BS BC 08

Chemistry 135: Chemistry of Natural Products

EXAM 1
OUTLINE
I. Methodology A. Basic extraction and analysis B. Crude extraction C. Lipid extraction D. Autoxidation E. Prepartion of derivatives of FA F. Separation methods G. Common extraction protocols for FA H. Identification and analysis I. Qualitative testing for FA II. Recent Studies A. The First Total Synthesis of ()-4methoxydecanoic acid B. A Flexible Synthesis of C33-C39 Polyketide Region of Apratoxin

FATTY ACIDS AND POLYKETIDES

August 5, 2011 Barba, Biadomang, Narciso

I. METHODOLOGY
Isolation of FA and PK follows extraction-purificationanalysis procedure 2 metabolites 0.01% dry weight of the plant Varied origin May be unstable and complex Microorganisms: metabolites produced depending on medium and fermentation details Fungi: metabolites found in mycelium, excreted in broth or medium Plants, insects, &marine organisms: stored, modified compounds from food/nutrient intake A. BASIC EXTRACTION AND ANALYSIS Basic isolation scheme o Crush material with solven o Sequential extraction if necessary o Separation of specific compounts Solvents used differ in polarity Action of solvent o Diffuse into cells o Solubilize metabolites o Diffuse out of cells enriched in extracted metabolites Commercial extraction: use of kits and complicated apparatuses Acidic, basic, and neutral fractions are separated in order for purification o Treat with base: remove carboxylic acids as their water soluble salts o Treat with acid: remove bases as their salts o Neutral compounds remain behind Artefact (compounds arising from human intervention) formation: ester hydrolysis, auto-oxidation, rearrangement during extraction Separation needed to characterize and examine minor components Natural products occur in closely related species: mono, di, trihydroxy
Figure 1 General schematic of lipid extraction and analysis

B. CRUDE EXTRACTION Solid Sources o Maceration Pound source in solvent Filter & centrifuge Time and solvent consuming May lead to potential loss of metabolites Some compounds cannot be extracted if poorly soluble in room temperature Small risk of degradation of thermolabile metabolites o Ultrasound-assisted solvent extraction Apply ultrasound to plant powder in vial in an ultrasonic bath Mechanical stress induced by cavity production Increases solubilization of metabolites in extraction solvent Applied for the extraction of intracellular metabolites from plant cell cultures o Percolation Powdered source or plant source soaked in a solvent in a percolator May cause clogging in source is too fine or swells excessively Material may not be distributed homogenously Solvent may not reach all areas Time and solvent consuming o Soxhlet extraction Plant powder placed in thimble in an extraction chamber Suitable solvent is added and heated in reflux at the boiling point of the solvent Continuous process Less time and solvent consuming Page 1 of 10

CHEM 135

FATTY ACDS AND POLYKETIDES

Possible degradation of thermolabile compounds and formation of artefacts o Pressurized solvent extraction Used for rapid and reproducible initial extraction Powdered material placed in oven Solvent is pumped from a reservoir to fill the cell where the sample is located Solvent is heated and pressurized at programmed levels for a certain period of time Purge sample by Nitrogen gas High temperatures and pressures increase penetration of solvent into material Need small amount of solvent Repeated extraction are possible o Extraction under reflux i.e. Steam distillation Collects vapors condensed when sample is immersed in a solvent heated until boiling point Liquid Sources o Partition between immiscible or miscible solvents o Extraction with solvents of increasing polarity to obtain different fractions that can be resuspended in other solvents

Repeated washing or percolation with solvent under reflux i.e. Soxhlet extraction Continuous solvent extraction Solvent trickles through the sample Reduces the amount of time the extraction must be carried out Channeling of solvent may occur

D. AUTOXIDATION PUFA and PUPK autoxidize rapidly in air Free radicals attack the sample Exacerbated by light and metal ions Autocatalytic A double bond increased the rate of destruction by 2-3 fold Antioxidants protect lipid extracts Butylated hydroxy toluene (BHT): commonly used synthetic antioxidant Added at a level of 10-100 mg/L relative to lipid concentration Too much? May act as pro-oxidant Can be removed with solvents by evaporation in stream of nitrogen

