Application of the Solid-Supported Glaser-Hay Reaction to Natural Product Synthesis

Article abstract of DOI:10.1021/acs.joc.6b02407

The Glaser-Hay coupling of terminal alkynes is a useful synthetic reaction for the preparation of polyynes; however, chemoselectivity issues have precluded its widespread utilization. Conducting the reaction on a solid-support provides a mechanism to alle

Full text of DOI:10.1021/acs.joc.6b02407

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Note
Application of the Solid-Supported Glaser-
Hay Reaction to Natural Product Synthesis
Jessica S. Lampkowski, Diya M. Uthappa, John F. Halonski, Johnathan C. Maza, and Douglas D. Young
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b02407 • Publication Date (Web): 30 Nov 2016
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Application of the Solid-Supported Glaser-Hay Reaction to Natural
Product Synthesis
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Jessica S. Lampkowski, Diya M. Uthappa, John F. Halonski, Johnathan C. Maza, and Douglas D.
Young*
Department of Chemistry, College of William & Mary, P.O. Box 8795, Williamsburg, VA 23187 USA
HO
CuI
CH3
R2
HO
TMEDA
O
O
ABSTRACT: The GlaserꢀHay coupling of terminal alkynes is a useful synthetic reaction for the preparation of polyynes; however,
chemoselectivity issues have precluded its widespread utilization. Conducting the reaction on a solidꢀsupport provides a mechanism
to alleviate the chemoselectivity issues and provide products in high purities and yields. Moreover, the polyyne core is a key comꢀ
ponent to several natural products. Herein, we describe the application of a solidꢀsupported GlaserꢀHay reaction in the preparation
of several natural products. These compounds were then screened for antibacterial activity, illustrating the utility of the methodoloꢀ
gy.
1-3
Polyyne core structures are prevalent in various natural products, and consist of a series of conjugated acetylenic units. Over
2
1
,000 of these naturally occurring molecules have been isolated from organisms such as plants, fungi, and coral. These structures
3ꢀ7
exhibit numerous biological activities including antibacterial, antifungal, antiꢀHIV, and anticancer properties. Therefore, synthetꢀ
ic routes to the preparation of these structures are necessary to further study their benefits and develop novel therapeutic analogs.
One approach to access these conjugated alkyne cores involves the GlaserꢀHay reaction. This reaction involves the coupling of
8
two terminal alkynes and was developed in the 1800s. The reaction was later optimized to lower the temperature and increase the
rate of the reaction; however, the lack of chemoselectivity precluded its use as a mixture of three coupling products could be obꢀ
9, 10
tained when using two unique terminal alkyne reagents (Figure 1).
This has been synthetically addressed via the transition to the
11, 12
CadiotꢀChodkiewicz reaction between a haloꢀalkyne and a terminal alkyne to differentiate the reaction partners.
While this apꢀ
proach does proffer a degree of chemoselectivity, homoꢀcoupling is still observed and additional synthetic effort must be employed
13ꢀ15
to prepare the haloꢀalkynes.
Based on the asymmetrical nature of many of these natural product derivatives, a more efficient
mechanism to address chemoselectivity issues is required. Recently, we reported the solidꢀsupported GlaserꢀHay reaction as a
16ꢀ18
mechanism to address several key pitfalls associated with the GlaserꢀHay reaction (Figure 1).
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Figure 1. The GlaserꢀHay coupling of terminal alkynes. A) Traditional GlaserꢀHay reactions result in three diynes. B) Solidꢀsupported
GlaserꢀHay reaction yields only the heterodimeric product after resin cleavage.
The solidꢀsupported GlaserꢀHay coupling requires the immobilization of one terminal alkyne on a polystyrene resin. Subsequent
reaction with another soluble alkyne affords the asymmetrical diyne in high yield and purity. Homodimerization of the immobilized
alkyne is precluded by the pseudoꢀhigh dilution conditions of the solid support, and homodimerization of the soluble alkyne (used
in excess) can simply be washed away from the solid support. Moreover, we developed a methodology to perform these solidꢀ
1
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supported couplings with TMSꢀacetylene to sequentially add alkyne units, yielding highly conjugated asymmetrical polyynes.
Based on the success of this methodology, we became interested in employing the solidꢀsupported GlaserꢀHay reaction towards the
synthesis of biologically relevant natural products. Herein we report the application of the reaction to the preparation of 4 natural
products and their subsequent screening for antibacterial activity.
