Synthesis and Biological Evaluation of 6-[(1 R)-1-Hydroxyethyl]-2,4a(R),6(S),8a(R)-tetrahydropyrano-[3,2- b]-pyran-2-one and Structural Analogues of the Putative Structure of Diplopyrone

Article abstract of DOI:10.1021/acs.joc.8b02490

The phytotoxin diplopyrone is considered to be the main phytotoxin in a fungus that is responsible for cork oak decline. A carbohydrate-based synthesis of the enantiomer of the structure proposed for diplopyrone has been developed from a commercially available derivative of d-galactose. Key steps in the synthesis are a highly stereoselective pyranose chain-extension based on methyltitanium, preparation of a vinyl glycoside via Isobe C-alkynylation-rearrangement/reduction, and RCM-based pyranopyran construction. Crystallographic and NMR analysis confirms an earlier report that the structure originally proposed for diplopyrone may require revision. Structural analogues were prepared for biological evaluation, the most promising being a pyranopyran nitrile synthesized from tri-O-acetyl-d-galactal by Ferrier cyanoglycosidation, Wittig chain extension, and lactonization. Biological assays revealed potent antibacterial activity for the nitrile analogue against common bacterial pathogens Edwardsiella ictaluri and Flavobacterium columnare that cause enteric septicemia (ESC) and columnaris disease, respectively, in catfish. The IC50 value of 0.002 against E. ictaluri indicates approximately 100 times greater potency than the antibiotic florfenicol used commercially for this disease. Phytotoxic activity for all three target compounds against duckweed was also observed. The antibiotic and phytotoxic activities of the new pyranopyrans synthesized in this study demonstrate the potential of such compounds as antibiotics and herbicides.

Full text of DOI:10.1021/acs.joc.8b02490

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Article
Synthesis and Biological Evaluation of 6-[(1R)-1-
hydroxyethyl]-2,4a(R),6(S),8a(R)-tetrahydropyrano-[3,2-b]-pyran-2-
one and Structural Analogs of the Putative Structure of Diplopyrone
Nicholas C. Lazzara, Robert J. Rosano, Purav P. Vagadia, Matthew T. Giovine, Mark
W. Bezpalko, Nicholas A. Piro, Wm. Scott Kassel, Walter J. Boyko, Deanna Lynn
Zubris, Kevin K. Schrader, David E. Wedge, Stephen O. Duke, and Robert M. Giuliano
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b02490 • Publication Date (Web): 14 Dec 2018
Downloaded from http://pubs.acs.org on December 17, 2018
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Synthesis and Biological Evaluation of 6-[(1R)-1-hydroxyethyl]-
2,4a(R),6(S),8a(R)-tetrahydropyrano-[3,2-b]-pyran-2-one and Structural
Analogs of the Putative Structure of Diplopyrone
9
Nicholas C. Lazzara,† Robert J. Rosano,† Purav P. Vagadia,† Matthew T. Giovine,†
Mark W. Bezpalko,† Nicholas A. Piro,† Wm. Scott Kassel,† Walter J. Boyko,† Deanna L. Zubris,†
Kevin K. Schrader,‡ David E. Wedge,‡ Stephen O. Duke,‡ and Robert M. Giuliano†*
†Department of Chemistry, Villanova University, Villanova, PA 19085
‡Natural Products Utilization Research Unit, Agricultural Research Service,
U.S. Department of Agriculture, P. O. Box 1848, University, MS 38677
robert.giuliano@villanova.edu
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OR
O
HO
HO
OH
OH
H
H
O
O
O
O
O
OH OH
H
O
(-)-Diplopyrone
C-Vinyl Glycoside
D-Galactose
ABSTRACT
The phytotoxin diplopyrone is considered to be the main phytotoxin in a fungus that is
responsible for cork oak decline. A carbohydrate-based synthesis of the enantiomer of the structure
proposed for diplopyrone has been developed from a commercially available derivative of D-
galactose. Key steps in the synthesis are a highly stereoselective pyranose chain-extension based
on methyltitanium, preparation of
a
vinyl glycoside via Isobe C-alkynylation-
rearrangment/reduction, and RCM-based pyranopyran construction. Crystallographic and NMR
analysis confirms an earlier report that the structure originally proposed for diplopyrone may
require revision. Structural analogs were prepared for biological evaluation, the most promising
being
a
pyranopyran nitrile synthesized from tri-O-acetyl-D-galactal by Ferrier
cyanoglycosidation, Wittig chain extension, and lactonization. Biological assays revealed potent
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antibacterial activity for the nitrile analog against common bacterial pathogens E. ictaluri and F.
columnare that cause enteric septicemia (ESC) and columnaris disease, respectively, in catfish.
The IC50 value of 0.002 against E. ictaluri indicates approximately 100 times greater potency than
the antibiotic florfenicol used commercially for this disease. Phytotoxic activity for all three target
compounds against duckweed was also observed. The antibiotic and phytotoxic activities of the
new pyranopyrans synthesized in this study demonstrate the potential of such compounds as
antibiotics and herbicides.
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INTRODUCTION
Diplopyrone is a phytotoxin isolated from the fungus Diplodia mutila and reported by
Evidente and coworkers in 2003.1,2 An endophytic fungus, meaning it can reside in the host plant
for a period of time before causing damage,3 D. mutila is thought to be responsible for cork oak
decline in parts of southern Europe where the disease has caused large and negative economic and
environmental impacts. Diplopyrone causes necrosis and wilting of cork oak (Quercus suber)
cuttings and is considered the main phytotoxin responsible for the observed pathogenesis. Strains
of this fungus also infect other oak species as well as cypress.2 Recently, there have been studies
of structure-activity relationships of fungal phytotoxins, in particular, the relationship of
stereochemistry to biological activity.4,5 These studies provide insight into the mechanisms of
action of microbially-derived natural products and may also lead to the identification of new
molecular targets for the development of environmentally benign herbicides.6-8
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OH
OH
5
H
H
H
H
4
S
O
O
3
2
9
4a
R
6
S
S
R
S
R
R
10
8a
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O
O
O
O
8
1
H
H
8
9
(+)-Diplopyrone (1)
Pyranopyran (2)
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AcO
H
H
H
H
H
H
O
NC
O
O
O
O
O
Pyranopyran Acetate (3)
Pyranopyran Nitrile (4)
Figure 1. Putative (+)-Diplopyrone and Related Compounds in this Study
The proposed structure of diplopyrone contains an unusual cis-fused pyranopyran core
and was assigned on the basis of spectroscopic data and theoretical calculations of its optical
properties as 6-[(1S)-1-hydroxyethyl]-2,4a(S),6(R),8a(S)-tetrahydropyrano-[3,2-b]-pyran-2-one
(1) (Figure1).2 Pyranopyrans9 such as those found in brevitoxin and other ladder ethers as well as
in okadaic acid, thyrsiferol and other natural products are typically trans-fused, consistent with
proposed biosynthetic origins by nucleophilic ring opening of polyepoxides.10 The first synthesis
of the putative structure of (+)-diplopyrone was achieved in 17 steps from cis-1,4-butenediol in
2017 by Mohaptra and coworkers.11 Key steps in their synthesis are the Sharpless asymmetric
epoxidation, Pd-catalyzed regioselective epoxide opening, tandem iodine-catalyzed
allylation/cyclization for dihydropyran synthesis, and a-aminohydroxylation in the presence of L-
proline to introduce the C-9 hydroxyl group stereoselectively (7:3 ratio). A comparison of NMR
spectral data for their synthetic diplopyrone with that reported for the natural product revealed
discrepancies in proton and carbon chemical shifts and led the authors to suggest that revisions to
the proposed structure of diplopyrone are necessary. Natural material is scarce,1,2 thereby
increasing the need for compounds to be made available through synthesis to support structural
verification and biological studies.
