheptose core (Figure 2), and a second aqueous acid
by homologation and application of the Acton and Fleet approach hydrolysis to recover the free aminoheptoses. We noted that
should afford septacidin (Scheme 1). Ultimately, however, we attempted strong acid hydrolysis of the adenosyl-aminoheptoses leads
selected a third approach starting from the readily available predominantly to a 1,7-anhydro-4-aminoheptose, presumably via a
N-acetylneuraminic acid by simple removal of carbons 1 and 9, 1,7-dehydration reaction (Supplementary Data).
inversion at C-7 and functionalization at C-3 (Scheme 1). This
The free 4-aminoheptoses from septacidin and spicamycin, and
approach was particularly attractive in view of recent improvements that from the synthetic septacidin glycone 13 were derivatized to
to the decarboxylation15 of N-acetylneuraminic acid reported by the form aldononitrile acetate derivatives (peracetylated aldononitriles
Gervay-Hague’s group16 with modifications by the Wong laboratory,17 (PAANs)) suitable for analysis by GC/MS.26 To aid in the EI-MS
and because of our earlier experience with application of the Barton fragmentation assignments (necessary for the later isotopic
decarboxylation reaction18 to the ulosonic acid glycosides.19
enrichment experiments), we also included 4-amino-D-glucose in
In the event, reaction of the known neuraminic acid derivative 520 this study, obtained from hydrolysis of apramycin. The authentic
with benzoyl chloride at 01C enabled selective esterification on the less 4-amino-L-gluco-heptose PAAN from the synthetic standard eluted at
hindered 4-hydroxyl group, leaving the 7-hydroxyl group free for 17.4min. This coeluted with the 4-amino-L-heptose PAAN from
inversion. Direct inversion of 6 by the Mitsunobu protocol, or by hydrolyzed native septacidin (Figure 3). In contrast with this, the
triflation followed by displacement with various nucleophiles, was 4-aminoheptose derivative from spicamycin eluted at 17.7min.
not productive; therefore, an oxidation reduction sequence was The three samples had identical EI-MS spectra, giving rise to stable
undertaken. Among the several oxidation procedures investigated, ions that predominantly arise from C3–C4 (m/z 288, 168, 128
the Parikh–Doering protocol21,22 with the sulfur trioxide–pyridine and 84) and C4–C5 (m/z 241, 199 and 139) cleavage either side of
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Septacidin and spicamycin glycone 2-epimers
NPJ Price et al
407
H
N
N
HO
HO
OH
OH
OH
HO
N
O
HO
N
OH
O
CO2H
AcHN
OH
O
NH
R1
HO
3: L-fucose
ROCHN
HO
4: NeuAc
HO
R2
(5-acetylamino-3-deoxy-D-glycero
-D-galactononulosonic acid)
1: R1 = OH, R2 = H, Septacidin
(4-amino-L-glycero-L-gluco)
2: R1 = H, R2 = OH, Spicamycin
(4-amino-L-glycero-L-manno)
CO2H
decarboxylate
functionalize
CHO
H
CHO
H
O
H
HO
HO
H
H
AcHN
HO
introduce N
with inversion
OH
OH
H
H
H2N
HO
OH
H
H
H
H
OH
H
H
OH
OH
deacetylate
HO
invert
HO
H
H
H
functionalize
and extend
OH
glycol cleavage
OH
Scheme 1 Retro-synthesis of the 4-amino-4-deoxy-L-gluco-pyranose core heptose of septacidins from L-fucose and N-acetylneuraminic acid.
Scheme 2 Synthesis of an authentic septacidin glycone starting from neuramic acid thioglycoside 5.
the N-acetyl group, and from a minor loss of the C7 carbon unit, as of 11 different 13C-enriched biosynthetic precursors to the cultures
discussed below.
after 3 days. After a further 5 days of growth, the cultures were
acidified and the labelled cells were harvested by centrifugation. The
labelled septacidins were extracted from cell pellets with butanol, and
evaporated to dryness. The incorporation of 13C into the septacidins
was verified by MALDI-time-of-flight/MS (MALDI-TOF/MS). The
samples were treated with the methanolic HCl to give adenine-
heptose, plus a mixture of fatty acids and glycyl-fatty acids as their
methyl esters. After partitioning between chloroform and water, the
13C-labelled adenine-heptoses in the aqueous phase were analyzed
by MALDI-TOF/MS to confirm the presence of label (Figure 2).
The chloroform-extracted acyl component was analyzed by electron
Biosynthesis studies
Having established that the aminoheptose sugar from native septaci-
din is 4-amino-gluco-heptose, we sought to apply this information to
understanding its biosynthetic pathway. The biosynthesis of higher
(long-chain) sugars generally involves the ligation of 2 or 3 carbon
units (via the pentose phosphate pathway) or by the chain extension
of pre-existing hexosyl or pentosyl intermediates.27,28 To determine
which, Streptomyces fimbriatus, cultures were grown on glucose-
containing medium and metabolically labelled by the introduction
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408
impact GC/MS. The labelled adenine-septoses were further hydro- Isotopic incorporations into the N-acylglycine structural motifs
lyzed with aqueous trifluoroacetic acid (2M, 100 1C, 30min) to give The fatty acid methyl esters from septacidins eluted from the GC
free adenine and heptose (4-amino-4-deoxyheptose). This was con- between 11 and 20min as 11 major peaks, plus 8 other minor
firmed by peracetylation of the labelled heptoses and analysis by components. Seven major saturated, branched-chain fatty acids were
GC/MS and MALDI-TOF/MS. To analyze the positional incorpora- assigned by the presence of characteristic m/z 74 and 87 MS ions,
tion of 13C, the labelled heptoses were converted to aldononitrile approximately corresponding to the eight major septacidins detected
acetate derivatives (PAANs) and analyzed by electron impact by LC/MS and by MALDI-TOF/MS (Supplementary Data).
GC/MS.26
Unsaturated fatty acids were also detected, as characterized by
additional m/z 55 and 69 ions. Fourteen N-acylglycine methyl esters
also eluted from the GC between 20 and 30min, after the acyl esters.
The major EI/MS fragment ions arising from these acylglycine methyl
esters were m/z 144, 131 and 90, which are characteristic of this group
of compounds.29 Those with a saturated acyl chain gave m/z 131 as
the largest fragment ion, formed by b-cleavage to the amide carbonyl
and a McLafferty-type rearrangement. Noticeably, the septacidin
acylglycine components with unsaturated acyl chains generated m/z
131 as only minor ions. This indicates that the double bond present in
these compounds is positioned either a or b to the acyl carbonyl,
thereby impeding the McLafferty rearrangement mechanism.