Many corals have been found to be rich in natural products that contain antibacterial, antifungal and cytotoxic properties. Specifꢀ
19
ically, numerous acetylenic polyynes have been isolated from the genus Montipora, a velvet coral. The most directly accessible
natural product that lends itself to this technology is 2,4ꢀdodecadinyl alcohol (3), an asymmetrical diyne shown to exhibit cytotoxiꢀ
2
20ꢀ24
city against human tumor lines. Numerous research groups have described the synthesis of this natural product.
For example,
24
Stefani et al. report this synthesis via a CadiotꢀChodkiewicz coupling with a reported yield of 76%. However, this reported synꢀ
thesis requires hazardous reagents, and requires a preliminary synthetic step to synthesize a halogenated alkyne. Another synthesis
2
3
reported by Fiandanese et al. requires numerous and sometimes harsh reagents, six synthetic steps, and has a 42% yield. Both
syntheses also required tedious purification steps postꢀreaction. We hypothesized that the solidꢀsupported GlaserꢀHay reaction
would be optimal to obtain this product in fewer steps using milder conditions, and afford higher yields.
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Immobilization of propargyl alcohol on a trityl chloride polystyrene resin (1) at ~ 0.7 mmol/g as previously described, facilitatꢀ
ed the subsequent GlaserꢀHay reaction with 1ꢀnonyne (2) in the presence of a CuI/TMEDA catalyst system (Scheme 1). Following
successive DCM/MeOH washes of the resin, 3 was cleaved from the resin using 2% TFA in DCM for 1 hour. After a silica plug, 3
1
13
was obtained in a 75% yield in high purity after essentially a single synthetic step. Analysis via H NMR, C NMR, and GC/MS
23
confirmed its identity and was in accordance with previously reported literature values. Overall, utilizing the solidꢀsupported Glaꢀ
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serꢀHay methodology to synthesize this product eliminated harsh reagents, halogenated precursor compounds, and synthetic steps
required in previously reported syntheses, and produced the product in a better or comparable yield.
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Scheme 1.
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A derivative of 3, Montiporic Acid A (5) is another common polyyne isolated from this velvet coral species. Montiporic Acid A
has been isolated from the eggs of this coral and was shown to possess significant cytotoxicity against Pꢀ338 murine leukemia cells.
23
It has also been proven to be an efficient antibacterial agent against E. coli. Conveniently, 5 can be accessed directly from 3 via a
SN2 reaction with 2ꢀbromoacetate (4) followed by an ester hydrolysis (Scheme 1) as previously reported. Synthesis of 5 was
1
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achieved in good yield (49%) and analyzed via H NMR, C NMR, and GC/MS to confirm its purity and identity. Thus, our methꢀ
odology allowed us to prepare 5 in three synthetic steps and in comparable yield to the previously reported synthesis that required 6
steps.
This methodology can also readily be employed towards triyne natural products. Towards this end, we initially targeted the prepꢀ
25
aration of octatriynꢀ1ꢀol (8), originally isolated from a fungus, Kuehneromyces mutabilis, in 1973. This compound, as well as
similar derivatives were shown to possess antibacterial activities. Previous syntheses have utilized a Fritsch ButtenbergꢀWiechell
2
6
rearrangement to prepare the triyne core. Overall, this synthesis requires eight synthetic steps to generate 8. This reported methꢀ
odology also requires numerous reagents, including some that are hazardous, and careful reaction temperature control leading to an
2
6
overall yield of only 3%.
16
Building on our previously reported methodology for extending the acetylenic scaffold, we developed a synthetic route toward
octatriynꢀ1ꢀol (Scheme 2), decreasing the synthetic steps and number of reagents used from previous reports as well as increasing
yield. Beginning with the previously described propargyl alcohol polystyrene resin (1), TMSꢀacetylene (6) was coupled using the
GlaserꢀHay conditions. The TMS group was then removed using a TBAF/DCM solution, regenerating the terminal alkyne. Propyne
(7) was then coupled to the resin using the GlaserꢀHay reaction and the product was cleaved with a 2% TFA solution, affording the
desired asymmetrical triyne natural product, 8. Following a silica plug purification, 8 was obtained in a 68% yield and analyzed by
NMR and MS, which corresponded to previously reported values. This is a dramatic increase over the current literature synthesis,
26
which was performed with only a 3% overall yield and in an additional four steps.
Scheme 2.
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Finally, another triyne natural product is readily accessible using a similar methodology to the preparation of octariynꢀ1ꢀol. Pheꢀ
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nylheptaꢀ2,4,6ꢀtrinyl acetate (10) was originally isolated from several species of Bidens in the Aster family of plants and has shown
2
antibacterial properties. A previously reported synthesis starting from 1,4 butynediol, and following the additon of a TBDPS proꢀ
tecting group, it is reacted with a lithiated phenylacetylene and undergoes a FritchꢀButenbergꢀWiechell rearrangement to produce
26
the triyne core. This synthetic strategy requires 8 total steps and the use of harsh chemical conditons, affording 10 in a 14% yield.