E. PREPARATION OF DERIVATIVES OF FA
Fatty acid must be converted to low molecular weight, non-polar derivatives = FAME Hydrolysis/Saponification o Reduces MW and increases volatility o Release bound fatty acids o Heat lipid mixture under reflux with en excess of dilute aqueous ethanol alkaline o Mechanism: cleavage of ester bond between the fatty acid and the glycerol moiety under heat o Products: fatty acids, diethyl ether-soluble nonsaponifiable materials, other non-saponifiable materials o Followed by methylation Methylation and Esterification o Acid-catalyzed esterification & transesterification Methylation reduces the polarity and increases volatility of the lipid Esterification: for free fatty acid Transesterification: for fatty acids linked by ester bonds to glycerol and cholesterol Heat with a large amount of methanol in the presence of acid catalyst Anhydrous HCl in MetOH Bubble HCl gas to dry MetOH Mechanism: Protonation of the acids to give an oxonium ion Undergo an exchange reaction with an alcohol to give the intermediate Intermediate loses a proton to become an ester

Figure 2 Sample schematic of liquid-liquid extraction

C. LIPID EXTRACTION Simple lipid: part of large aggregates in storage tissues Complex lipids: constituents of membrane and closely associated to proteins and polysaccharides IFA involved: weak hydrophobic, VDW, H-bonding, ionic bonding Solvent o Readily dissolve the lipid o Overcome the interaction between the lipids and matrix tissue o Like dissolves like o For low polarity: use hydrocabons, i.e. hexane, toluene, cyclohezane, chloroform o For high polarity: alcohols o For polyketides: ethyl acetate o Isopropanol-hexane (2:1 v/v) used for limited toxicity o Chloroform-methanol (2:1 v/v) is more versatile Washing contaminant via chloroformmethanol (2:1 v/v) and equilibrate of its volume with saline solution Two layers forms: chloroform-methanol-water layer with lipids (lower) and contaminants (upper) Water causes swelling of biopolymers to assist extraction Denaturation techniques target cell walls Lipid extraction methods o Batch solvent extraction Mix the sample and solvent in container (i.e. separatory funnel) Continuous mixing Applied for solvent mixtures that form polar and non-polar phase o Semi-continuous solvent extraction

Scheme 1 Acid catalyzed esterification

Page 2 of 10

CHEM 135

FATTY ACDS AND POLYKETIDES

F. SEPARATION METHODS Distillation o Separate mixtures of FA and esters derived from natural fats o Applied due to thermolability o Difference in boiling points or volatility of FA depends on the difference in chain length o Ease in obtaining enough amounts of sample/fractions to be used for further chemical and biological processes o Disadvantage: cant distinguish between sat and unsat compound of the same chain length o Kugelrohr Distillation: Short-path vacuum distillation apparatus used to distill small amounts of compounds with high boiling points under reduced pressure Distills with minimal hold-up & sample loss. Removes color and particulates Can be used for sublimation and solvent evaporation o Purification method for polyketides (not much data) Crystallization o Salt-solubility Methods Lipid form salts with metallic ions Salts formed have varying solubilities in water and organic solvents (factors: nature of metallic ion, chain length, degree of unsaturation, other characteristics of acid radicals) Use of lead salts or some of fatty acid in ether or ethanol The solid or saturated acids are regenerated in insoluble lead salts by boiling the fatty acids with dilute HCl o Low-temperature Crystallization Utilizes the melting point of the lipid which depends on the chain lengths and degree of unsaturation Increasing chain length, increasing melting point For long chain FA: unsaturation decreases melting point At low temperatures short chain FA crystallize and PUFA can be isolated from the rest of the FA Hexane and acetone facilitate separation Some polyketides crystallize upon standing at 4C, and can be separated with ether or hexane o Urea Complexation Urea forms crystalline inclusion compounds with straight-chain compounds in water, methanol, and ether The longer the chain length, the more stable the urea complex Resist autoxidation The length of the carbon chain can be told in terms of the formation of the urea complexes and their dissociation temperature No recorded study of use of urea complexes for polyketides

