Structural Elucidation and Total Synthesis

Structural Elucidation and Total Synthesis
Azaspiracid-1 Structural Elucidation and Total Synthesis Good Afternoon to all! Today I will be talking to you about a Marine Toxin called Azaspiracid. There are two aspects of this talk. First the focus is on the creative work that has been done by Nicolaou in elucidating the following structure and the second focus shift to the total synthesis of one of the analogues that has been done by David Evans, which has a great contribution of our very own professor, Andre Beauchemin. Thivisha Rajagopal January 29, 2009 University of Ottawa

A New Marine Toxin Occurrence
Mussels (Mytilus edulis) cultivated at Killary Harbour, Symptoms: Nausea, vomiting, severe diarrhea, and stomach cramps Similar to diarrhetic shellfish poisoning, DSP A new marine toxin In Nov 1995, 8 people became ill in Netherland after eating mussels that were cultivated at Killary Harbor, west coast of Ireland The symptoms associated with this illness were nausea, vomiting, severe diarrhea and stomach cramps which are similar to the existing diarrhetic shellfish poisoning called DSP. The two major toxins in DSP are Okadaic acid when R=H and Dinophysistoxin when R=Me, however the levels of these toxins were low which indicated an evolution of new marine toxin.

Discovery of Azaspiracid
Produced by marine dinoflagellate, Protoperidinium crassipes Absorbed by mussels, oysters, scallops, clams and cockles Discovered in Europe Harmful to the environment and human health Damage to lung, liver, spleen, and lymphocyte, as well as lung tumor formation in mice This new marine toxin orginated from dinoflagellate, Protoperidinium Crassipes and are absorbed by mussels, oysters, scallops, clams and cockles. It started off in the west coast of Ireland and expanded through out Europe, specifically England, Norway, Spain, France and Italy. Toxicology study in mice reveals a great risk to human health and our environment. It has shown to damage lung. Liver, spleen, lymphocyte and lung tumour formation in mice. These observations prompted many scientists to explore this causative toxin in mussels for structural studies

Azaspiracid-1 Structural Elucidation
Structure Elucidation by NMR Stereochemistry via NOE Along these footsteps, the first person to report the structure of Azaspiracid is Satake’s group in 1998. They extracted 2mg of Azaspiracid, colorless solid from 20Kg of mussel meat, elucidated the structure by NMR and confirmed the stereochemistry of the proposed structure via NOE. Azaspiracid is composed of double spiroketal fused to tetrahydrofuran moeity, 6-membered hemiketal bridge, spiroaminal ring fused to bicyclic ketal system, and a gamma, delta unsaturated terminal carboxylic acid It total, there are 9 rings and 20 stereogenic centers within this synthetically challenging and novel molecular architecture.

AZA Analogues AZA 1 is the predominant toxin in shellfish
Eleven different analogues of AZA were determined via Liquid Chromatography with tandem mass spectrometry. These analogues vary in 4 positions, R1 to R4. AZA-1 is the predominant toxin in shellfish, in which R3-Me. The other analogues are either hydroxylated or methylated version of the AZA-1. AZA 1 is the predominant toxin in shellfish Analogues determined by tandem mass spectrometry (LC-MS)

Closer Look at Azaspiracid-1 Structure
Despite the heroic spectroscopic attempts to elucidate the structure of Azaspiracid by Satake’s group, its daunting molecular framework yielded neither its relative nor its absolute stereochemistry b/w ABCDE and FGHI domains. Therefore, to clarify its molecular structure, Nicolaou’s group initiated the program toward its total synthesis. As a start point, they tackled the total synthesis of the proposed structure by Satake.

Nicolaou - Retrosynthesis
Nicolaou’s proposed synthetic strategy was based on a two fold disconnection of the polycylcic array of Azaspiracid-1. 1st disconnection at C20-C21 via lithiated dithiane coupling gave ABCD fragment 2nd disconnection at C27-C28 via Pd-mediated Sp2-Sp3 Stille Coupling gave E and FHI fragments This disconnection allowed the necessary flexibility to address the relative stereochemical issues b/w the two domains. For Nicolaou’s structural elucidation, I will only focus on the key points of the synthesis of the important fragments which lead to the determination of the correct structure Lets look at the brief forward synthesis.