The odd-electron ion appearing at m/z 131 is very characteristic
of a-unsubstituted, saturated N-acylglycine methyl esters, and hence
contains the carbonyl and methylene carbons from the original glycine
motif plus carbons 1 and 2 of the acyl chain (Scheme 3). Examination
of the 13C-enrichment into the m/z 131 ion showed that the major
incorporation occurs from carbons
3 to 6
of [U-13C]glucose
(Supplementary Data). The 13C-enrichment is less from [1-13C]glu-
cose- or [2-13C]glucose precursors, and very little incorporation was
observed from labelled acetates or pyruvate. Glycine is generally
biosynthesized from serine, and the initial three-carbon precursor of
serine and glycine, 3-phosphoglycerate, is derived from the glycolytic
pathway, where it arises from carbons 4, 5 and 6 of glucose. Hence, this
is consistent with the observed 13C incorporations of carbons 3–6 from
[U-13C]glucose into the glycine motif of the septacidin N-acylglycines.
The N-acylglycine esters also generated minor fragment ions
because of the carbon–carbon bond cleavage in the acyl backbone.
Thus, the major GC peak at 25.9min corresponds to the 16-carbon
Figure 2 MALDI/MS of (a) septacidins, (b) adenine-heptose and
(c) 13C-enriched adenine-heptose.
Figure 3 GC traces of 4-aminoheptosyl aldononitrile acetate derivatives: (a) Synthetic, RT ¼ 17.4min; (b) from septacidin, RT ¼ 17.4 min; (c) from
spicamycin, RT ¼ 17.7 min; and (d) overlaid chromatographs of synthetic and spicamycin PAANs. A full color version of this figure is available at The
Journal of Antibiotics journal online.
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Septacidin and spicamycin glycone 2-epimers
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409
H
H
C
R
R
O
C
CH
OH
C
+
COOMe
NH
COOMe
CH2
H2C
NH2 CH2
CH2
H2C
C
H2
m/z 131
Scheme 3 The m/z 131 fragment ion from McLafferty rearrangement of N-acylglycine methyl esters is comprised of two carbons from glycine, and two from
the N-acyl chain.
N-glycosidically linked adenine-heptoses. These were acid hydrolyzed
to the free aminoheptose, and analyzed as their aldononitrile acetate
derivatives by GC/MS. Ion extraction of characteristic ion m/z 139
revealed a GC peak at 17.4 min corresponding to the aldononitrile
acetate derivative of 4-amino-4-deoxy-L-glucoheptose. The EI/MS
spectra are shown in Figure 5. Examination of the 13C-labelled sugar
components showed that seven fragment ions are useful for tracing
isotopic incorporations. Ions m/z 241, 199, 139 and 97 originate from
carbon 1 to 4 of the parent aminoheptose, and ions m/z 288, 228 and
168 originate from carbons 4 to 7 (Figure 5).
These ions were well resolved from neighboring peaks, allowing us
to assess isotopomer incorporations into these carbons. The enrich-
ment from [1-13C]fructose, [2-13C]fructose and [U-13C]fructose was
at relatively low level, and the isotopomer patterns were similar. By
comparison, enrichment from the labelled glucose precursors was
clearly apparent (Figure 5). Ions m/z 288 and 228 have less 13C
incorporated than m/z 241, 199 and 139 (Figure 5). This was also
apparent for m/z 60. This suggests that the six glucose carbons are
preferentially incorporated into the aminoheptose at carbon 1–4,
rather than at carbons 4–7. Carbons 1 and 2 from [1-13C]glucose and
[2-13C]glucose, respectively, are also incorporated into most of the
fragment ions, but again enrichment was greater than carbon 1–4.
Noticeably, the scrambling of 13C isotopomers into all of these ions
suggests that the glucose is first metabolized to small catabolites
before being reincorporated into the aminoheptose backbone. This
scrambling of labelled carbon atoms is characteristic of metabolism
via the pentose phosphate recovery pathways, and is in contrast to an
Figure 4 EI-MS spectra of the septacidin glycyl-C16-acyl Me esters,
isotopically enriched from (a) [U-13C]glucose and (b) [2-13C]glucose
accompanying GC/MS peak due to microbial trehalose, where
M þ 6 ions clearly show the incorporation of [U-13C]glucose or
[U-13C]mannose as intact six-carbon units (Supplementary Data).
precursors.
acylglycine chain of the major septacidin, and contained the fragment Noticeably, [U-13C]xylose, 13C-acetates and [3-13C]pyruvate do not
ions m/z 327 (o), 312 (o ꢀ1), 296 (o ꢀ2), 284 (o ꢀ3) and 268 incorporate into the trehalose to any great extent. [U-13C]mannose is
(o ꢀ4) (Figure 4). Similarly, the GC peaks at retention time 24.3 and also incorporated into the septacidins via 2- or 3- carbon precursors
24.5min are due to N-acylglycine esters with 15 acyl carbons, and it in a manner analogous to glucose. This leads to the conclusion that
gives rise to the corresponding series of MS fragment ions at m/z small labelled metabolites arising selectively from glucose (or man-
313 (o), 298 (o ꢀ1), 282 (o ꢀ2), 270 (o ꢀ3) and 254 (o ꢀ4). These nose) are the metabolic precursors of the seven-carbon amino sugar.
are 14Da smaller than the ions from the GC peak at 25.9min, due to The low-level incorporation and scrambling of 13C into septacidins
the one less -CH2- in the acyl chain. Isotopic enrichment from from [1-13C]acetate, [2-13C]acetate and [3-13C]pyruvate also tends to
[U-13C]glucose and [2-13C]glucose was detected in all of these acyl support this conclusion.
group ions (Figure 4). The two-carbon unit incorporations from
The known biosynthetic pathways for seven-carbon sugars are via
[U-13C]glucose into these ions suggest that the biosynthesis occurs via the pentose phosphate recovery pathway intermediate sedoheptulose-
acetyl-CoA. Moreover, the acyl o ꢀ2 and o ꢀ4 ions are more heavily 7-P. In several Gram-negative bacteria, sedohepulose-7-P is converted
labelled by [1-13C]glucose, suggesting that labelled acetyl-CoA arising to ADP-D-glycero-D-manno-heptose sugar nucleotide. A 6-epimerase
from [1-13C]glucose is more selectively incorporated into the activity (RfaD)30 inverts the chirality at C-6 to generate
glycyl-fatty acids. This was also apparent from 13C-acetate labelling, ADP-b-L-glycero-D-manno-heptose, which is incorporated into
where [2-13C]acetate gave greater enrichment than [1-13C]acetate bacterial lipopolysaccharide.31,32 In Gram-positive bacteria, four
(Supplementary Data).
