A "Motif-Oriented" Total Synthesis of Nannocystin Ax. Preparation and Biological Assessment of Analogues.

A“Mo i -O ien ed”To al Syn hesis o Nannocys in Ax. P epa a ion and Biological Assessmen o Analogues Zhanchao Meng, † Lae i ia Souilla , † B endan Monks, † Nikolas Huwyle , † Jenni e He mann, ‡ Rol Mulle , ‡ and Alois Fu s ne * ,† † Max-Planck-Ins i u u Kohlen o schung, D-45470 Mulheim/Ruh , Ge many ‡ Helmhol z Ins i u e o Pha maceu ical Resea ch Saa land, Saa land Uni e si y, 66123 Saa b ucken, Ge many * SSuppo ing In o ma ion ABSTRACT: The highly cy o oxic cyclodepsipep ides o he nannocys in amily a e known o bind o he euka yo ic ans- la ion elonga ion ac o 1α(EF-1α). Analysis o he docking pose, as p oposed by a p e ious in silico s udy, sugges ed ha he isubs i u ed alkene moie y and he neighbo ing me hyl e he o m a domain ha migh be closely co ela ed wi h bio- logical ac i i y. This hypo hesis sponso ed a syn he ic cam- paign which was designed o be “mo i -o ien ed”: specifically, a sequence o ing closing alkyne me a hesis (RCAM) ollowed by hyd oxy-di ec ed ans-hyd os anna ion o he esul ing cyclo- alkyne was concei ed, which allowed his po en ially ancho ing subs uc u e o be sys ema ically add essed a a la e s age. This inhe en ly flexible app oach opened access o nannocys in Ax (1) i sel as well as o 10 non-na u al analogues. While he biological da a confi med he ema kable po ency o his class o com- pounds and showed ha he domain in ques ion is indeed an inna e pa o he pha macopho e, he specific s uc u e/ac i i y ela ionships can only pa ly be econciled wi h he o iginal in silico docking s udy; he e o e, we conclude ha his model needs o be ca e ully e isi ed. ■INTRODUCTION Myxobac e ia a e a p olific sou ce o bioac i e na u al p oduc s. 1 In acco d wi h his no ion, he hi he o ai ly un apped genus Nannocys is sp. has ecen ly been shown o p oduce a small amily o cyclodepsipep ides endowed wi h ema kable an i ungal and cy o oxic p ope ies. 2,3 An ex ensi e sc eening exe cise e ealed ha he na u ally occu ing nannocys ins exhibi desi able di - e en ial ac i i y ac oss a comp ehensi e panel o up o 472 cance cell lines, including cell lines ha a e esis an o clinically app o ed d ugs. Impo an ly, hey seem o in e e e nei he wi h he ac in no he ubulin cy oskele on and do no inhibi a numbe o ep esen a i e kinases ei he ; 2,3 a he , he euka yo ic ansla ion elonga ion ac o 1α(EF-1α) was iden ified as he p ima y biological a ge . 3 Apa om he po en ial ele ance in a medicinal chemis y con ex , 4,5 he nannocys ins a e he e o e aluable p obe molecules o chemical biology o in e oga e ansla ion and p o ein anspo p ocesses in euka yo ic cells. As he sou ce o ganism p oduces a small compound collec- ion, some p elimina y insigh s in o s uc u e/ac i i y ela ion- ships (SAR) ha e been es ablished by he wo independen isola ion eams (Scheme 1). 2,3 Specifically, nannocys in Ax (1) and nannocys in A (2) we e ound almos equipo en agains he HCT-116 colon ca cinoma cell line, which sugges s ha he epoxide ing migh no be equi ed o high ac i i y 3 (al hough one o he isola ion eams had o iginally concluded o he wise). 2 The chlo ine subs i uen s on he D- y osine uni a e no essen ial ei he and can be eplaced by −Ho −B wi hou much loss in po ency. 2,3 In his con ex , i is in e es ing o no e ha epoxida ion and chlo ina ion occu la e in he biosyn hesis pa hway, only a e he mac ocyclic amewo k has been o ged Special Issue: Syn hesis o An ibio ics and Rela ed Molecules Recei ed: No embe 13, 2017 Published: Decembe 21, 2017 A icle pubs.acs.o g/joc Ci e This: J. O g. Chem. 2018, 83, 6977−6994 © 2017 Ame ican Chemical Socie y 6977 DOI: 10.1021/acs.joc.7b02871 J. O g. Chem. 2018, 83, 6977−6994 Downloaded ia HELMHOLTZ INST PHARM RSRCH SAARLAND on Oc obe 24, 2018 a 09:02:28 (UTC). See h ps://pubs.acs.o g/sha ingguidelines o op ions on how o legi ima ely sha e published a icles. by lac oniza ion wi h concomi an clea age o he mixed polyke ide/pep ide chain off he ca ie p o ein. 3 In silico docking s udies sugges ed ha nannocys in A (2)binds oa a he shallow ca i yon he su ace o EF-1α, 6 whe eby hyd ophobic in e ac ions seem o p e ail o e hyd ogen bonding be ween gues and hos . 3 In line wi h his analysis, de i a iza ion o he phenol −OH g oup o exchange o L-isoleucine o L- aline did no al e he cy o- oxici y by much. 2,3 Fo hei po ency and ele ance, he nannocys ins immedia ely caugh he a en ion o he syn he ic communi y; no less han six diffe en o al syn heses ha e been epo ed in sho o de . 7−12 While wo o hem eso ed o mac olac amiza ion o o m he 21-membe ed backbone, 10,12 i is chemically elling ha all o he success ul app oaches a ge ed he conspicuous diene uni embedded in o he polyke ide sec o , using ei he obus c oss- coupling 7,9,11 o equally well-es ablished alkene me a hesis o o m he ing (al hough he la e was no s e eoselec i e). 8,13 The o al syn hesis ou lined below is concep ually diffe en in ha i was no designed o he sake o apid conques o a single ep esen a i e o his amily bu a he mean o open access o s uc u al a ian s ha allow he pha macopho e o be mapped a a po en ially c i ical bu as ye uncha ed domain. Al hough ce ainly in keeping wi h he gene al concep o “di e ed o al syn hesis”, 14,15 he logic o he app oach is mo e ocused in ha i is s ic ly o ien ed owa d a po en ial key mo i wi hin he pha macopho e. ■RESULTS AND DISCUSSION S a egic Conside a ions. As alluded o abo e, he com- pu ed docking pose o nannocys in A (2) seems o esul om weak bu likely addi i e hyd ophobic in e ac ions wi h i s EF-1α p o ein hos . 3 A close look e eals only wo somewha deepe subpocke s wi hin an o e all a he shallow binding si e: one o hem accommoda es he gem-dime hyl g oup o he 3-hyd oxy-L- aline uni , whe eas he o he one emb aces he C6a-Me subs i- uen on he diene; he neighbo ing C7−OMe g oup, in con as , was compu ed o poin away om he p o ein su ace, jus as he epoxide ing does, which is known no o be manda o y o high ac i i y. 3 One can he e o e expec ha he allylic me hyl e he is also a po en ially o gi ing si e: i so, i migh quali y o chemical modifica ion in o de o adjus he physicochemical p ope ies o he compounds o o a ach app op ia e linke s. On he o he hand, excision o he C6a and he C19b,c me hyl subs i uen s is o ecas ed o be de imen al, while hei o mal eplacemen by o he hyd ophobic esidues migh allow po ency and lipophilici y o be fine- uned. Based on his analysis, we conside ed he isubs i u ed C5−C6 double bond flanked by he −OMe g oup o be he s a egic si e o disconnec ion (Scheme 1): i is his subs uc u e which we wished o add ess o he pu pose o la e-s age di e sifica ion. The s e eoselec i e anda he same imeflexible o ma ion o highlysubs i u ed alkenes embedded in oa mac ocyclicscaffold, howe e , is a om i ial. Ring closing alkyne me a hesis (RCAM) 16 ollowed by egioselec i e ans-hyd ome ala ion 17,18 o he ensuing alkyne migh allow his challenge o be me , e en hough his ac ic has no ye been applied wi hin a simila ly challenging chemical en i onmen . Al hough RCAM had p e iously excelled wi h complex pep idic subs a es, 19,20 i was by no means clea a he ou se o his in es iga ion whe he ans-hyd ome ala ion qualifies in he p esen con ex : his ans- o ma ion gains high egioselec i i y only i a p o ic subs i u- en on he subs a e s ee s he incoming [Cp*Ru−Cl] ca alys ia hyd ogen bonding o he chlo ide ligand and, in doing so, imposes di ec ionali y on he ansi ion s a e (Scheme 2). 