our synthesis was initiated with chromone 12 which was prepared in greater than 50 gram batches and purified by recrystallization ( figure 2 ) .
utilizing diisopropylsilyl ditriflate to activate chromone 12 to a siloxybenzopyrylium species , vinylogous addition of 2-trimethylsiloxyfuran was achieved which was followed by hydrogenation to afford chromone lactone 13 on adecagram scale ( 89 % yield , dr > 10:1 ) after purification by trituration ( supplementary information s4s19 ) .
further treatment of 13 with nah in thf led to the production of epi - blennolide c 14 through dieckmann cyclization . due to the propensity of thevinylogous acid moiety of tetrahydroxanthone 14 to both epimerization and tetrahydroxanthone - to - chromone lactone rearrangement
, we utilized the neutral methylating reagent trimethylsilyldiazomethane to obtain the desired o - methylated product 16 in 61 % yield along with the separable isomer 15 ( 20 % yield ) .
after phenol - directed ortho - iodination of 16 , the derived aryl iodide 17 was subjected to kinetic acylative resolution using birman 's catalyst   on a gram scale ( s factor > 200 ) .
chiral monomer ( )-17 was isolated in 46 % yield ( 99 % ee ) and the o - acylated product ( + ) -18 was obtained in 46 % yield ( 99 % ee ) after recrystallization . after obtaining the two enantiopure tetrahydroxanthone monomers ,
our initial plan was to employ our stannane dimerization strategy which successfully led to syntheses of secalonic acids a and d. however , after evaluation of various conditions , we observed only trace stannylation of iodides 17 or 18 ( table 1 ) which may be due to steric hindrance of the aryl iodide substrate .
gratifyingly , iodide ( )-17 was found to be stable under basic conditions and to produce an inseparable mixture of dimeric products ( dr = 88 : 12 ) under one - pot suzuki coupling conditions using pd(oac)2/sphos and bis ( pinacolato ) diboron .
we subsequently attempted to establish the stereochemistry of the major atropisomer by nmr analysis and by acid - mediated deprotection of both atropisomers 19 and 20 to 1 and 23 ( figure 4 ) .
however , both efforts failed as the axial chirality center is remote from existing stereocenters leading to almost identical h nmr spectra for both a tropisomers of rugulotrosin a. fortunately , a single crystal of the major atropisomer ( )-19 was obtained by recrystallization ( ch2cl2/meoh ) from its mixture with 20 .
x - ray crystal structure analysis of ( )-19 ( ccdc#1022610 ) was achieved which verified that the relative stereochemistry was identical to that found in natural rugulotrosin a. during our evaluation of dimerization conditions , we found that water was essential and that the ratio of atropisomers was dependent on ligands for palladium rather than solvent , temperature , or reaction time .
accordingly , we focused our efforts on the influence of ligands on atropisomeric selectivity ( table 1 ) .
after extensive ligand screening , we found that monophosphines were crucial for the success of there action , and that bidentate ligands such as xantphos or dpephos ( not shown ) failed to provide the desired dimeric products .
besides sphos(l1 ) , the related ligand ruphos ( l2 ) provided dimeric products in even higher atropselectivity ( 93:7 ) .
interestingly , we found when the 2 ' position of the monophophine ligand did not contain an oxygen atom ( e.g. carbon atom in xphos l2 or a nitrogen atom ( davephos , not shown ) ) , dimeric products were obtained in trace amounts . after experiments with the achiral ligand l4 , which provided similar results to sphos , we anticipated that related chiral ligands may achieve efficient control of atropselectivity .
we began this investigation with ( r ) and ( s)-bi - dime ligands l5 , which recently have shown excellent atropselectivities in biaryl couplings .
however , neither ligand was able to perform the one - pot dimerization of ( )-12 .
gratifyingly , chiral versions of the sphos ligands , s - cy - mop ( l6 ) and r - cy - mop ( l7 ) , both successfully produced dimeric products .