Forward Synthesis Starting from cyclization precursor, the cascade deprotection leads to the following intermediate which undergo a cascade cyclization to afford the double spiroketal, further manipulations furnish the desired ABCD fragment as pentafluorophenyl ester in less 20 steps

This ester is immediately employed in the coupling reaction with the lithiated form of dithiane fragment to afford ketone at C20, which is stereoselectively reduced via DIBAL-H to an alcohol. Added manipulations furnish the primary allylic acetate at C27 as the most suitable site for the projected Stille Coupling

FHI vinyl stannane couples to the primary allylic acetate via Pd-catalyzed Stille Coupling to assemble the complete carbon framework of the proposed Azaspiracid-1 structure. The TES selectively removed and subjected to iodoetherification with N-iodosuccinimide to form the iodide which is carefully removed by tributyl stannane to afford the G ring

Following the remaining operations unveil the following the structure which is one step away from the target molecule. Removal of the secondary acetate group by the action of LiOH in MeOH, forms the E ring and accomplish the synthetic product.

Structural Comparison
Synthetic NMR DOES NOT match the Natural Azaspiracid-1 Upon the excitement of constructing the Azaspiracid-1, it was compared to the Natural Product. However, it was more disturbing to realize that neither TLC, HPLC, and 1H NMR data matched the naturally derived Azaspiracid-1. More specifically when comparing the NMR of synthetic vs natural, a clear differences arise in the upfield region of the spectrum along with the different splitting patterns for the protons In light of these studies, Nicolaou’s group concluded the proposed structure is in error. Synthetic NMR Natural NMR

Structural Comparisons
Synthetic NMR DOES NOT match the Natural Azaspiracid-1 Having already eliminated the synthetic compound as true structure, a few more logical targets were tested as they were easy to assemble via the two key disconnection method mentioned earlier. FGHI-epimer synthesized by joining the FHI ent via stille coupling. Both synthetic and FGHI epimer existed as inseparable isomers, unlike the natural AZA exist as a single compound. This observation again highlighted the structural discrepancy, in addition the NMR did not match the natural product. Two more possible alternatives moved to the front table to test if the structural discrepancy is imbedded in the stereochemistry at C20 for both synthetic and its FGHI epimer since it the key disconnection b/w the two domains Theses analogs were prepared along the same lines. However, also proved them to be incorrect. At this point the plan for determining the true structure of Azaspiracid called for the degradation of the natural material, which required the collaboration study with the Satake’s group

Chemical Degradation Natural Azaspiracid-1 was derivatized by exposure to trimethylsilyldiazomethane to obtain methyl ester which was treated with sodium periodate, resulting in the cleavage of the diol bond and the formation of corresponding amino lactone A and aldehyde which is reduced to an alcohol B. We will start our investigation to determine both relative and absolute stereochemistry of amino lactone A.

Structural Identity of Amino Lactone A
The allylic acetate lactone synthesized from dihydroxy dithiane in 3 steps undergo Stille Coupling with both enantiomers of FHI stannannes to produce the two possible amino lactones.

Natural vs Synthetic Amino Lactone
MATCH In comparing the 1H NMR of both products, the NMR spectral data for only one of these synthetic materials matched those of the degrative amino lactone These results concluded the relative stereochemical arrangement for the EFGHI domain of azaspiracid-1 is that depicted by this structure However, the absolute stereochemistry remains unknown. How do we determine absolute stereochemistry of organic molecules?

Absolute Stereochemistry
Synthetic interconversions and comparison of optical rotation NMR based methods – Mosher Ester Analysis For Carboxylic Acids Alcohols and Amines There are two major ways of determining the absolute stereochemistry. Correlation with compounds of known configuration by synthetic interconversions and comparison of optical rotation by polarimetric methods 2. NMR based methods more specifically Mosher Ester Analysis which is discovered by Mosher in 1973 and modified in late 1980. This method employs the use of chiral enantiomerically pure esters such as Pheny glycine methy ester for caraboxylic acids and α-methoxy-α-trifluoromethyl phenylacetic acids for alcohols and amines The analyzing procedure for both CA and Alcohols are similar. Step1: The acid of interest is coupled with each enantiomer of Mosher’s Ester, R-Phenyl glycine and S-Phenyl glycine, in two separate but analogous experiments to form the respective diastereomeric amide Step2: Diastereomers have different physical and spectroscopic properties. Therefore, they can be separated by chromatography and 1H NMR spectra can be compared to identify the differences in chemical shifts employing the diamagnetic anisotropy effect of the phenyl ring. For the purpose of this part of the seminar, we are interested in comparing the synthetic diastereomers with natural to elucidate further proof in the absolute stereochemistry.