Scheme 2 Acid catalyzed transesterification

Solvent with 1-2% (w/v) H2SO4(conc) in MetOH Same manner and rate as anhydrous HCl Not for PUFA, H2SO4 is a strong antioxidant Long reflux times and high [H2SO4(conc)] can lead to the formation of colored products and destruction of polyenoic fatty acids Boron Trifluoride-Methanol Powerful Can be used with a wide range of lipid classes Can produce methoxy artefacts across double bonds, especially when not freshly prepared Base-catalyzed transesterification Mechanism: Esters form an anionic intermediate in the presence of an alcohol anion Intermediate can dissociate back to the original ester or new ester In the presence of large excess of alcohol, the equilibrium point of the reaction will be displaced until the sole product in the new ester

Scheme 3 Base catalyzed transesterification

ESTERIFICATION CANNOT OCCUR: An unesterified FA is converted to a carboxylate ion in a basic solution, cannot be subjected to Nu- attack because of negative charge Sodium/potassium methoxide in anhydrous MetOH Additional solvent such as toluene or THF is needed to solubilize non-polar lipids Prolonged of careless use of basic reagents can cause alterations to FA Methyl iodide reacts with Na or K salts of FA in the presence of a polar aprotic solvent such as dimethylacetamide to form methyl esters Amide-bound fatty acids are not affected by alkaline transesterification reagents under mild conditions Diazomethane Esterification, not transesterification

Scheme 4 Reaction of diazomehane with FA

Diazomethane Prepared in ethereal solution by the action of alkali of nitrosamide

Figure 3 Urea complexation of free fatty acids

Chromatography Page 3 of 10

CHEM 135

FATTY ACDS AND POLYKETIDES

Adsorption Chromatography Separation using charcoal, silicic acid, alumina, or even filter paper Utilize difference in polarity Polar lipid binds to the polar phase (usually stationary phase), neutral lipid pass through and emerge in first wash Polar lipids are then eluted, in order of increasing polarity why washing the column with solvents of increasing polarity o Thin-Layer Chromatography (TLC) Same principle of adsorption chromatography Uses a chamber saturated with organic/nonpolar solvent Less polar lipids moves fastest Plate can be dyed with iodine fumes or rhodamine for lipid visualization by fluorescence Regions containing the separated lipid can be scraped of the plate and recovered by solvent extraction

Figure 4 Separation of methyl ester derivative of unsaturated FA by TLC on silica gel with 10% (w/w) silver nitrate. Mobile phase is hexane-diethyl ether, where plate A is 9:1 (v/v) and plate B is 2:3 (v/v)

Column Chromatography (CC) More often used for the purification of liquid The components of the sample solution separate from each other by partitioning the stationary packing material and the mobile eluent Pattern of migration of lipid will be same as TLC Flash Column Chromatography (FCC) Rapid form of CC Based on optimized pre-packed columns where solvent is pumped at a high flow rate Uses a plastic column pack with solid support (i.e. silica gel) Sample is placed on top of the support The column is filled with an isocractic solvent which enables the sample to run through the column in the presence of pressure of pumps to speed up separation Reversed Phase Partition Chromatography (RPPC) Polar mobile phase, non-polar stationary phase Used for very small samples Found to separate C6-C12 FA with methanol and acetone or methanol and water as mobile solvents Found to separate C4-C29 with acetone and water as mobile solvents High Performance Liquid Chromatography (HPLC) Utilizes different stationary phases, a pump that mobilized the mobile phase/s and analyte through the column, and a detector to provide a characteristic retention time for the analyte with the stationary and mobile phases Operates at ambient temperature so the degradation of heat-sensitive functional groups is prevented