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We have been seeking to develop an asymmetric, carbohydrate-based synthesis of
enantiomeric (-)-diplopyrone and analogs for the past few years (Figure 1). There are two reasons
for the choice of the enantiomer as the initial target. First, viewing diplopyrone as a “C-glycoside
problem”12 opens up multiple synthetic routes from common D sugars. Marine polyethers such as
brevitoxin and the anticancer drug Halaven® have been synthesized by pathways that rely on a
pyranopyran core structure accessible from carbohydrates via C-glycosides as intermediates.9 Our
design utilizes C-glycosides as precursors to the lactone ring of diplopyrone. The second reason,
of a less practical nature but enticing nonetheless, stems from phytochemistry. It has been
demonstrated that stereoisomers of phytotoxins may possess different biological acitivity.4,5 For
example, the dihydroxynaphthaleneone (-)-regiolone is produced by Botrytis fabae, which is a
much more virulent pathogen than Botrytis cinerea. The latter produces the enantiomeric
phytotoxin (+)-isosclerone.13 Enantiomers of the phytotoxic lichen metabolite usnic acid possess
different levels of activity, with (-)-usnic acid having more phytotoxic activity.14 Sesquiterpenes
(+)- and (-)-gossypol have divergent activities across a range of bioassays with the (-)-enantiomer
showing more potency in most instances.15 Will (-) diplopyrone exhibit necrotic effects in
bioassays similar to the naturally occurring (+)-diplopyrone? Will it exhibit inverse agonism by
blocking the action of (+)-diplopyrone, or antimicrobial activity against strains of D. mutila that
infect other species? The compounds in this study will be used to determine structure-activity
relationships for diplopyrone and to obtain biological data for assays against invasive plants,
bacteria, and other pathogenic organisms.
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Two carbohydrate-based routes to the enantiomer of the structure proposed for diplopyrone
(pyranopyran 2) are shown retrosynthetically in Scheme 1. Part of the appeal to a carbohydrate-
based strategy for the enantioselective synthesis of diplopyrones lies in the pathways can be
developed from simple starting materials. Six of the ten carbons of diplopyrone and two of its four
chirality centers are derived from common carbohydrates in our approach. Viewing the target
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from the standpoint of C-6 Wittig chain-extension16 (Scheme 1, Type 1 approach) allows for
formation of the pyranopyran core by intramolecular esterification of a chain-extended substrate,
which is a “higher-carbon sugar.”17 In this approach, the chirality centers at C-4a and C-8a are
derived from C-5 and C-4 of the carbohydrate starting material, D-galactose, and the hydroxyethyl
side-chain is derived from a group that is introduced at the anomeric center by C-glycosidation.
In an alternative approach, the pyranopyran core can potentially be formed from ring-closing
metathesis (RCM) using a C-vinyl glycoside18 (Scheme 1, Type 2 approach). In a Type 2
approach, the chirality centers at C-6 and C-8a as numbered in diplopyrone originate from C-5and
C-2 of either D-glucose or D-galactose. C-glycosidation is required to introduce the vinyl group
needed in the RCM precursor, while carbohydrate chain extension is required to create the
hydroxyethyl side-chain. The approaches outlined in Scheme 1 present opportunities for the
development of synthesis methodology in the areas of chain-extension and in C-vinyl glycoside
synthesis, both of which are useful methods with well-known applications in synthetic
carbohydrate chemistry.19-24
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Type 1 Approach
O
3
O
2
8
2
3
OH
HO
Lactonization
4
7
6
1
O
4
5
O
HO1
4
OR
O
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8a
5
4a
O
1
8a
6
9
HO
4a
2
6
3
OH OH
8
7
9
HO
HO
Galactose
(sugar numbering)
(-)-Diplopyrone
(proposed)
Chirality from sugar: (4a, 8a)
C-Glycosidation: (6)
Type 2 Approach
OH
OR
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9
OH
HO
HO
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RCM
O
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O
8a
O
4
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4a
3
1
8
2
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4
2
4
3
OH OH
1O
1
O
3
2
O
O
(-)-Diplopyrone
(proposed)
Chirality from sugar: (6, 8a)
C-Glycosidation: (4a)
Glucose or Galactose
(sugar numbering)
Scheme 1. C-Glycoside-based approaches to pyranopyran 2
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In this paper we describe the first synthesis of enantiomeric (-)-diplopyrone, pyranopyran
2, based on the structure originally proposed for the natural product. The synthesis was carried
out in 13 steps from a commercially available derivative of D-galactose by the RCM (Type 2)
approach. We also describe the syntheses of pyranopyran nitrile 4 in 8 steps from tri-O-acetyl-D-
galactal using a Type 1 approach. Both syntheses are highly stereoselective and both are amenable
for the preparation of additional analogs for use in biological studies. ‘X-ray crystallographic
analyses has been obtained for 3 and 4. The crystallographic analyses coupled with NMR data
provide further insight into the structure assignments for natural and synthetic diplopyrones in the
literature. Biological assays were carried out at the USDA (Natural Products Utilization Research
Unit). Compounds 2, 3, and 4 were tested for phytotoxic, fungicidal, and antibacterial activity,
the latter against common bacterial pathogens in fish. It is of interest that compound 4 is
significantly more active than a commercially used antibiotic used against E. ictaluri, a common
pathogen in catfish. Results of these bioassays are presented herein.
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RESULTS AND DISCUSSION
In our first approach to pyranopyran 2, we chose a Type 1 strategy in which a nitrile group
would be introduced by a Ferrier rearrangement to the anomeric center of D-galactal (Scheme 2).
The nitrile group would then be converted to the required hydroxyethyl side-chain prior to chain
extension and lactonization. Tri-O-acetyl-D-galactal 5 was treated with trimethyl-
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t-BuMe2SiCl,
DMF, imidazole
CN
CN
O
O
O
TsOH
CH3OH, Δ
TMSCN
HO
AcO
AcO
AcO
BF3 OEt2
CH2Cl2
60%
HO
AcO
94%
97%
OAc
5
7
6
8
9
CN
CN
O
CN
O
SO3 pyr
O
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PPTS, CH3OH
81%
TBSO
TBSO
HO
O
DMSO-CH2Cl2
DIPEA, 0 oC
86%
TBSO
TBSO
10
8
9
H
CN
O
CN 1. TsOH, CH3OH
O
Ph3P CHCO2Et
CH3OH, 0 oC, 61%
EtO2C
TBSO
2. TsOH, toluene, Δ
O
O
H
48h, 88%
11(Z only)
4
Scheme 2. Synthesis of pyranopyran nitrile 4
silyl cyanide in the presense of boron trifluoride diethyl etherate to provide crystalline glycosyl
nitrile 6 in 60% yield on a 20-gram scale.25 Deacylation of 6 with p-toluenesulfonic acid in
methanol gave diol 7 which was converted to di-O-TBS ether 8 in 91% overall yield. The nitrile
was expected to be a suitable precursor for the hydroxyethyl side-chain by either of two sequences,
one involving reduction to the corresponding aldehyde followed by methylation and the other
consisting of prior methylation to give a methyl ketone which could be reduced. Our attempts at
conversion of the nitrile to the hydroxyethyl side chain of pyranopyran 2 by both of these
sequences were unsuccessful. Compound 8 and similar compounds were found to be prone to
double bond migration, epimerization at the anomeric center in the presence of base, and
vinylogous b-elimination. For example, addition of methyllithium to 8 followed by hydride
reduction of the resulting ketone gave a hydroxyethyl derivative in 14% overall yield, along with
products of elimination. In spite of these difficulties, it was possible to construct the pyranopyran
core of diplopyrone from 8, leaving the nitrile group intact. Selective deprotection of the 6-
hydroxyl group of 8 was achieved using PPTS in methanol at room temperature. Migration of the
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4-O-TBS group in 9 to the C-6 hydroxyl group was observed to occur upon standing at room
temperature so it was necessary to carry out the next two steps in quick succession. Oxidation of
the primary alcohol 9 by the Parikh-Doering method gave aldehyde 10 in 86% yield. Attempted
Wittig reactions of 10 with methylenetriphenylphosphorane failed as did Peterson alkenation,
again likely the result of the tendency for 10 to undergo vinylogous b-elimination of the 4-O-TBS
group. Tebbe methylenation was also unsuccessful. The reaction of 10 with the stabilized Wittig
reagent (carbethoxymethylidene)triphenylphosphorane under conditions described by Valverde
and coworkers26 gave ester 11 exclusively as the Z isomer. The cyclization of 11 to diplopyrone
nitrile 4 proved more difficult than expected perhaps because the double bond in the pyranose ring
places the C-4 hydroxyl and ester groups in poor proximity for ring closure. However, we were
able to obtain 4 in 88% overall yield from 11 by a two-step procedure consisting of deprotection
in methanol then heating in toluene, both in the presence of an acid catalyst. The overall yield of
nitrile 4 from galactal derivative 5 is 20%. Nitrile 4 was crystalline and suitable for analysis by
X-ray diffraction, which supports the assignment of its structure. The ORTEP diagram of 4 is
included in the Supporting Information.