enzymes convert D-sedoheptulose-7-P into GDP-D-glycero-a-D-
manno-heptose,33 which in Campylobacter can be further converted
into GDP-6-deoxy-D-manno-heptose and GDP-6-deoxy-D-altro-
heptose.34,35 For the biosynthesis of septacidin and spicamycin,
Isotopic incorporations into the 4-acetylamino-4-deoxy-L-heptose
sugar
Following methanolysis of the isotopically labelled septacidin and we also predict N-heptosyl glycosyltransferase, 4-O-oxidoreductase
washing with chloroform, the aqueous portions contained the and 4-amidotransferase, glycosyltransferase activities and enzymes
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Septacidin and spicamycin glycone 2-epimers
NPJ Price et al
410
Figure 5 GC/MS of 13C-enriched aminoheptose from septacidins. (a) Unlabelled control; (b) [U-13C]Glc; (c) [2-13C]Glc; (d) [U-13C]Xyl; (e) [U-13C]Man.
Fragment ions are from 13C-aminoheptosyl aldononitrile acetates (PAANs).
CH2OH
O
HO
HO
HO
OH
O
HO
IV
O
III
I
OH
OH
OH
OHHO
II
OHHO
ADP
HO
ADP
HO
CH O-P
2
D, D-sedoheptulose-7-P
ADP-D-glycero-
ADP-L-glycero-
(D-altoheptulose-7-P)
D-mannoheptose
D-mannoheptose
HO
O
OH
O
OH
H N
OH
OH
2
VI
V
OH
O
O
HO
HO
ADP
ADP
O
OHHO
OH
OH
ADP
ADP-4-keto-
ADP-4-keto-
ADP-4-amino-
L, D-mannoheptose
L, L-glucoheptose
L, L-glucoheptose
O
O
OH
OH
OH
NH
NH
VII
VIII
OH
HO
HO
O
NH
O
H N
2
ADP
ADP
R
O
OH
OH
ADP-4-N-glycyl-
ADP-4-N-glycyl-N-acyl
L-glycero-L-glucoheptose
L-glycero-L-glucoheptose
IX
Septacidin
Scheme 4 Proposed nine-step biosynthetic pathway from sedo-heptulose-7-phosphate to septacidin. I. sedoheptulose-7-phosphate isomerase;
II. D-glycero-b-D-manno-heptose-1-phosphate adenylyltransferase (RfaE); III. 6-epimerase (RfaD); IV. 4-oxidase; V. 3,5-epimerase; VI. 4-aminotransferase;
VII. N-glycyltransferase; VIII. N-acyltransferase; IX. adenine N-glycosyltransferase.
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Septacidin and spicamycin glycone 2-epimers
NPJ Price et al
411
involved in biosynthesis and transfer of N-acyl groups (Scheme 4). partitioning between chloroform and water, the 13C-labelled adenine-septoses
in the aqueous phase were analyzed by MALDI-TOF/MS to confirm the
presence of label. The chloroform-extracted acyl component was analyzed by
electron impact GC/MS. The labelled adenine-septoses were further hydrolyzed
with aqueous trifluoroacetic acid (2M, 1001C, 30 min) to give free adenine and
septacidose (4-amino-4-deoxyheptose). This was further confirmed by per-
acetylation (acetic anhydride: pyridine 1:1 (vv), 601C, 30 min) of the labelled
septacidoses and analysis by GC/MS and MALDI-TOF/MS. To analyze the
positional incorporation of 13C, the labelled septacidoses were converted to
aldononitriles acetate derivatives (PAANs) and analyzed by electron impact
GC/MS.
Assuming an L-heptose 2-epimerase that acts via an 1,2-enediol
intermediate, this would suggest that the epimerization occurs on the
free heptose sugar, before the formation of the adenine glycoside. The
heptose (or 4-aminoheptose) would subsequently form an activated
heptosyl sugar nucleotide (either L-gluco- or L-manno-), before a
heptosyltransferase-catalyzed transfer to the adenine acceptor. This
suggests that diverse biosynthetic pathways occur for septacidins or
spicamycins after the 2-epimerase step, and will likely involve either
L-gluco-heptosyl- or L-manno-heptosyl-specific enzymes. In keeping
with other natural products, this probably occurs before the differ-
ential N-acylations and active cellular transport of the diversified
septacidins or spicamycins.
Synthesis and spectral assignments of 4-deoxy-4-amino-L-glycero-L-
glucoheptose and intermediates
Compound 6. Methyl (phenyl 5-acetamido-4-O-benzoyl-3,5-dideoxy-8,9-O-
isopropylidene-2-thio-D-glycero-alpha-D-galacto-2-nonulopyranosid)onate (6).
To a solution of compound 51 (4.0g, 8.8 mmol) in dichloromethane (80 ml)
and pyridine (20 ml), benzoyl chloride (2.6ml, 22.0 mmol) was added at 01C.
The mixture was stirred for 1 h at 0 1C with monitoring of the reaction by TLC
(hexane: EtOAc ¼ 1:1, Rf ¼ 0.2). The reaction was quenched with MeOH
(10 ml) at 0 1C. Then, the reaction mixture was diluted with EtOAc, washed
with H2O, 1 N HCl, sat. NaHCO3, brine, dried over Na2SO4, filtered and
concentrated. The residue was purified by column chromatography on silica
gel (hexane: EtOAc¼ 1:1–1:2) to give 4-O-benzoate 6 (4.12 g, 84%) as a white
solid.
METHODS
Chemicals used for culturing and chemical analysis were obtained from Sigma-
Aldrich (St Louis, MO, USA). The septacidin-producing streptomyces strain,
S. fimbriatus NRRL B-3175, was obtained from the Agricultural Research
Service Microbial Collection housed at the NCAUR laboratory in Peoria,
Illinois, USA. The strain was cultured as described below. Spicamycin
(KRN5500) was obtained from the Developmental Therapeutics Program of
the National Cancer Institute, National Institutes of Health (Bethesda, MD,
USA). Isotopically enriched sugars were purchased from Omicron Bio
chemicals (South Bend, IN, USA).