HPLC on columns of silica gel is used to tell the presence of polar functional groups (I,e, oxygenated moieties) Isomers differing in the position of hydroperoxy or hydroxy groups on an aliphatic chain can be separated Gas Chromatography (GC) Uses a high-boiling liquid as stationary phase and inert gas as the mobile phase to analyze compounds that can be vaporized without decomposition Separates volatile components of a mixture according to their relative tendencies to dissolve the inert material in the chromatography column and volatilize and move through the column, carried by a current of an inert gas (i.e. Helium or Nitrogen) Methylation of FA prior to separation improves FC FAME loaded onto GC column Column is heated to volatilize the material The more soluble the molecules are in the material, they more they partition to the column The less soluble are carried by the stream of helium/nitrogen and emerge first from the column Order of elution will depend on the nature of the solid adsorbent in the column and the boiling point of the components of the liquid mixture As the chemicals exit the column, they are detected and identified electronically Found to separate FFA from 1-12 carbon atoms No record of using GC for separation of polyketides in solution Silver Ion Chromatography Uses silver ions that are bound to an ionexchange support Complexation are of charge-transfer type: unsat compounds acts as electron donors, silver ion acts as electron acceptor Bond formation s type bond between the 2p of an olefinic double bond and the free 5s and 5p orbitals of the silver ion a acceptor backbone between the occupied 4d orbitals of the solver ion and the free antibonding 2p* orbitals of the olfenic bond Conclusions unsaturated acyclic and carbocyclic compounds form more stable complexes than do aromatioc stability decreases with increasing chain length stability decreases with an increasing number of substituents at the double bond in the order: stability increases when a hydrogen from a molecule of the olefinic bond is replaced wit a deuterium or tritium because or greater electron release cis olefinic double bonds forming stronger complexes in comparison with the corresponding trans molecules and the longer the chain length, the lower the stability of the complexes formed The supporting material is also a factor to the migration order of lipids Silica gel: posses appreciable polarity and absorption activity Product of a mixed retention mechanism Position of the double bond when the unsaturated lipid complexes with the silver Page 4 of 10

RCH=CH2 > R2C=CH2 > cis RCH=CHR > trans RCH=CHR > R2C=CHR > R2C=CR2

CHEM 135

FATTY ACDS AND POLYKETIDES

ions will influence the conformation of the molecule The strength at which the molecule adheres to the support will depend on interaction between the functional groups and silanol moiety

Figure 5 Principle behind Silver Ion Chromatography

Figure 6 The Dewar model of interaction between a silver ion and an olefinic double bond

G. COMMON EXTRACTION PROTOCOLS FOR FA The Folch Procedure o Solvent: 8;4;3 of chloroform, methanol, saline solution o Tissue sample homogenized with chloroform and methanol (may be methanol first, then chloroform) o 2-3 extractions o For wet bacterial cells: heat solvents o Extractability of the tissue is variable & will depend on the nature of the tissue and lipids The Bligh and Byer Method o Tissue sample + chloroform + methanol combined to give a single-phase system for homogenization o Filter o The residue was re-homogenized with fresh chloroform to ensure that the simple lipids were extracted completely o Combined organic layers added to fresh saline solution to produce biphasic mixture o Chloroform layer produced contains the lipid Extraction of Plant Tissues o Lipid in plant material are very liable because they can undergo extensive enzyme catalyzed degradation o Preliminary extraction with propan-2-ol will overcome such degradation o n-Propanol is able to relieve fatty acid amylose inclusion complexes or those linked via ionic or Hbonding to hydroxyl groups of the starch components o Re-extraction of the solid residue with chloroformmethanol o Solvent may be evaporated o The crude extract can be taken up in fresh chloroform-methanol and given a Folch wash to eliminate non-lipid contaminants