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The pyranopyran nitrile 4 turned out to be an interesting analog, especially in view of its
biological activity discussed later in this paper. Our lack of success in converting the nitrile group
to a hydroxyethyl side-chain prompted us to explore an alternative (Type 2) strategy for the
synthesis of pyranopyran 2. In this approach, the hydroxyethyl group is introduced first, by chain
extension at C-6 of a hexopyranoside. While many methods exist for the synthesis of higher-
17
carbon sugars, a challenge that emerges in the route to our target compounds is the control of
stereochemistry at the off-ring, C-9 chirality center. Additions of organometallic reagents to
dialdopyranosides I typically favor products II that result from chelation-control and which
27
possess the incorrect stereochemistry for diplopyrone (Figure 2). Felkin-Anh stereoselectivity
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(III) is required for our synthesis. A second challenge in this approach is the need for an efficient
synthesis of a C-vinyl glycoside precursor for the RCM-based route to the
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9
OH
H
OH
H
O
R
H
R
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R-M
O
O
O
III (Felkin-Anh)
II (Chelation control)
I
Figure 2. Chelation vs. Felkin –Anh Stereoselectivity in Dialdohexopyranosides
lactone (Scheme 1). C-Vinyl glycosides have been used as precursors to carbon-linked
20
glycoconjugates, for example, C-galactosyl ceramides. However, unlike their C-allyl glycoside
28-30
counterparts, simple, unsubstituted C-vinyl glycosides have proven to be more difficult to
synthesize as illustrated in recent approaches to C-alkyl, C-aryl, and (substituted) C-vinyl
22-24
glycosides, some of which are based on modern catalytic methods. We were able to solve both
the chain-extension and C-vinyl glycoside problems in our approach.
Commercially available 1,2:3,4-di-O-isopropylidene-a-D-galactopyranose 12 was
oxidized with catalytic TEMPO in the presence of bis(acetoxy)iodo-benzene (BAIB) on a 15-gram
scale to give aldehyde 13 which was used immediately in the next step (Scheme 3).31 This
oxidation was also carried out using a polymer-bound reagent,32 which gave improved yields on
smaller (1-2 mmol) scales, but turned out to be less desirable for scaled-up preparations of 13. The
addition of organometallic reagents to aldehyde 13 has provided products that have found use as
building blocks for trisaccharides used to study monoclonal antibodies,33 enzyme inhibitors,34 and
higher-carbon sugars.17 However, most of the addition reactions of organometallic reagents to 13
provide mixtures of diastereomers with the highest selectivity for 14 on the order of 2.5:1 to
3.0:1.27 Our previous studies35 of the addition of organometallic reagents to pentodialdo-1,4-
furanoses
revealed
high
levels
of
Felkin-Anh
stereoselectivity
when
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methyl(triisopropoxy)titanium36,37 was used instead of methyllithium or Grignard reagents.
Methyl(triisopropoxy)titanium has received limited attention in natural product synthesis, there
being only a few reported examples.38-41 In need of a highly stereoselective route to 14, we decided
to examine the addition of methyl(triisopropoxy)titanium to 13 and were able to achieve a d.r. of
19:1 in favor of desired 14 on a 4-gram scale.42 In the larger-scale preparation included in the
experimental section of this paper, the minor isomer was estimated at 0.7%. The structure of 14
was assigned based on comparison of NMR data with that published by Lemieux.33 Further
confirmation of the structure of 14 was provided by x-ray analysis of the corresponding p-
bromobenzoate, which shows that the configuration at the newly formed C-9 chirality center is
R.42 The yield and stereoselectivity of the methyltitanium-based route to chain-extension were
critical to the overall success of our synthesis. Two of the four chirality centers of pyranopyran 2,
C-6 and C-9, are established in 14, without the production of diastereomers.
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OH
OH
O
O
O
O
O
PhI(OAc)2 (1.1 eq)
(BAIB)
O
O
O
O
O
CH3Li (6.2 equiv)
ClTi(Oi-Pr)3 (6.2 eq)
O
O
H
H
O
diethyl ether;
CH2Cl2, 74%
TEMPO (10 mol%)
CH2Cl2, 74%
O
O
14
O
12
13
AcO
AcO
1. 60% HOAc
reflux
DBU (2.75 equiv)
CH3CN
O
O
OAc
OAc
33% HBr/HOAc
0 oC→rt
Br
2. Ac2O, pyr
0 oC, 89%
H
AcO
H
AcO
n-Bu4NBr (1 equiv)
20 min, 73%
85%
OAc
OAc
OAc
15 (α/β = 1:1.3)
16
AcO
Me Si
SiMe3
1.
AcO
3
SiMe3
SnCl4 (4 eq each)
CH2Cl2, -20 →0 oC
2. Na, K-tartrate, NaHCO3
O
O
H
H
OAc
AcO
OH
3.
NaBH4,CeCl3, CH3OH
88% overall
OAc
17
18
Scheme 3. Chain extension and C-glycosidation for pyranopyran synthesis
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The second challenge in a Type 2 route to pyranopyran 2 was the synthesis for the C-vinyl
glycoside needed for the lactone by RCM. Progress in the development of methods for the
synthesis of C-alkynyl glycosides43-45 suggested that a sequence consisting of C-alkynylation
followed by Lindlar reduction would provide the vinyl glycoside needed as the RCM precursor.
This approach was investigated by Franck and coworkers in their synthesis of C-galatosyl
ceramides;20 however, low overall yields C-vinyl glycoside from methyl galactopyranoside led the
authors to pursue an alternative synthesis. We were encouraged by the recent work of Isobe in
which the C-alkynylation of glycals with bis(trimethylsilyl)acetylene was carried out in good yield
and high stereoselectivity.43 As shown in Scheme 3, glycal 17 was prepared from 14 by a four-
step sequence consisting of deisopropylidenation, acylation, and bromination to give glycosyl
bromide 16, which was converted to 17 by a modification of the procedure of Ferrier.46,47
Substitution of DBU for diethylamine in the elimination step gave fewer side products. The C-
alkynylation of 17 with bis(trimethylsilyl)acetylene, using conditions described by Isobe for
glycals, followed by hydrolytic workup and sodium borohydride reduction as described gave
exclusively alkynyl alcohol 18 in 88% overall yield. Four transformations occur in this two-step
sequence: Ferrier rearrangement, enol acetate hydrolysis, b-elimination of the C-4 acetoxy group
and ketone reduction. The C-3-C-4 double bond in 18 is in the correct location of diplopyrone and
the configurations at C-1 and C-2 are introduced with complete stereoselectivity. At this point in
the synthesis, all four of the chirality centers are established.
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Final transformations culminating in a successful synthesis of the target pyranopyran 2 are
shown in Scheme 4. Desilylation of alkyne 18 with TBAF gave alkynyl glycoside 19 in 68% yield.
