Accep ed Manusc ip
A syn he ic and in silico s udy on he highly egioselec i e Diels-Alde eac ion
o he polyenic an i ungal an ibio ics na amycin and la o ungin
Zsol Fejes, A ila Mándi, Is án Komá omi, László Majo os, Gyula Ba a, Pál
He czegh
PII: S0040-4039(10)01229-3
DOI: 10.1016/j. e le .2010.07.049
Re e ence: TETL 38087
To appea in: Te ahed on Le e s
Recei ed Da e: 24 Feb ua y 2010
Re ised Da e: 29 June 2010
Accep ed Da e: 9 July 2010
Please ci e his a icle as: Fejes, Z., Mándi, A., Komá omi, I., Majo os, L., Ba a, G., He czegh, P., A syn he ic and
in silico s udy on he highly egioselec i e Diels-Alde eac ion o he polyenic an i ungal an ibio ics na amycin and
la o ungin, Te ahed on Le e s (2010), doi: 10.1016/j. e le .2010.07.049
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G aphical Abs ac
A syn he ic and in silico s udy on he highly
egioselec i e Diels-Alde eac ion o he
polyenic an i ungal an ibio ics na amycin
and la o ungin
Zsol Fejes, A ila Mándi, Is án Komá omi,
László Majo os, Gyula Ba a and Pál He czegh
The e aenic mac olide an ibio ic na amycin and he pen aenic mac olide
la o ungin ga e monoadduc s on Diels-Alde eac ion wi h 4-phenyl-
1,2,4- iazoline-3,5-dione. The egioselec i i y o he eac ion as well as
he con o ma ion o he p oduc s we e s udied using heo e ical calcula ions.
Lea e his a ea blank o abs ac in o.
Te ahed on Le e s
1
A syn he ic and in silico s udy on he highly egioselec i e Diels-
Alde eac ion o he polyenic an i ungal an ibio ics na amycin
and la o ungin
Zsol Fejes,a A ila Mándi,b,c Is án Komá omi,d,* László Majo os,e Gyula Ba ac and Pál He czegha,*
aDepa men o Pha maceu ical Chemis y, Uni e si y o Deb ecen, H-4032 Deb ecen, Hunga y
bResea ch G oup o Ca bohyd a es o he Hunga ian Academy o Sciences, Uni e si y o Deb ecen, H-4032 Deb ecen, Hunga y
cDepa men o O ganic Chemis y, Uni e si y o Deb ecen, H-4032 Deb ecen, Hunga y
dHaemos asis, Th ombosis and Vascula Biology Resea ch G oup o he Hunga ian Academy o Sciences, Uni e si y o Deb ecen, H-4032
Deb ecen, Hunga y
eDepa men o Medical Mic obiology, Uni e si y o Deb ecen, H-4032 Deb ecen, Hunga y
Dedica ed o P o esso Ká oly Lempe on he occasion o his 85 h bi hday
Abs ac — The e aenic mac olide an ibio ic na amycin and he pen aenic mac olide la o ungin ga e monoadduc s on Diels-Alde
eac ion wi h 4-phenyl-1,2,4- iazoline-3,5-dione. The egioselec i i y o he eac ion as well as he con o ma ion o he p oduc s we e
s udied using heo e ical calcula ions. © 2010 Else ie Science. All igh s ese ed
———
* Co esponding au ho . Tel.: +36-52-512-900 ex . 55178; ax: +36-52-340-011; e-mail: koma [email protected]
* Co esponding au ho . Tel.: +36-52-512-900 ex . 22895; ax: +36-52-512-914; e-mail: he cze[email p o ec ed].
Polyene mac olide an ibio ics such as ampho e icin B,
nys a in and na amycin a e impo an he apeu ics o he
ea men o ungal in ec ions.1 Thei s uc u e consis s o
ou o eigh conjuga ed ca bon-ca bon double bonds wi h
E-con igu a ion. This s uc u al uni esul s in he od-
shaped o m o hese molecules.2 This con o ma ion
p obably plays an impo an ole in binding o ungal s e ol
molecules esul ing in he an i ungal ac ion o hese
an ibio ics. In his wo k wo ep esen a i es o he polyene
mac olide an ibio ic amily, na amycin (1) and la o ungin
(2) ha e been chosen o s udy a Diels-Alde (DA) eac ion
on hei polyenic moie y, as well as he in luence o he
newly o med py idazine ing annela ion on he
con o ma ion o he mac ocycle and on he an i ungal
ac i i y.