Degradative Fragments
With that said, in an effort to determine the absolute stereochemistry, the Natural amino lactone was oxidized to an amino acid by chemical means and secured the optical rotation as

MATCH Therefore, for comparison purposes, the amino acid was synthesized. Much to our delight the NMR data matched the natural amino acid. Furthermore, two samples exhibited comparable rotations, but with opposite signs. Synthetic +49.6, Natural This confirms the absolute stereochemistry of FGHI domain of azaspiracid-1 as the antipode of the synthetic amino acid. However as a further proof, the synthetic amino acid was treated with both R and S Phenyl glycine methyl esters to produce the respective diastereomers, which was compared to the synthetic diastereomers. Indeed, the synthetically derived R-PGME exhibited a 1H spectrum identical to the S-PGME of the natural amino acid.

Confirmation of EFGHI Absolute Stereochemistry
These studies together provided a complete picture for the structure of the EFGHI domain of azaspiracid-1.

Chemical Degradation With the establishment of both the relative and absolute stereochemistry of EFGHI domain, the attention was then turned to the remaining segment, namely ABCD framework of the natural product in which they suspected the structural discrepancy to be embedded

Comparison of Degradation vs Synthetic Fragment
Synthetic Compound NMR Does Not Match the Degradation Fragment H Degradation Fragment Synthetic Compound Δ (1H) [ppm] 6 4.79 4.50 7 2.48 2.06 2.03 2.04 8 5.74 6.07 9 5.62 5.79 Investigation began by comparing the 1H NMR of the initially synthesized compound to the degradation fragment, spectroscopic data differered significantly. The main differences b/w the two spectra were associated with the protons located at C6 to C9. Despite these clear differences, the true architecture of this region of the molecule remained elusive.

Position of the Double Bond
Degradation Fragment Weak HMBC correlation b/w C10 and 7-H Weak COSY correlation b/w 6-H and 9-H H Degradation Fragment Synthetic Structure Lissoketal Revised Degradation Fragment Δ (1H) [ppm] 6 4.79 4.50 4.34 7 2.48 2.06 5.80 5.62 2.03 2.04 8 5.74 6.07 9 5.79 2.57 1.91 The first clue to this puzzle came from a marine natural product, Lissoketal, discovered by Hopmann and late Faulkner in 1997. Lissoketal bears a close resemblance to the A ring of Azaspiracid-1, except for the fact that the endocyclic double bond in ring A resides b/w C7 and C8 rather than C8 and C9 in the degradation fragment. A close examination of the 1H and 2D NMR showed remarkable similarities b/w lissoketal and degradation fragment. In particular the weak HMBC correlation b/w C10 and 7H and a weak COSY correlation b/c 6H and 9-H for the degradation fragment was in line with the observations for Lissoketal. As a result the endocylcic double bond was relocated b/w C7 and C8, this further explains the downfield shift of the 6-H on the spectrum as it placed in a doubly allylic environment. To test this hypothesis, a chemical synthesis of the newly proposed structure was required.

Double Bond Correction: Synthesis
Hence, starting from double spiroketal intermediate, arrive at trimethylsilyl enol ether, whose oxidation to the corresponding enone via Pd(Oac)2, reduction with NaBH4, and acetylation furnished the allylic acetate. Finally, catalytic amounts of Pd2dba in the presence of tributylphosphine and NaBH4 afford the desired olefin. Treatment with TBAF leads to the target synthetic compound. Much to our disappointment, the spectral data of synthetic compound did not match the degradation fragment. Therefore, confirming there is something more than the double bond location was wrong with the synthetic compound Synthetic Compound DID NOT MATCH Degradation Fragment Something more than the double bond location was wrong with the synthetic compound…

Degradation ABCD fragment is Thermodynamically Stable
Two Key Observations: Degradation ABCD fragment is Thermodynamically Stable NOE between H-6 and C-14 Methyl At this point we were still faced with 128 possible structures for the ABCD domain arising from 7 stereogenic centers. However, a second clue to this mystery puzzle is in observing the thermodynamic stability of the double spiroketal in the ABCD degradation fragment under acidic conditions. Thus, having to know the relative and absolute stereochemistry of the tetrahydrofuran from the starting material, the focus was on the two stereogenic centers in the double spiroketal In addition to this information, realizing a strong NOE interaction b/w 6-H and C-14 methyl group allowed us to narrow down the possibilities. But at first, we need understand what is meant by thermodynamically stable for spiroketals.

Anomeric Effect

Two Possible Structures
The first to be targeted for the synthesis is the following fragment.

