Selective molecular sequestration with concurrent natural product functionalization and derivatization: From crude natural product extracts to a single natural product derivative in one step

Article abstract of DOI:10.1021/jo201361s

A resin-bound nitroso compound sequestered a single unexpected component from crude plant seed extracts. Several plants, including Piper nigrum, Eugenia caryophyllata, and Pimenta dioica, were extracted with organic solvent in the presence of a nitroso-co

Full text of DOI:10.1021/jo201361s

Note
pubs.acs.org/joc
Selective Molecular Sequestration with Concurrent Natural Product
Functionalization and Derivatization: From Crude Natural Product
Extracts to a Single Natural Product Derivative in One Step
Viktor Krchnak, Jaroslav Zajícek, Patricia A. Miller, and Marvin J. Miller*
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́
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Department of Chemistry and Biochemistry, 251 Nieuwland Science Hall, University of Notre Dame, Notre Dame, Indiana 46556,
United States
S
* Supporting Information
ABSTRACT: A resin-bound nitroso compound sequestered a single unexpected component from crude plant seed extracts.
Several plants, including Piper nigrum, Eugenia caryophyllata, and Pimenta dioica, were extracted with organic solvent in the
presence of a nitroso-containing resin. The nitroso resin selectively sequestered a single compound, β-caryophyllene, via a
chemo- and regioselective ene reaction. The ene product was released from the resin, and proper selection of the solid-phase
linker and cleavage cocktail allowed concomitant further transformation of the primary ene product to a novel functionalized
polycycle. Preliminary studies indicate that the new hydroxylamine-containing natural product derivatives have antibiotic activity.
any current drugs originated from structural types
created by nature.1,2 A typical scenario of natural
products-based drug discovery includes identification of the
producing source, isolation of particular components, assessing
their activities, and chemical transformation of natural products
in order to improve pharmacological properties.
Nitroso derivatives are known to selectively react with
compounds containing carbon−carbon double bonds: reactions
of isolated alkenes with allylic C−H bonds form ene products,3
reactions with conjugated dienes lead to hetero-Diels−Alder
adducts.4,5 We previously reported that the nitroso hetero-
Diels−Alder (HDA) reaction is effective for simultaneous
functionalization and derivatization of a number of structurally
varied and highly functionalized diene-containing natural
products to promote Modular Enhancement of Nature’s
Diversity (MEND) in solution6 and on solid phase.7−9 The
HDA reaction with even relatively simple diene-containing
natural products, like piperine, provided access to structurally
diverse compounds, including structurally unrelated hetero-
cycles.10
compounds toward (di)enes and immense differences in
reactivity among individual (di)enes, we decided to evaluate
this reaction for direct and selective sequestration of individual
components from crude extracts obtained from natural sources.
Herein we report the use of a solid-phase nitroso agent for
isolation and modification of a single natural product from
crude extracts using a very efficient and operationally simple
procedure.
To demonstrate the selective sequestration concept, we
turned our attention to natural products from herbs and spices:
their extracts contain complex mixtures of natural products
including diene- and ene-containing species. The first experi-
ments were carried out with extracts from black peppercorn
(Piper nigrum). The main component (∼2−4% by weight,
depending on the source and processing) of peppercorn is
piperine (1, Figure 1), responsible for the pungent taste of
pepper.11 We have previously shown that piperine reacted with
resin-bound nitroso dienophiles and provided access to a
diverse set of derivatives including heterocyclic compounds.8
Black peppercorn also contains piperettine (2), a vinylogous
homologue of piperine.11,12 The amount of piperettine is about
M
The relative reactivity of dienes with nitroso compounds
extends over several orders of magnitude. For example, 1,3-
cyclohexadiene is >100 times more reactive than 2,4-
hexadiene.8 Because of the high selectivity of nitroso
Received: June 29, 2011
Published: November 7, 2011
© 2011 American Chemical Society
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Note
selectivity (only traces of piperine HDA adduct were detected).