Only a ew examples ha e been published on Diels-Alde
eac ions whe e he eac ing diene is pa o a polyene. In
hese cases he polyene can be ega ded as a subs i u ed
diene o which he egio- and/o s e eoselec i i y is
de e mined by i s subs i uen s (e.g. he emainde o he
polyene). A de ailed expe imen al and heo e ical analysis
o DA eac ions o his ype wi h polyenes possessing a
ca boxyla e and hyd oxyl g oup a he end was epo ed by
Tu ne e al.3 They ound ha he dienophile always eac s
wi h he ou e mos diene uni o he polyene, and he
heo e ical calcula ions ca ied ou o a model compound
suppo ed hei indings. They also ound ha a simple
model based on he on ie molecula o bi al coe icien s
o he p edic ion o egioselec i i y o DA eac ions was
no always applicable o such sys ems. No examples ha e
been ound, howe e , in he li e a u e whe e geome ical
cons ain s exis o he polyene, e.g. when a linea polyene
is assembled in a cyclic compound as in he case o polyene
mac olide an ibio ics.
Pe
g
amon
TETRAHEDRON
LETTERS
Te ahed on Le e s
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Na amycin4 (1) has a mac olac one s uc u e consis ing o
26 a oms. The conjuga ed e aenic pa o he molecule
was he a ge o a DA eac ion wi h he highly eac i e
diazadienophile 3. The DA eac ion o 1 wi h 3 a oom
empe a u e led o a single p oduc which was isola ed by
adso p ion ch oma og aphy. On he basis o NMR, MS and
UV-VIS spec oscopic da a ( he con igu a ion o C-20 and
C-23 was assigned on he basis o TS calcula ions) he
s uc u e 4 was assigned o his adduc (Scheme 1) as
de ailed in he supplemen a y ma e ial oge he wi h he
obse ed an i ungal ac i i y (Figu e S1*, Table S1). In an
an i ungal es agains Candida s ains, p oduc 4 exhibi ed
no ac i i y.
Scheme 1. Diels-Alde eac ion o na amycin (1) and 3.
Na amycin heo e ical model (1a)(Scheme 2): S a ing om
a plausible all- ansoid s uc u e,5 cons an empe a u e
(500 K) molecula dynamics (200 ns o al ime, 1 s ime
s ep, GAFF o ce ield6) simula ions we e ca ied ou in
o de o explo e he s uc u es a ailable wi h non-negligible
popula ions o DA eac ions. A o al o 200,000 s uc u es
we e sa ed, and each minimized and classi ied in o clus e s
ega ding only he polyene chain con o ma ion. The lowes
ene gy ep esen a i e om each clus e was hen submi ed
o semiempi ical (AM1, PM3, PM6 and RM1) and densi y
unc ional (B3LYP/6-31G) calcula ions. T ansi ion s a es
o cisoid s uc u es which con ain only a single cisoid
con o ma ion ( o bo h possible o ien a ion o he
dienophile) we e calcula ed a AM1, PM3, PM6, RM1 and
ONIOM(B3LYP/6-31G(d):AM1) le els o heo y.
Classi ying he op imized s uc u es om he
con o ma ional sea ch esul ed in eigh clus e s wi hin a
na ow ene gy ange which means ha any cisoid
con o ma ion sui able o a DA eac ion can be o med
wi h su icien p obabili y. These clus e s can ea u e om
ze o o h ee cisoids and wo o i e ansoids (i.e. he Ci+k–
C i+1+k–Ci+2+k–Ci+3+k o sion angles close o ze o o 180
deg ees, espec i ely, whe e i=15 and k=1 and/o 3 and/o
———
* Figu es and ables s a ing wi h “S” can be ound in he supplemen a y
ma e ial.
5, see Figu e S2). The desc ip ion o hese clus e s as well
as he ene gy alues ob ained o hei lowes ene gy
ep esen a i es a a ious le els o heo y can be ound in
he Supplemen a y Ma e ial (Table S2). The ansi ion s a e
(TS) calcula ions be ween 3 and 1a wi h single cisoid
geome ies (a k=1 o 3 o 5) a he AM1, RM1 and PM6 as
well as ONIOM(B3LYP/6-31G(d):AM1) le els o heo y
show he TS o cisoid diene s uc u es co espond o k=5,
being he mos s able in all ypes o compu a ions. I is in
pe ec ag eemen wi h he p epa a i e indings whe e only
his p oduc was o med. The g aphical ep esen a ions o
he TSs calcula ed a ONIOM(B3LYP/6-31G(d):AM1 le el
a e shown in Figu es S3-S8 while he ela i e TS ene gies
o all he me hods and o all he single-cisoid diene
subsys ems a e gi en in Table S3. The mos s able
ansi ion s a e ob ained om ONIOM(B3LYP/6-
31G(d):AM1) calcula ions is shown in Figu e 1.
