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
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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.
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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
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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.
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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
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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%.
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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
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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
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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
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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.
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