Thus, the ene reaction of β-caryophyllene with the nitroso resin
is >1000 times faster than the [4 + 2] cycloaddition reaction
with the major component, piperine. In order to further
evaluate the relative reactivity of β-caryophyllene with respect
to dienes, we performed a competitive reaction of the nitroso
agent 3 with equimolar amounts of β-caryophyllene (5) and
cyclohexadiene (9), one of the most reactive dienes in nitroso
cycloadditions.7 As now expected, after cleavage from the resin,
a product ratio of 1:4 in favor of the β-caryophyllene product
(4) was obtained (Scheme 3).
Although the rearrangement of the initially formed ene
product produced a structurally very different and interesting
scaffold, we anticipated that repetition of the experiments with
a nitroso resin amenable to product release under nonacidic
conditions would allow isolation of the ene product itself. Thus,
a silicon-based linker (11), from our previous studies,5,6 was
used, and the ene product (12) was released by tetrabutyla-
monium fluoride (Scheme 4). We also demonstrated that the
rearranged product (13) could be produced by TFA-mediated
release. Thus, two different cleavage conditions provided access
to two structurally diverse scaffolds from a single resin-bound
precursor. Independently, product 13 was also prepared from
resin 14 using the TFA cleavage cocktail.
While pyridylnitroso reagents, such as 11, can be generated
and separately reacted with enes and dienes, acylnitroso agents
are unstable and typically are generated in situ by oxidation of
their hydroxamic acid precursor. To avoid problems with
oxidatively sensitive substrates, acylnitroso agents can be
thermally regenerated from dimethylanthracene14 or α-
terpinene cycloadducts (15).15 Indeed, exposure of β-
caryophyllene to a resin-bound HDA α-terpinene adduct
(15), even at 100 °C as required for the retro HDA reaction,
produced the ene product cleanly (Scheme 5). Interestingly,
after TFA-mediated release from the resin, we were able to
isolate and characterize the rearranged product (16). However,
the 1,2-oxazetidine ring-containing product (16) hydrolyzed in
aqueous solutions and afforded 17, analogous to compounds
obtained from arylnitroso resins.
Similar reactions with a variety of peppercorns (see the
Supporting Information) all yielded the β-caryophyllene ene
product, although the amounts varied and the yields where
reduced by ∼50% when 24-h-old ground rather than freshly
ground peppercorn was used. Reactions of crude extracts of
freshly ground clove (Eugenia caryophyllata),16 known to
contain 1.4% of β-caryophyllene, and allspice (Pimenta
dioica),17 where the main essential oil is eugenol, also produced
the β-caryophyllene ene reaction product.
Figure 1. Structures of piperine (1) and piperettine (2).
15% of that of piperine; however, on the basis of our previous
results, it was anticipated to be more reactive than piperine in
the HDA reactions. Analysis of extracts from 10 peppercorns
that originated from different geographical regions revealed
insignificant variation in piperine and piperettine content (see
the Supporting Information). The sequestration experiments
were then arbitrarily carried out with Lampong Black
peppercorn.
The crude dichloromethane extract of Lampong Black
peppercorn was exposed to 6-nitrosonicotinic acid attached
to a Rink amide resin (3). The resin was separated and
product(s) released by treatment with a TFA-containing
cleavage cocktail.8,10 LCMS analysis revealed the presence of
two major components: an alcohol and its TFA ester (the later
eluting ester was then hydrolyzed to the alcohol). However, the
MS spectrum did not correspond to the expected HDA adduct
of either piperine or piperettine. Isolation and full character-
ization revealed that the product was a totally unrelated
cyclobutane-containing tricyclic product (4) derived from a
selective ene reaction (Scheme 1).