Utilizing our previously described solidꢀsupported strategy the triyne core is readily accessible in minimal steps and can be further
elaborated to generate the acetate (Scheme 3).
Scheme 3.
The previously described GlaserꢀHay coupling with TMS followed by TBAF induced TMS deprotection was employed to preꢀ
pare the immobilized polyyne core, followed by the capping of the polyyne with phenylacetylene (9). The solidꢀsupport was then
cleaved to afford the triynol product. The alcohol was acetylated with acetic anhydride in the presence of DMAP to afford the deꢀ
sired product. Upon acetylation, the reaction was extracted and washed with DCM/H O and dried with MgSO . The triyne 10 was
2
4
then purified on a silica column, affording the desired natural product in a 46% yield, a marked improvement. The product was then
analyzed via NMR and MS and matched to previously reported spectra. Utilizing the solid supported GlaserꢀHay methodology we
were able to eliminate synthetic steps as well as harsh and excessive reagents, and drastically improve the yield of this natural
product.
With the natural products in hand we wanted to quickly assess their antibacterial properties, as some have already been classified
as antibacterial, while others exhibited other biological relevance, but all harbor a similar alkynyl core. Each of the products was
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dissolved in DMSO to generate stock solutions at 50 mg/mL. The compounds were then introduced in triplicate at various concenꢀ
trations to E. coli cultures at different cellular densities to assess if the compounds either prevented bacterial growth (assay at low
density) or simply induced cell death (assay at high density). The cellular density of the bacteria was then observed over a 48ꢀhour
period. When added to dense cultures (OD600 ~ 0.5), no decrease in cell density was observed with any of the natural products, even
at 5 mM concentrations (See Supporting Information Figure S10). Chloramphenicol, a wellꢀdocumented antibiotic, was used as a
positive control, and a significant decrease in bacterial density was observed at much lower concentrations of 0.05 mM (See Supꢀ
porting Information). However, when the natural products were introduced to newly inoculated E. coli cultures, 5 and 8 prevented
bacterial growth at 2 µM concentrations, and 10 at 3 mM concentrations. No growth inhibition was observed for 3 at even high
concentrations (Figure 2).
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Control
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Figure 2. E. coli screens with natural products prepared via the solidꢀsupported GlaserꢀHay methodology. Cultures were innoculated at an
OD600 of 0.1 and grown in the presence of the compounds at approximately 2 µM in a 96 well plate. The bacterial growth was monitored
at 6 h, 8h, and 12 h. A DMSO control exhibited significant culture growth, while a positive control of chloramphenicol at 0.05 mM exhibꢀ
ited no growth. All cultures were grown in triplicate to establish experimental error.
This suggests that these natural products do indeed possess antibacterial properties, and can be easily prepared with the solidꢀ
supported GlaserꢀHay methodology. Future work includes the use of more sophisticated bacterial assays to determine their mechaꢀ
nism of action and their investigation for other biological activity. Moreover, due to the modular synthesis, various analogs can be
rapidly generated to conduct an extensive SAR study to further optimize their antibacterial properties. The application of the solid
support also facilitates a wide range of combinatorial strategies to be employed towards the facile generation of large and varied
polyyne libraries.
Overall, we have demonstrated that solidꢀsupported Glaser Hay reaction is a useful methodology in the synthesis of natural prodꢀ
ucts. The methodology has been employed to access 4 key natural products in fewer synthetic steps, higher yields, and with less
purification. These natural products have been assessed for their antibacterial properties and found to be comparable in efficacy to
traditional antibacterials such as choramphenicol.
Experimental Section
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Immobilization of Alcohol onto Trityl Chloride Resin in Low Loading Conditions. Trityl chloride resin (200 mg, 0.36 mmol,
1 equiv.) was added to a flame dried vial charged with dichloromethane (5 mL). The resin was swelled at room temperature with
gentle stirring for 15 min. Alcohol (25.0 µL, ~1.2 equiv.) was added to reaction, followed by triethylamine (10.0 µL, 0.072 mmol,
0.2 equiv). The mixture was stirred at room temperature for 16 h. The resin was transferred to a syringe filter and washed with
DCM and MeOH (5 alternating rinses with 5 mL each). The resin was swelled in CH Cl and dried under vacuum for 45 min before
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further use.