General methods
[a]RDT ꢀ43.5 (c 0.67, CHCl3)
Solvents and other reagents for chemical synthesis were purchased from
Aldrich Chemical (Milwaukee, WI, USA) and Tokyo Chemical Industry
(Portland, OR, USA), and used without further purification. The reactions
were monitored by TLC (20 ꢁ 20cm2; layer thickness, 0.25mm; Silica Gel HL
TLC Plates w/UV254; Sorbent Technologies, Norcross, GA, USA). Visualiza-
tion was accomplished with UV light (at 254nm) and exposure to sulfuric acid
in methanol (5:95 (vv)) or ceric ammonium molybdate solution (Ce(SO4)2:
4 g; (NH4)6Mo7O24: 10 g; H2SO4: 40ml; H2O: 360 ml), followed by heating.
Purifications were performed on silica gel columns (Sorbent Technologies,
Standard Grade, 32–63mm). 1H NMR and 13C NMR spectra were recorded
with Agilent 600 (1H: 600 MHz; 13C: 150 MHz) and Varian-500S (1H:
500 MHz; 13C: 125MHz) spectrometers; multiplicities are given as singlet
(s), broad (br), doublet (d), double doublets (dd), triple doublets (td),
septuplet (sept), triplet (t), quintet (q) or multiplet (m). Assignments in 1H
and 13C NMR were made by first-order analysis of the spectra by using ACD/
NMR processor software (Advanced Chemistry Development, Toronto, ON,
Canada) and were verified by H ꢀH COSYand 13C-edited HSQC experiments.
Residual solvent signals were used as an internal reference (CDCl3: d
7.26 p.p.m. 1H NMR, d 77.16 p.p.m. 13C NMR). Specific optical rotations
were measured on an Autopol III polarimeter (Rudolph Research Analytical,
Hackettstown, NJ, USA) with a path length of 10 cm. HR-MS ESI-TOF mass
spectra were recorded using a Micromass LCT (Waters, Milford, MA, USA)
instrument.
1H NMR (600MHz, CDCl3): d ¼ 7.98–7.97 (d, 2H, J ¼ 7.3 Hz: Ar-H),
7.60–7.57 (m, 3H: Ar-H), 7.44–7.39 (m, 3H; Ar-H), 7.36–7.33 (t, 2H,
J ¼ 7.7, 7.4 Hz: Ar-H), 6.17–6.15 (d, 1H, J ¼ 7.7 Hz: NH), 5.26–5.21 (td, 1H,
J ¼ 4.8, 6.2 Hz: H-4), 4.39 (br, 1H: 7-OH), 4.29–4.25 (q, 1H, J ¼ 6.7 Hz: H-8),
4.11–4.05 (m, 2H: H-5, H-9a), 3.99–3.97 (dd, 1H, J ¼ 6.6, 1.9 Hz: H-9b), 3.53
(s, 3H: COOMe), 3.47–3.46 (d, 1H, J ¼ 7.3Hz: H-7), 3.34–3.33 (dd, 1H,
J ¼ 1.2, 9.5 Hz: H-6), 2.98–2.95 (dd, 1H, J ¼ 5.1, 7.7 Hz: H-3eq), 2.20–2.16
(t, 1H, J ¼ 12.2Hz: H-3ax), 1.89 (s, 3H: AcNH), 1.36 (s, 3H: Me) and 1.25
(s, 3H: Me).
13C NMR (150MHz, CDCl3): d ¼ 172.8 (C ¼ O), 169.0 (C ¼ O), 167.7
(C ¼ O), 136.9 (Ar), 134.0 (Ar), 130.0 (Ar), 129.9 (Ar), 128.8 (Ar), 128.7 (Ar),
108.8 (CMe2), 87.3 (C2), 77.1 (C6), 74.7 (C8), 70.5 (C7), 70.1 (C4), 67.6 (C9),
52.5 (COOMe), 52.1 (C5), 37.8 (C3), 27.0 (CMe2), 25.5 (CMe2) and 23.2 (Ac).
HR-MS (ESI): m/z calcd for: C28H34O9NS, [Mþ H]þ 560.1954 found:
560.1975.
Compound 7. Methyl (phenyl 5-acetamido-4-O-benzoyl-3,5-dideoxy-8,9-
O-isopropylidene-7-oxo-2-thio-D-glycero-alpha-D-galacto-2-nonulopyranosi-
d)onate (7). To a solution of compound 6 (5.5g, 9.84mmol) in DIPEA
(diisopropylethylamine;40 ml) and dimethylsulfoxide (40 ml), SO3 ꢂ pyridine
(11.7 g, 73.8 mmol) was added at ambient temperature. The mixture was
stirred for 1.5 h with monitoring of the reaction by TLC (toluene: EtOAc¼ 1:1,
Rf ¼ 0.25), diluted with EtOAc, washed with H2O, 1 N HCl, sat. NaHCO3,
brine, dried over Na2SO4, filtered and concentrated. The residue was purified
by column chromatography on silica gel (hexane: EtOAc¼ 2:1–1:1) to give
7-keto 7 (5.2g, 95%) as a white solid.
Culturing and metabolic labelling experiments
S. fimbriatus NRRL B-3175 was grown for 3 days in culture on glucose-
containing medium (tryptone–yeast extract–glucose) and metabolically
labelled after this time by the introduction of different 13C-enriched
biosynthetic precursors to the cultures. The enriched precursors used were:
[a]RDT 4.52 (c 0.67, CHCl3)
[1-13C]glucose, [2-13C]glucose, [U-13C]glucose, [1-13C]fructose, [2-13C]fruc- 1H NMR (600MHz, CDCl3): d ¼ 7.99–7.98 (m, 2H: Ar-H), 7.57–7.52 (m, 3H:
tose, [U-13C]fructose, [1-13C]mannose, [U-13C]mannose, [U-13C]xylose, Ar-H), 7.43–7.40 (m, 3H: Ar-H), 7.35–7.33 (m, 2H: Ar-H), 5.92–5.91 (d, 1H,
[1-13C]ribose, [U-13C]erythrose, [3-13C]pyruvate, [1-13C]acetate or
J ¼ 8.1 Hz: NH), 5.24–5.20 (td, 1H, J ¼ 4.7, 6.6, 3.3, 3.4, 4.8: H-4), 4.99–4.96
[2-13C]acetate. After a further 5 days of growth, the cultures were acidified (t, 1H, J ¼ 7.3 Hz: H-8), 4.39–4.37 (t, 1H, J ¼ 7.7, 8.1 Hz: H-9a), 4.26–4.17
and the labelled cells were harvested by centrifugation. The isotopically (m, 3H: H-5, H-6, H-9b), 3.57(s, 3H: COOMe), 3.08–3.05 (dd, 1H, J ¼ 4.7,
enriched septacidins were extracted from the cell pellets with butanol, which 8.1 Hz: H-3eq), 2.17–2.13 (t, 1H, J ¼ 11.7Hz: H-3ax), 1.76 (s, 3H: AcNH),
was centrifuged and evaporated to dryness. The incorporation of 13C into the 1.46 (s, 3H: Me) and 1.45 (s, 3H: Me).