Black peppercorn contains essential oils, including potential
ene-reactive terpenoids with the total amount of oil reportedly
being 0.6 mg/g of pepper (0.06%).13 However, none of the
known components of pepper contained the carbon skeleton
found in the isolated ene product. Pepper does contain one
known cyclobutane-containing component, β-caryophyllene
(5); however, its content may vary from 1 to 70%.13 β-
Caryophyllene is a sesquiterpene containing two isolated
double bonds amenable to ene reactions, not cycloadditions,
with nitroso compounds. On the basis of detailed structural
analyses, a selective ene reaction occurred at the C5-carbon of
β-caryophyllene, and subsequent treatment with TFA formed a
bridged scaffold (Scheme 2). Acid-mediated rearrangement of
β-caryophyllene is known.11 The rearrangement was remark-
ably clean as no other side product was detected. The identity
of the ene product was also confirmed by reacting authentic,
commercially available β-caryophyllene with resin-bound nitro-
so species.
The structure and stereochemistry (Figure 2) of all
compounds was determined by measuring and analyzing 1D
1H and 13C NMR, and 2D homo- (gCOSY, TOCSY) and
Despite the molar ratio of piperine/caryophyllene in pepper
being ∼150:1, the reaction of the nitroso resin with the crude
extract afforded the β-caryophyllene product with exquisite
Scheme 1. Reaction of Nitroso Resin with Peppercorn Extract
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Scheme 2. Nitroso Ene Reaction and Acid-Mediated Rearrangement of the β-Caryophyllene Skeleton
teria, including M. vaccae, a common model for M. tuberculosis.
The generally strong activity against M. luteus is notable since
this pathogen is resistant to the broad-spectrum antibiotic,
ciprofloxacin, the control antibiotic (Table 4, Supporting
Information). Subsequently, we have determined that the
nitroso chemistry can be broadly applied to ene substrates to
generate libraries of antibiotics that are selectively active against
cipro-resistant M. luteus.19
Scheme 3. Evaluation of Relative Reactivity of
Cyclohexadiene and β-Caryophyllene with a Resin-Bound
Nitroso Agent
In conclusion, we demonstrated a very efficient and
operationally simple procedure for sequestration and con-
current transformation of nitroso-reactive natural products.
Upon exposure of solutions of complex mixtures from crude
natural product extracts to nitroso resin under mild conditions,
a single olefinic natural product, β-caryophyllene, present in
black peppercorn only in 0.06%, was sequestered. Use of
different cleavage conditions allowed transformation of the
primary ene products to structurally diverse molecules.
Preliminary screening indicated that the new derivatives have
antibiotic activity. Overall, the process further demonstrates the
utility of the MEND process by providing a direct method for
enhancement of nature’s diversity.
The process is also a form of molecular fishing. To portray
the parallelism with traditional fishing, our fishing rod is a resin
used for solid-phase synthesis, the bait is a nitroso species, the
river/sea is a crude extract from a natural source and the fish is
a (di)ene-containing natural product. In addition, we added
processing, i.e., chemical transformation of the “caught” natural
product.
1
heteronuclear H−13C (gHSQC, gHMBC, gHSQC-TOCSY)
spectra (Supporting Information).
Compounds 4, 12, 13, 16, and 17 were tested for their
antibacterial activities against various strains of Gram-positive
and Gram-negative bacteria as well as Mycobacterium smegmatis
and Mycobacterium vaccae, using agar diffusion assays (Table 4,
Supporting Information). In general, all compounds, except β-
caryophyllene itself, exhibited moderate to good activity against
the Gram-positive strains and slightly less against Gram-
negative strains. All compounds had activity against mycobac-
Scheme 4. Product Diversification by Different Cleavage Conditions
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Scheme 5. Retro-HDA Reaction with β-Caryophyllene
stream of nitrogen, frozen, and lyophilized. The yield was calculated
with respect to linker loading and was found to vary considerably
depending on the spice and whether it was freshly ground before the
reaction (Table 2, Supporting Information).
Reaction with Authentic β-Caryophyllene. A solution of β-
caryophyllene (1 mmol, 254 μL) in 3 mL of DCM was added to the
nitroso resin 14, 300 mg, and the slurry was shaken for 1 h. The resin
was washed 5× with DCM. The product was cleaved from the resin by
10% TFA in DCM for 1 h, and the crude product was purified as
described.