21,22 While he C7−OMe e he o 1can ob iously be aced back o a p opa gylic −OH g oup which hen p o ides he necessa y handle, we el unable o assess wi h any le el o ce ain y whe he o no he p o ic amide linkages in ansannula p oximi y in a subs a e o ype Bin e e e wi h o po en ially e en dis up his c ucial p eo ganiza ion. 23 I egioselec i e ans-hyd ome ala ion is success ul, howe e , he −OH subs i uen in he esul ing p oduc Ap o ides an addi ional oppo uni y o la e-s age modifica ion: unde he p emise ha he −OR g oup uly poin s away o he p o ein su ace once he compound is bound o EF-1α, 3 i should no ma e much whe he his g oup emains unp o ec ed, is con e ed in o he pa en al me hyl e he , o is ans o med in o ano he biologically iable subs i uen . P epa a ion o he Building Blocks and F agmen Coupling. Since one o he isola ion eams had shown ha he epoxide ing is no necessa y o high po ency, 3 we chose nannocys in Ax (1) as ou lead a ge . Fo he p epa a ion o he equi ed enyne agmen E(Scheme 1), we adap ed he coppe - ca alyzed asymme ic ca bonyl addi ion chemis y ecen ly de eloped by Buchwald and co-wo ke s. 24 This ans o ma ion was desc ibed only o ke one subs a es, mos likely because aldehydes a e subjec o compe ing educ ion by he coppe hyd ide species gene a ed in si u. As he p ojec ed applica ion equi ed no hing bu benzaldehyde, i was deemed accep able o Scheme 1. Colo -Coded Summa y o Confi med and An icipa ed (“?”) SAR (G een/Red = Pe missi e/P ohibi i e Si e) a a See ex ; e osyn he ic analysis o nannocys in A ocusing on he p esumably c i ical isubs i u ed alkene en i y; “DOS”indica es he en isaged si es o “mo i -o ien ed”di e si y. Scheme 2. Di ec ing Effec Exe ed by a P opa gylic −OH G oup on o he Regiochemical Cou se o ans- Hyd ome ala ion Ca alyzed by [Cp*Ru−Cl] a a In he Newman- ype p ojec ion o he pu a i e loaded ca alys , “•” deno es he Me subs i uen s o he Cp* ing. The Jou nal o O ganic Chemis y A icle DOI: 10.1021/acs.joc.7b02871 J. O g. Chem. 2018, 83, 6977−6994 6978 d i e he con e sion by using his cheap subs a e in excess. In doing so, op ically ac i e 4(ee = 99.5%) became a ailable in only wo s eps om inyl b omide (Scheme 3). The b e i y o he app oach, he a ac i e ca alys loading (0.5 mol %, unop imized), and he excellen op ical pu i y o p oduc 4clea ly ou weighed he modes dias e eoselec i i y (d = 2.8:1), no leas because he an i-isome could be emo ed by flash ch oma og aphy. Alkyne 4 was hen c oss-coupled wi h iodop opyne, 25 ollowed by a egio- and s e eoselec i e semi educ ion o he p opa gylic iple bond o 1,3-diyne 5 hus o med. 26 The esul ing p oduc 6was es e ified wi h p o ec ed 3-hyd oxy alina e 10, which in u n was eadily a ailable om D-se ina e 8by aking ad an age o he hidden symme y (Scheme 4). 27 Since he asymme ic allyla ion o p opynal de i a i es such as 12 end o be unsa is ac o y unde a mul i ude o condi ions, 28 he p epa a ion o he acid agmen commenced wi h enzyma ic esolu ion o ac-13, which u nished op ically pu e 14 (ee >99%) on mul ig am scale (Scheme 5); 29 he eco e ed alcohol (S)-13 can also be con e ed in o 14 by a Mi sunobu eac ion. 30 The elabo a ion o 14 in o acid 16 in ol ed selec i e ozonolysis o he double bond in he p esence o he alkyne, Wi ig olefina ion, 31 saponifica ion o he wo es e uni s, and a achmen o a TBS p o ec ing g oup. Ghosez’s chlo oenamine eagen p o ed mos adequa e o he con e sion o he acid 16 in o he equi ed acid chlo ide 17. 32,33 The syn hesis o he dipep ide agmen 22 la gely ollowed es ablished ou es (Scheme 6). Specifically, selec i e dichlo ina- ion o me hyl y osina e 18 and condensa ion wi h comme cially a ailable N-Boc-N-me hyl isoleucine 20 u nished 21. 34,35 An ace yl g oup was ini ially conside ed as he p o ec ing g oup o he phenol o a oid p oblems in he subsequen es e ifica ion and pep ide coupling e en s; his g oup, howe e , la e u ned ou o engage in ansannula acyl mig a ion (see below). The phenacyl g oup p o ed o be a p ac ical and mo e s able al e na i e. 36 Whe eas coupling o amine 7wi h dipep ide 22 p oceeded wi hou inciden , he missing amide linkage a he s e ically hinde ed N-me hyl-L-isoleucine e minus was mo e difficul o o m (Scheme 6). 37 To his end, 23 was ea ed wi h a la ge excess o TBSOT /lu idine because (pa ial) silyla ion o he amides p eceded clea age o he N-Boc esidue; wo kup o he c ude ma e ial wi h TBAF ga e he desi ed ee amine; his compound eac ed well wi h acid chlo ide 17, which was p epa ed om 16 on demand as men ioned abo e. Mac ocycliza ion and Comple ion o he To al Syn- hesis. Wi h compound 24 in hand, he s age was se o ing closu e by RCAM and downs eam elabo a ion o he esul ing cycloalkyne in o nannocys in Ax (1)(Scheme 7). While me a hesis o wo e minal alkynes emains e a ic in ou hands, 38 p e ious wo k om his labo a o y has shown ha eac ions o subs a es comp ising one e minal and one in e nal alkyne a e obus and scalable. 39,40 Indeed, he molybdenum Scheme 3. P epa a ion o he Enyne Building Block Scheme 4. P epa a ion o he 3-Hyd oxy alina e Building Block Scheme 5. P epa a ion o he Acid Segmen Scheme 6. P epa a ion o he Cycliza ion P ecu so The Jou nal o O ganic Chemis y A icle DOI: 10.1021/acs.joc.7b02871 J. O g. Chem. 2018, 83, 6977−6994 6979 alkylidyne ca alys 25 41 endowed wi h silanola e ligands con e ed compound 24 wi hin no mo e han 15 min a ambien empe - a u e in o he co esponding mac ocyclic enyne, which was desilyla ed o gi e 26 in eadiness o he p ojec ed ans-hyd o- s anna ion. This example highligh s he eac i i y and unc ional g oup ole ance o 25 as he p o o ype membe o he a guably mos selec i e gene a ion o alkyne me a hesis ca alys s cu en ly a ailable. Since Sch ock alkylidynes a e inhe en ly nucleophilic a ca bon, 42 he compa ibili y wi h p o ic si es as well as diffe en ca bonyl g oups is by no means ob ious. 43 Equally impo an in he p esen case is he abili y o 25 o dis inguish be ween iple and double bonds: whe eas alkynes eac smoo hly, olefins a e ine independen o hei elec onic na u e, deg ee o subs i u- ion, and chemical mic oen i onmen . 16,44,45 A e he p opa gylic −OH g oup had been un eiled, which is needed o impose di ec ionali y on he p ojec ed ans-hyd o- s anna ion (see Scheme 2) and has o o e w i e any de imen- al influence o he wo o he p o ic si es embedded in o he mac ocyclic a ay, 21,22 alkyne 26 was eac ed wi h Bu3SnH and ca aly ic amoun s o [Cp*RuCl]4in CH2Cl2. G a i yingly, his ans o ma ion p oceeded cleanly, p o ided ha he s annane was slowly added o he eac ion mix u e. Unde hese condi- ions, p oduc 27 was basically o med as a single egio- and s e eoisome , 46 which was isola ed in 80% yield. The s uc u e o he enyne subs a e 26 in he solid s a e (Figu e 1) migh help o explain why his ans o ma ion p oceeded so selec i ely: i shows ha he di ec ing hyd oxy g oup a C7 as well as he flank- ing C5−C6 alkyne o be hyd ome ala ed a e well exposed o he s e ically demanding [Cp*RuCl] ca alys . 47 In any case, his elabo a e example illus a es he obus ness o his eme ging me hodology ha has al eady se ed o al syn hesis well on se e al o he occasions. 48 Scheme 7. Comple ion o he To al Syn hesis o Nannocys in Ax (1) Figu e 1. S uc u e o cycloalkyne 26 in he solid s a e; he compound c ys allized as a monohyd a e (no shown o cla i y); numbe ing scheme as in oduced by he isola ion eam and used h oughou his pape . Aniso opic displacemen pa ame e s a e shown a 50% p obabili y le el. The Jou nal o O ganic Chemis y A icle DOI: 10.1021/acs.joc.7b02871 J. O g. Chem. 2018, 83, 6977−6994 6980 The alkenyls annane uni in 27a (R1= phenacyl) was amenable o C-me hyla ion unde condi ions p e iously de eloped in ou labo a o y; 49 hus, ea men wi h CuTC, [Ph2PO2][NBu4], and MeI u nished he desi ed alkene 30 in good yield wi hou sc ambling o he double bond geome y. I was a his s age ha he phenacyl p o ec ing g oup p o ed necessa y: compound 27b (R1= Ac) diffe ing only in he p esence o an ace yl moie y on he y osine’s phenolic −OH eac ed much less cleanly; ac ually, allene o ma ion p e ailed (wi h o wi hou concomi an me hy- la ion o he libe a ed phenol). We eel confiden o asc ibe his ou come o ansannula acyl mig a ion p io o/concomi an wi h in/coppe exchange; he p esence o he good lea ing g oup a he allylic posi ion o he pu a i e o ganocoppe in e - media e 28 en ails apid elimina ion ha ou compe es he desi ed C-me hyla ion. Al hough allene o ma ion was undesi able in he p esen con ex , i p o ides an oppo uni y o al e na i e down- s eam p ocessing o he p oduc s a ailable by hyd oxy-di ec ed ans-hyd ome ala ion. S