On the way to 3rd Possible Fragment
Starting from a similar intermediate as shown earlier, followed by cascade cyclization afford the double spiroketal with the opposite stereocenter at C13. Exposure to TFA in DCM for 4h at rt resulted in an equilibrium mixture in which the C-13 epimer, the desired stereocenter predominated in a 55:45 ratio. A strong NOE effect was observed for 6-H and C-14 Me which supported the structure, but its relative thermodynamic stability of the desired stereocenter was problematic, since the degradation fragment is firmly anchored in one stereochemistry and presumed to be thermodynamically most stable. In view of these inconsistencies, the pursuit of the proposed structure was abadonded at this stage and turned the attention toward the construction of the fourth possible fragment

Construction of 4th Possible Fragment
MATCH Which began with the cyclization of a similar intermediate with the opposite stereochemistry at C-6 and C-14 Me group leading to double spiroketal with the correct stereocenter at C13, which undergo further modification to furnish the synthetic compound. Delightfully, the 1H NMR spectrum of the synthetic was identical to the degradation fragment. Unfortunately no optical rotation was gathered for the degradation fragment due to insufficient amounts obtained. Hence, the absolute stereochemistry remained unknown at this point. Absolute Stereochemistry was unknown

To determine the absolute stereochemistry, both enantiomers of the ABCD domain was synthesized and combined with the EFGHI domain whose relative and absolute stereochemistry is confirmed to synthesize the Azaspiracid-1.

MATCH One of the two possible structures matched the naturally derived AZA-1 by 1H NMR. Thereby confirming the correct structure in 2004.

Initial Structure vs Correct Structure
In comparing the initial vs the correc structure of Azaspiracid-1, there are 3 key differences. The position of the double bond shifted one carbon down, the relative configuration of CD domain in blue and FGHI domain in red are opposite in the correctly assigned natural product. “Like several other total syntheses, this endeavor demonstrated once again the continued role of chemical synthesis in structural elucidation of natural products and its indispensable nature as a source of scarce, but highly valuable, substances for biological investigations.”

(-) and (+)-Azaspiracid-1
Now we will shift our focus onto pursuing the total synthesis of the enantiomer of the correct structure of AZA-1 by David Evans. This target required only minimal changes in the original synthesis plan designed to pursue the initially proposed incorrect structure of AZA-1.

EVANS Total Synthesis Development of C2-symmetric CuII-complexes
Integration of 3 key catalytic enantioselective processes Hetero-Diels Alder Glyoxylate-Ene Mukaiyama Aldol

Retrosynthetic Analysis
The principal disconnection at C20 and C21 provide the electrophilic ABCD aldehyde at C20 and EFGHI sulfone at C21. This strategy provide a convergent approach to AZA-1 since all 9 rings are formed prior to the final sulfone anion addition

ABCD-aldehyde ABCD aldehyde involves a thermodynamically controlled spiroketalization event to give the intermediary ketone. An addition of Sulfone at C12 provide the AB and CD ring fragments CD ring fragment is accessed via enantioselective Cu2+ catalyzed glyoxylate-ene reaction to provide the initial C17 stereocenter So lets start the synthesis of CD ring fragment via Glyoxylate-ene employing the Cu2+ catalyst

Glyoxylate-ene Reaction
The general mechanism for carbonyl ene reaction involves LUMO of carbonyl to react with the HOMO of ene to form an alcohol, a new db and sigma bond. Lewis acid can used to lower the energy of the LUMO of the enophile. Evans, employed his developed Cu(II) complex as a chiral lewis acid to catalyze the carbonyl-ene reaction using glyoxylate esters with variety of olefin substrates to produce enantioselective alcohol. The following two catalysts exhibited high reactivity affording the ene products in excellent enantioselectivity What is the explanation for the observed enantioselectivity?

Glyoxylate-ene: Stereochemistry
Ene (Re face) Ene (Si face) The Cu complex forms a bidentate coordination to the glyoxylate ester via chelating to the O, there by forming a distorted square planar geometry at the Cu center as shown by the 3D which creates the facial bias for the Nuc attack The Re-face is encumbered by the t-Bu group on the ligand, hence favoring the Si face attack which result in the generation of S-configured alcohol Bidentate coordination Distorted square planar Cu(II) ligand complex

CD Ring Reaction of ethyl glyoxylate with silyl protected 2-methyl-2-propen-1-ol in the presence of optimal bis(aquo) Cu complex provide the desired stereochemistry at C17 of alpha-hydroxy ester. Which is converted into the corresponding weinreb amide followed by the PMB protection of the alcohol Metalation of the readily available iodide with t-BuLi followed by the addition of the weinreb amide introduce the remaining 4 carbon subunit Ketone selectively reduced via L-selectride to an alcohol