septacidins was verified by MALDI-TOF/MS and NMR. The samples were
treated with dry methanolic HCl (1M, 601C, 1 h) to give adenine-septacidose, (C ¼ O), 166.4 (C ¼ O), 136.7 (Ar), 133.7 (Ar), 130.4 (Ar), 129.9 (Ar), 129.1
plus a mixture of fatty acids and glycyl-fatty acids as their methyl esters. After (Ar), 129.0 (Ar), 128.7 (Ar), 128.4 (Ar), 111.0 (CMe2), 87.7 (C2), 78.8 (C6),
13C NMR (150MHz, CDCl3): d ¼ 201.3 (C ¼ O: C7), 170.7 (C ¼ O), 167.9
The Journal of Antibiotics

Septacidin and spicamycin glycone 2-epimers
NPJ Price et al
412
78.5 (C8), 69.3 (C4), 66.1 (C9), 53.0 (COOMe), 51.0 (C5), 38.1 (C3), 25.8 (Ar), 87.1 (C2), 77.4 (C7), 77.3 (C6), 70.1 (C4), 53.0 (COOMe), 50.5 (C5),
(CMe2), 25.7 (CMe2) and 23.1 (Ac).
37.9 (C3) and 23.3 (AcNH).
HR-MS (ESI): m/z calcd for: C31H29NO9SNa, [Mþ Na] þ 614.1461 found:
HR-MS (ESI): m/z calcd for: C28H31NO9SNa, [Mþ Na]þ 580.1617 found:
580.1617.
614.1469.
Compound 10. To a solution of compound 9 (1.70 g, 2.88mmol) in MeOH
(15 ml), NaBH4 (153mg, 4.02mmol) was added at 0 1C. The mixture was
stirred for 1 h at 0 1C with monitoring of the reaction by TLC (toluene:
EtOAc ¼ 1:1, Rf ¼ 0.2), diluted with EtOAc, washed with sat. NaHCO3, brine,
dried over Na2SO4, filtered and concentrated. The resulting residue was
dissolved in tetrahydrofuran (20 ml), followed by the addition of 1 N NaOH
(20 ml). The reaction mixture was stirred for 3 h at an ambient temperature,
quenched by 1 N HCl (solution color changes from pale yellow to transparent
by the neutralization), concentrated and coevaporated with toluene three
times. The residue was dissolved in acetic anhydride (10 ml) and pyridine
(15 ml), stirred for 2 h at an ambient temperature with monitoring of the
reaction by TLC (chloroform: MeOH¼ 5:1, Rf ¼ 0.5), filtered by celite and
concentrated. The residue was purified by column chromatography on silica
gel (chloroform: MeOH ¼ 10:1–5:1) to give carboxylic acid 10 (1.20 g, 84%) as
a white solid.
Compound 8. Methyl [phenyl (7S)-5-acetamido-4,7-O-dibenzoyl-3,5-
dideoxy-8,9-O-isopropylidene-2-thio-D-glycero-alpha-D-galacto-2-nonulopyra-
nosid]onate (8). To a solution of compound 7 (110mg, 0.20mmol) in MeOH
(4ml), CeCl3 ꢂ 7H2O (220mg, 0.59mmol) was added at 0 1C. The mixture was
stirred for 1 h at 0 1C, and then NaBH4 (11 mg, 0.30mmol) was added. After
stirring for 1 h at 0 1C with monitoring of the reaction by TLC (toluene:
EtOAc ¼ 2:3, Rf ¼ 0.15), the reaction mixture was roughly concentrated in
reduced pressure. Then, the reaction mixture was diluted with EtOAc, washed
with sat. NaHCO3, brine, dried over Na2SO4, filtered and concentrated. The
residue (S:R417:1) was dissolved in dichloromethane (1.6ml) and pyridine
(0.4ml), and then benzoyl chloride (72 ml, 0.62mmol) was added. The mixture
was stirred for 3 h at 0 1C with monitoring by TLC (hexane: EtOAc ¼ 1:1,
Rf ¼ 0.5). Then, the reaction mixture was diluted with EtOAc, washed with
H2O, 1 N HCl, sat. NaHCO3, brine, dried over Na2SO4, filtered and
concentrated. The residue was purified by column chromatography on silica
gel (hexane:EtOAc ¼ 2:1–1:1) to give (7S)-O-benzoate 8 (90 mg, 66%) as a
white solid.
[a]RDT ꢀ48.5 (c 0.67, CHCl3)
1H NMR (600MHz, CDCl3): d ¼ 7.60–7.59 (d, 2H, J ¼ 7.4 Hz: Ar-H),
7.38–7.36 (m, 1H: Ar-H), 7.33–7.31 (m, 2H: Ar-H), 6.56–6.55 (d, 1H, J ¼ 9.2
Hz: NH), 5.08–5.07 (d, 1H, J ¼ 7.7 Hz: H-7), 4.73–4.71 (td, 1H, J ¼ 3.7,
5.4Hz: H-4), 4.51–4.42 (m, 2H: H-8a,b), 4.08–4.05 (q, 1H, J ¼ 9.7, 9.9 Hz:
H-5), 3.84–3.82 (d, 1H, J ¼ 10.6Hz: H-6), 2.75–2.72 (dd, 1H, J ¼ 3.6, 8.0 Hz:
H-3eq), 2.07 (s, 3H: Ac), 2.02 (s, 3H: Ac), 1.95 (s, 3H: Ac), 1.90–1.86 (t, 1H,
J ¼ 12.3Hz: H-3ax) and 1.83 (s, 3H: AcNH).