udies along hese lines a e unde way and will be epo ed in due cou se. Wi h access o 30 secu ed, he comple ion o he o al syn- hesis appea ed o be s aigh o wa d. Ye , he seemingly i ial O-me hyla ion p o ed o be a he challenging. Al hough he desi ed p oduc 31 could be o med unde a a ie y o condi ions (e.g., MeI/Ag2O, [Me3O]BF4/p o on sponge, MeOTs/K2CO3), he eac ions we e no o e ly clean and he yields ai ly e a ic. A e conside able expe imen a ion, we concei ed o a somewha uno hodox ye po en ially widely applicable al e na i e me hod (Scheme 8): he equi ed [H3C+] equi alen was gene a ed in si u by a gold-ca alyzed cycliza ion o 32; 50 in his case, addi ion o an ex e nal base is no necessa y as he p o on o he alcohol o be me hyla ed ge s apped upon p o odeau a ion o in e - media e 33, which eleases he [LAu+] agmen and closes he ca aly ic cycle. When applied o 30, his me hod p o ed indeed mo e eliable han he classical al e na i es; he esul ing c ude me hyl e he 31 was subjec ed o educ i e clea age o he phenacyl g oup wi h zinc dus in acidic medium 36 o u nish nannocys in Ax (1). Al hough he spec a o ou syn he ic samples we e in good acco d wi h he published da a, we no iced he p esence o a second se o signals which had no been desc ibed by he iso- la ion eam. 3 Va iable- empe a u e NMR p o ed ha his cha - ac e is ic spec al signa u e is caused by a second con o me and no by any isome ha migh ha e gone un ecognized h ough- ou he syn hesis. Liu and co-wo ke s ha e ecen ly desc ibed a simila obse a ion. 11 La e-S age Di e sifica ion. Fo i s con e gence, he ou e o nannocys in Ax (1) desc ibed abo e p o ides many oppo - uni ies o s uc u al modifica ions o he skele on and a ia ion o he s e eos uc u e, i desi able. In a fi s o ay, we in ended o al e he isubs i u ed alkene and i s flanking −OR subs i uen o p obe whe he his subs uc u e is c i ical o he biological ac i i y o no . I is emphasized, howe e , ha none o he ans- o ma ions leading o he analogues shown in Scheme 9 has been ully op imized a his poin . Dep o ec ion o compound 30 p io o O-me hyla ion affo ded p oduc 35,diffe ing om he na u al p oduc 1only by he absence o he me hyl e he ; he compa ison should hence e eal i his subs i uen , which is supposed o poin away om he binding side, 3 exe s any no iceable influence on cy o oxici y. In con as , he C6a-me hyl g oup b anching off he alkene was compu ed o be imme sed in o one o only wo deepe hyd o- phobic subpocke s o he binding si e; 3 o mal dele ion should he e o e ha e a qui e p onounced effec . This s uc u al modifi- ca ion was eadily a ained by p o odes anna ion o 27,whichpa ed he way o he desi ed no -me hyl compound in O-unp o ec ed (36) as well as O-me hyla ed o ma (37). Along he same lines, o mal eplacemen o he C6a-me hyl g oup by a fluo ine a om was deemed in e es ing. 51 To his end, fluo o-des anna ion o 27 acco ding o a p ocedu e ecen ly de eloped in ou labo a o y 52 u nished he fluo oalkene ana- logue in he o m o he co esponding alcohol 38 and he de i ed me hyl e he 39. In hispa icula case, O-me hyla ion had ac ually been ca ied ou be o e he new gold-ca alyzed p ocedu e was de eloped, using MeOTs/K2CO3in ace one. Unde hese basic condi ions, pa ial clea age o he e -alcohol g oup o he 3-hyd oxy aline uni ook place. This unexpec ed bu ce ainly no implausible e o-aldol eac ion wa an s u he op imiza- ion because i migh allow o p o ound modifica ions o he skele on a a la e s age. Compa ison o he fluo ina ed analogues 40 and 39 wi h a unca ed and in ac backbone, espec i ely, should show whe he his si e is c i ically impo an o ac i i y as he in silico docking s udy insinua es. Addi ional analogues o es ing we e he chlo o-olefin41 53 as well as he alkynylogous nannocys ins 42 and 43,diffe ing only in he p esence o absence o he me hyl e he cap. Finally, a Lindla - ype semi educ ion o 43 u nished he geome ical isome 44, whichupon compa ison wi h 35 and 36should indica e whe he he s e eochemical in eg i y o he diene is ele an o no . F om he concep ual iewpoin , we like o emphasize ha all compounds desc ibed he ein we e ai ly s aigh o wa d o make; ye , hey in a iably ea u e deep-sea ed s uc u al modifica ions ha could no be eachedwi hou undue effo by chemical de i a iza ion o he na u al p oduc . The e o e, his se o 10 non-na u al analogues exemplifies he concep o “di e ed o al syn hesis”as a means o explo e chemical space su ounding a p e alida ed na u al lead. 14,15 Ye , he modifica ions a e no andom bu “mo i -o ien ed”in ha hey exclusi ely add ess a p esumably ele an domain. Biological Assessmen . The fi s ound o sc eening assessed he cy o oxici y o his panel o compounds using he HCT-116 human colon ca cinoma and he HL-60 human p omyelocy ic leukemia cell lines. Syn he ic nannocys in Ax (1) allowed o compa ison wi h he li e a u e and hence se ed as he calib a ion poin . Ac ually, he IC50 o syn he ic 1was lowe han ha epo ed o na u al 1in he li e a u e (Table 1), 3 bu bo h da a poin s lie in he low single-digi nanomola ange. Whe he his diffe ence is due o diffe en assay condi ions o is caused by o he easons canno be decided; in his con ex , howe e , we like o poin ou ha he wo diffe en isola ion eams epo ed a simila diffe en ial in he IC50 o he sis e com- pound 2. 2,3 The e o e, we eel confiden ha he da a a e ele an and compa able. Scheme 8. Base-F ee, Gold-Ca alyzed Fo ma ion o Me hyl E he s The Jou nal o O ganic Chemis y A icle DOI: 10.1021/acs.joc.7b02871 J. O g. Chem. 2018, 83, 6977−6994 6981 The esul s compiled in Table 1 show se e al clea -cu ends: nannocys in Ax (1) i sel p o ed o be he mos po en com- pound o he se ies, bu he desme hyl de i a i e 37 as well as he fluo o-analogue 39 a e almos as ac i e; e en he alkynylogous de i a i e 42 e ains app eciable po ency, mos no ably agains he HCT-116 cell line. Howe e , hese findings a e difficul o econcile wi h he o ecas om he in silico docking expe i- men s, which had sugges ed ha he C6a-Me g oup occupies a p i ileged si e wi hin an o he wise a he ea u eless and shallow binding pocke . 3 I s excision, as mani es in 37, o i s eplacemen by a s ongly pola ized C−F uni as in 39 had he e o e been expec ed o en ail a s onge biological esponse. Equally i no e en mo e su p ising was he o he ema kable pa e n mani es in he da a: nannocys in Ax (1) is mo e han 2 o de s o magni ude mo e po en han i s alcohol sibling 35, al hough he −OMe subs i uen had been compu ed o poin ou o he binding pocke . 3 The e o e, his d ama ic diffe ence was unexpec ed bu is consis en ly ound o all alcohol/e he pai s (35/1,36/37,38/39). None o he o he C7-OH de i a i es (41,44) showed app eciable ac i i y ei he . Wi hin he −OH subse ies, howe e , 35 and 36 comp ising he E,E-configu ed diene a e mo e po en han is he geome ical isome 44 wi h an E,Z-en i y. O a guably e y high ele ance is he ac ha he unca ed fluo oalkene analogue 40 is >2.6 ×103 imes less ac i e han fluo oalkene 39 ea u ing he in ac backbone. 40 is ac ually he leas cy o oxic compound o he en i e se ies, which ad o- ca es he no ion ha 3-hyd oxy aline is a e y c i ical seg- men wi hin he pha macopho e, likely because he wo me hyl g oups engage in hyd ophobic con ac s wi h he p o ein hos . 