Careful ozonolysis of the 1,1-disubstituted olefin allowed the oxidative cleavage of the olefin and the formation of ketone at C19 Subsequent ketalization afforded the methyl ketal in the presence of pyridinium para toluene sulfonate in MeOH. Stereoselective reduction with triethylsilane mediated by trifluoroborane etherate allows the inside Nu attack on oxocarbenium ion to provide the desired trisubstituted tetrahydrofuran Following simple modifications leads to the target CD ring

ABCD-aldehyde

AB Ring Racemic acetylene readily prepared from acrolein by conjugate addition of thiphenol, followed by addition of allenyl grignard reagent The anion of the acetylene coupled to the weinrebamide which is synthesized via cross metathesis to form the ene-ynone

General Mechanism for CBS Reduction
Borane complex to the N atom of the catalyst to form the active reducing agent. Once the carbonyl oxygen complex to the other borane of the catalyst, it becomes electrophilic enough to be reduced by the weak hydride source. The hydride transfer occur via a 6-membered cyclic transition state. The enantioselectivity arise from the preference of the larger substituent on the ketone for the pseudoequatorial position on the ring to provide the desired product

ABCD-Aldehyde

Retrosynthetic Analysis

EFGHI-Sulfone Boron aldol addition Mukaiyama Aldol

Breakdown of E, HI and FG Fragments

Hetero Diels -Alder Reaction

Reaction Stereochemistry
α-Re-face Bidentate coordination Distorted Square planar Cu(II) substrate complex

E Ring

HI Ring Sequential acetal and ester reduction followed by Iodine installation leads to the Iodide Intermediary alkoxide was formed and trapped with TsCl in sity to the form the following intermediate Terminal olefin was smoothly oxidized to methyl ketone following an introduction of azide to furnish the HI ring fragment

Breakdown of E, HI and FG Fragments

Mukaiyama Aldol

Optimal Catalyst

FG Ring

EFGHI-Sulfone

FG and HI coupling

E +FGHI

FG Bicyclic Ketal

EFGHI-Sulfone

Final Fragment Coupling

Synthesized 75mg of the target molecule 2.8% yield for 26 linear steps
(+)-Azaspiracid-1 (+)-Azaspiracid-1 Synthesized 75mg of the target molecule 2.8% yield for 26 linear steps “Frontiers of organic synthesis defined by one’s ability to successfully handle the challenges of escalating architectural and dynamic molecular complexity.”

CONCLUSION Azaspiracid (AZA) is a marine toxin from dinoflagellate and absorbed by mussels AZA-1 is the dominant toxin and its was first synthesized by K. C. Nicolaou in 2003 Correct structure was elucidated in 2004 via degradation study

CONCLUSION Integration of catalytic enantioselective processes

CONCLUSION Total synthesis of (+)-Azaspiracid-1 in 26 linear steps with 2.8% overall yield

Acknowledgements Anthony Pianosi Dr. W. Ogilvie Jojo Jiang
Special thanks to Dr. A. Beauchemin Dr. W. Ogilvie Dr. A. Flynn Alex Bush Daniel Carter Ramirez

Shielding Deshielding S Shielding Deshielding R L1L2 C(H)-O-C(O)-C-CF3
COPLANAR Mosher: “these conformations are intended to represent a model which successfully correlates the known results” Shielding Deshielding L2 CF3 Ph L1 MeO S Shielding Deshielding L2 CF3 OMe L1 Ph R When we analyze these diastereomeric esters, we assume that they exist in one major conformation in which the ester adopts a s-trans arrangement, while carbinyl-H, O, C-O and CF3 are coplanar.

ΔδSRL2 = δL2(S) - δL2(R) < 0 ΔδSRL1 = δL1(S) - δL1(R) > 0
Shielding Deshielding L2 CF3 Ph L1 MeO S Shielding Deshielding L2 CF3 OMe L1 Ph R δL2(S) < δL2(R) and δL1(S) > δL1(R) ΔδSRL2 = δL2(S) - δL2(R) < 0 and ΔδSRL1 = δL1(S) - δL1(R) > 0 Kakisawa Model

Determination of Absolute Stereochemistry of FGHI

Preparation of Ligand

Preparation of the Catalyst
Bis(oxazoline) ligand with Copper dichloride forms the cupric chloride complex which upon treatment with silver hexafluoroantimonate followed by filtration through celite to remove the precipitated AgCl, results in the active catalyst as a deep-green solution. Exposure to atomospheric moisture afford the bench stable deep blue crystals of the bis(aquo) hexafluoroantimonate complex

Wacker Oxidation

Corey Bakshi Shibata (CBS) Reduction

Observed Stereochemistry
Nu (Si face) Bidendate coordination of the substrate Square Pyramidal Copper Geometry

Structural Elucidation and Total Synthesis

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