[a]RDT ꢀ3.90 (c 0.67, CHCl3)
1H NMR (600MHz, CDCl3): d ¼ 8.03–8.02 (d, 2H, J ¼ 7.3 Hz; Ar-H),
7.99–7.97 (d, 2H, J ¼ 7.4 Hz: Ar-H), 7.65–7.64 (d, 2H, J ¼ 7.4 Hz: Ar-H),
7.57–7.54 (m, 2H: Ar-H), 7.44–7.39 (m, 5H: Ar-H), 7.37–7.34 (t, 2H, J ¼ 7.7
Hz: Ar-H), 5.70–5.69 (d, 1H, J ¼ 9.1Hz: NH), 5.27–5.26 (dd, 1H, J ¼ 3.3,
1.5 Hz: H-7), 5.09–5.04 (td, 1H, J ¼ 4.4, 6.4Hz: H-4), 4.72–4.69 (dd, 1H,
J ¼ 6.8, 5.5 Hz: H-8), 4.25–4.20 (q, 1H, J¼ 9.9 Hz: H-5), 3.94–3.92 (t, 1H,
J ¼ 7.9 Hz: H-9b), 3.81–3.79 (dd, 1H, J ¼ 3.0, 2.0Hz: H-6), 3.33 (s, 3H:
COOMe), 3.00–2.98 (dd, 1H, J ¼ 4.9, 7.7 Hz: H-3eq), 2.13–2.09 (t, 1H, J ¼ 12.1
Hz: H-3ax), 1.70 (s, 3H: AcNH), 1.43 (s, 3H: CMe2) and 1.42 (s, 3H: CMe2).
13C NMR (150MHz, CDCl3): d ¼ 170.6 (C ¼ O), 168.5 (C ¼ O), 166.6
(C ¼ O), 166.3 (C ¼ O), 136.9 (Ar), 133.6 (Ar), 133.3 (Ar), 130.0 (Ar), 130.0
(Ar), 128.8 (Ar), 128.6 (Ar), 128.5 (Ar), 109.5 (CMe2), 87.1 (C2), 77.3 (C6),
74.2 (C8), 73.3 (C7), 70.8 (C4), 66.4 (C9), 52.6 (COOMe), 51.3 (C5), 37.8
(C3), 26.5 (CMe2), 26.1 (CMe2) and 23.2 (Ac).
13C NMR (150MHz, CDCl3): d ¼ 172.1 (C ¼ O), 171.6 (C ¼ O), 171.2
(C ¼ O), 171.0 (C ¼ O), 136.7 (Ar), 130.0 (Ar), 128.9 (Ar), 86.2 (C2), 75.3
(C6), 71.9 (C7), 70.7 (C4), 61.8 (C8), 49.6 (C5), 37.4 (C3), 23.0 (AcNH), 21.2
(OAc) and 21.0 (OAc).
HR-MS (ESI): m/z calcd for: C22H27NO10SNa, [Mþ Na]þ 520.1253 found:
520.1251.
Compound 11. To a solution of compound 10 (1.20 g, 2.40mmol) and
1-oxa-2-oxo-3-thiaindolizinium chloride (2.30 g, 12.1mmol) in dichloro-
methane (50 ml), triethylamine (2.7ml, 19.3mmol) was added at 0 1C under
argon atmosphere. The mixture was stirred for 5 h at 0 1C, and
then photolyzed (254 nm, Rayonnet photoreactor, Southern New England
Ultraviolet Co., Branford, CT, USA) at 0 1C for 2 h. The reaction mixture
was diluted with EtOAc, washed with H2O, sat. NaHCO3, brine, dried
over Na2SO4, filtered and concentrated. The residue (a:b ¼ 1:2 from 1H
NMR of crude mixture) was purified by column chromatography on silica gel
(toluene: isopropanol ¼ 40:1–30:1) to give b-thioglycoside 11 (527mg, 38%)
as a yellow solid.
HR-MS (ESI): m/z calcd for: C35H37NO10SNa, [Mþ Na]þ 686.2036 found:
686.2025.
Compound 9. A solution of compound 8 (415mg, 0.63mmol) in 60% AcOH
(9ml) was stirred at 601C for 2 h. After full consumption of starting material
8, the reaction mixture was concentrated in reduced pressure, coevaporated
with toluene three times and kept in vacuo for 1 h. Then, to a solution of
resulting diol in tetrahydrofuran (4.5ml) and H2O (1.5ml), NaIO4 (535mg,
2.5 mmol) was added. The reaction mixture was stirred for 12h with
monitoring by TLC (toluene: EtOAc ¼ 1:1, Rf ¼ 0.3, spots shows orange color
with only heating), diluted with EtOAc, washed with H2O, sat. NaHCO3,
brine, dried over Na2SO4, filtered and concentrated. The residue was purified
by column chromatography on silica gel (hexane: EtOAc¼ 2:1–1:1) to give
aldehyde 9 (310mg, 84%) as a white solid.
[a]RDT ꢀ17.0 (c 0.67, CHCl3)
1H NMR (600MHz, CDCl3): d ¼ 7.54–7.53 (m, 2H: Ar-H), 7.33–7.32 (m, 3H:
Ar-H), 5.53–5.51 (d, 1H, J ¼ 8.8 Hz: NH), 5.12–5.11 (m, 1H: H-6), 4.95–4.91
(td, 1H, J ¼ 4.7, 5.9, 4.8, 5.8, 4.8 Hz: H-3), 4.69–4.66 (dd, 1H, J ¼ 1.8, 9.9 Hz:
H-1), 4.50–4.41 (m, 2H: H-7a,b), 3.96–3.91 (q, 1H, J ¼ 9.9 Hz: H-4), 3.56–3.54
(dd, 1H, J ¼ 1.5, 9.1 Hz: H-5), 2.32–2.29 (ddd, 1H, J ¼ 1.1, 3.6, 1.5, 6.3, 1.8,
3.3, 1.5 Hz: H-2eq), 2.09 (s, 3H: Ac), 2.07 (s, 3H: Ac), 2.05 (s, 3H: Ac), 1.99
(s, 3H: AcNH) and 1.85–1.79 (q, 1H, J ¼ 11.8, 12.1, 12.1 Hz: H-2ax).
13C NMR (150MHz, CDCl3): d ¼ 171.3 (C ¼ O), 170.9 (C ¼ O), 170.8
(C ¼ O), 170.7 (C ¼ O), 133.0 (Ar), 132.6 (Ar), 129.0 (Ar), 128.3 (Ar), 82.2
(C1), 79.2, 71.6, 71.3, 61.9, 51.0 (C4), 36.6 (C2), 23.3 (AcNH), 21.2 (OAc), 21.1
(OAc) and 21.0 (OAc).