3 Scheme 9. P epa a ion o a Focused Lib a y o Analogues (S uc u al Modifica ions Rela i e o he Pa en Compound 1 A e Indica ed in Red) a a Reagen s and condi ions: (a) Zn, HOAc, THF, 78% (35), 75% (36), 37% (37) (o e s eps ,a), 87% (38), [34% (39) + 28% (40)] (o e s eps d,a), 45% (42, o e s eps d,a), 80% (43); (b) CuTC, [Ph2PO2][NBu4], DMF, 86%; (c) AgOP(O)Ph2, F-TEDA-PF6, ace one, 54%; (d) K2CO3, MeOTs, ace one; (e) CuCl2, lu idine, THF, 82%; ( ) 32,Ph 3PAuCl, AgOT , benzene; (g) Zn(Cu/Ag), aq MeOH/1,4-dioxane, 62%. The Jou nal o O ganic Chemis y A icle DOI: 10.1021/acs.joc.7b02871 J. O g. Chem. 2018, 83, 6977−6994 6982 This conclusion wa an s a much mo e de ailed assessmen in u u e s udies. Nannocys in Ax (1) and he h ee single mos po en ana- logues we e hen sc eened mo e b oadly. As can be seen om Table 2, he anking in e ms o po ency be ween he fluo o-analogue 39 and he no -me hyl de i a i e 37 obse ed wi h HCT-116 was in e ed in all o he cell lines, al hough he diffe ences a e a he small and mus no be o e in e p e ed. Impo an ly, he sensi i i y o he chosen six human cance cell lines owa d he indi idual compounds p o ides a consis en pic u e. The e o e, i seems ha he isubs i u ed alkene mo i o nannocys in Ax is a mo e pe missi e si e o s uc u al modi- fica ion han an icipa ed, whe eas changes o he flanking −OMe e he subs i uen a e much mo e c i ical han o ecas ed by he cu en ly only binding model published in he li e a u e. 3 Al hough ou da a sugges ha his pu ely compu a ional p oposal needs o be e isi ed and calib a ed in mo e de ail, we like o emphaisze ha cell oxici y is he o al sco e o a ious ac o s. The e o e, addi ional SAR da a a e necessa y be o e a final con- clusion can be d awn. ■CONCLUSIONS Incon as o all p e ious syn heses o membe so he nannocys in amily, 7−12 he app oach desc ibed he ein is “mo i - ocused” a he han pu ely “ a ge -o ien ed”in concep ual e ms. 54 I was delib- e a ely designed o al e and hence in e oga e a subsi e embedded in o he molecula ame ha was sugges ed o play a c i ical ole in he binding o hese highly cy o oxic agen s o EF-1αas hei p ima y biological a ge . This goal was accomplished wi h he aid o a eac ion sequence comp ising ing closing alkyne me a hesis ollowed by hyd oxy-di ec ed ans-hyd os anna ion o he esul ing mac ocyclic p opa gyl alcohol de i a i e. This ac ic opened a selec i e ye flexible en y in o di- as well as isubs i u ed alkenes including nannocys in Ax i sel and a se o 10 non- na u al analogues; hese de i a i es a e dis inguished by deep- sea ed s uc u al “poin mu a ions” ha would no be accessible by de i a iza ion o he na u al lead. Mo eo e , he chosen s a - egy is almos ce ainly o in e es in en i ely diffe en chemical con ex s, oo. Assessmen o he cy o oxici y o he syn he ic compound collec ion p o ided impo an insigh s in o he SAR o he c i ical mo i , which calls o eassessmen o he only a ailable model mean o desc ibe he binding o he cyclodepsipep ides o he nannocys in amily o hei p o ein hos . 3 ■EXPERIMENTAL SECTION Cy o oxic Ac i i y (IC50). Cell lines we e ob ained om he Ge man Collec ion o Mic oo ganisms and Cell Cul u es (Deu sche Sammlung u Mik oo ganismen and Zellkul u en, DSMZ) o he Ame ican Type Cul u e Collec ion (ATCC). All cell lines we e cul u ed unde condi- ions ecommended by he deposi o . Cells we e seeded a 5 ×104cells pe well in 96-well pla es in 180 μL o medium supplemen ed wi h 10% FBS (McCoy’s 5A modified medium o HCT-116, U-2OS, and KB-3.1 cells; RPMI 1640 medium o HL-60, THP-1, and U937 cells) and ea ed wi h nannocys ins dissol ed in DMSO in se ial dilu ion a e 2 h o equilib a ion. Cells we e ea ed o 5 days. Fo adhe en cells, 20 μL o 5 mg/mL MTT ( hiazolyl blue e azolium b omide) in PBS (phospha e-buffe ed saline; pH 7.4) was added pe well, and cells we e incuba ed o an addi ional 2 h a 37 °C and 5% CO2. The medium was disca ded, and 100 μL o 2-p opanol/HCl (10 M, 250:1) was added in o de o dissol e o mazan g anules. The abso bance a 570 nm was measu ed using a mic opla e eade (Tecan M200P o). Fo suspension cell lines, 20 μL o 0.2 mg/mL alama blue ( esazu in sodium sal ) in PBS was added pe well, and cells we e incuba ed o an addi ional 24 h a 37 °C and 5% CO2. The fluo escence in ensi y a 570 nm (exci a ion wa eleng h: 540 nm) was measu ed using a mic opla e eade (Tecan M200P o). Cell iabili y co ela es wi h he abso bance and fluo es- cence in ensi y alues and was exp essed as pe cen age ela i e o he espec i e sol en con ol. Hal -inhibi o y concen a ions (IC50) we e de e mined by sigmoidal cu e fi ing. Gene al Rema ks. Unless s a ed o he wise, all eac ions we e ca ied ou unde a gon in flame-d ied glasswa e using anhyd ous sol en s. The sol en s we e pu ified by dis illa ion o e he ollowing d ying agen s and we e ans e ed unde A : THF, E 2O (Mg/an h acene), CH2Cl2, oluene (Na/K), MeOH (Mg, s o ed o e 3 Å MS); DMF, DMSO, E 3N, 1,4-dioxane, and py idine we e d ied by an adso p ion sol en pu ifica ion sys em based on molecula sie es; anhyd ous (99.9%) cyclopen yl me hyl e he (CPME) pu chased om Ald ich was kep in a flame-d ied Schlenk flask con aining 4 Å MS unde a gon. Thin laye ch oma og aphy (TLC): Mache ey-Nagel p ecoa ed pla es (POLY- GRAMSIL/UV254). P epa a i e TLC: Mache ey-Nagel p ecoa ed pla es (SIL G-100 UV 254; silica gel laye : 1.0 mm). Flash ch oma- og aphy: Me ck silica gel 60 (40−63 μm) wi h p edis illed o HPLC g ade sol en s; Celi e was d ied a 170 °C o 48 h unde high acuum (1 ×10−3mba ) and s o ed unde a gon. NMR: Spec a we e eco ded on B uke DPX 300, AV 400, AV 500, o AVIII 600 spec ome e s in he sol en s indica ed; chemical shi s (δ) a e gi en in pa s pe million ela i e o TMS, and coupling cons an s (J) a e in Hz. The sol en signals we e used as e e ences, and he chemical shi s con e ed o he TMS scale (CDCl3,δC= 77.16 ppm; esidual CHCl3in CDCl3,δH= 7.26 ppm; CD3OD, δC = 49.0 ppm; esidual CHD2OD, δH= 3.31 ppm; (CD3)2CO, δC= 29.8, 206.3 ppm; esidual CD3CHD2OD, δH= 2.05 ppm, (CD3)2SO, δC= 39.5 ppm; esidual CD3CHD2SO, δH= 2.50 ppm). IR: Spec um One (Pe kinElme ) spec ome e , wa e- numbe s (ν)incm −1. MS (EI): Finnigan MAT 8200 (70 eV, doubly ocused sec o field MS), ESI-MS: ESQ3000 (B uke , ion ap), accu a e mass de e mina ions; B uke APEX III FTMS (7 T magne , ion cyclo on esonance MS) o Ma 95 (Finnigan, doubly ocused sec o field MS). Op ical o a ions ([α]D20) we e measu ed wi h a Pe kinElme Table 2. Hal -Inhibi o y Concen a ions (IC50 [nM]) o he Mos Po en Nannocys in De i a i es on a Panel o Six Cance Cell Lines o Human O igin a cmpound HCT-116 HL-60 KB-3.1 THP-1 U-2 OS U937 10.8 5.9 1.7 1.3 0.2 5.0 39 1.5 58.6 7.8 33.4 11.2 141 37 4.3 46.4 5.9 20.5 3.5 33.5 42 22.2 245 83.6 81.2 20.2 198 a The his o ypes a e as ollows: HCT-116, colon ca cinoma; HL-60, p omyelocy ic leukemia; KB-3.1, ce ical ca cinoma; THP-1, acu e mono- cy ic leukemia; U-2 OS, os eosa coma; U937, his iocy ic lymphoma. Table 1. Ini ial P ofiling o Nannocys ins in Te ms o Cy o oxic Ac i i y (Hal -Inhibi o y Concen a ions a e 5 days o Incuba ion, IC50 [nM]) on HCT-116 and HL-60 Cells compound HCT-116 HL-60 10.8 [5.4] 3 5.9 2[1.2] 2 /[5.1] 3 [12] 2 39 1.5 58.6 37 4.3 46.4 42 22.2 245 35 198 767 41 1190 2964 38 1345 2702 36 1549 2108 43 1761 4254 44 2472 4366 40 3918 10229 The Jou nal o O ganic Chemis y A icle DOI: 10.1021/acs.joc.7b02871 J. O g. Chem. 2018, 83, 6977−6994 6983 model 343 pola ime e . LC-MS analyses we e conduc ed on a Shimadzu LCMS 2020 ins umen (pumps LC-20AD, au osample SIL-20AC, column o en CTO-20AC, diode a ay de ec o SPD-M20A, con olle CBM-20A, ESI de ec o , and Labsolu ions so wa e) wi h an ZORBAX Eclipse Plus C18 1.8 μm, 3.0 o 4.6 mm i.d. ×50 mm (Agilen ). A bina y g adien o MeCN o MeOH in wa e o aqueous ie hylammonium ace a e buffe (10 mmol, pH 8) was used a a flow a e o 0.5 (3.0 mm i.d.) o 0.8 (4.6 mm i.d.) mL/min. The o en empe a u e was kep a 35 °C and he de ec ion wa eleng h a 254 nm. P epa a i e LC was pe o med wi h a Shimadzu LC-20A p ominence sys em (pumps LC-20AP, column o en CTO-20AC, diode a ay de ec o SPD-M20A, ac ion collec o FRC-10A, con olle CBM-20A and LC-solu ion so wa e); condi ions o each compound a e specified below. De e - mina ions o he enan iome ic excess (ee) we e pe o med by HPLC o GC using he chi al s a iona y phases and condi ions specified below. Unless s a ed o he wise, all comme cially a ailable compounds (Al a Aesa , Ald ich, TCI, S em Chemicals) we e used as ecei ed. Bu -3-en-1-yn-1-yl iphenylsilane (3). 