Selective reduction of the alkyne in 19 proved challenging with initial attempts with Lindlar’s
catalyst in methanol producing mixtures of C-vinyl and C-ethyl glycosides. Careful optimization
of this reaction in terms of catalyst preparation (pre-treatment with quinoline under hydrogen),
o
solvent (ethyl acetate), and temperature (0 C) led to an efficient reduction of 19 to C-vinyl
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glycoside 20 with only trace amount of over-reduced product. Acryloylation of 20 and RCM using
Grubbs II catalyst in toluene gave pyranopyran acetate 3. The addition of phenol to the RCM, as
suggested by Forman for electron-deficient alkenes,48 resulted in an improvement in yield (36%
to 59%) and a significantly reduced reaction time (41 h to 1 h). Deacylation of 3 was expected to
be challenging due to the presence of the unsaturated lactone that would preclude the use of sodium
methoxide in methanol, which is known to undergo conjugate addition with carbohydrate enones.49
Deacylation with methanol and p-toluenesulfonic gave (-)-diplopyrone that was contaminated with
a side-product that appeared to be the result of conjugate addition of methanol to the unsaturated
lactone. The ratio of the desired alcohol to the methanol adduct was estimated at 3:1. Lipases have
been shown to catalyze selective deacylations in carbohydrate esters and we had some previous
success in kinetic resolutions in our laboratory.50 Several lipases were assayed against 3 and
acceptable results were obtained with Amano PS-30 lipase which gave pyranopyran 2 in 63% yield
(80% based on recovered 3).
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AcO
AcO
H
O
n-Bu4NF, THF
H2, Lindlar Pd
O
0 oC
O
Cl
18
H
EtOAc, 0 oC,
1 h, 71%
NEt3, DMAP
69%
OH
10 min, 68%
OH
20
19
AcO
H
AcO
HO
Grubbs II (10 mol%)
phenol
H
O
H
H
H
O
O
lipase PS 30
O
1 h, toluene, Δ
O
O
O
O
THF-H2O
63%
H
H
59%
O
2
21
3
Scheme 4. Completion of the synthesis of pyranopyran 2
Not surprisingly, enzyme-catalyzed deacylation of 3 was slower than that reported for primary
alcohols, but nonetheless was reproducible and less complicated by side-product formation than
methanolysis. The route from commercially available 12 to pyranopyran 2 requires 13 steps, is
highly stereoselective, and proceeds in 4% overall yield. Our route compares favorably with the
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published route from butanediol (17 steps).11 The final product 2 obtained in our synthesis was
crystalline, while natural and synthetic diplopyrone 1 were reported as oils. Acetate 3 was also
crystalline and suitable for x-ray analysis. The ORTEP diagram for 3 is shown in Figure 3.
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AcO
H
H
5
4
O
9
3
2
4a
8a
6
10
7
O
O
8
1
H
Figure 3. ORTEP diagram of 3
One of the goals of this study was to attempt to clarify the differences in analytical data, in
particular NMR data, for diplopyrone isolated from the natural source with data obtained for
material in the Mohaptra synthesis of “putative (+)-diplopyrone.” Table 1 shows a comparison
of NMR data of our synthetic pyranopyran 2 with the NMR data reported for natural, (+)-
diplopyrone along with NMR data for material synthesized by Mohaptra.11 The NMR data of 2
synthesized in this study match that reported for putative (+)-diplopyrone by Mohaptra and
coworkers. The same discrepancies are observed when the data for 2 are compared to that reported
for the natural product, most notably in proton chemical shifts for H-4a and H-8a, but also in other
peak values. The structure was originally assigned on the basis of computational
methods and NMR data.1,2 While the structure is in need of revision, it is difficult to determine
exactly where it is incorrect. The 2D 1H NOE (NOESY) data did not show an effect from H-4a
to H-6, which would be consistent with a trans relationship of H-6 to both H-4a and H-8a.1
It is interesting to compare the conformations of compounds 3 and 4 obtained by x-ray
analysis with the calculated conformation reported in the literature for (+)-diplopyrone 1.2 To
facilitate this comparison, ORTEP structures of 3 and 4 can be inverted to show corresponding
absolute stereochemistry (Figure 4). On the basis of molecular mechanics (MMII) calculations
performed on the proposed structure of 1 two main conformers were predicted, in which the major
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differences were in the conformation of the hydroxyethyl side chain and the ring to which it is
attached. The major conformation predicted from these calculations is indicated as (a) in Figure
4, while the minor conformation is (b). Our crystallographic analyses of pyranopyran acetate 3
and nitrile 4 indicate solid state conformations for both that more closely match (b).
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Figure 4. Comparison of conformations of pyranopyran acetate 3 and nitrile 4 from
crystallographic data with calculated structures reported for diplopyrone 1
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1H
1H
1H
13C
13C
13C
OH
9
5
H
H
H
4
(natural) (synthetic) (synthetic) (natural) (synthetic) (synthetic)
Ref. 2
O
3
2
Ref. 11
This work
Ref. 2
Ref. 11
This work
4a
6
7
10
8a
O
O
8
1
8
9
2
3
162.7
124.8
161.7
123.3
161.8
123.2
6.23 (d, J
= 9.8 Hz)
6.07 (dd,
J = 10.0,
1.6 Hz)
6.07 (dd,
J = 10.0,
1.8 Hz)
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4
6.88 (dd, 6.88 (ddd,
6.88 (dd,
J = 10.0,
2.8 Hz)
140.0
64.9
144.2
64.9
144.3
64.8
J = 9.8,
5.8 Hz)
J = 10.0,
3.1, 1.0
Hz)
4a
6
4.09 (dd, 4.92 (ddd,
4.91 (cm,
5.8, 2.8,
1.8 Hz)
J = 5.8,
2.8 Hz)
J = 5.6,
3.1, 1.6
Hz)
4.16 (brs, 4.08 (ddt,
4.07 (cm,
J = 4.1,
3.0, 1.8
Hz)
6.01 (cm,
1H, J =
10.7, 1.8
Hz)
78.9
74.8
74.8
J = 4.2,
3.5, 1.8
Hz)
6.18 (d, J
= 10.4
Hz)
J = 4.2,
2.6, 2.6,
1.6 Hz)
6.06 (td,
J = 10.5,
1.6, 1.6
Hz)
7
132.6
123.0
69.7
129.6
124.7
70.0
129.6
124.6
70.0
8
6.14
6.04 (td,
J = 10.5,
2.6, 2.6
Hz)
4.97 (m,
J = 5.6,
2.6, 2.6,
1.6, 1.0
Hz)
6.05 (cm,
J = 2.0 Hz)
(ddd, J =
10.4, 4.6,
1.8 Hz)
4.65
(ddd, J =
4.6, 3.5,
2.8 Hz)
8a
4.97 (cm,
J = 10.7,
5.8, 2.0
Hz)
9
3.92 (dq,
J = 6.5,
4.2 Hz)
3.96 (dd,
J = 6.4, 4.2
Hz)
3.96 (dq,
1H, J =
6.4, 4.1
Hz)
69.0
17.8
69.7
18.7
69.6
18.7
10
1.21 (d, J 1.25(d, J = 1.24 (d, J
= 6.5 Hz) 6.4 Hz) = 6.5 Hz
Table 1. Comparison of NMR data of proposed diplopyrone and synthetic products
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BIOLOGICAL EVALUATION
4
5
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9
Pyranopyran 2, its acetyl derivative 3, and nitrile analog 4 were evaluated for phytotoxic
and antibacterial activity. Experimental procedures for the bioassays are included in the
Supporting Information. Antibacterial activity was evaluated using common pathogenic bacteria
in catfish. The largest segment of aquaculture in the United States of America (USA) is pond-
raised catfish, with most production occurring in Mississippi, and USA pond-raised catfish are
rated “Best Choice” by the Monterey Bay Aquarium Seafood Watch. The most common bacterial
diseases in pond-raised catfish are enteric septicemia in catfish (ESC) and columnaris disease
which are caused by Edwardsiella ictaluri and Flavobacterium columnare, respectively. These
diseases can cost the catfish industry over US$100 million annually.51 Catfish producers use
numerous approaches to manage bacterial diseases in catfish ponds including the use of medicated
feed containing the antibiotic florfenicol, live attenuated vaccines, and chemical therapeutants such
as peroxides, copper sulfate (CuSO4·5H2O), and potassium permanganate (KMnO4). However, the
use of chemicals in catfish ponds must be carefully monitored because these compounds have
broad-spectrum toxicities toward non-target species, such as catfish and planktons. The discovery
of efficacious natural or natural-based compounds that are toxic towards E. ictaluri and F.
columnare would provide catfish producers with another beneficial management approach.