Scheme 2. Na amycin heo e ical model (1a) and i s heo e ical adduc
model (4a).
Figu e 1. Mos s able ONIOM(B3LYP/6-31G(D):AM1) ansi ion s a e
o he Diels-Alde eac ion be ween 1a and 3.
The s uc u e o na amycin heo e ical model DA adduc
(4a)(Scheme 2): The s a ing p oduc geome y was
ob ained by minimizing he adduc de i ed di ec ly om
he mos s able ansi ion s a e (Figu e 1). Using his
“ ansi ion s a e like p oduc geome y”, 5 ns cons an
empe a u e molecula dynamics simula ion was pe o med
Te ahed on Le e s
3
a 900 K sa ing 20 geome ies being equidis an om he
ajec o y and each o hese was submi ed o 10 ns cons an
empe a u e (T=400 K) simula ions. The mos s able
geome ies om he co esponding ajec o ies we e
minimized a he HF/6-31G and B3LYP/6-31G le els o
heo y.
Depic ion o he ene gy and con o ma ional changes du ing
one ep esen a i e molecula dynamics simula ion leading
o he mos s able con o me a e gi en in Figu es 2 and S9.
Al hough he mac ocyclic ing in he mos s able TS
(Figu e 1) ob ained om he single cisoid con o me and in
he nex local minimum (Figu e S10) shows app eciable
simila i y in shape o hose obse ed a he global minimum
o na amycin, i can be seen om hese Figu es ha p oduc
4 does no p e e he co esponding elonga ed od-shaped
geome y. Ins ead, i adop s a mo e compac con o ma ion
(Figu e S12), which was eached h ough an in e media e
p oduc (Figu e S11). O he 20 sho molecula dynamics
simula ions ca ied ou o he DA p oduc 4a, 19 led o he
same con o ma ion (Figu e S12) which was signi ican ly
di e en om he s a ing “TS-like p oduc ” (Figu e S10)
and was lowe in ene gy (Table S4) han was ob ained o
he geome y om he 20 h simula ion (Figu e S13).
Figu e 2. To al ene gy o he Diels-Alde adduc 4a du ing 10 ns
molecula dynamics simula ion o one o he ep esen a i e ajec o ies
leading o he mos equen ly ound con o ma ion.
Fla o ungin7 (2)(Scheme 3) was disco e ed 50 yea s ago a
Deb ecen Uni e si y. I s s uc u e consis s o a 32-
membe ed mac ocycle wi h a conjuga ed pen aenic lac one
moie y.8 On he eac ion wi h an equimola quan i y o 3
he majo p oduc isola ed, on he basis o spec oscopic
da a, p o ed o ha e he s uc u e 5(Scheme 3). The
numbe ing, de ails on he s uc u al elucida ion as well as
he obse ed an i ungal ac i i y o compound 5 a e gi en in
he Supplemen a y Ma e ial (Figu e S1, Table S1). The
an i ungal ac i i y o 5 was e alua ed agains Candida
yeas s ains and his compound p o ed o be inac i e.
Scheme 3. Diels-Alde eac ion o la o ungin (2) and 3.
I should be men ioned ha al hough in bo h cycloaddi ions
wo new chi al cen e s we e o med, by ca e ul analysis o
he NMR spec a o 4 and 5, only a single dias e eome
could be obse ed.
Theo e ical calcula ions on la o ungin (2): The
compu a ional p o ocols ou lined in he case o he
na amycin heo e ical model we e epea ed wi h he
excep ions ha in his case he e we e mo e clus e s ound
due o he longe polyene chain and he ansi ion s a es
we e de e mined using AM1, PM3, PM6, RM1 as well as
ONIOM (B3LYP/6-31G:AM1:AMBER, B3LYP/6-
31G:HF/6-31G:AMBER, B3LYP/6-31G(d):HF/6-
31G:AMBER, HF/6-31G(d):HF/6-31G:AMBER, MP2/6-
31G:HF/6-31G:AMBER and MP2/6-31G(d):HF/6-
31G:AMBER) me hods.
This molecule has many o a able dihed al angles and
he e o e is oo lexible o ind he global and all he low
ene gy local minima wi h ull ce ain y. Conside ing,
howe e , only he polyene pa o he la o ungin (Figu e
S2), i can be classi ied in o 16 clus e s acco ding o i s
pen aenic s uc u e. These clus e s can be cha ac e ized
om ze o o ou cisoids and h ee o se en ansoids.