55 E hynyl iphenylsilane (5.00 g, 17.6 mmol) and inyl b omide (1.0 M in THF, 22.9 mL, 22.9 mmol) we e added o a solu ion o CuI (67.0 mg, 0.352 mmol) and [Pd(Ph3P)4](101mg,87.4μmol)in die hylamine(8.7 mL). The esul ing mix u e was s i ed a ambien empe a u e o 24 h be o e he eac ion was quenched wi h wa e . The aqueous laye was ex ac ed wi h pen ane/ die hyl e he (1:1, 100 mL), and he combined ex ac s we e washed wi h HCl (1 M), d ied o e Na2SO4, and e apo a ed. The esidue was pu ified by flash ch oma og aphy on silica gel (hexanes) o affo d he i le compound as a whi e solid (3.41 g, 63%): mp = 99.0−99.8 °C; 1H NMR (400 MHz, CDCl3)δ= 7.71−7.66 (m, 6H), 7.48−7.37 (m, 9H), 5.98 (dd, J= 17.6, 11.0 Hz, 1H), 5.87 (dd, J= 17.6, 2.4 Hz, 1H), 5.64 (dd, J= 11.0, 2.4 Hz, 1H) ppm; 13C NMR (101 MHz, CDCl3)δ= 135.7, 133.6, 130.1, 129.4, 128.1, 117.3, 108.2, 90.0 ppm; IR (film) ν= 3068, 2152, 1428 cm−1; MS (EI) m/z(%) 105 (94), 129 (50), 155 (41), 181 (100), 203 (20), 232 (83), 310 (29); HRMS (ESI) m/zcalcd o C22H18Si [M+] 310.1172, ound 310.1177. (1S,2R)-2-Me hyl-1-phenylbu -3-yn-1-ol (4). Dime hoxyme hyl- silane (1.3 mL, 10.5 mmol) was added a 0 °C o a s i ed solu ion o Cu(OAc)2(1.9 mg, 10.5 μmol), (R,R)-Ph-BPE (6.4 mg, 12.6 μmol), 3 (650 mg, 2.09 mmol), benzaldehyde (640 μL, 6.30 mmol), and -BuOH (200 μL, 2.10 mmol) in cyclohexane (4.2 mL). The mix u e was s i ed a his empe a u e o 10 h. The eac ion was ca e ully quenched wi h NaOH solu ion in MeOH (2 M, ca. 30 mL) (Cau ion: gas e olu ion) and s i ing con inued o 10 h be o e he mix u e was dilu ed wi h H2O. The aqueous laye was ex ac ed wi h E OAc (3 ×100 mL), and he combined o ganic phases we e washed wi h b ine (5 mL), d ied o e Na2SO4,fil e ed, and concen a ed. A e ec ys alliza ion om hexane, he c ude ma e ial was pu ified by flash ch oma og aphy on silica gel (hexanes/ e -bu yl me hyl e he , 10:1 o 8:1) o affo d he i le compound 4(165 mg, 49%, 99.5% ee) and he an i-isome (61.4 mg, 18%, 98.7% ee), each as a colo less oil. [The ee was de e - mined by HPLC analysis: Daicel Chi alpak IA (4.6 mm ×250 mm), n-hep ane/2-p opanol = 98/2, =1.0mL·min−1,λ=220nm, (mino ) = 13.42 min, (majo ) = 14.81 min; HPLC analysis o he an i-isome : Daicel Chi alpak IC-3 (4.6 mm ×150 mm), n-hep ane/2-p opanol = 99.5/ 0.5, = 1.0 mL·min−1,λ= 220 nm, (mino ) = 10.33 min, (majo ) = 14.92 min.] Analy ical da a o 4:[α]D20 =−47.6 (c1.0, CHCl3); 1H NMR (400 MHz, CDCl3)δ= 7.42−7.28 (m, 5H), 4.75 (dd, J= 5.5, 3.6 Hz, 1H), 2.88 (qdd, J= 7.0, 5.5, 2.5 Hz, 1H), 2.22 (d, J= 3.6 Hz, 1H), 2.12 (d, J= 2.4 Hz, 1H), 1.14 (d, J= 6.9 Hz, 3H) ppm; 13C NMR (101 MHz, CDCl3)δ= 141.3, 128.3, 128.0, 126.6, 86.0, 76.3, 71.0, 34.2, 15.7 ppm; IR (film) ν= 3292, 2977, 2936 cm−1; MS (ESI) m/z178 [M + NH4+], 183 [M + Na+]; HRMS (ESI) m/zcalcd o C11H12ONa [M + Na+] 183.0780, ound 183.0782. Analy ical da a o he an i-isome : [α]D20 = +68.3 (c1.1, CHCl3); 1H NMR (400 MHz, CHCl3)δ= 7.42−7.28 (m, 5H), 4.52 (dd, J= 7.2, 3.6 Hz, 1H), 2.81 (pd, J= 7.1, 2.4 Hz, 1H), 2.50 (d, J= 3.8 Hz, 1H), 2.22 (d, J= 2.4 Hz, 1H), 1.11 (d, J= 7.0 Hz, 3H) ppm; 13C NMR (101 MHz, CDCl3)δ= 141.4, 128.5, 128.2, 126.8, 85.6, 77.6, 71.5, 35.3, 17.5 ppm; IR (film) ν= 3293, 2977, 2936 cm−1; MS (EI) m/z(%)79 (100), 107 (74); HRMS (ESI) m/zcalcd o C11H12ONa [M + Na+] 183.0780, ound 183.0781. (1S,2R)-2-Me hyl-1-phenylhep a-3,5-diyn-1-ol (5). 56 CuI (24.9 mg, 0.131 mmol) was added o a s i ed solu ion o 4(140 mg, 0.874 mmol) and eshly p epa ed iodop opyne (excess, ca. 10 equi ) 25 in degassed py olidine (44 mL), causing an immedia e colo change o g een. A e being s i ed o 10 h, he hen yellow mix u e was dilu ed wi h HCl (2 M, 50 mL) and ex ac ed wi h e -bu yl me hyl e he (3 ×100 mL). The combined o ganic phases we e washed wi h b ine (15 mL), d ied o e MgSO4,fil e ed, and concen a ed. The esidue was pu ified by flash ch oma og aphy (hexanes/ e -bu yl me hyl e he , 10:1) o affo d he i le compound as a pale yellow oil (159 mg, 92%): [α]D20 =−9.0 (c2.2, CHCl3); 1H NMR (400 MHz, CDCl3)δ= 7.39−7.33 (m, 4H), 7.32−7.27 (m, 1H), 4.72 (d, J= 5.5 Hz, 1H), 2.95−2.86 (m, 1H), 2.17 (d, J= 3.2 Hz, 1H), 1.90 (d, J= 1.1 Hz, 3H), 1.13 (d, J= 7.0 Hz, 3H) ppm; 13C NMR (101 MHz, CDCl3)δ= 141.3, 128.3, 128.0, 126.6, 77.6, 76.4, 74.7, 68.1, 64.3, 35.0, 15.6, 4.4 ppm; IR (film) ν= 3420, 2975, 2932, 2914, 1453 cm−1; MS (ESI) m/z198 [M + NH4+], 221 [M + Na+]; HRMS (ESI) m/z calcd o C14H14ONa [M + Na+] 221.0937, ound 221.0938. (1S,2R,E)-2-Me hyl-1-phenylhep -3-en-5-yn-1-ol (6). Red-Al (3.5 M in oluene, 0.57 mL, 2.0 mmol) was added o a s i ed solu ion o 5(100 mg, 0.504 mmol) in THF (1.0 mL) a oom empe a u e. The mix u e was s i ed a 65 °C o 2 h be o e he eac ion was ca e ully quenched wi h HCl (1 M, 4.0 mL). The aqueous phase was ex ac ed wi h E OAc (3 ×10 mL), and he combined ex ac s we e washed wi h b ine and d ied wi h Na2SO4. The sol en was e apo a ed and he esidue pu ified by ch oma og aphy on silica gel (hexanes/E OAc, 20:1) o affo d he i le compound as a colo less oil (71.3 mg, 71%): [α]D20 = +8.5 (c0.8, CHCl3); 1H NMR (400 MHz, CDCl3)δ= 7.39−7.24 (m, 5H), 5.97 (ddd, J= 16.0, 7.6, 0.8 Hz, 1H), 5.44 (dqd, J= 16.0, 2.3, 1.3 Hz, 1H), 4.60 (dd, J= 5.6, 3.6 Hz, 1H), 2.69−2.54 (m, 1H), 1.91 (d, J= 2.3 Hz, 3H), 1.87 (d, J= 3.6 Hz, 1H), 1.01 (d, J= 6.8 Hz, 3H) ppm; 13C NMR (101 MHz, CDCl3)δ= 144.2, 142.4, 128.3, 127.7, 126.6, 111.1, 85.4, 78.2, 44.4, 14.5, 4.4 ppm; IR (film) ν= 3428, 3029, 2964, 2916, 1494, 1453, 1376 cm−1; MS (EI) m/z(%) 79 (100), 94 (38), 105 (20), 107 (37); HRMS (ESI) m/zcalcd o C14H16ONa [M + Na+] 223.1093, ound 223.1095. (1S,2R,E)-2-Me hyl-1-phenylhep -3-en-5-yn-1-yl (S)-2-(( e - Bu oxyca bonyl)amino)-3-(( e -bu yldime hylsilyl)oxy)-3- me hylbu anoa e (S1). N-E hyl-N′-(dime hylamino-p opyl)-ca bodiimide hyd ochlo ide (143.6 mg, 0.750 mmol) was added o a s i ed solu ion o 6 (100 mg, 0.500 mmol), 10 (208 mg, 0.600 mmol), and DMAP (12.2 mg, 0.100 mmol) in CH2Cl2(0.66 mL) a 0 °C. A e being s i ed o 15 min a 0 °C and o 5 h a ambien empe a u e, he mix u e was pa i ioned be ween E OAc and sa u a ed aqueous NH4Cl. The aqueous laye was ex ac ed wi h E OAc (3 ×10 mL) be o e i was acidified o pH 2 wi h HCl (2 M) and ex ac ed again wi h E OAc (3 ×10 mL). The combined o ganic laye s we e washed wi h b ine (20 mL), d ied o e Na2SO4, fil e ed, and concen a ed. The c ude ma e ial was pu ified by flash ch oma og aphy on silica gel (hexanes/E OAc, 10:1) o affo d he i le compound as a colo less oil (206 mg, 78%): [α]D20 =−17 (c1.0, CHCl3); 1H NMR (400 MHz, CDCl3)δ= 7.33−7.22 (m, 5H), 5.79 (dd, J= 16.0, 7.7 Hz, 1H), 5.62 (d, J= 7.7 Hz, 1H), 5.33 (ddd, J= 16.0, 2.4, 1.3 Hz, 1H), 5.26 (d, J= 9.5 Hz, 1H), 4.10 (d, J= 9.5 Hz, 1H), 2.84−2.75 (m, 1H), 1.87 (d, J= 2.4 Hz, 3H), 1.44 (s, 9H), 1.25 (s, 3H), 1.17 (s, 3H), 1.06 (d, J= 6.8 Hz, 3H), 0.77 (s, 9H), 0.01 (s, 3H), −0.12 (s, 3H) ppm; 13C NMR (101 MHz, CDCl3)δ= 170.2, 155.9, 142.4, 137.9, 128.2, 128.2, 127.9, 111.5, 85.4, 79.9, 79.8, 78.2, 75.0, 62.9, 42.2, 28.5, 28.4, 27.6, 25.8, 18.1, 16.2, 4.4, −2.2, −2.4 ppm; IR (film) ν= 3453, The Jou nal o O ganic Chemis y A icle DOI: 10.1021/acs.joc.7b02871 J. O g. Chem. 2018, 83, 6977−6994 6984 2930, 2956, 2857, 1802, 1720, 1495 cm−1; MS (ESI) m/z530 [M + H+], 547 [M + NH4+], 552 [M + Na+]; HRMS (ESI) m/zcalcd o C30H47NO5SiNa [M + Na+] 552.3116, ound 552.3118. (1S,2R,E)-2-Me hyl-1-phenylhep -3-en-5-yn-1-yl (S)-2- Amino-3-(( e -bu yldime hylsilyl)oxy)-3-me hylbu anoa e (7). HCl (4 M in 1,4-dioxane, 7.5 mL, 30.0 mmol) was added o a solu ion o compound S1 (200 mg, 0.370 mmol) in 1,4-dioxane (3.8 mL) a 0 °C. The mix u e was s i ed a 0 °C o 15 min and hen a oom empe a- u e o 5 h. The eac ion was quenched wi h sa u a ed aqueous Na2CO3, and he aqueous laye was ex ac ed wi h E OAc (3 ×10 mL). The combined ex ac s we e washed wi h b ine (40 mL), d ied o e Na2SO4,fil e ed, and concen a ed. The esidue was pu ified by flash ch oma og aphy on silica gel (hexanes/E OAc, 4:1) o affo d he i le compound as a colo less oil (154 mg, 95%): [α]D20 =−2.0 (c2.2, CHCl3); 1H NMR (400 MHz, CDCl3)δ= 7.34−7.24 (m, 5H), 5.81 (ddd, J= 16.0, 7.7, 0.8 Hz, 1H), 5.63 (d, J= 7.4 Hz, 1H), 5.33 (ddd, J= 16.0, 2.3, 1.2 Hz, 1H), 3.36 (s, 1H), 2.84−2.76 (m, 1H), 1.94−1.83 (m, 3H), 1.64 (s, 2H), 1.25 (s, 3H), 1.09−1.04 (m, 6H), 0.81 (s, 9H), 0.06 (s, 3H), −0.01 (s, 3H) ppm; 13C NMR (101 MHz, CDCl3)δ= 172.6, 142.4, 137.9, 128.3, 128.2, 127.8, 111.5, 85.6, 79.5, 78.1, 75.3, 65.1, 42.1, 28.2, 25.9, 25.5, 18.2, 16.2, 4.4, −2.1, −2.1 ppm; IR (film) ν= 3394, 2929, 2955, 2856, 1738, 1687, 1461 cm−1; MS (ESI) m/z430 [M + H+]; HRMS (ESI) m/zcalcd o C25H40NO3Si [M + H+] 430.2772, ound 430.2772. e -Bu yl (R)-(1,3-Dihyd oxy-3-me hylbu an-2-yl)ca bama e (9). 