During our initial screening to discover efficacious antibacterial compounds against E. ictaluri and
F. columnare, we evaluated pyranopyran 2 and derivatives 3, and 4 using a rapid bioassay (See
Supporting Information).
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Pyranopyran nitrile 4 was the most active of the three test compounds against F. columnare
ALM-00-173, with a MIC of 17.7±0 mg/L, 24-h IC50 of 7.5±0.5 mg/L, and 24-h cellular viability
IC50 of 16.8±0.9 mg/L (Table 2). The 24-h IC50 RDCF value of 14.0±0.9 indicates moderate
activity of nitrile 4 against F. columnare ALM-00-173 when compared to the drug control
florfenicol. Pyranopyran acetate 3 was only slightly active against F. columnare ALM-00-173
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based on a MIC at the highest test concentration of 238 mg/L. Similar results were observed for
evaluation of the three test compounds against E. ictaluri S02-1039, with 4 also as the most active
compound based on a MIC of 0.002±0 mg/L and 24-h IC50 of 7.7±1.2 mg/L (Table 3). In
comparison to the drug control, pyranopyran nitrile 4 had stronger activity than the drug control
florfenicol against E. ictaluri S02-1039 based on the MIC RDCF value of 0.5±0 but was
moderately active based on the 24-h IC50 RDCF value of 85.2±3.3. Pyranopyran 2 was moderately
toxic against E. ictaluri S02-1039 based on a MIC of 19.6±0 mg/L, but it was less toxic than the
nitrile with a 24-h IC50 of 46.1±1.0 mg/L. Because pyranopyran nitrile 4 had strong activity against
E. ictaluri S02-1039, the minimum bactericidal concentration (MBC) was evaluated and
determined to be >177.0 mg/L (the highest test concentration). These results indicate that
pyranopyran nitrile 4 is more likely to be bacteriostatic against E. ictaluri S02-1039 rather than
completely lethal at the active test concentrations.
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The current study is the first to evaluate and report the antibacterial activities of pyranopyrans
2, 3, and 4, against F. columnare and E. ictaluri, and, more specifically, the strong activity of 4
against E. ictaluri S02-1039 based on MIC results. Prior to efficacy studies (e.g., challenge tests),
additional studies need to be performed such as the determination of fish toxicity (e.g., LC50) for
the nitrile. Following positive efficacy results, other studies would need to be performed such as
the determination of pyranopyran nitrile 4 toxicity towards other non-target organisms in the
environment. In addition, approval by the United States Food and Drug Administration would
need to occur before aquaculturists could potentially use 4 as an alternative to currently approved
therapeutants and antibiotics.
Compounds 2, 3, and 4 were also evaluated for phytotoxic activity using the aquatic plant
Lemna paucicostata (L.) Hegelm. (duckweed). Only 4 was significantly phytotoxic, with an IC50
of less than 1 μM (Figure 5). At 33 μM some of the plants had no chlorophyll and were necrotic.
At 100 μM most of the plants were devoid of chlorophyll and necrotic. The phytotoxicity of the
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nitrile analog in the duckweed bioassay is similar to that of the commercial herbicides Isoproturon
(IC50 = 0.35 μM), imazethapyr (IC50 = 0.93 μM), and pendimethalin (IC50 = 0.38 μM) in the same
bioassay.52 A figure showing the visual symptoms of the effects of 4 on the growth of L.
paucicostata is included in the Supporting Information.
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Table 2. Results of bioassay evaluations of diplopyrone derivatives for antibacterial
activity against Flavobacterium columnare ALM-00-173. Numbers in parentheses are the
standard error of the means.
MICa
MTT 24-h IC50 RDCFd
24-h IC50
RDCFd
b
Test material
MICa
24-h IC50
0.54
b
c
Florfenicol
0.36
Pyranopyran (2)
Pyranopyran
acetate (3)
196.0 (0) 76.4 (2.0) 78.4 (0)
544.4 (0) 141.6 (3.6)
661.1 (0) >440.7
238.0 (0) >238.0
>238.0
16.8 (0.9)
b
Pyranopyran
nitrile (4)
17.7 (0)
7.5 (0.5)
49.2 (0)
14.0 (0.9)
aMIC = Minimum inhibitory concentration in mg/L. 24-h IC50 = 50% inhibition concentration
in mg/L.
c24-h IC50 (mg/L) as measurement of cell viability using the tetrazolium bromide dye MTT.
dRDCF = Relative-to-drug-control florfenicol; values closer to 1.0 indicate higher antibacterial
activity compared to florfenicol.
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Table 3. Results of bioassay evaluations of diplopyrone derivatives for antibacterial
4
5
activity against Edwardsiella ictaluri S02-1039. Numbers in parentheses are the standard
6
7
error of the means.
8
9
MICa
MTT 24-h IC50 RDCFd
24-h IC50
RDCFd
b
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b
c
Test material
MICa
24-h IC50
0.14
Florfenicol
0.36
Pyranopyran (2)
Pyranopyran
acetate (3)
19.6 (0)
46.1 (1.0)
22.1 (9.3)
54.4 (0)
661.1 (0)
0.5 (0)
329.0 (7.0)
1,181.5 (8.5)
85.2 (3.3)
238.0 (0) 165.4 (1.2) 90.4 (4.8)
Pyranopyran
nitrile (4)
0.002 (0) 7.7 (1.2)
64.6 (4.4)
b
aMIC = Minimum inhibitory concentration in mg/L. 24-h IC50 = 50% inhibition concentration
in mg/L.
c24-h IC50 (mg/L) as measurement of cell viability using the tetrazolium bromide dye MTT.
dRDCF = Relative-to-drug-control florfenicol; values closer to 1.0 indicate higher antibacterial
activity compared to florfenicol.
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350
300
250
200
150
100
50
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60
0
-50
0.1
1
10
100
1000
Concentration (µM)
Figure 5. Dose/response curves for the nitrile pyranopyran (4) after 6 days of exposure. Error
bars are ± 1 SE. The control (no test compound) is at 0.3 μM on the graph. The Y axis is the
percent growth increase after exposure to the test compounds.
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In a recent study, the antimicrobial activities of secondary metabolites, including diplopyrone,
produced by the oak pathogenic fungus Diplodia corticola were isolated and determined to
possess antifungal activities against plant pathogens.53 Compounds 2, 3, and 4 were evaluated as
fungicides using isolates of the fungal plant pathogens Colletotrichum acutatum Simmonds, C.
fragariae Brooks, and C. gloeosporioides. Details of the fungicide bioassay are provided in the
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Supporting Information. None of the diplopyrone analogs were active as fungicides.
CONCLUSION
Highly stereoselective syntheses of the enantiomer of the structure originally proposed for
the phytotoxin diplopyrone and structural analogs have been achieved using carbohydrate starting
materials in two complementary approaches. Key steps in the synthesis of pyranopyran 2, the
enantiomer of putative diplopyrone, are a highly stereoselective pyranose chain-extension based
on methyltitanium, preparation of a vinyl glycoside via Isobe C-alkynylation-rearrangment and
chemoselctive reduction, and RCM-based pyranopyran construction. The synthesis of nitrile
analog 4 is based on an alternative strategy using a Ferrier C-glycosidation to introduce the nitrile
group and Wittig chain-extension, both of which are completely stereoselective. X-ray
crystallographic analyses were obtained for the pyranopyran acetate 3 and the nitrile analog 4. Our
syntheses coupled with new crystallographic and NMR data provide further confirmation that the
structure originally proposed for (+)-diplopyrone may require revision. Biological assays carried
out at the USDA-ARS-Natural Products Utilization Research Unit revealed potent antibacterial
activity for pyranopyran nitrile 4 against common bacterial pathogens in fish. Compound 4 is
significantly more active in vitro than a commercially used antibiotic used against E. ictaluri, a
common pathogen in catfish and it is also phytotoxic as determined by bioassay using L.
paucicostata (duckweed). The antibiotic and phytotoxic activities of 2, 3, and 4 demonstrate the
potential of these and similar pyranopyrans as antibiotics and herbicides. Significantly, the
bioassay results show an intriguing structure-activity relationship for the C-6 position on the
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pyranopyran scaffold that can be exploited using our synthetic methodology to generate additional
analogs for biological testing. Both the routes to 2 and 3 (Type 1) and the route to 4 (Type 2) are
amenable to modification to introduce other functionality at this key position. These studies and
further biological evaluation are currently in progress.