H. IDENTIFICATION AND ANALYSIS Spectroscopy o IR

Can detect FA and PK in free state, bound state, or as methyl ester derivatives Determine important and distinguishing functional groups Esterified version is preferec for FA because a bad due to carboxyl group at 10-11 m can obscure other important features in the spectra 5.75 & 8.6 m (sharp): esterified carbonyl function 5.9 m (sharp) and 3. 5 & 10.7 m (broad): FFA 3.3 and 6.1 m (small): cis-Double bonds 10.3 m: trans-Double bonds The remaining bands are absorption frequencies of the hydrocarbon chain o Raman Exploits the characteristic vibrational frequencies of chemical bonds Influenced by which atoms are bound, the bonds saturation, and its molecular environment. -1 1656 cm :cis-double bonds -1 1670 cm :trans-double bonds -1 2232 and 2291 cm :triple bonds -1 2120 cm : terminal triple bonds o UV-Vis Detect or confirm the presence of fatty acids and polyketides containing conjugated double bond systems or chromophores observe chemical or enzymatic isomerization of fatty acid and polyketide double bonds in which conjugated systems are found. The greater the number of double bonds, the higher the absorbance Different geometrical isomers have different spectra The greater the number of trans-double bonds, the higher the extinction coefficient, and the shorter the wavelengths of the maximum band o NMR Identification of lipid structures in terms of detection and location of double bond systems in fatty acid chain and in their methyl ester derivatives and in polyketides Proton NMR: the number of peaks and their respective positions in the spectra aids in determining the conjugation of the fatty acids and polyketides Integration of the signals assists in confirming the assignments to particular protons Free hydroxyl groups gives rise to two separate signals that are caused the OH molecule itself, its intensity and position that may vary due to hydrogen bonding effects, and the CHO- proton Methyl braches on aliphatic chains do not give signals that are helpful in locating their positions, unless the branch is immediately adjacent to either end of the molecule Cis- and trans- isomers of unsaturated fatty acids are readily distinguished by the use of lanthanide shift reagent 13 C NMR spectroscopy has been suggested as a means of quantification of trans-unsaturation in lipid mixtures 13 It is best to couple C NMR studies with MS 13 because C NMR is somehow insensitive Mass Spectrometry o Gives the location of double bonds, molecular weight, retention times, branched functional groups, and oxygenated substituents in the fatty acid sample o able to give information on the fatty acid and polyketides with regard to its structure whether they be saturated strain-chains, unsaturated, branched chain, carbocyclic, oxygenated, and other miscellaneous classification of such lipid class Page 5 of 10

CHEM 135

FATTY ACDS AND POLYKETIDES

Scheme 5 Example of fatty acid fragmentation

Atmospheric Pressure Ionization MS LC-MS method Able to extend analysis of analytes that are high in MW, thermally labile, and polar Gives a spectrum rich in fragments (thus rich in structural information) Uses high flow rates without losing a large part of the fraction/sample to waste Mobile phase: eluting analyte Mobile phase is heated to high temperatures, spreayed with high flow rates of nitrogen by means of an electrospray ionization technique (ESI) The entire aerosol cloud formed is subjected to a corona discharge that creates ions Skimmers are used to transmit ions to the high vacuum region and subjected to the mass analyzer

Polyketides Used in structure elucidation of a sample that has undergone biosynthesis Polyketides are made up of acetate units Methyl branchings may occur from the incorporation of propionate instead of acetate or the result of a subsequent transfer of a methyl group of S-adenosyl methionine (SAM) Determined by fermenting Phoma betae Fr. 13 from a media that contained C-labeled 13 13 substrate [1- C] acetate, [2- C] acetate, or 13 [Me- C] methionine 13 Each betaenone B inspected with C NMR Results: 13 Betaenone B is made up of C-labeled acetate units the methyl branching were due to the methyl transfer from SAM instead of the incorporation of propionate, as the 13 corresponding C NMR signals of the methyl groups were amplified after the cultivation in the nutrient medium 13 containing [Me- C] methionine

Figure 7 Schematic of API-MS

Isotopic Labeling o Tracks the passage of a sample through a system or a process by labeling or including less abundant isotopes in chemical composition o The less common isotopes are detected later on. This will indicate that it came from the labeled substance. o Easy detection due to the difference in weight and the consequences of having molecules that contain the isotopes --- different vibrational modes (use MS and IR) o Can also be a means to study chemical reactions because specific atoms from the reactant are replaced by their isotope o Easy detection of the location of a particular molecular fragment in the reactant in terms of its location in one of the products o Fatty Acids Elucidation of the mechanism of the acidcatalyzed esterification begs the question of which starting product the oxygen atom of the product water molecule comes from or which oxygen is in the ester 18 Alcohol can be labeled with O

Scheme 7 Biogenesis of betaenone B

Scheme 6 Esterification with an O-labelled alcohol

18

Chemical Degradative Procedures o Chain length determination Catalytic hydrogenation to form saturated compounds Use GC or HPLC Carry out the reaction on the single FA of interest o Location of double bonds Oxidative fission and GC or HPLC for identification of products Permanganate-periodate Oxidation (von Rudloff oxidation) Oxidizes the methyl ester of the unsaturated FA or PK in tert-butanol solution Done by a solution containing a small amount of potassium permanganate with a larger amount of sodium metaperiodate Sodium metaperiodate: regenerates the permanganate as it is reduced Potassium carbonate: buffer Completion of reaction o Solution is acidified o Excess antioxidant is destroyed by sodium bisulfite o Products extracted with diethyl ether Ozonolysis and Reductive or Oxidative Cleavage Page 6 of 10