the ligand r - cy - mop provided a satisfactory dr ( 95.5:4.5 ) and the mismatched ligand s - cy - mop afforded a lower dr ( 81:19 ) , both favoring the same major atropisomer .
even with the increased bulk of ligand l8 , the dr did not noticeably improve . in order to enhance product yield
, we prepared the buchwald 3rd generation precatalysts with sphos , r - cy - mop , and s - cy - mop .
indeed , use of the precatalyst increased yields for these cases and maintained at ropselectivity .
use of bis ( neopentyl glycolato ) diboron ortetrahydroxydiboron as boron transfer reagents led only to trace amounts of dimeric products .
the acylated , enantiopure monomer ( + ) -18 was also investigated in the suzuki dimerization which generated a separable 3:1 mixture of atropisomersin 36 % yield ( figure 3 ) .
fortunately , an x - ray crystal structure of the minor atropisomer ( )-22 ( ccdc#1022611 ) was obtained to verify relative and absolute stereochemistry .
introduction of a bulk yester onto the secondary alcohol of the monomer ( e.g. ocoph ) did not affect the ratio of atropisomers .
after obtaining and establishing stereochemistry for several protected dimers , we anticipated that we could access all atropisomers and enantiomers of rugulotrosin a. dimer ( )-19 was treated with 3 m hcl / meoh for 10 min which afforded rugulotrosin a ( ( )-1 ) in 89 % yield ( figure 4 , a ) . in a similar manner , ( + ) -rugulotrosin a was obtained from acidic hydrolysis of ( + ) -21 ( figure 4 , b ) . with both enantiomers of rugulotrosin a in hand , natural rugulotrosin a was unambiguously assigned as ( + ) -1 though comparison of rotation data .
similarly , atrop - rugulotrosin a ( )-23 and entatrop rugulotrosin a ( + ) -23 were synthesized using similar protocols from the protected precursors ( )-22 and ( + ) -22 ( figure 4 , c and d ) . to verify the stability of the axially chiral biaryl moiety
, we heated rugulotrosin a ( + ) -1 in toluene ( 1 mg / ml ) at 100 c for 12 h and 150 c for 3 h. under both conditions , we did not observe formation of atrop rugulotrosin .
this is supported by a high barrier of rotation of rugulotrosin a , calculated as 47.7 kcal / mol using the b3lyp/6 - 31g(d ) level of theory ( see supplementary information s52s58 ) . in order to rationalize the axial chirality selectivity observed in the dimerization
, we modeled the geometry of the expected intermediate diaryl palladium ( ii ) complexes using sphos ( l1 ) as ligand ( figure 5 ) .
conformational analysis was carried out by systematic six - fold rotation about the c - pd bonds , followed by optimization of candidate structures at the b3lyp / lanl2dz level of theory .
the three lowest energy structures 24a  24c all have dihedral angles ( c1 , c2 , c2 , c1 ) for the monomer fragments which would afford atropisomer ( )-19 ( precursor to rugulotrosin ) after stereospecific reductive elimination ( figure 5,a ) .
the next two higher energy structures 25a and 25b display a reversed dihedral angle which should lead to atropisomer 20 ( table 1 ) .
space filling models of structures 24a - c and 25a - b ( supplementary information s34s52 ) show a more crowded arrangement of the latter , in part because of steric interactions between the methyl ester and the cyclohexyl group .
the distances between the hydrogen of the methyl ester to the hydrogen on the cyclohexyl of the sphos ligand are 2.4 and 2.5  for conformers 25a and 25b , respectively . based on literature precedent
, we assume that these catalyst - reactant complexes are under thermodynamic equilibration ; additional computational studies will be required to predict barriers to interconversion .
it thus appears that product stereochemistry is determined by reactant and catalyst assembly in the pd ( ii ) complex ; this provides a basis for prediction of stereochemistry in future atropselective couplings . after assigning the absolute configuration of rugulotrosin a , we employed our library of synthetic rugulotrosins to determine whether penicillium nov .