[a]RDT ꢀ55.8 (c 0.67, CHCl3)
1H NMR (600MHz, CDCl3): d ¼ 9.75 (s, 1H: CHO), 8.16–8.15 (d, 2H, J ¼ 7.3
Hz: Ar-H), 8.01–7.99 (d, 2H, J ¼ 7.4 Hz: Ar-H), 7.64–7.58 (m, 2H: Ar-H),
7.51–7.44 (m, 6H: Ar-H), 7.38–7.35 (m, 1H: Ar-H), 7.28–7.25 (m, 2H: Ar-H),
5.67–5.66 (d, 1H, J ¼ 1.9 Hz: H-7), 5.64–5.63 (d, 1H, J ¼ 9.5 Hz: NH),
5.17–5.12 (td, 1H, J ¼ 4.8, 5.4, 4.0, 6.4, 4.8 Hz: H-4), 4.60–4.55 (q, 1H,
J ¼ 10.2, 10.0Hz: H-5), 4.17–4.14 (dd, 1H, J ¼ 1.8, 9.2 Hz: H-6), 3.53 (s, 3H:
COOMe), 3.03–3.00 (dd, 1H, J ¼ 4.8, 7.7 Hz: H-3eq), 2.16–2.12 (t, 1H, J ¼ 12.1,
12.5Hz: H-3ax) and 1.82 (s, 3H: AcNH).
HR-MS (ESI): m/z calcd for: C21H27NO8SNa, [Mþ Na] þ 476.1355 found:
476.1358.
Compound 12. To a solution of compound 11 (50 mg, 0.11mmol) in
13C NMR (150MHz, CDCl3): d ¼ 195.8 (CHO), 170.7 (C ¼ O), 168.4 dichloromethane (1ml), 77% meta-chloroperoxybenzoic acid (26 mg,
(C ¼ O), 166.9 (C ¼ O), 165.8 (C ¼ O), 136.8 (Ar), 133.9 (Ar), 133.8 (Ar), 0.12 mmol) was added at ꢀ781C. The mixture was stirred for 7.5 h at
130.4 (Ar), 130.4 (Ar), 130.0 (Ar), 130.0 (Ar), 128.9 (Ar), 128.7 (Ar), 128.7
ꢀ781C with monitoring of the reaction by TLC (toluene: isopropanol ¼ 7.5:1,
The Journal of Antibiotics

Septacidin and spicamycin glycone 2-epimers
NPJ Price et al
413
Rf ¼ 0.2), and then quenched by the addition of sat. aq. NaHCO3 (1ml) at ACKNOWLEDGEMENTS
ꢀ201C. The reaction mixture was diluted with EtOAc, washed with sat.
NPJP thanks Trina Hartman for excellent technical assistance. We thank the
NaHCO3, brine, dried over Na2SO4, filtered and concentrated. The residue was
coevaporated by toluene (2ml) two times and dried in vacuo for 1.5 h. The
resulting solid was dissolved to dry toluene (2ml) and stirred for 4 h at 901C
under argon atmosphere. The reaction was monitored by TLC (EtOAc only,
Rf ¼ 0.35), diluted with EtOAc, washed with H2O, sat. NaHCO3, brine, dried
over Na2SO4, filtered and concentrated. The residue was purified by column
chromatography on silica gel (hexane: EtOAc ¼ 1:2–1:4) to give glycal 12
(9.4mg, 25%) as a yellow syrup.
Developmental Therapeutics Program of the National Cancer Institute,
National Institutes of Health, for providing us with spicamycin (KRN5500).
NMR and MS spectral data for the products, and additional isotopic
enrichment data are available via the internet.
1
2
3
Dutcher, J. D., Von Saltza, M. H. & Pansy, F. E. Septacidin, a new antitumor and
antifungal antibiotic produced by Streptomyces fimbriatus. Antimicrob. Agents Che-
mother. 161, 83–88 (1963).
Hayakawa, Y. et al. Studies on the differentiation inducers of myeloid leukemic cells.
III. Spicamycin, a new inducer of differentiation of HL-60 human promyelocytic
leukemia cells. J. Antibiot. 36, 934–937 (1983).
Hayakawa, Y. et al. Spicamycin, a new differentiation inducer of mouse myeloid
leukemia cells (Ml) and human promyelocytic leukemia cells (HL-60). Agric. Biol.
Chem. 49, 2685–2691 (1985).
[a]RDT 16.0 (c 0.23, CHCl3)
1H NMR (500MHz, CDCl3): d ¼ 6.51–6.49 (d, 1H, J ¼ 6.2 Hz: H-1), 5.69–5.68
(d, 1H, J ¼ 9.2 Hz: NH), 5.39–5.35 (m, 1H: H-6), 5.09–5.07 (m, 1H: H-2), 4.91
(m, 1H: H-3), 4.55–4.52 (m, 2H: H-4, H-7a), 4.33–4.30 (dd, 1H, J ¼ 3.3,
5.9Hz: H-5), 4.20–4.16 (dd, 1H, J ¼ 4.0, 7.9, 4.4 Hz: H-7b), 2.11 (s, 3H: Ac),
2.09 (s, 3H: Ac), 2.02 (s, 3H: AcNH) and 1.99 (s, 3H: Ac).
13C NMR (125MHz, CDCl3): d ¼ 170.9 (C ¼ O), 170.3 (C ¼ O), 169.9
(C ¼ O), 169.5 (C ¼ O), 145.4 (C1), 99.6 (C2), 73.6 (C5), 67.9 (C6), 64.8 (C3),
61.9 (C7), 45.7 (C4), 23.5 (AcNH), 21.2 (OAc), 21.0 (OAc) and 21.0 (OAc).
HR-MS (ESI): m/z calcd for: C15H21NO8Na, [M þ Na]þ 366.1165 found:
366.1180.
4
5
Kamishohara, M. et al. Structure–antitumor activity relationship of semi-synthetic
spicamycin analogues. J. Antibiot. 46, 1439–1446 (1993).
Weinstein, S. M. et al. A spicamycin derivative (KRN5500) provides neuropathic pain
relief in patients with advanced cancer: a placebo-controlled, proof-of-concept trial.