CHEM 135

FATTY ACDS AND POLYKETIDES

Produce ozonides as ozone attacts olefins Ozonide can be cleaved reductively to aldehydes and aldehyde-esters by dimethyl sulfide in methanol Breaking of carbon-carbon double bonds: O3 molecules binds to both molecules at the separation point At separation o Alcohol o Deprotonated alcohol o Aldehyde o Carboxylic acids The functional groups found at separation are used as markers for the location and number of double bonds found in the FA sample Reduction by hydrazine Hydrazine: reduce FA without causing any double bond migration or stereomutation Monoenes are formed Isomers are then found with double bonds in each of the positions in which they were present in the original PUFA Cis- and trans- monoenes are separated by silver ion chromatography Location of other functional groups Triple bonds A TLC spray composed 4-(4-nitrobenzyl)pyridine (5%) in acetone gives off a violet color Use MS to detect position Permanganate-periodate reagent readily cleaves triple bonds Ozone can react with triple bonds and the products of the reductive cleavage are mono- and dibasic acids rather than aldehydes and aldehyde-esters, so that double and triple bonds in a single fatty acids can be differentiatied Allenic groups Have distinctive IR and NMR spectra Exhibit marked optical activity Partial reduction by hydrazine and oxidation of the monoene fragments by ozone can tell its position n the FA Oxygenated functional groups GC can detect hydroxyl groups Hydrogenation can eliminate any multiple bonds before free hydroxyl or epoxyl groups are converted to iodide by means of iodine and red phosphorous Hydrogenolysis with zinc and hydrochloric acid in methanol removes the iodine atom in the aliphatic chain Borohydride can reduce keto groups to hydroxyl groups Resulting saturated FA can be analyzed by GC MS can be used to determine position of hydroxyl group Periodic acid in halogenated solvents or in diethyl ether can cleave epoxyl groups Furanoid fatty acids, that has been separated by TLC, can be detected by spraying the sample with a 2% solution of tetracyanoethylene in acetone Cyclopropane Cyclopropane FA can be mistaken for normal saturated fatty acids in silver ion chromatography Boron trifluoride-methanol reagent: form methoxy derivative of cyclopropane FA The methoxy derivative can be converted to methyl-branched fatty acids by vigorous catalytic hydrogenation

MS for analysis Methyl branches Methyl branches are inert to most chemical reagents Acidic potassium permanganate: oxidize the lipids to determine position

I. QUALITATIVE TESTING FOR FATTY ACIDS Solubility test o Degree of solubility of a lipid or fatty acid will depend on the relative amounts of the elemental composition, and structural positions of the sample o Like dissolve like o pH can change ionic character of a lipid o If the sample contains a strong acid, hydrolysis of some of the ester bonds may occur o The products of hydrolysis may have solubility properties different from the original molecule Emulsification test o A lipid with a non-polar hydrocarbon portion and a polar portion are usually good emulsifying agents o The non-polar portion solubilizes with the less polar layer o The polar portion will be attracted to the polar molecules in the solution Halogenation test for Unsaturated Lipids o Double bonds have the capability of undergoing addition reactions to become saturated o The amount of halogen, such bromine, added to a lipid molecule will give a quantitative measure of the degree of unsaturation in the lipid

II. RECENT STUDIES A. THE FIRST TOTAL SYNTHESIS OF ()-4

METHOXYDECANOIC ACID "The First Total Synthesis of ()-4-methoxydecanoic acid: A Novel Antifungal Fatty Acid," N. Caballeria, C. Miranda, and K. Parang Mid-chain methoxylated fatty acids are valuable compounds that can be used to develop more potent antifungal agents The synthesis of ()-4-methoxydecanoic acid (1) was done in six steps with a 25% overall yield from the commercially available 4-penten-1-ol. The synthesized compound demonstrated 17-fold higher antifungal activity against Candida albicans ATCC 60193 and Cryptococcus neoformas ATCC 66031 when compared to an unsubstituted n-decanoic acid. Significance: use fatty acid amide analogues to find new ways to cure fungal infections.