( mst - f8741 ) was capable of producing atrop - rugulotrosin a. to this end , separate 21 day fermentations of mst - f8741 were extracted with either mecn ( figure 4,e(a ) ) or meoh ( figure 4 , e(b ) ) and were subsequently analyzed using an hplc - dad method optimized for the resolution of rugulotrosin a ( figure 4 , e(c , d ) ) and atrop - rugulotrosin a ( figure 4 , e(e ) ) . to further enhance sensitivity ,
these analyses were also carried out using hplc - dad - esim sprotocols augmented by single ion extraction ( sie ) ( supplementary information , s22 ) . as these analytical studies failed to detect any trace of atrop - rugulotrosin in mst - f8741 extracts , we conclude that the rugulotrosin biosynthetic pathway present in penicillium nov .
a had previously been reported to exhibit antibacterial activity , we also tested the synthetic rugulotrosins ( ( + ) -1 , ( )-1 , ( )-23 , and ( + ) -23 ) against several strains of gram - positive and gram - negative bacteria .
consistent with earlier reports,(+)-1 exhibited antibacterial activity against bacillus subtilis ( atcc 6633 ) ( ic50 = 2.1 m ) and staphylococcus aureus ( atcc 25923 ) ( ic50 = 6 m ) . of note , the enantiomer ( )-1 was also active against b. subtilis ( atcc 6633 ) ( ic50 = 0.7 m ) and s. aureus ( atcc 25923 ) ( ic50 = 18 m ) , whereas the antibacterial activity of the atropisomer ( )-23 was weak and limited to b. subtilis ( atcc 6633 ) ( ic50 = 10 m ) and ( + ) -23 lacked any appreciable antibacterial activity .
none of the rugulotrosins ( + ) -1 , ( )-1 , ( )-23 , or ( + ) -23 exhibited activity against the gram - negative bacteria escherichia coli ( atcc 25922 ) and pseudomonas aeruginosa ( atcc 27853 ) , nor did they exhibit cytotoxicity ( ic50>30 m ) against human colon ( sw620 ) and lung ( nci - h460 ) cancer cells .
in conclusion , we have developed a concise , atropselective approach to rugulotrosin a and stereoisomers through point - to - axial chirality transfer which has facilitated determination of the absolute configuration of rugulotrosin a. computational studies modelling the geometry of intermediate diaryl palladium ( ii ) complexes have provided a rationale for the atropselectivity observed in the key suzuki dimerization .
these studies highlight the utility of sphos and related bulky monophosphine ligands in palladium couplings both to bring monomers in proximity and enhance remote steric effects leading to atropselectivity . through hplc analysis of fungal extracts and synthetic samples ,
. moreover , the atropisomers and enantiomers of rugulotrosin a were found to have different activities against gram - positive bacteria illustrating the importance of stereochemistry on target selectivity .
further studies regarding atropselective syntheses of dimeric natural products are currently under investigation and will be reported in due course .
 chiral , dimeric natural products containing complex structures and interesting biological properties have inspired chemists and biologists for decades . a seven step total synthesis of the axially chiral , dimeric tetrahydroxanthone natural product rugulotrosin a is described . 
 the synthesis employs a one - pot suzuki coupling / dimerization to generate the requisite 2,2'-linked biaryl linkage . 
 highly selective point - to - axial chirality transfer was achieved using palladium catalysis with achiral phosphine ligands . 
 single x - ray crystal diffraction data was obtained to confirm both the atropisomeric configuration and absolute stereochemistry of rugulotrosin a. computational studies are described to rationalize the atropselectivity observed in the key dimerization step . 
 comparison of the crude fungal extract with synthetic rugulotros in a and its atropisomer verified that nature generates a single atropisomer of the natural product . 
Results and Discussion
Conclusion
Supplementary Material