Based on he ene gy alues (Table S5) ob ained o each o
he 16 geome ies, he p obabili y o he p esence o any
cisoid con o ma ion sui able o a DA eac ion is non-
negligible.

Te ahed on Le e s
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Figu e 3. ONIOM(B3LYP/6-31G(d):HF/6-31G:AMBER) ansi ion s a e
o he Diels-Alde eac ion be ween la o ungin (2) and 3 a k=7.
Rega ding he DA ansi ion s a e ene gies and he
co esponding single cisoid diene subsys em geome ies in
he polyene chain, he PM3, as well as he B3LYP/6-
31G:HF/6-31G:AMBER, MP2/6-31G:HF/6-31G:AMBER,
HF/6-31G(d):HF/6-31G:AMBER, B3LYP/6-31G(d):HF/6-
31G:AMBER and MP2/6-31G(d):HF/6-31G:AMBER
ONIOM me hods p edic he mos s able TS wi h a
pa icipa ing cisoid diene con o ma ion a C8=C9–
C10=C11. In con as , he AM1 and PM6 me hods p e e
DA eac ion a C6=C7–C8=C9, while he RM1 and
B3LYP/6-31G:AM1:AMBER ONIOM me hods indica e
eac ion a he C4=C5–C6=C7 a oms. Only he PM3 and
ONIOM calcula ions wi h ab ini io HF/6-31G
“in e media e le el” o heo y esul ed in a TS geome y in
which he pa icipa ion o he cisoid diene si ua ed u hes
om he lac one g oup is p e e ed in ag eemen wi h he
expe imen al inding. Ene gy alues and geome ies o he
possible ansi ion s a es o he single cisoid polyene can
be ound in he supplemen a y ma e ial (Table S6, Figu es
S14-S21) while he mos s able ONIOM(B3LYP/6-
31G(d):HF/6-31G:AMBER) TS is shown in Figu e 3.
Polyenoic acids as la o ungin heo e ical models: he DA
eac ion o e inoic acid was s udied expe imen ally and
heo e ically by Yamada e al.9 Since la o ungin can be
ega ded as “subs i u ed” e inoic acid, in his wo k, we
a emp ed o analyze he di ec ing e ec o he ca boxyl
unc ion by means o mo e sophis ica ed calcula ions.
Conjuga ed i-, e a- and pen aenes wi h a ca boxyl g oup
a he end o he polyene chain (wi h he es ic ion ha
only one cisoid diene subsys em exis s a he same ime in
he chain) we e op imized a AM1, HF/6-31G(d),
B3LYP/6-31G(d) and MP2/6-31G(d) le els o heo y. The
MO coe icien s we e calcula ed a HF/STO-6G and
B3LYP/6-31G(d) le els. The AM1, HF/6-31G(d),
B3LYP/6-31G(d) and MP2/6-31G(d) ansi ion s a es o
DA eac ions be ween hese ca boxylic acids (as dienes)
and he dienophile (3) we e also calcula ed.
The calcula ions ou lined o model polyenoic acids we e
epea ed o ienoic acid sys ems also possessing a –CH2–
CH2–OH g oup a he opposi e end o polyene chain o he
ca boxyl g oup. The TSs we e de e mined o he ω-Me
polyenoic acids a ONIOM(MP2/6-31G(d):HF/6-31G)
le el o heo y, as well. The ONIOM(MP2/6-31G(d):HF/6-
31G) me hod, applying HF le el only he phenyl g oup,
was alida ed agains he pu e MP2/6-31G(d) me hod a ω-
Me subs i u ed and ω-unsubs i u ed ienoic acids (Table
S10) and was p o en o gi e quali a i ely he same esul s.
T ansi ion s a e calcula ions o he DA eac ions be ween
he model polyenoic acids (as dienes) and 3 showed ha he
TSs in ol ing he Cω-3–Cω-2–Cω-1–Cω (numbe ing
s a ed a he ca boxyl ca bon) a oms we e he mos s able
o all polyenoic acids and o all bu he MP2/6-31G(d)
le els o heo y applied. A he MP2/6-31G(d) le el he
TSs in ol ing he Cω-5–Cω-4–Cω-3–Cω-2 a oms we e
sys ema ically he mos s able one. E en hough he
B3LYP me hod was shown o be ema kably success ul o
pe icyclic eac ions,3,10 addi ional se ies o calcula ions
we e ca ied ou in o de o esol e he disc epancy in he
p edic ed (by means MP2 e sus o he dependable
me hods) DA egioselec i i y. Model eac ions be ween he
Me subs i u ed (a he ω posi ion ela i e o he ca boxyl
g oup) polyenoic acids and 3 a ONIOM(MP2/6-
31G(d):HF/6-31G) le el p e e again a DA eac ion
in ol ing he a oms o he a hes cisoid con o ma ion
om he ca boxyl g oup. Since he ω-Me subs i u ed model
is expec ed o mi o he p ope ies o la o ungin in a
g ea e ex en , he esul s o calcula ions a e in a nice
ag eemen wi h ou expe imen s whe e he p oduc
co esponding o his adduc (a “subs i u ed pen aenoic
acid”) was ob ained exclusi ely. I is also wo h
men ioning ha his ansi ion s a e co esponds o a a he
asynch onous eac ion s ep since he bond be ween Cω and
he N o he dienophile p eceed he o ma ion o he Cω-3–
N bond, like hose obse ed by Tu ne 3 e al. om hei
heo e ical calcula ions on symme ic polyenes (Figu e 4).