27 Asolu iono MeMgB inE 2O(3.0MinE 2O, 29.0 mL, 86.7 mmol) was added d opwise o a solu ion o N-Boc-L-Se -OMe (8) (4.80 g, 21.7 mmol) in E 2O (108 mL) a −78 °C. The mix u e was allowed o each oom empe a u e and was s i ed o 1 h. A e being cooled o 0 °C, he eac ion was quenched wi h sa u a ed aqueous NH4Cl. The aqueous phase was ex ac ed wi h die hyl e he (3 ×25 mL), and he combined o ganic laye s we e washed wi h b ine, d ied o e MgSO4, fil e ed, and concen a ed. The esidue was pu ified by flash ch oma- og aphy on silica gel (hexanes/E OAc, 1:3) o affo d he i le compound as a whi e solid (4.33 g, 91%): [α]D20 =−23 (c1.1, CHCl3); mp = 63−64 °C; 1H NMR (300 MHz, CDCl3)δ= 5.45 (d, J= 9.0 Hz, 1H), 3.98 (d , J= 11.3, 3.6 Hz, 1H), 3.84−3.71 (m, 1H), 3.52−3.38 (m, 1H), 3.27−3.18 (m, 1H), 3.16 (s, 1H), 1.43 (s, 9H), 1.33 (s, 3H), 1.22 (s, 3H) ppm; 13C NMR (75 MHz, CDCl3)δ= 156.6, 79.7, 73.9, 63.5, 57.8, 28.5, 27.7, 27.4 ppm; IR (film) ν= 3350, 2977, 2934, 1684, 1505 cm−1; MS (EI) m/z242 [M + Na+]; HRMS (ESI) m/zcalcd o C10H21NO4Na [M + Na+] 242.1363, ound 242.1362. N-Boc-L-Val-OH (S2). 27 Phospha e buffe (pH 6.7, 30 mL), PhI(OAc)2 (290 mg, 0.900 mmol), and TEMPO (142 mg, 0.909 mmol) we e added o a solu ion o alcohol 9(2.80 g, 12.6 mmol) in MeCN (36 mL). The mix u e was cooled o 0 °C be o e sodium chlo i e (1.3 g, 15 mmol) was added. S i ing was con inued a 0 °C o 2 h and a oom empe a- u e o 16 h. The eac ion was quenched wi h sa u a ed aqueous NH4Cl, and he aqueous laye was acidified o pH 2 and ex ac ed wi h E OAc (3 ×25 mL). The combined ex ac s we e washed wi h b ine, d ied o e MgSO4,fil e ed, and concen a ed. The esidue was ec ys- allized om E OAc/hexanes o affo d he i le compound in he o m o whi e c ys als (2.58 g, 88%): [α]D20 =−3.0 (c0.9, CHCl3); mp = 123−124 °C; 1H NMR (300 MHz, CDCl3)δ= 5.44 (s, 1H), 4.25 (d, J= 8.5 Hz, 1H), 1.46 (s, 9H), 1.38 (s, 3H), 1.29 (s, 3H) ppm; 13C NMR (126 MHz, CDCl3)δ= 173.9, 156.4, 80.9, 72.6, 61.2, 28.4, 27.3, 25.9 ppm; IR (film) ν= 3337, 2979, 2936, 2583, 1694, 1508 cm−1; MS (ESI) m/z256 [M + Na+]; HRMS (ESI) m/zcalcd o C10H19NO5Na [M + Na+] 256.1155, ound 256.1155. N-Boc-L-Val-OTBS (10). E 3N (1.40 mL, 10.0 mmol), TBSCl (1.10 g, 7.30 mmol), and Ve kade’s base 11 (146 mg, 0.675 mmol) we e added o a s i ed solu ion o S2 (785 mg, 3.37 mmol) in DMF (3.0 mL), and he esul ing mix u e was s i ed a 80 °C o 48 h. The eac ion was quenched wi h HCl (2 M, 10 mL), and he aqueous laye was ex ac ed wi h E OAc (3 ×5 mL); he combined ex ac s we e washed wi h b ine (1 mL) and d ied o e Na2SO4. The sol en was e apo a ed, and he esidue was pu ified by flash ch oma og aphy on silica gel (hexanes/ E OAc, 5:1 o 3:1) o affo d he i le compound as a whi e solid (830 mg, 71%): [α]D20 = +31 (c1.5, CHCl3); mp = 93.6−94.9 °C; 1H NMR (400 MHz, CDCl3)δ= 5.26 (d, J= 9.0 Hz, 1H), 4.18 (d, J= 9.0 Hz, 1H), 1.44 (s, 9H), 1.41 (s, 3H), 1.28 (s, 3H), 0.86 (s, 9H), 0.13 (s, 3H), 0.12 (s, 3H) ppm; 13C NMR (101 MHz, CDCl3)δ= 174.8, 156.0, 80.2, 76.2, 62.5, 28.4, 27.7, 26.8, 25.8, 18.1, −2.1, −2.2 ppm; IR (film) ν= 3453, 3091, 2928, 2856, 1717, 1501, 1473, 1463 cm−1; MS (EI) m/z(%) 173 (100), 190 (28), 234 (26); HRMS (ESI) m/zcalcd o C16H33NO5SiNa [M + Na+] 370.2020, ound 370.2023. 1-(T ime hylsilyl)hex-5-en-1-yn-3-ol (13). 29 A solu ion o allyl- magnesium chlo ide (2 M in THF, 27.4 mL, 54.8 mmol) was added o e 30 min o a solu ion o 12 (6.02 g, 47.7 mmol) in THF (95 mL) a 0 °C. A e he addi ion was comple e, he mix u e was allowed o each ambien empe a u e. A e being s i ed o ano he 2 h, he eac ion was quenched wi h sa u a ed aqueous NH4Cl. The aqueous laye was ex ac ed wi h e -bu yl me hyl e he (3 ×50 mL), and he combined o ganic phases we e washed wi h b ine (5 mL), d ied o e MgSO4, fil e ed, and concen a ed. The esidue was pu ified by flash ch oma- og aphy on silica gel (hexanes/ e -bu yl me hyl e he , 5:1) o affo d he i le compound as a colo less oil (7.9 g, 98%): 1H NMR (400 MHz, CDCl3)δ= 5.96−5.80 (m, 1H), 5.24−5.17 (m, 1H), 5.17 ( , J= 1.2 Hz, 1H), 4.41 (q, J= 6.1 Hz, 1H), 2.47 (ddq, J= 7.1, 5.9, 1.2 Hz, 2H), 1.91 (dd, J= 6.1, 1.2 Hz, 1H), 0.17 (s, 9H) ppm; 13C NMR (101 MHz, CDCl3)δ= 133.1, 119.2, 106.0, 90.0, 62.1, 42.2, 0.0 ppm; IR (film) ν= 3330, 3080, 2960, 2175, 1643 cm−1; MS (EI) m/z(%) 75 (32), 83 (9), 99 (100), 127 (64); HRMS (ESI) m/zcalcd o C9H16OSiNa [M + Na+] 191.0863, ound 191.0863. (R)-1-(T ime hylsilyl)hex-5-en-1-yn-3-yl ace a e (14) 29 .Me hod A: Molecula sie es (4 Å, 500 mg), Amano lipase PS (395 mg), and inyl ace a e (15.0 mL, 164 mmol) we e added o a solu ion o compound 13 (7.90 g, 47.0 mmol) in pen ane (313 mL). The suspension was gen ly s i ed o 56 h be o e i was fil e ed h ough a pad o Celi e. The fil a e was e apo a ed, and he esidue was pu ified by flash ch oma og aphy on silica gel (hexanes/E 2O, 10:1) o affo d he i le compound as a colo less oil (4.65 g, 47%, 99% ee). [Condi ions o GC analysis: column 30.0 m, BGB-17/BGB-15, G/698; 0.50 ba H2; 230/50 min iso, 80 4/min 220, 5/min iso 350, (mino ) = 30.75 min, (majo ) = 32.67 min.] Me hod B: Ace ic acid (1.9 mL, 32.6 mmol) and iphenylphosphine (8.54 g, 32.6 mmol) we e added o a solu ion o (S)-13 (3.73 g, 21.7 mmol) in E 2O (109 mL) a 0 °C. A e being s i ed o 5 min a 0°C, diisop opylazodica boxyla e (6.4 mL, 32.6 mmol) was added d opwise, and s i ing was con inued o 1.5 h a his empe a u e. The eac ion was quenched wi h sa u a ed aqueous NaHCO3, and he aqueous laye was ex ac ed wi h e -bu yl me hyl e he ; he combined ex ac s we e washed wi h b ine, d ied o e MgSO4, and e apo a ed. The esidue was pu ified by flash ch oma og aphy on silica gel (hexanes/ e -bu yl me hyl e he , 20:1) o gi e he i le compound as colo less oil (4.13 g, 91%): [α]D20 = +100 (c1.3, CHCl3); 1H NMR (400 MHz, CDCl3)δ= 5.81 (dd , J= 17.3, 10.3, 7.0 Hz, 1H), 5.43 ( , J= 6.5 Hz, 1H), 5.18−5.14 (m, 1H), 5.14−5.11 (m, 1H), 2.51 ( d, J= 6.5, 1.2 Hz, 2H), 2.08 (s, 3H), 0.17 (s, 9H) ppm; 13C NMR (101 MHz, CDCl3)δ= 170.0, 132.3, 118.9, 102.1, 91.0, 63.7, 39.5, 21.2, −0.1 ppm; IR (film) ν= 2961, 1746 cm−1; MS (EI) m/z(%) 43 (100), 75 (45), 99 (20), 117 (17), 127 (11), 135 (16), 169 (35); HRMS (ESI) m/zcalcd o C11H18O2SiNa [M + Na+] 233.0968, ound 233.0970. (S)-1-(T ime hylsilyl)hex-5-en-1-yn-3-ol ((S)-13)). 29 The com- pound was ob ained as he second ac ion om he enzyma ic esolu- ion desc ibed abo e: colo less oil (3.73 g, 47%, 99% ee). [Condi ions o GC analysis: column 24.5 m, hyd odex-be a-TBDAC; G/589; 0.80 ba H2; 220/10 min iso, 105 6/min 220, 5 min iso/350, (mino ) = 3.57 min, (majo ) = 3.73 min]: [α]D20 =−29 (c1.1, CHCl3); 1H NMR (400 MHz, CDCl3)δ= 5.96−5.80 (m, 1H), 5.24−5.17 (m, 1H), 5.17 ( , J= 1.2 Hz, 1H), 4.41 (q, J= 6.1 Hz, 1H), 2.47 (ddq, J= 7.1, 5.9, 1.2 Hz, 2H), 1.91 (dd, J= 6.1, 1.2 Hz, 1H), 0.17 (s, 9H) ppm; 13C NMR (101 MHz, CDCl3)δ= 133.1, 119.2, 106.0, 90.0, 62.1, 42.2, 0.0 ppm; IR (film) ν= 3330, 3080, 2960, 2175, 1643, cm−1; MS (EI) m/z(%) 75 (32), 99 (100), 127 (64); HRMS (ESI) m/zcalcd o C9H16OSiNa [M + Na+] 191.0863, ound 191.0863. E hyl (R,E)-5-Ace oxy-2-me hyl-7-( ime hylsilyl)hep -2-en-6- ynoa e (15). A solu ion o 14 (2.0 g, 9.5 mmol) and Sudan ed III (5.0 mg, 14 μmol) in CH2Cl2(38 mL) was cooled o −78 °C. Ozone gas was bubbled h ough he ed solu ion a −78 °C un il he colo aded away. A his poin , excess ozone was emo ed by bubbling a gon The Jou nal o O ganic Chemis y A icle DOI: 10.1021/acs.joc.7b02871 J. O g. Chem. 2018, 83, 6977−6994 6985 171.1, 171.0, 170.6, 148.6, 148.0, 145.4, 139.8, 139.1, 135.6, 135.3, 132.7, 131.7, 130.3, 130.0, 129.0, 128.7, 128.3, 128.2, 127.7, 126.8, 126.5, 126.1, 122.4, 122.3, 111.2, 110.1, 91.7, 85.1, 83.2, 80.2, 76.6, 72.6, 71.7, 67.5, 62.2, 61.9, 61.3, 61.1, 60.7, 55.5, 54.2, 43.2, 43.0, 