Relative Reactivity of β-Caryophyllene and 1,3-Cyclohex-
adiene. A solution of β-caryophyllene (0.5 mmol, 127 μL) and 1,3-
cyclohexadiene (0.5 mmol, 50 μL) in 2 mL of DCM was added to the
nitroso resin 3 (50 mg). The resin slurry was shaken for 2 h. The resin
was washed 5× with DCM. The products were cleaved form the resin
by 50% TFA in DCM, and the cleaved product was analyzed on
LCMS. The relative ratio of products was calculated from the UV
response at 230 nm. The ratio of HDA cyclohexane adduct/β-
caryophyllene ene product was 1:4.2 (average from three experi-
ments).
Figure 2. Stereochemistry of tricyclic skeleton.
EXPERIMENTAL SECTION
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General information related to sources of materials, solvent purity, and
generalized conditions is provided in the Supporting Information.
Analysis of Extracts from Various Peppercorns. Pepper, 3 g,
was ground and extracted with 10 mL of DCM at ambient temperature
overnight. The solution was filtered, evaporated, and lypholized. The
quantity of piperine and piperettine was calculated from LC traces at
350 nm using piperine as a standard. The amounts of total material
extracted and amount of piperine and piperettine obtained depended
somewhat on the source and type of peppercorn (see Table 1,
Supporting Information)
Retro-Hetero-Diels−Alder (HDA) Reaction. Polymer-sup-
ported HDA adduct with α-terpinene 15 (50 mg) was reacted with
β-caryophyllene (0.5 mmol, 127 μL) in 1 mL of DMF at 100 °C
overnight. The resin was washed 3× with DMF and 5× with DCM and
treated with 50% TFA in DCM for 30 min. The crude reaction
mixture contained two major components, 16 and 17, which were
isolated after HPLC purification. Compound 16 was not stable in
aqueous solutions, and after isolation, it was lyophilized from benzene
to remove residual water.
Nitroso Resins. Nitroso resins 3, 11, and 14 were prepared
following our previously described protocol.8 Resins were stored in the
form of hydroxylamine derivatives and oxidized prior to their use.
Molecular Sequestration. Typically, nitroso resin 3, 50 mg, and
ground peppercorn were placed into a plastic reaction vessel for solid-
phase synthesis, and 10 mL of dichloromethane (DCM) was added.
The slurry was shaken vigorously for 1 h and transferred into a
separation funnel, and the resin was separated from the ground pepper
based on different density of the resin and ground pepper by adjusting
the ratio of DCM (resin floats) and methanol (resin sinks). The resin
was transferred into a plastic reaction vessel and washed 5 times with
DCM.
Compound Isolation and Purification. Resin-bound com-
pounds, 50 mg, in a fritted polypropylene reaction vessel were treated
with 1 mL of 50% TFA in DCM for 1 h, unless stated otherwise. The
TFA solution was collected. The resin was washed three times with
10% TFA, washes were collected, and the combined extracts were
evaporated by a stream of nitrogen. Compounds synthesized on silicon
linkers were cleaved by reaction with 1 mL of 0.1 M TBAF solution in
THF for 30 min. The THF solution was collected, resin was washed
three times with THF, washes were collected, and the combined
extracts were evaporated by a stream of nitrogen. The crude product
was purified by HPLC. Collected fractions were concentrated by a
6-[Hydroxy-N-(8-hydroxy-1,4,4-trimethyltricyclo[6.3.1.02,5]dodec-
9-yl)amino]pyridine-3-carboxamide (4). The yield from 50 mg of
nitroso resin 3 was 5.2 mg (46%). Repetition with a different batch of
resin 1 gave 5.7 mg (37%): HRMS (ESI) m/z calcd for C21H32N3O3
[M + H]+ 374.2399, found 374.2469.