J. Pain Sympt. Manage. 43, 679–693 (2012).
6
Agahigian, H. et al. Nuclear magnetic resonance spectroscopy of acetylated
methyl glycopyranosides of aminohexoses. Characterization of an aminohexose from
septacidin. J. Org. Chem. 30, 1085–1088 (1965).
7
8
Sakai, T. et al. Absolute configuration of spicamycin, an antitumor antibiotic produced
by Streptomyces alanosinicus. J. Antibiot. 48, 899–900 (1995).
Compound 13. To a solution of compound 12 (9.4mg, 27.4mmol) in
tetrahydrofuran (140ml) and H2O (30 ml), pyridine (5ml) and freshly prepared
OsO4 (10 mgmlꢀ1 tBuOH soln.: 140 ml) were added at 501C. The mixture was
stirred for 1 h at 50 1C with monitoring of the reaction by TLC (CHCl3:
MeOH ¼ 7.5:1, Rf ¼ 0.35). Then, solvents were removed under reduced
pressure and the residue was coevaporated with toluene two times. The
resulting residue was dissolved in pyridine (300ml) and Ac2O (200ml), stirred
for overnight and concentrated. The residue was purified by column
chromatography on silica gel (hexane: EtOAc¼ 1:2–0:1) to give gluco-
hexaacetate 13 (8.1mg, 64%, a:b ¼ 10:7, Glc: Man¼ 25:1) as a yellow oil
and an a, b-mixture.
Igarashi, Y. et al. Anicemycin,
a new inhibitor of anchorage-independent
growth of tumor cells from Streptomyces sp. TP-A0648. J. Antibiot. 58, 322–326
(2005).
Suzuki, T. et al. Total synthesis of spicamycin. J. Org. Chem. 67, 2874–2880
(2002).
9
10 Chida, N., Suzuki, T., Tanaka, S. & Yamada, I. Pd-catalyzed coupling reaction of
glycosylamines with 6-chloropurines: synthesis of 6-(b-D-mannopyranosylamino)-9H-
purine and its b-D-gluco isomer, N-glycoside models for spicamycin and septacidin.
Tetrahedron Lett. 40, 2573–2576 (1999).
11 Acton, E. M., Ryan, K. J. & Luetzow, A. E. Antitumor septacidin analogues. J. Med.
Chem. 20, 1362–1371 (1977).
´
12 Martın, A., Butters, T. D. & Fleet, G. W. J. Synthesis and biological evaluation of
enantiomeric rhamnose analogues of the antitumour agent spicamycin. Tetrahedron
10, 2343–2360 (1999).
13 Mons, S. & Fleet, G. W. J. An approach to the generation of simple analogues of the
antitumor agent spicamycin. Org. Biomol. Chem. 1, 3685–3691 (2003).
[a]RDT ꢀ48.3 (c 0.54, CHCl3: a, b ¼ 10:7 mixture)
HR-MS (ESI): m/z calcd for: C19H27NO12Na, [M þ Na]þ 484.1431 found:
484.1439.
14 Frihed, T. G., Heuckendorff, M., Pedersen, C. M.
& Bols, M. Easy access to
L-mannosides and L-galactosides by using C–H activation of the corresponding
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16 Horn, E. J., Saludes, J. P. & Gervay-Hague, J. Investigation into an efficient synthesis of
2,3-dehydro-N-acetyl neuraminic acid leads to three decarboxylated sialic acid
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Compound 13a. 1H NMR (600MHz, CDCl3): d ¼ 6.34 (d, 1H, J ¼ 3.7 Hz:
H-1),
5.65–5.63
(d, 1H, J ¼ 8.8 Hz: NH), 5.29–5.25 (t, 1H, J ¼ 9.9Hz; H-3), 5.12–5.10 (m,
1H: H-6), 5.05–5.03 (dd, 1H, J ¼ 3.7, 6.6 Hz: H-2), 4.36–4.35 (m, 2H: H-7a,b),
4.25–4.20 (q, 1H, J ¼ 10.2Hz: H-4), 4.03–4.01 (dd, 1H, J ¼ 1.5, 9.9Hz: H-5),
2.14 (s, 3H: Ac), 2.09 (s, 3H: Ac), 2.07 (s, 3H: Ac), 2.06 (s, 3H: Ac), 2.00 (s, 3H:
Ac) and 1.99 (s, 3H: AcNH).
17 Shie, J.-J. et al.
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A practical synthesis of zanamivir phosphonate congeners
13C NMR (150MHz, CDCl3): d ¼ 171.6 (C ¼ O), 170.8 (C ¼ O), 170.8
(C ¼ O), 170.4 (C ¼ O), 169.7 (C ¼ O), 168.6 (C ¼ O), 89.2 (C1), 73.3 (C5),
70.6 (C6), 69.7 (C3), 69.3 (C2), 61.3 (C7), 50.9 (C4), 23.3 (AcNH), 21.2 (OAc),
21.0 (OAc), 21.0 (OAc), 20.9 (OAc) and 20.6 (OAc).
19 Crich, D. & Ritchie, T. J. Stereoselective free radical reactions in the preparation
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20 Ando, H. et al. Studies on the alpha-(1-4)- and alpha-(1-8)-fucosylation of sialic
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Hz: NH), 5.62–5.61 (d, 1H, J ¼ 8.1Hz: H-1), 5.11–5.04 (m, 3H; H-2, H-3,
H-6), 4.38–4.31 (m, 2 H: H-7a,b), 4.21–4.16 (q, 1H, J ¼ 9.9 Hz: H-4), 3.76–3.74
(dd, 1H, J ¼ 1.8, 8.8Hz: H-5), 2.12 (s, 3H: Ac), 2.10 (s, 3H: Ac), 2.07 (s, 3H:
Ac), 2.05 (s, 3H: Ac), 2.03 (s, 3H: Ac) and 1.99 (s, 3H: AcNH).
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(C ¼ O), 170.5 (C ¼ O), 169.2 (C ¼ O), 169.1 (C ¼ O), 92.0 (C1), 75.9 (C5),
72.6 (C3), 70.5 (C6), 70.1 (C2), 61.3 (C7), 50.9 (C4), 23.3 (AcNH), 21.2 (OAc),
21.0 (OAc), 21.0 (OAc), 20.8 (OAc) and 20.7 (OAc).
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CONFLICT OF INTEREST
The authors declare no conflict of interest.
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Supplementary Information accompanies the paper on The Journal of Antibiotics website (http://www.nature.com/ja)
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