Polyyne Extension Protocol. Trimethylsilylacetylene (160 µL, 1.05 mmol, 15 equiv.) was added to a flame dried vial containing
the alcohol derivatized trityl resin (100 mg, 0.07 mmol, 1 equiv.), and tetrahydrofuran (2.0 mL). The CuI (20 mg, 1.06 mmol) and
tetramethylethylenediamine (20 µL, 0.132 mmol.) were added to a separate flameꢀdried vial then dissolved in tetrahydrofuran (2.0
mL). The catalyst mixture was then added to the resin in one portion and stirred at 60 ˚C for 16 h. The resin was transferred to a
syringe filter and washed with DCM and MeOH (5 alternating rinses with 5 mL each). The TMS group was then cleaved by incubaꢀ
tion in 1M tetraꢀnꢀbutylammonium fluoride trihydrate in DCM (TBAF, 1 mL, 1 h). Then the reaction was again transferred to a
syringe filter and washed with DCM and MeOH (5 alternating rinses with 5 mL each) and dried under vacuum for 45 minutes.
Product was weighed and transferred to flame dried vial for future use.
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Dodeca-2,4-diyn-1-ol (3). 1ꢀNonyne (115 ꢁL, 0.70 mmol, 10 equiv.) was added to a flame dried vial containing the propargyl
alcohol derivatized trityl resin (100 mg, 0.070 mmol, 1 equiv.), and tetrahydrofuran (2 mL). CuI (10 mg, 0.053 mmol, ~0.7 equiv.)
and tetramethylethylenediamine (30 µL) were added to a separate flameꢀdried vial then dissolved in tetrahydrofuran (2 mL). The
catalyst mixture was then added to the resin in one portion and stirred at 60 ˚C for 16 h. The resin was transferred to a syringe filter
and washed with DCM and MeOH (5 alternating rinses with 5 mL each). The product was then cleaved from the resin by treatment
with 1 mL 2% TFA (DCM, 1 h), and filtered into a vial. A short silica plug was utilized to remove unreacted starting material (1:1
1
EtOAc/Hex) and pure product was obtained (0.010 g, 0.052 mmol, 75%). H NMR (CDCl , 400 MHz): δ 4.27 (s, 2H), 2.24
(
3
13
t, J = 7.2 Hz, 2H), 1.55 (quint, J = 7.2 Hz, 2H), 1.39–1.31 (m, 2H), 1.30–1.19 (m, 6H), 0.90 (t, J = 6.9 Hz, 3H). + C NMR (CDCl3,
00 MHz): δ = 51.7, 31.8, 29.5, 28.7, 28.1, 22.4, 19.2, 14.0. GC: t = 10.43 min; MS: m/z calcd for C H O [M ]: 178.136; found:
R 12 18
4
1
78.092.
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Montiporic Acid A (5). Ethyl bromoacetate (10 ꢁL, 0.1 mmol, 2 equiv.) was added at 10 ˚C to a vial containing 3 (0.010 g,
0
.052 mmol, 1 equiv.) dissolved in toluene (1 mL), 50% KOH (200 ꢁL), and tetraꢀbutyl ammonium sulfate (10 mg, 0.03 mmol,
~
0.5 equiv.). The reaction was then vigorously stirred at 10 ˚C for 3 hours. Upon completion, the reaction was quenched with dilute
HCl (5 mL), extracted with EtOAc, and washed with H O. (3x5 mL) The product was then dried over anhydrous MgSO and solꢀ
2
4
vent removed in vacuo. Purification was performed via column chromatography (hexanes: EtOAc 10:1 to 1:3) to yield the desired
1
product (8 mg, 0.039 mmol, 49%). H NMR (CDCl : δ 4.38 (s, 2H), 4.21 (s, 2H), 2.24 (t, J = 7.2 Hz, 2H), 1.49 (quint, J = 7.2 Hz,
3
13
2H), 1.34–1.31 (m, 2H), 1.29–1.19 (m, 6H), 0.84 (t, J = 6.9 Hz, 3H); C NMR (CDCl , 400 MHz): δ 74.5, 71.1, 64.8, 64.6, 58.0,
51.5, 4.8; GC: t = 11.02 min; MS: m/z calcd for C H O [M ]: 236.141; found: 236.172
3
+
R
14 20
3
2
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Octatriyn-1-ol (8). The described polyyne extension protocol was used to obtain the immobilized terminal alkyne diyne. 1ꢀ
Propyne (53 ꢁL, 0.70 mmol, 10 equiv.) was added to a flame dried vial containing the immobilized resin (100 mg, 0.07 mmol, 1
equiv.) and tetrahydrofuran (2 mL). The copper catalyst (10 mg, 0.053 mmol, ~0.7 equiv.) and tetramethylethylenediamine (30 µL)
were added to a separate flameꢀdried vial then dissolved in tetrahydrofuran (2 mL). The catalyst mixture was then added to the resin
reaction in one portion and stirred at 60 ˚C for 16 h. The resin was transferred to a syringe filter and washed with DCM and MeOH