6-[N-(10,10-Dimethyl-2,6-dimethylidenebicyclo[7.2.0]undecan-5-
yl)hydroxyamino]-N-(2-hydroxyethyl)pyridine-3-carboxamide
(12). The yield was 4.9 mg (18%): HRMS (ESI) m/z calcd for
C23H34N3O3 [M + H]+ 400.2600, found 400.2595
6-[Hydroxy-N-(8-hydroxy-1,4,4-trimethyltricyclo[6.3.1.02,5]-
dodecan-9-yl)amino]-N-(2-hydroxyethyl)pyridine-3-carboxamide
(13). The yield from 300 mg of nitroso resin 14 was 33.5 mg (89%):
HRMS (ESI) m/z calcd for C23H36N3O4 [M + H]+ 418.2706, found
418.2700
[4-[(2-Hydroxyethyl)carbamoyl]phenyl]methyl 5,5,8-Trimethyl-
13-oxa-12-azatetracyclo- [6.5.1.01,11.04,7]tetradecane-12-carboxy-
late (16). The yield was 8.3 mg (18%): HRMS (ESI) m/z calcd for
C26H37N2O5 [M + H]+ 457.2683, found 457.2697
[4-[(2-Hydroxyethyl)carbamoyl]phenyl]methyl N-Hydroxy-N-(8-
hydroxy-1,4,4-trimethyltricyclo[6.3.1.02,5]dodecan-9-yl)carbamate
(17). The yield was 2.0 mg (4%): HRMS (ESI) m/z calcd for
C26H38N2NaO6 [M + H]+ 497.2593, found 497.262.
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Proton connectivities were derived by examination of the gCOSY
and TOCSY spectra. Signals of all carbons with directly attached
protons were assigned using the gHSQC spectra. Finally, the gHMBC
and gHSQC-TOCSY spectra were used to assign quaternary carbons
and to check the correctness of the connectivities established by the
interpretation of the other spectra. The stereochemistry at C-20 and
C-21, originated from caryophyllene, is known,18 C-12, C-15, and C-
17 were determined by analyzing the 1D proton (magnitudes of the
1H-1H spin−spin coupling constants; J) and 2D ROESY spectra of
compound 17. Its six-membered-ring adopts a chair conformation as
evident from the following facts. The equatorial position of the
substituent originated from the nitroso species at C-12 is supported by
the fact that the Ha-12 signal (δ 4.02) exhibits a large diaxial spin−spin
coupling of 12.8 Hz and an axial−equatorial spin−spin coupling of 4.6
Hz with the Ha-13 (δ 2.32) and He-13 (δ 1.57) methylene proton
resonances, respectively. Additionally, the 14,16-diequatorial protons
are connected by four bonds in a W arrangement. Their corresponding
signals show a mutual spin−spin coupling of 2.9 Hz. Moreover, the
ROESY spectrum displays a crosspeak between the He-16 proton
resonance (δ 1.86) and the H-20 signal (δ 1.69), which indicates that
both the CH3-26 (δ 0.84) at C-15 and OH group at C-17 are in the
equatorial positions.
(15) Yang, B.; Lin, W.; Krchnak, V.; Miller, M. J. Tetrahedron Lett.
2009, 50, 5879−5883.
(16) Chaieb, K.; Hajlaoui, H.; Zmantar, T.; Kahla-Nakbi, A.;
Rouabhia, M.; Mahdouani, K.; Bakhrouf, A. Phytother. Res. 2007, 21,
501−506.
(17) Marongiu, B.; Piras, A.; Porcedda, S.; Casu, R.; Pierucci, P. J.
Essent. Oil Res. 2005, 17, 530−532.
(18) Barton, D. H. R.; Nickson, A. J. Chem. Soc. 1954, 4665−4669.
(19) Wencewicz, T. A.; Yang, B.; Rudloff, J. R.; Oliver, A. G.; Miller,
M. J. J. Med. Chem. 2011, 54, 6843−6858.
ASSOCIATED CONTENT
■
S
* Supporting Information
Full experimental details, descriptions of biological assays, and
1
copies of all H NMR, 13C NMR, and multidimensional NMR
spectra. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: mmiller1@nd.edu.
■
ACKNOWLEDGMENTS
■
This work was supported by the Department of Chemistry and
Biochemistry at the University of Notre Dame and the NIH
(GM075855). We gratefully appreciate the use of the NMR
facility at the University of Notre Dame.
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