37.8, 37.4, 36.8, 35.2, 34.6, 32.7, 31.6, 28.4, 28.3, 27.8, 26.3, 26.0, 25.7, 25.2, 16.1, 15.5, 15.2, 14.8, 14.4, 13.5, 11.7, 10.6, 10.1 ppm; IR (film) ν= 3342, 2970, 2931, 2877, 1738, 1605, 1521 cm−1; MS (ESI) m/z770 [M + H+], 787 [M + Na+], 792[M + Na+]; HRMS (ESI) m/zcalcd o C40H49N3O8Cl2Na [M + Na+] 792.2789, ound 792.2795. Compound 36. The compound was p epa ed analogously om S7 (4.0 mg, 4.5 μmol) as a whi e solid (2.6 mg, 75%): p epa a i e HPLC (150 mm YMC Pack P o C18, 5 μm, ⌀10 mm, MeOH/H2O = 80:20, 4.7 mL/min, 9.6 MPa, 308 K, UV, 220 nm); [α]D20 =−91.0 (c0.2, ace one); 1H NMR (600 MHz, MeOH-d4, mix u e o o ame s, ca. 3.5:1) δ=7.42(d,J= 7.4 Hz, 1.56H, majo ), 7.39 (d, J= 7.9 Hz, 0.44H, mino ), 7.35−7.30 (m, 2H), 7.27−7.24 (m, 1.44H), 7.22 (s, 1.56H), 6.24−6.09 (m, 2H), 5.99 (s, 0.22H, mino ), 5.94 (dd, J= 15.3, 7.3 Hz, 0.22H, mino ), 5.85−5.79 (m, 1.56H, majo ), 5.75 (dd, J= 15.3, 5.4 Hz, 0.22H, mino ), 5.65 (dd, J= 14.8, 6.6 Hz, 0.78H, majo ), 5.55 ( d, J= 6.8, 6.1, 3.4 Hz, 1H), 4.76 (dd, J= 9.3, 6.2 Hz, 1H), 4.63 (d, J= 11.2 Hz, 0.78H, majo ), 4.60 (s, 0.78H, majo ), 4.56 (s, 0.22H, mino ), 4.41−4.32 (m, 0.22H, mino ), 4.2−4.13 (m, 0.78H, majo ), 4.07 (d, J= 10.8 Hz, 0.22H, mino ), 3.14 (dd, J= 14.3, 4.6 Hz, 0.22H, mino ), 3.00 (dd, J= 13.8, 6.2 Hz, 0.78H, majo ), 2.96 (s, 0.66H, mino ), 2.92−2.84 (m, 0.22H, mino ), 2.78 (s, 2.34H, majo ), 2.73 (dd, J= 13.8, 9.4 Hz, 0.78H, majo ), 2.69−2.62 (m, 1.22H), 2.55−2.50 (m, 1H), 2.41−2.35 (m, 0.78H, majo ), 2.04−1.95 (m, 0.78H, majo ), 1.87 (d, J= 1.5 Hz, 2.34H, majo ), 1.83 (s, 0.88H), 1.39−1.31 (m, 1H), 1.29 (s, 1H), 1.24 (s, 0.66H, mino ), 1.20 (s, 0.66H, mino ), 1.16 (s, 2.34H, majo ), 1.06 (s, 2.34H, majo ), 1.06−1.00 (m, 1H), 0.97 (d, J= 6.9 Hz, 2.34H, majo ), 0.95 (d, J= 7.1 Hz, 0.66H, mino ), 0.87 ( , J= 7.4 Hz, 3H), 0.69 (d, J= 6.5 Hz, 2.34H, majo ), 0.44 (d, J= 6.5 Hz, 0.66H, mino ) ppm; 13C NMR (151 MHz, MeOH-d4)δ= 177.3, 176.8, 172.4, 172.4, 172.1, 171.4, 171.3, 171.3, 149.7, 140.3, 140.2, 137.2, 137.0, 135.1, 134.5, 134.2, 133.4, 132.1, 132.1, 131.4, 131.3, 131.1, 130.8, 130.5, 130.4, 129.5, 129.2, 129.0, 128.5, 128.4, 128.4, 127.7, 127.3, 123.4, 123.3, 81.4, 79.3, 73.1, 72.3, 72.2, 71.3, 68.4, 62.1, 61.5, 61.3, 55.5, 55.0, 43.0, 42.8, 38.0, 36.5, 36.2, 35.1, 34.8, 33.1, 32.5, 29.4, 28.2, 27.5, 26.6, 26.4, 25.7, 25.4, 15.9, 15.3, 15.1, 14.5, 12.2, 12.0, 11.6, 10.7 ppm; IR (film) ν= 3369, 2969, 2929, 2875, 1735, 1663, 1604, 1490 cm−1; MS (ESI) m/z772 [M + H+], 789 [M + NH4+], 794 [M + Na+]; HRMS (ESI) m/zcalcd o C40H51N3O8Cl2Na [M+ Na+] 794.2945, ound 794.2952. Compound 44. Zn(Cu/Ag) alloy (20 mg) was added o a solu ion o 43 (3.7 mg, 4.8 μmol) in MeOH/dioxane/H2O (300 μL, 1:1:1), and he esul ing suspension was s i ed a 60 °C o 12 h. All insoluble ma e ials we e fil e ed off h ough a pad o Celi e. The fil a e was e apo a ed and he c ude p oduc pu ified by p epa a i e HPLC (150 mm YMC ODS-A 5 μm, ⌀20 mm, MeOH/H2O = 75:25, 15 mL/min, 10.8 MPa, 298 K, UV, 220 nm) o affo d he i le compound as a whi e solid (2.3 mg, 62%): [α]D20 =−113.5 (c0.23, ace one); 1H NMR (600 MHz, MeOH-d4), mix u e o o ame s, ca. 2.6:1) δ= 7.41−7.36 (m, 2H), 7.35−7.31 (q, J= 8.8, 7.7 Hz, 2H), 7.29−7.25 (m, 1H), 7.24 (s, 2H), 6.34 (dd, J= 16.2, 5.8 Hz, 0.28H, mino ), 6.17−6.09 (m, 1H), 5.91−5.85 (m, 0.28H, mino ), 5.74 (s, 0.72H, majo ), 5.64 (d , J= 8.0, 4.4 Hz, 0.72H, majo ), 5.56 (d, J= 16.0 Hz, 0.72H, majo ), 5.51 (d, J= 16.2 Hz, 0.28H, mino ), 4.85−4.81 (m, 1H), 4.77 (dd, J= 9.3, 6.1 Hz, 1H), 4.72 (dd, J= 11.2, 3.8 Hz, 1H), 4.66 (d, J= 11.3 Hz, 0.72H, majo ), 4.63 (s, 0.72H, majo ), 4.53 (s, 0.28H, mino ), 4.52−4.48 (m, 0.72H, majo ), 4.14 (d, J= 10.8 Hz, 0.28H, mino ), 3.26 (d, J= 15.7 Hz, 0.28H, mino ), 3.04−3.01 (m, 0.72H, majo ), 3.01 (s, 0.84H, mino ), 2.96 (dd, J= 14.6, 11.4 Hz, 0.28H, mino ), 2.87 (s, 2.16H, majo ), 2.79−2.72 (m, 2H), 2.59−2.49 (m, 1.44H, majo ), 2.43 (d, J= 15.0 Hz, 0.28H, mino ), 2.06−1.97 (m, 0.72H, majo ), 1.94 (s, 2.16H, majo ), 1.87 (d, J= 27.2 Hz, 0.28H, mino ), 1.84 (s, 0.84H, majo ), 1.40−1.33 (m, 1H), 1.24 (s, 0.84H, mino ), 1.20 (s, 0.84H, mino ), 1.15 (s, 2.16H, majo ), 1.08 (s, 2.16H, majo ), 1.07−0.97 (m, 1H), 0.93 (d, J= 6.9 Hz, 2.16H, majo ), 0.91−0.86 (m, 3.84H), 0.72 (d, J= 6.5 Hz, 2.16H, majo ), 0.44 (d, J= 6.5 Hz, 0.84H, mino ) ppm; 13C NMR (151 MHz, MeOH-d4)δ= 177.1, 176.9, 172.6, 172.6, 172.2, 171.4, 171.3, 171.2, 149.9, 148.5, 145.9, 139.7, 139.0, 135.7, 134.8, 130.7, 130.4, 130.1, 129.3, 129.0, 128.8, 128.5, 128.4, 128.0, 127.1, 127.0, 111.4, 110.3, 90.8, 89.4, 85.3, 84.1, 81.2, 77.8, 72.9, 72.2, 68.3, 62.5, 62.0, 61.8, 61.5, 61.5, 56.2, 55.1, 43.8, 43.4, 37.9, 37.2, 37.2, 35.2, 35.2, 33.1, 32.4, 29.2, 28.0, 27.4, 26.4, 26.4, 25.7, 25.4, 15.8, 15.4, 15.4, 14.6, 13.7, 11.7, 10.7, 10.1 ppm; IR (film) ν= 2923, 2853, 1659, 1604, 1455 cm−1; MS (ESI) m/z772 [M + H+], 794 [M + Na+]; HRMS (ESI) m/zcalcd o C40H51N3O8Cl2Na [M + Na+] 794.2945, ound 794.2945. ■ASSOCIATED CONTENT * SSuppo ing In o ma ion The Suppo ing In o ma ion is a ailable ee o cha ge on he ACS Publica ions websi e a DOI: 10.1021/acs.joc.7b02871. CCDC-1584397 con ains he supplemen a y c ys allog aphic da a o his pape . These da a can be ob ained ee o cha ge om he Camb idge C ys allog aphic Da a Cen e ia he In e ne a www.ccdc.ca.ac.uk/da a_ eques /ci . C ys allog aphic abs ac , abula compa ison o he NMR da a o syn he ic nannocys in Ax wi h hose o he na u al p oduc , copies o HPLC aces, and copies o spec a o new compounds (PDF) X- ay da a o compound 26 (CIF) ■AUTHOR INFORMATION Co esponding Au ho *E-mail: [email p o ec ed]. ORCID Rol Mulle : 0000-0002-1042-5665 Alois Fu s ne : 0000-0003-0098-3417 No es The au ho s decla e no compe ing financial in e es . ■ACKNOWLEDGMENTS Gene ous financial suppo by he Swiss Na ional Science Founda ion ( ellowship o L.S.), he Alexande - on-Humbold Founda ion ( ellowship o B.M.), and he MPG is g a e ully acknowledged. We hank he analy ical depa men s o ou ins i u e o expe suppo , especially D . R. Godda d o sol ing he X- ay s uc u e. The c ys allog aphic da ase was eco ded a PETRA III a DESY, a membe o he Helmhol z Associa ion (HGF); we would like o hank Anja Bu kha d and Alke Meen s o assis ance in using he P11 beamline. ■REFERENCES (1) (a) He mann, J.; Abou Fayad, A.; Mulle , R. Na . P od. Rep. 2017, 34, 135−160. (b) Weissman, K. J.; Mulle , R. Na . P od. Rep. 2010,27, 1276−1295. (c) Schabe le, T. F.; Loh , F.; Schmi z, A.; Konig, G. M. Na . P od. Rep. 2014,31, 953−972. (2) Ho mann, H.; Kogle , H.; Heyse, W.; Ma e , H.; Caspe s, M.; Schumme , D.; Klemke-Jahn, C.; Baue , A.; Pena ie , G.; Debussche, L.; B ons up, M. Angew. Chem., In . Ed. 2015,54, 10145−10148. (3) K as el, P.; Roggo, S.; Schi le, M.; Ross, N. T.; Pe uccio, F.; Aspesi, P.; Aus , T.; Bun in, K.; Es oppey, D.; Liech y, B.; Mapa, F.; Memme , K.; Mille , H.; Pan, X.; Riedl, R.; Thibau , C.; Thomas, J.; Wagne , T.; Webe , E.; Xie, X.; Schmi , E. K.; Hoep ne , D. Angew. Chem., In . Ed. 2015,54, 10149−10154. (4) Al hough EF-1 is ubiqui ous in mammalian cells, EF-1 le els and mu a ions a e hough o play an impo an ole in ce ain cance s; o a leading e e ence, see he ollowing and li e a u e ci ed he ein: Lee, M.- H.; Su h, Y.-J. Ann. N. Y. Acad. Sci. 2009,1171,87−93. (5) Didemnin B, ano he po en inhibi o o EF-1, en e ed clinical phase II ials in he U.S.; o a e iew, see: Lee, J.; Cu ano, J. N.; Ca oll, P. J.; Joullie,M.M.Na . P od. Rep. 2012,29, 404−424. (6) The binding si e was shown o o e lap wi h ha o didemnin B; see e 3. The Jou nal o O ganic Chemis y A icle DOI: 10.1021/acs.joc.7b02871 J. O g. Chem. 2018, 83, 6977−6994 6992 (7) Liao, L.; Zhou, J.; Xu, Z.; Ye, T. Angew. Chem., In . Ed. 2016,55, 13263−13266. (8) Huang, J.; Wang, Z. O g. Le . 2016,18, 4702−4705. (9) Yang, Z.; Xu, X.; Yang, C.-H.; Tian, Y.; Chen, X.; Lian, L.; Pan, W.; Su, X.; Zhang, W.; Chen, Y. O g. Le . 2016,18, 5768−5770. (10) Liu, Q.; Hu, P.; He, Y. J. O g. Chem. 2017,82, 9217−9222. (11) Zhang, Y.-H.; Liu, R.; Liu, B. Chem. Commun. 2017,53, 5549− 5552. (12) Poock, C.; Kalesse, M. O g. Le . 