(5 alternating rinses with 3 mL each). The product was then cleaved from the resin by treatment with 1 mL 2% TFA (DCM, 1 h),
and filtered into a vial. A short silica plug was performed to remove unreacted starting material (5:1 EtOAc/Hex), affording product
1
13
8
. (4 mg, 0.040 mmol, 68%) H NMR (CDCl , 400 MHz) δ 4.70 (s, br, 1H), δ = 4.34 (s, 2H), 1.96 (s, 3H); C NMR (CDCl , 400
3 3
+
MHz): δ 74.2, 71.1, 65.0, 64.8, 58.4, 51.7, 4.5; GC: t = 10.99 min; MS: m/z calcd for C H O [M ]: 118.042; found: 118.051
R
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6
2
6
Phenylhepta-2,4,6-triynyl acetate (10). The described polyyne extension protocol was used to obtain the immobilized termiꢀ
nal alkyne diyne. Phenylacetlyene (0.70 mmol, 10 equiv.) was added to a flame dried vial containing the starting material (100 mg,
0
.070 mmol, 1 equiv.) and tetrahydrofuran (2 mL). The CuI (10 mg, 0.053 mmol) and tetramethylethylenediamine (30 µL) were
added to a separate flameꢀdried vial then dissolved in tetrahydrofuran (2 mL). The catalyst mixture was then added to the resin reꢀ
action in one portion and stirred at 60 ˚C for 16 h. The resin was transferred to a syringe filter and washed with DCM and MeOH (5
alternating rinses with 5 mL each). The product was then cleaved from the resin by treatment with 1 mL 2% TFA (DCM, 1 h), and
filtered into a vial. Solvent was removed in vacuo to afford the free alcohol (10 mg, 0.056 mmol, 80%) Acetic anhydride (1 mL)
and a catalytic amount of DMAP were added and dissolved in 1 mL DCM. The reaction was allowed to stir at room temperature for
3
hr, followed by an extraction using DCM/H O (3x5 mL) and drying with MgSO4. The product was then purified on a silica gel
2
1
column using 5:1 Hex:EtOAc yielding 10 (9 mg, 0.041 mmol, 46%), which was then analyzed via H NMR (CDCl , 400 MHz):
δ 7.70ꢀ7.61 (m, 2H), 7.56ꢀ7.32 (m, 3H), 4.85 (s, 2H), 2.16 (s, 3H); C NMR (CDCl , 400 MHz): δ 171.0, 134.5, 131.5, 129.9,
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3
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3
+
21.2, 78.5, 76.3, 74.3, 70.8, 66.2, 63.8, 53.1, 20.9; GC: t = 9.89 min; MS: m/z calcd for C H O [M ]: 222.068; found: 222.079.
R 15 10 2
Low Cell Density Absorbance Assay. LuriaꢀBertani (LB) media (10ml) was inoculated with Escherichia coli Novagen
BL21(DE3) strain of cells and then incubated for 16 h at 37°C. Optical density measurements on a spectrophotometer at 600nm
OD 600 ) was used to assess the density of the starter culture. The culture was diluted to an OD 600 of 0.1 (low density) by addition
(
of fresh LB media. In a new 96 well microplate (Greiner Bioꢀ One), the solutions of varying concentration for each product from
the working plate were plated including chloramphenicol and DMSO (20ꢁL). Subsequently, the low density cell solution was added
to each well in which solution had been previously added. An initial absorbance was read using a Synergy HT Microplate Reader
set to shake the plate for 10 seconds prior to reading the OD 600. An absorbance reading was taken again at 2, 4, 6, 8, 12, 24 h. Beꢀ
tween OD 600 readings the microplate was allowed to shake at 37°C.
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Supplemental experimental protocols, H and C NMR data, GC data, and low and high density biological assay results. The Supporting
Information is available free of charge on the ACS Publications website.
AUTHOR INFORMATION
Corresponding Author
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Douglas D. Young, eꢀmail: dyoung01@wm.edu
Author Contributions
The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript.
ACKNOWLEDGMENT
The authors would like to acknowledge the College of William & Mary, the National Institute of General Medical Sciences of the NIH
(R15GM113203), and the Beckman Foundation for funding.
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