PREVIOUS STUDIES ()-4-hydroxydecanoic acid o Can cyclize easily to y-decalactone o Known to have antibacterial activity o The presence of a hydroxyl group in the acyl chain decreases toxicity towards fungi o y-decalactone has higher antibacterial acivity because it inhibits the growth of bacteria and fungi (i.e. Bacillus subtilis and Trichothecium roseum) 3-(R)-hydroxydecanoic has antifungal activity -methoxylation increases the antifungal activity of fatty acids No reports on antifungal activity after mid-chain methoxylation

Hypothesis: that ()-4-methoxydecanoic acid could be a good starting template for better antifungal activity as compared to ()-4-hydroxydecanoic acid 10 carbon chain: capric acid kills Candida albicans at 10 mM by fungal plasma membrane disintegration It will not cyclize to a y-lactone Mid-chain methoxylation vs. -methoxylation Page 7 of 10

CHEM 135

FATTY ACDS AND POLYKETIDES

o SYNTHESIS

Fragments containing the carboxyl end (at m/z = 117 corresponding to C5H9O3+ and at m/z = 85 corresponding to C4H5O2+) were the most abundant.

Table 1 Antifungal activity (MIC values, uM) against Candida albicans (SDB) and Crytococcus neoformans (SDB) at 35-37C after 24-48 hours

Scheme 8 The synthesis of ()-4-methoxydecanocic acid. Reagents and conditions: (i) DHP/PTSA, CHCl3, room temperature, 5 hours, 88%; (ii) MMPP/EtOH, 48 hours, 89%; (iii) CH3(CH2)3CH2MgBr, Cu(I)/THF, -78C to -30C, 81%; (iv) NaH/CH3I, THF. OC to room temperature, 2 hours, 88%; (v) PTSA, CHCl3, 45C, 2 hourse, 73%; (vi) PDC/DMF, 24 hours, 63%

1. Protection of 4-penten-1-ol (2) a. Reagents: dihydropyran (DHP) and catalytic amounts of p-toluenesulfonic acid (PTSA) in CHCl3 b. Details: room temperature for 5 hours c. Product: 1-[(tetrahydropyran-2-yl)oxy]-2-pentene (3) d. Yield: 88% yield 2. Epoxidation of the double bond a. Reagents: magnesium monoperoxyphthalate (MMPP) in EtOH; MMPP was observed to be more efficient than the m-chloroperoxybenzoic acid (m-CPBA) in epoxidizing these alkenes b. Details: 48 hours c. Product: 4,5-epoxy-1-[(tetrahydropyran-2yl)oxy]pentane (observed after purification of the crude extract by silica gel CC and elution with hexane and diethyl ether) d. Yield: 89% yield 3. Opening of the tetrahydropyran (THP) protected epoxide 3 a. Reagents: 1-pentylmagnesium bromide assisted by catalytic amounts of copper (I) chloride in THF b. Details: temperature range of (-78C to -30C) c. Product: 4-hydroxy-1[(tetrahydropyran-2yl)oxy]decane (4) (purified by silica gel CC) d. Yield: 81% yield 4. Methylation of the free hydroxyl group in 4 a. Reagents: methyl iodide in the presence of sodium hydride in THP 5. Deprotection of the primary alcohol a. Reagents: PTSA in CHCl3 b. Details: at 45C for two hours c. Product: ()-4-methoxydecan-1-ol 6. Oxidation of alcohol a. Reagents: pyridinium dichromate in DMF b. Details: 24 hours c. Product: ()-4-methoxydecanoic acid d. Yield: 61% yield of 1 and overall 25% yield ANALYSIS NMR Analysis o ()-4-methoxydecanoic acid: absorption for carbon and hydrogens with methoxy functional group Methoxy hydrogen: 3.32 ppm Methoxy carbon: 56.5 ppm Methine (CHOCH3) hydrogen: 3.20 ppm Methine carbon: 79.9 ppm Characteristic of saturated mid-chain methoxylated fatty acids Mass Spectrum o McLafferty rearrangement of fatty acids at m/z=60 was greatly reduced by the presence of the methoxy functional group o Predominant -fragmentation at both sides of the methoxylated carbon