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EXPERIMENTAL SECTION
General Methods.
Melting points were recorded on a Thomas-Hoover apparatus and they are uncorrected.
Thin-layer chromatography was carried out on aluminum foil-backed silica gel plates coated with
a fluorescent indicator. Plates were developed with cerium molybdate stain. Flash chromatography
was carried out using 230-400 mesh silica gel. Optical rotations were recorded on a Perkin-Elmer
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Model 341 polarimeter at 20 or 23 C as indicated. HRMS analyses were conducted at the
University of Illinois Mass Spectrometry Laboratory. NMR spectra were recorded on a Varian
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(Agilent) Mercury 300 Plus spectrometer in CDCl3 for H NMR at 300.0 MHz, tetramethysilane
13
reference, δ = 0.0 ppm, and, C NMR 75.4 MHz, CDCl3 reference, δ = 76.9 ppm. Spectral
assignments were confirmed using COSY and DQCOSY experiments. Data were collected on a
Brüker-AXS Kappa APEX II CCD diffractometer with 0.71073 Å Mo-Ka radiation (See
Supporting Information). Reagents and starting materials were purchased from Sigma-Aldrich
with the exception of 5% Pd/CaCO3 (Alfa Aesar), chlorotitanium triisopropoxide (Strem), and tri-
O-acetyl-D-galactal (TCI).
a
2,3-Dideoxy- -D-threo-hex-2-enopyranosyl cyanide (7). To a solution of diester 6 365
mg (1.5 mmol) in methanol (15 mL) was added p-toluenesulfonic acid monohydrate (16 mg, 0.085
mmol). The mixture was stirred under reflux for 7 h and monitored by TLC (50% ethyl
acetate/hexanes) until starting material was consumed (Rf ~ 0, 50% ethyl acetate/hexanes). The
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reaction mixture was concentrated to a small volume (2-3 mL) and cooled to -20 C to give
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crystalline diol 7 (120 mg, 52%). mp 134–137 C; lit54 mp 134– 135 C; [a]D -351.3 (c 0.97,
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5
EtOH); lit [a]D -362.4 (c 1.0, EtOH). HRMS (ESI): m/z calcd for C7H9NO3 [M + Na]+:
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178.0480. Found 178.0486. Larger scale deacylation of 6 (10.9 g, 45.8 mmol) was carried out by
the same procedure in anhydrous methanol (140 mL) with p-toluenesulfonic acid monohydrate
(430 mg, 2.3 mmol, 5 mol%) to afford crude diol 7 (6.91 g 97%) as an amorphous solid after
concentration of the reaction mixture and drying. This product was suitable for use without further
purification in the silylation step.
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a
4,6-di-O-tert-butyldimethylsilyl-2,3-dideoxy- -D-threo-hex-2-enopyranosyl cyanide
(8). To a stirring solution of diol nitrile 7 (6.13 g, 39.5 mmol) in anhydrous DMF (15 mL) was
added imidazole (13.5 g, 198 mmol, 5 eq) and tert-butyldimethylsilyl chloride (15.7 g, 104 mmol,
2.6 eq). The reaction was stirred at rt 12 h then quenched with sat’d aq NH4Cl sol’n and extracted
with ethyl acetate (3 x 30 mL). The combined organic extracts were washed with water (2 x 20
mL), brine (20 mL) then dried (Na2SO4) and concentrated to a syrup that was purified by flash
chromatography with 5% ethyl acetate/hexanes to give 14.3 g (94%) of 8 as an oil that solidified.
Rf = 0.79 (20% ethyl acetate/hexanes); mp 31 – 33 oC; [a]D23 -181.2 (c 0.96, CH2Cl2); 1H NMR
(CDCl3): d 6.12 (ddd, 1H, J2,3 = 10.0, J3,4 = 5.4, J1,3 = 2.0 Hz, H-3), 5.84 (dd, 1H, J2,3 = 10.0, J1,2
= 3.8 Hz, H-2), 5.08 (dd, 1H, J1,2 = 3.8, J1,3 = 2.1 Hz, H-1), 4.03 (dd, 1H, J3,4 = 5.4, J4,5 = 1.9 Hz,
H-4), 3.86 – 3.70 (m, 3H, H-5,6,6’), 0.90, (t-BuMe2Si), 0.89
(t-BuMe2Si), 0.08 (t-BuMe2Si), 0.07 (t-BuMe2Si); 13C{1H} NMR( CDCl3): d 130.4, 123.5, 115.9,
76.8, 62.7, 61.9, 61.2, 25.8, 25.7, 18.2, -4.1, -4.8, -5.2, -5.3. HRMS (CI-magnetic sector): m/z: [M
+ H]+ Calcd for C19H38NO3Si2 384.2390; Found 384.2389.
(2S,5R,6S)-5-(tert-butyldimethylsilyl)oxy)-6-formyl-5,6-dihydro-2H-pyran-2-
carbonitrile (10). A mixture of di-O-TBS ether 8 (1.40 g, 3.65 mmol) and PPTS (185 mg, 0.74
mmol, 20 mol %) in anhydrous methanol (20 mL) was stirred at rt for 10 h, until TLC showed
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consumption of the starting material. The reaction mixture was concentrated rapidly under
reduced pressure using a high vacuum pump connected to a rotary evaporator without heating and
the resulting oil diluted with dichloromethane (45 mL) and washed with cold water (15 mL), cold
sat’d aq NaHCO3 sol’n (10 mL), dried (Na2SO4) and concentrated to yield 800 mg (81%) alcohol
9 as an oil. Rf = 0.60 (25% ethyl acetate/hexanes); HRMS (CI): m/z calcd for C13H24NO3Si [M +
H]+: 270.1525. Found 270.1533. The crude product alcohol (630 mg, 2.34 mmol), was dissolved
in anhydrous dichloromethane (5 mL) and to this solution was added anhydrous DMSO (2.0 mL,
2.2 g, 28 mmol, 12 eq), and diisopropylethylamine (2.5 mL, 1.9 g, 14 mmol, 6 eq). The mixture
was cooled to 0 oC and SO3•pyridine (1.86 g, 11.7 mmol, 5 eq) was added and the reaction stirred
for 2 h. Water (30 mL) was added and the mixture was extracted with ethyl acetate (3 x 20 mL .
The combined extracts were washed with water (4 x 10 mL), brine (10 mL), dried (Na2SO4), and
concentrated under reduced pressure. Flash chromatography (16% ethyl acetate/hexanes) gave 540
mg (86%) of 10 as an oil.
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Rf = 0.70 (25% ethyl acetate/hexanes); [a]D -282 (c 1.1, CHCl3); H NMR (CDCl3): d 9.61 (s,
1H, CHO), 6.15 (ddd, 1H, J2,3 = 10.1, J3,4 =5.1, J1,3 = 2.1 Hz, H-3), 5.93 (ddd, 1H, J2,3 = 10.1, J1,2
= 3.7, J2,4 = 0.7 Hz, H-2), 5.28 (dd, 1H, J1,2 = 3.5, J1,3 = 1.9 Hz, H-1), 4.45 (ddd, 1H, J3,4 = 5.3, J4,5
= 3.0, J = 0.6 Hz, H-4), 4.30 (d, 1H, J4,5 = 2.9 Hz, H-5), 0.84 (t-BuMe2Si), 0.08 (t-BuMe2Si);
13C{1H} NMR (CDCl3): d 198.4, 129.4, 123.5, 115.1, 79.4, 62.3, 62.1, 25.5, 17.9, -4.2, -5.0.
HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C13H21NO3SiNa 290.1188; Found 290.1194.
Ethyl (Z)-3-((2R,3R,6S)-3-((tert-butyldimethylsilyl)oxy)-6-cyano-3,6-dihydro-2H-pyran-2-
yl)acrylate (11). To a stirring solution of aldehyde 10 (402 mg, 1.5 mmol), in anhydrous methanol
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(25 mL), at 0 C was added (carbethoxymethylene)-triphenylphosphorane (651 mg, 1.86 mmol,
1.5 eq) and the reaction was stirred 3 h. Concentration of the mixture and flash chromatography
(5% ethyl acetate/hexanes) gave 310 mg (61%) of cis ester 11 as a colorless oil. Rf = 0.64 (10%
ethyl acetate/hexanes); [a]D20 -194 (c 1.8, CHCl3); 1H NMR (CDCl3): d 6.22 (dd, 1H, J6,7 = 11.9,
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J5,6 = 6.8 Hz, H-6), 6.13 (ddd, 1H, J2,3 = 10.4, J3,4 = 5.4, J1,3 = 2.1 Hz, H-3), 5.93 (dd, 1H, J6,7
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11.8, J5,7 = 1.4 Hz, H-7), 5.87 (ddd, 1H, J2,3= 10.1, J1,2 = 4.0, J2,4 = 0.6 Hz,H-2), 5.36 (ddd, 1H,
J5,6 = 6.7, J4,5 = 2.5, J5,7 = 1.7 Hz, H-5), 5.12 (dd, 1H, J1,2 = 3.9, J1,3 = 2.1 Hz, H-1), 4.37 (dd, 1H,
J 3,4= 5.4, J4,5 = 2.5 Hz, H-4), 4.20 (q, 2H, J = 7.2 Hz), 1.30 (t, 3H, J = 7.2 Hz), 0.85 (t-BuMe2Si),
0.018 (t-BuMe2Si), 0.012 (t-BuMe2Si); 13C{1H} NMR (CDCl3): d 165.3, 145.6, 130.6, 122.8,
120.6, 115.9, 73.9, 63.0, 62.2, 25.7, 18.1, 14.2, -4.34, -4.85.
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HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C17H27NO4SiNa 360.1607; Found 360.1615.
(4aR,6S,8aR)-6-Cyano-6,8a-dihydropyrano-[3,2-b]pyran-2(4aH)-one (Diplopyrone
nitrile 4). Ester 11 (130 mg, 0.385 mol), and TsOH (11 mg, 15 mol%) were stirred under reflux
in anhydrous methanol (10 mL) for 4.5 h. The mixture was then concentrated under reduced
pressure and dried briefly under vacuum to remove trace methanol. Toluene (10 mL) was added
and the mixture stirred overnight under reflux. The crude product was purified by flash
chromatography (30% ethyl acetate/hexanes) to give 60 mg (88%, 34% overall from 6) of
crystalline 4. Rf = 0.23 (30% ethyl acetate/hexanes); mp 162 – 164 oC; [a]D20 -279o (c 1.0 CHCl3);
1H NMR (CDCl3): d 6.91 (dd, 1H, J3,4 = 9.8, J4,4a = 5.8 Hz, H-4), 6.33 (m, 1H, H-8), 6.31 (dd, 1H,
J3,4 = 9.8, J3,4a = 0.6 Hz, H-3), 6.17 (ddd, 1H, J7,8 = 10.1, J6,7 = 4.0, J6,8 = 0.8 Hz, H-7), 5.18 (ddd,
1H, J6,7 = 4.1, J6,8 = 2.0, J = 0.5 Hz, H-6), 4.74 (dd, 1H, J8,8a = 5.3, J4a,8a = 3.0 Hz, H-8a), 4.45
13
(ddd, 1H, J4,4a = 5.8, J4a,8a = 2.9, J4a,8 = 0.5 Hz, H-4a); C{1H} NMR (CDCl3): d 161.3, 138.3,
126.6, 125.6, 125.0, 114.9, 67.5, 62.7, 62.3. HRMS (ESI-TOF) m/z: M+ Calcd for C9H7NO3
177.0426; Found 177.0431.
1,2;3,4-di-O-isopropylidene-α-D-galacto-1,6-dialdo-hexopyranose (13). A mixture of
1,2:3,4-diisopropylidene-α-D-galactopyranose 12 (14.99 g, 57.6 mmol) and TEMPO (900 mg,
5.76 mmol, 10 mol %) was stirred in dichloromethane (62 mL, anhydrous) at room temperature as
diacetoxyiodobenzene (BAIB) (20.42 g, 63.4 mmol, 1.1 eq,) was added to the reaction mixture.
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After several minutes of stirring, all the BAIB dissolved in the clear, bright orange reaction
mixture. After stirring 4 h the reaction mixture was diluted with dichloromethane (35 mL), washed
with sat. aq Na2S2O3 (100 mL), and the aqueous layer was extracted with dichloromethane (4 X
50 mL). The combined organic layers were then washed with sat. aq NaHCO3 sol’n (100 mL)
followed by brine (100 mL), dried via sodium sulfate (anhydrous) and concentrated via rotary
evaporation to yield a bright orange liquid that was purified by flash chromatography (40:60 ethyl
acetate/hexanes) yielding 10.99 (74 %) of 13 as a bright yellow oil that was stored at -23 °C. Rf =
0.67 (1:1 ethyl acetate/hexanes); [a]D20 -125.5 (c 0.98, CHCl3); lit55 [a]D20 -131o (c 0.9, CHCl3);
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lit56 [a]D21 -113o (c 3.4, CHCl3). The 1H NMR spectra of 13 matched that reported.32
7-Deoxy-1,2;3,4-di-O-isopropylidene-D-glycero-α-D-galacto-heptopyranose (14).
In a nitrogen glove box, chlorotitanium triisopropoxide (83 g, 318.1 mmol, 6.2 eq) was added to a
Schlenk flask equipped with a teflon plug and a magnetic stir bar. The Schlenk flask was fitted
with an addition funnel, sealed with a rubber septum, and removed from the glove box. The
Schlenk flask was then connected to a Schlenk line, and the flask was placed under argon. Diethyl
ether (220 mL, anhydrous) was added to the flask via a cannula transfer. The mixture was stirred
at room temperature until all the solids dissolved, then the reaction mixture was cooled to -50 °C
(CO2(s)/acetone bath). Methyllithium (199 mL, 1.6 M in diethyl ether, 318.1 mmol, 6.2 eq) was
added to the dropping funnel via cannula transfer. The methyllithium was then added to the
reaction mixture at a rate so that the cooling bath temperature did not rise above -50 °C. Following
this addition, the light-yellow reaction mixture was stirred for 1 h (the reaction warmed to -25 °C
during this time) and a white precipitate formed. Under an argon counter flow, the addition funnel
was replaced with a rubber septum. While maintaining the cooling bath temperature at -25 °C,
the diethyl ether was removed in vacuo (using the Schlenk line, with a liquid nitrogen cooled trap
to collect solvent). When the solvent was removed, the methyl(triisopropoxy)titanium thus formed
was kept under argon.
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Methyl(triisopropoxy)titanium was suspended in dichloromethane (220 mL, anhydrous),
and the Schlenk flask was cooled to -78 °C (CO2/acetone bath). A solution of 1,2:3,4-di-O-
aldehyde 13 (13.24 g, 51.3 mmol) in dichloromethane (50 mL, anhydrous) was added dropwise
via a syringe while maintaining a cooling bath temperature of -78 °C. The reaction mixture was
allowed to slowly warm over 22 h under argon (a needle was affixed to an argon balloon and was
inserted through the septum of the Schlenk flask in order to prevent pressure build up). At this
point, the cooling bath reached 20 °C. The reaction mixture was quenched via the addition of
water. The first 10 mL of water was added dropwise until vigorous effervescence subsided.