2017,19, 4536−4539. (13) Fo a discussion, see: Fu s ne , A. Science 2013,341, 1229713. (14) Wilson, R. M.; Danishe sky, S. J. Angew. Chem., In . Ed. 2010,49, 6032−6056. (15) (a) Szpilman, A. M.; Ca ei a, E. M. Angew. Chem., In . Ed. 2010, 49, 9592−9628. (b) Fu s ne , A. Is . J. Chem. 2011,51, 329−345. (c) Wach, J.-Y.; Gademann, K. Synle 2012,2012, 163−170. (16) Fu s ne , A. Angew. Chem., In . Ed. 2013,52, 2794−2819. (17) T os , B. M.; Ball, Z. T. Syn hesis 2005,2005, 853−887. (18) F ihed, T. G.; Fu s ne , A. Bull. Chem. Soc. Jpn. 2016,89, 135− 160. (19) (a) C omm, P. M.; Schaubach, S.; Spiegel, J.; Fu s ne , A.; G ossmann, T. N.; Waldmann, H. Na . Commun. 2016,7, 11300. (b) C omm, P. M.; Wall a en, K.; Glas, A.; Bie , D.; Fu s ne , A.; O mann, C.; G ossmann, T. N. ChemBioChem 2016,17, 1915−1919. (c) Ghali , N.; Poo , A. J.; Fu s ne , A.; Rijke s, D. T. S.; Liskamp, R. M. J. O g. Le . 2005,7, 2961−2964. (20) Bu nley, J.; Jackson, W. R.; Robinson, A. J. J. O g. Chem. 2015,80, 9057−9063. (21) (a) Rummel , S. M.; Radkowski, K.; Rosca, D.-A.; Fu s ne , A. J. Am. Chem. Soc. 2015,137, 5506−5519. (b) Rummel , S. M.; Fu s ne , A. Angew. Chem., In . Ed. 2014,53, 3626−3630. (22) Rosca, D.-A.; Radkowski, K.; Wol , L. M.; Wagh, M.; Godda d, R.; Thiel, W.; Fu s ne , A. J. Am. Chem. Soc. 2017,139, 2443−2455. (23) Amides and sul onamides we e p e iously shown o exe an app eciable s ee ing effec ; see e 21. (24) Yang, Y.; Pe y, I. B.; Lu, G.; Liu, P.; Buchwald, S. L. Science 2016, 353, 144−150. (25) Ba ko, S. G.; Deng, J.; Danheise , R. L. O g. Syn h. 2016,93, 245− 262. (26) Denma k, S. E.; Jones, T. K. J. O g. Chem. 1982,47, 4595−4597. (27) De wile , J. E.; Lubell, W. D. J. O g. Chem. 2003,68, 177−179. (28) Fu s ne , A.; Schlecke , A. Chem. - Eu . J. 2008,14, 9181−9191. (29) (a) Bu gess, K.; Jennings, L. D. J. Am. Chem. Soc. 1991,113, 6129−6139. (b) Bu o a, S. A.; McDonald, F. E. J. Am. Chem. Soc. 2002, 124, 8188−8189. (c) Masuda, Y.; Mo i, K. Eu . J. O g. Chem. 2005,2005, 4789−4800. (d) Ma suda, M.; Yamazaki, T.; Fuhshuku, K.-i.; Sugai, T. Te ahed on 2007,63, 8752−8760. (30) Hughes, D. L. O g. Reac . 1992,42, 335−656. (31) Ma yano , B. E.; Rei z, A. B. Chem. Re . 1989,89, 863−927. (32) (a) De os, A.; Remion, J.; F isque-Hesbain, A.-M.; Colens, A.; Ghosez, L. J. Chem. Soc., Chem. Commun. 1979, 1180−1181. (b) Munyemana, F.; Geo ge, I.; De os, A.; Colens, A.; Bada au, E.; F isque-Hesbain, A.-M.; Loude , A.; Di e ding, E.; Damien, J.-M.; Remion, J.; Van Uy be gen, J.; Ghosez, L. Te ahed on 2016,72, 420− 430. (33) Fo an applica ion whe e he use o his eagen had been c i ical, see: Fu s ne , A.; Wein i , H. J. Am. Chem. Soc. 1998,120, 2817−2825. (34) Golako i, T.; Ogino, J.; Hel zel, C. E.; Le Husebo, T.; Jensen, C. M.; La sen, L. K.; Pa e son, G. M. L.; Moo e, R. E.; Moobe y, S. L.; Co be , T. H.; Vale io e, F. A. J. Am. Chem. Soc. 1995,117, 12030− 12049. (35) (a) Rachele, J. R. J. O g. Chem. 1963,28, 2898. (b) Nishiyama, S.; Suzuki, Y.; Yamamu a, S. Te ahed on Le . 1988,29, 559−562. (36) Hend ickson, J. B.; Kandall, C. Te ahed on Le . 1970,11, 343− 344. (37) Au elio, L.; B ownlee, R. T. C.; Hughes, A. B. Chem. Re . 2004, 104, 5823−5846. (38) Habe lag, B.; F ey ag, M.; Daniliuc, C. G.; Jones, P. G.; Tamm, M. Angew. Chem., In . Ed. 2012,51, 13019−13022. (39) (a) Willwache , J.; Fu s ne , A. Angew. Chem., In . Ed. 2014,53, 4217−4221. (b) Ungeheue , F.; Fu s ne , A. Chem. - Eu . J. 2015,21, 11387−11392. (40) (a) Lhe me , R.; Fu s ne , A. Chem. - Eu . J. 2014,20, 13188− 13193. (b) Pe sich, P.; Lla e ia, J.; Lhe me , R.; de Ha o, T.; S ade, R.; Kondoh, A.; Fu s ne , A. Chem. - Eu . J. 2013,19, 13047−13058. (41) (a) Heppekausen, J.; S ade, R.; Godda d, R.; Fu s ne , A. J. Am. Chem. Soc. 2010,132, 11045−11057. (b) Heppekausen, J.; S ade, R.; Kondoh, A.; Seidel, G.; Godda d, R.; Fu s ne , A. Chem. - Eu . J. 2012, 18, 10281−10299. (42) Sch ock, R. R. Chem. Re . 2002,102, 145−179. (43) Ce ain ungs en alkylidynes eac wi h benzaldehyde, ace one, e hyl o ma e, and DMF: F eudenbe ge , J. H.; Sch ock, R. R. O ganome allics 1986,5, 398−400. (44) Fo o he ins uc i e examples, see e 45 and he ollowing: (a) Rals on, K. J.; Rams adius, H. C.; B ews e , R. C.; Niblock, H. S.; Hulme, A. N. Angew. Chem., In . Ed. 2015,54, 7086−7090. (b) Guo, L.- D.; Huang, X.-Z.; Luo, S.-P.; Cao, W.-S.; Ruan, Y.-P.; Ye, J.-L.; Huang, P.-Q. Angew. Chem., In . Ed. 2016,55, 4064−4068. (c) He s ad, G.; Moleswo h, P. P.; Mille , C. M.; Benneche, T.; Tius, M. A. Te ahed on 2016,72, 2084−2093. (d) Ho ling, S.; Bi ne , C.; Tamm, M.; Dahn, S.; Colla z, J.; S eidle, J. L. M.; Schulz, S. O g. Le . 2015,17, 5004−5007. (e) Neuhaus, C. M.; Linige , M.; S iege , M.; Al mann, K.-H. Angew. Chem., In . Ed. 2013,52, 5866−5870. (45) Fo ep esen a i e examples om ou g oup, see he ollowing and li e a u e ci ed he ein: (a) Chaładaj, W.; Co be , M.; Fu s ne , A. Angew. Chem., In . Ed. 2012,51, 6929−6933. (b) Micoine, K.; Fu s ne , A. J. Am. Chem. Soc. 2010,132, 14064−14066. (c) Hickmann, V.; Kondoh, A.; Gabo , B.; Alca azo, M.; Fu s ne , A. J. Am. Chem. Soc. 2011,133, 13471−13480. (d) Benson, S.; Collin, M.-P.; A l , A.; Gabo , B.; Godda d, R.; Fu s ne , A. Angew. Chem., In . Ed. 2011,50, 8739− 8744. (46) The a io o egioisome s in he c ude p oduc is >20:1; o he s e eoisome s could no be de ec ed by NMR. (47) [Cp*RuCl]4disassembles on ea men wi h an alkyne subs a e o gi e [Cp*RuCl] as he ca aly ically ac i e agmen ; see e s 21 and 22; o he p epa a ion o his p eca alys , see: (a) Fagan, P. J.; Mahoney, W. S.; Calab ese, J. C.; Williams, I. D. O ganome allics 1990,9, 1843− 1852. (b) Fagan, P. J.; Wa d, M. D.; Calab ese, J. C. J. Am. Chem. Soc. 1989,111, 1698−1719. (48) (a) Rummel , S. M.; P eindl, J.; Somme , H.; Fu s ne , A. Angew. Chem., In . Ed. 2015,54, 6241−6245. (b) Somme , H.; Hamil on, J. Y.; Fu s ne , A. Angew. Chem., In . Ed. 2017,56, 6161−6165. (c) Somme , H.; Fu s ne , A. O g. Le . 2016,18, 3210−3213. (d) Schaubach, S.; Michigami, K.; Fu s ne , A. Syn hesis 2016,49, 202−208. (49) Huwyle , N.; Radkowski, K.; Rummel , S. M.; Fu s ne , A. Chem. - Eu . J. 2017,23, 12412−12419. (50) P eindl, J.; Jou in, K.; Lau ich, D.; Seidel, G.; Fu s ne , A. Chem. - Eu . J. 2016,22, 237−247. (b) Fu s ne , A.; Da ies, P. W. J. Am. Chem. Soc. 2005,127, 15024−15025. (51) (a) Fluo ine in Pha maceu ical and Medicinal Chemis y. F om Biophysical Aspec s o Clinical Applica ions; Gou e neu , V., Mulle , K., Eds.; Wo ld Scien ific: London, 2012; Vol. 6. (b) Fluo ine in Medicinal Chemis y and Chemical Biology; Ojima, I., Ed.; Wiley-Blackwell: Chiches e , U.K., 2009. (52) Somme , H.; Fu s ne , A. Chem. - Eu . J. 2017,23, 558−562. (53) Fo chlo ine/ in exchange, see: (a) Takeda, T.; Kanamo i, F.; Ma susi a, H.; Fujiwa a, T. Te ahed on Le . 1991,32, 6563−6566. (b) Madec, D.; Fe ezou, J.-P. Eu . J. O g. Chem. 2006,2006,92−104. (54) Fo o he s udies o his ype, see e 48a and he ollowing: (a) Mailhol, D.; Willwache , J.; Kausch-Busies, N.; Rubi ski, E. E.; Sobol, Z.; Schule , M.; Lam, M.-H.; Mus o, S.; Loganzo, F.; Made na, A.; Fu s ne , A. J. Am. Chem. Soc. 2014,136, 15719−15729. (b) Micoine, K.; Pe sich, P.; Lla e ia, J.; Lam, M.-H.; Made na, A.; Loganzo, F.; Fu s ne , A. Chem. - Eu . J. 2013,19, 7370−7383. (c) Fu s ne , A.; Ne ado, C.; Wase , M.; T emblay, M.; Che ie , C.; Teply, F.; Aïssa, C.; Moulin, E.; Mulle , O. J. Am. Chem. Soc. 2007,129, 9150−9161. (d) Fu s ne , A.; Ki k, D.; Fens e , M. D. B.; Aïssa, C.; De Souza, D.; Ne ado, C.; Tu le, T.; Thiel, W.; Mulle , O. Chem. - Eu . J. 2007,13, 135−149. The Jou nal o O ganic Chemis y A icle DOI: 10.1021/acs.joc.7b02871 J. O g. Chem. 2018, 83, 6977−6994 6993 (55) Cheng, J.-K.; Loh, T.-P. J. Am. Chem. Soc. 2015,137,42−45. (56) Wu, B.; Feas , G. C.; Thompson, A. L.; Robe son, J. J. O g. Chem. 2012,77, 10623−10630. (57) Li, Y.; Yang, X.; Liu, Y.; Zhu, C.; Yang, Y.; Yu, B. Chem. - Eu . J. 2010,16, 1871−1882. The Jou nal o O ganic Chemis y A icle DOI: 10.1021/acs.joc.7b02871 J. O g. Chem. 2018, 83, 6977−6994 6994