Antifungal Activity o Fungi: fluconazole-resistant strain of C. albicans (ATCC 60193) & C. neoformans (ATCC 66031). o Control: n-Decanoic acid o C-4 methoxylation increased the antifungal activity of the parent n-decanoic acid o Factors: Solubility Efficiency of disrupting fungal membranes Inhibition of fatty acid biosynthesis with the fungi and interact with some key enzymes

B. A FLEXIBLE SYNTHESIS OF C33-C39 POLYKETIDE REGION OF APRATOXIN "A Flexible Synthesis of C33-C39 Polyketide Region of Apratoxin: Synthesis of Natural and Unnatural Analogues," A. Giles, J. Martinez, F. Cavelier Syntheses of the polyketide moiety of apratoxins A and C and two other polyketide analogues and demonstrates the versatility of such synthesis in introducing different alkyl groups at C39 position. Significance: for future structure-activity relationship (SAR) studies of the potent antitumoral compound, apratoxin, by means of proposing a strategy to synthesize the oxazoline analogues of the said compound

PREVIOUS STUDIES Cytotoxic apratoxin A and analogues o Discovered cyclodespeptidases o Marine origin o Antitumoral activity

Figure 8 Apratoxin and analogues

Page 8 of 10

CHEM 135

FATTY ACDS AND POLYKETIDES

Hydrolytic kinetic resolution produced the pure (R)-epoxide 13 c. Regioselective nucleophilic substitution with homoallyl magnesium bromide to yield 14. 3. Analogue 4 a. Parent molecule: Hex-5-en-1-ol
b.

Figure 9 The four synthesized analogues

SYNTHESIS

Scheme 9 Synthesis of apratoxin A and C

1. Apratoxin A (1) and apratoxin C (2) a. Lists aldolisation of pivalaldehyde and butyraldehyde Butyraldehyde gave a lower yield. The diasteroselective reduction of the hydroxyketones 6 and 6 under Prasad reaction conditions produced the diols 7 and 7', with a better diastereoselectivity for the pivalaldehyde due to steric hindrance. b. Separation of the diastereomer products by silica gel CC c. Cyclic sulfite formation (8 and 8') with thionyl chloride. d. Oxidation with RuCl4 and NaIO4 as cooxidant to produce 9 and 9'. e. Regioselective nucleophilic substitution of the sulfate with allylmagnesium bromide. When R=iPr for 9', the regioselectivity of the reaction was not complete due to less steric hindrance with the yield of the formation of its regioisomer only being 30%. f. The substitution reaction (SN2) induced complete inversion of configuration for both cases. g. Final hydrolysis of the newly formed sulfate furnished the free alcohol. 2. Analogue 3

Scheme 10 Synthesis of Analogue 3 a.

Parent molecule: racemic epoxide in the molecule 11 Page 9 of 10

BS BC 08

Chemistry 135: Chemistry of Natural Products

EXAM 1

FATTY ACIDS AND POLYKETIDES

August 5, 2011 Barba, Biadomang, Narciso

4. The rest of the synthesis was identical for all the four analogues a. Silylation of free alcohol Reagent: TESCI (16) b. Oxidative cleavage of the double bond to give the corresponding aldehyde (17). c. Brown's crotylation reaction. This was a more efficient option in yielding the corresponding homoallylic alcohol. d. ()--methoxydiisopinocampheyl borane was used as chiral auxiliary to obtain the desired enantioselectivity at the C35 chiral center e. Trans-butene was used as adduct to give the anticompound with a good diastereoselectivity. f. Identification of syn diastereomers by silica gel chromatography. The compounds 17c and 17d did not possess a methyl group in the beta position from the aldehyde have less diastereoselectivity due to a loss of steric hindrance. g. Protection of 17d as TBDMS silyl ether h. Ozonolysis gave an aldehyde. i. Oxidation with NaClO2 into its corresponding polyketide moieties.
END OF TRANSCRIPTION

Page 10 of 10