Following this, an additional 240 mL of water was added to the reaction mixture followed by 300
mL of diethyl ether. The entire mixture (including solids formed) was transferred into an
Erlenmeyer flask and stirred vigorously for 20 min. After filtration, the solids were washed with
diethyl ether (200 mL), the filtrate was transferred to a separatory funnel, the organic and aqueous
layers were separated, and the aqueous phase was extracted with diethyl ether (2 X 200 mL). The
combined organic extracts were washed successively with H2O (250 mL), brine (400 mL), dried
via MgSO4 (anhydrous) and concentrated to yield 10.36 g (78 %) of a bright yellow oil that was
filtered through a pad of SiO2, washed with diethyl ether (300 mL), and concentrated under
reduced pressure to yield 10.34 g (74 %) of 14 as a pale yellow oil that was stored at -23 °C. Rf =
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0.45 (1:1 ethyl acetate/hexanes) and used without further purification. The H NMR spectrum
matched that reported,37 except for the presence of the minor b-L-glycero diastereomer that was
seen previously (19:1 dr) but in this larger-scale preparation was estimated at approximately 0.7%.
1,2,3,4,6-Penta-O-acetyl-7-deoxy-D-glycero-D-galacto-heptopyranoside (15).
A mixture of 1,2;3,4-di-O-isopropylidene-7-deoxy-D-glycero-D-galacto-heptopyranoside 14
(10.30 g, 37.55 mmol) and acetic acid/water (80 mL, 60 % v/v) was heated and stirred under reflux.
As the reaction progressed the reaction mixture became dark brown in color. After 2 h, TLC
analysis (1:1 ethyl acetate/hexanes) showed the disappearance of starting material and the reaction
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mixture was allowed to cool to room temperature. Once cooled the solvent was removed under
reduced pressure (vacuum pump) at a temperature no greater than 30 °C. The resulting brown oil
was taken up in ethanol (anhydrous), concentrated under reduced pressure, and dried under high
vacuum. The crude material was dissolved in pyridine (150 mL, anhydrous) and cooled to 0 °C.
Acetic anhydride (110 mL) was added via syringe. After five minutes of stirring at 0 °C, the
reaction mixture was allowed to warm to room temperature and stirred 22 h. The reaction was
quenched by the addition of 110 mL of H2O and stirred for 10 minutes. Following this, the reaction
mixture was taken up in 100 mL of dichloromethane and the organic and aqueous layers were
separated. The aqueous layer was extracted with dichloromethane (4 x 100 mL). The combined
organic layers were washed with sat’d aq CuSO4 sol’n (4 x 500 mL), sat’d aq NaHCO3 sol’n (250
mL), brine (250 mL), dried (Na2SO4), and concentrated under reduced pressure to yield 13.5 g (89
%) of 15 as a foam in a 1:1.3 mixture of α/β anomers that was stored at 0 °C. Rf = 0.46 (1:1 ethyl
acetate/hexanes). The 1H NMR spectrum of 15 matched that reported.41 13C{1H} NMR (CDCl3):
δ170.3, 170.3, 170.15, 170.00, 169.91, 169.74, 169.68, 169.37, 169.04, 168.89, 92.42, 92.42,
89.72, 77.23, 76.21, 73.12, 70.85, 67.75, 66.21, 65.90, 20.91, 20.87, 20.82, 20.67, 20.58, 17.5.
HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C17H24O11Na 427.1216; Found 427.1227.
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2,3,4,6-Tetra-O-acetyl-7-deoxy-D-glycero-α-D-galacto-heptopyranoside bromide (16).
1,2,3,4,6-Penta-O-acetyl-7-deoxy-D-glycero-α/β-D-galacto-heptopyranoside 15 (13.45 g, 33.26
mmol) was cooled to 0 °C then dissolved in 33 % wt HBr solution in glacial acetic acid (75 mL).
The dark brown reaction mixture was allowed to stir at 0 °C for 5 min. Following this the reaction
mixture was allowed to warm to room temperature. After 3 h, TLC analysis (1:1 ethyl acetate/
hexanes) showed the disappearance of starting material, the reaction mixture was taken up in
dichloromethane (250 mL) and washed with ice water (200 mL). The aqueous layer was extracted
with dichloromethane (2 x 200 mL), the combined organic layers were washed with cold water
until the washings were neutral (4 x 500 mL), dried (Na2SO4), and concentrated via reduced
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pressure to 12.01 g (85 %) of 16 as a foam that was stored at -23 °C. Rf = 0.63 (1:1 ethyl
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acetate/hexanes); [α]D +197.8 (c 1.4, CHCl3); 1H NMR (CDCl3): δ 6.66 (d, 1H, J1,2 = 3.97 Hz,
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H-1), 5.54 (dd, 1H, J3,4 = 3.29, J5,4 = 1.23 Hz, H-4), 5.39 (dd, 1H, J3,4 = 3.29, J3,2 = 10.63 Hz, H -
3), 5.02 (dq, 1H, J6,7 = 6.24, J6,5 = 9.38 Hz, H-6), 5.02 (dd, 1H, J1,2 = 3.97,J2,3 = 10.63, J2,7 = 6.24
Hz, H-2), 4.12 (ddd, 1H, J5,6 = 9.38 Hz, H-5), 2.11, 2.10, 2.00, 1.99 (4s, 12H, OCOCH3), 1.26 (d,
3H, J7,6 = 6.24, CH3), 13C{1H} NMR (CDCl3): δ 170.0, 169.7, 88.3, 75.1, 67.9, 67.8, 65.9, 65.7,
53.5, 20.8, 20.7, 20.5, 20.4, 17.3. HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C15H21O9BrNa
447.0267; Found 447.0274.
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2,3,4,6-Tetra-O-acetyl-7-deoxy-1,5-anhydro-D-gluco-hept-1-enitol (17). A mixture of
glycosyl bromide 16 (11.87 g, 27.70 mmol) and tetra-n-butylammonium bromide (8.93, 27.7
mmol, 1 eq) was stirred in acetonitrile (6 mL, anhydrous) as DBU (11.15 mL, 76.2 mmol, 2.75 eq)
was added via a syringe. Immediately following the addition of DBU the reaction became black.
After 20 min TLC analysis (1:1 ethyl acetate/hexanes) showed the disappearance of starting
material and the reaction mixture was diluted with 6 mL of dichloromethane and concentrated via
rotary evaporation. The resulting syrup was taken up in dichloromethane (150 mL) and washed
with 10 % HCl (150 mL), followed by saturated NaHCO3 (150 mL), dried (Na2SO4), and
concentrated via rotary evaporation to yield a dark oil that was purified by flash chromatography
(1:1 ethyl acetate/hexanes) to yield 6.94 g (73 %) of 17 as a yellow oil that was stored at 0 °C. Rf
20
= 0.50 (1:1 ethyl acetate/hexanes); [α]D + 57.7 (c 1.3, CHCl3); 1H NMR (300 MHz, CDCl3): δ
6.63 (d, 1H, J1,3 = 1.75 Hz, H-1), 5.89 (ddd, 1H, J3,4 = 5.04, J3,1 = 1.75 Hz, H-3), 5.53 (dd, 1H, J4,3
= 5.04, J4,5 = 0.95 Hz, H-4), 5.02 (dq, 1H, J6,7 = 6.25, J6,5 = 9.15 Hz, H-6), 3.99 (ddd, 1H, J5,6
=
9.15 Hz, H-5), 2.11, 2.02, 2.01, (3s, 12H, OCOCH3), 1.32 (d, 3H, J7,6 = 6.25, CH3); 13C{1H} NMR
(CDCl3): δ 170.3, 169.9, 169.7, 169.4, 138.8, 127.3, 77.6, 66.4, 64.0, 62.1, 20.9, 20.5, 20.4, 17.2.
HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C15H20O9Na 367.1005; Found 367.1005.
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