A aching he –CH2–CH2–OH g oup o he o he end o he
polyene ( iene) chain esul ed quali a i ely in he same
indings.
Ene gies o each o hese ansi ion s a es calcula ed a
a ious le els o heo y can be ound in Table S10 while
g aphical ep esen a ions o he co esponding s uc u es
a e gi en in Figu es S22-S39. I should be no ed, ha using
on ie MO coe icien s o p edic he egioselec i i y o
he DA eac ion seems o wo k o ienoic and e aenoic
acids while i equen ly ails (depending on he applied
me hod) o he pen aenoic acid. The p edic ing powe o
MO coe icien s on he di ec ing e ec can be e en mo e
doub ul in he ienoic acid case when he –CH2–CH2–OH
subs i uen is a he ω posi ion. Demons a i e MO
coe icien s and eigen alues a e lis ed in Tables S8 and S9.
Te ahed on Le e s
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Figu e 4. B3LYP/6-31G(D) ansi ion s a e o he Diels-Alde eac ion
be ween a pen aene ca boxylic acid ( la o ungin model) and 3.
In conclusion, he DA eac ions a compounds 1 and 2 ha e
been ca ied ou and he ob ained p oduc s we e
cha ac e ized expe imen ally. The heo e ical calcula ions
demons a ed ha despi e he cyclic s uc u es o 1 and 2
he lexibili y o hese molecules allows he p esence o he
cisoid con o ma ion a any posi ion o he polyene uni o
he mac ocycles.
In he case o he na amycin heo e ical model, all he
applied quan um chemical me hods suppo ed he same
egioselec i i y p e e ence in pe ec ag eemen wi h he
expe imen al indings despi e he missing bulky suga
g oup. The applied semiempi ical and ab ini io quan um
chemical calcula ions on polyenoic acids (as models o he
la o ungin polyene chain) also p edic a p oduc in
ag eemen wi h he expe imen s. F om hese ac s we
a ou a mainly elec onic na u e o he obse ed DA
egioselec i i y.
In con as , ca ying ou heo e ical calcula ions on
la o ungin i sel shows a somewha di e en pic u e since
in his case he p edic ed egioselec i i y showed me hod
dependency. Howe e , applying he ab ini io HF/6-31G
me hod as an “in e media e” le el o heo y o he
polyenoic acid pa in ONIOM calcula ions was su icien
o p edic he expe imen ally obse ed egioselec i i y.
In he case o na amycin he heo e ical calcula ions
demons a ed ha he od-shaped con o ma ion o he
an ibio ic was de o med as a consequence o he DA
eac ion, leading o a mo e compac con o ma ion o 4. The
elonga ed shape is p obably necessa y o s e ol complex
o ma ion, which migh explain he loss o he an i ungal
ac i i y o 4 compa ed o he pa en compound 1.
So wa e packages used in his wo k: The AMBER
so wa e package6 was used o empi ical o ce- ield
calcula ions while quan um chemis y calcula ions we e
ca ied ou using ei he MOPAC11 o Gaussian’0312 sui es.
The con o me s we e classi ied in o clus e s using in-house
so wa e13. The MOLEKEL14 so wa e package was used
o isualiza ion o he esul s.
Acknowledgemen s
This wo k was suppo ed by he Hunga ian Na ional
In as uc u e De elopmen P og am (G an : NIIF 1116)
and he Hunga ian Scien i ic Resea ch Funds (G an s:
OTKA 68578, NKTH-OTKA CK 77515, TÁMOP-4.2.2-
08/1/2008-0019, TÁMOP 4.2.1./B-09/1/KONV-2010-0007
and OTKA 79126). We a e g a e ul o he help ul
discussions wi h P o esso László Szilágyi in he ield o
NMR assignmen s.
Re e ences
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