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Ho s mann, N., Essig, S., Bockelmann, S., Wieczo ek,
H., Huss, M., Sasse, F., Menche, D.
A chazolid A-15-O-β-D-glucopy anoside and iso-
a chazolid B: Po en V-ATPase inhibi o y polyke ides
om he Myxobac e ia cys obac e iolaceus and
A changium gephy a
(2011) Jou nal o Na u al P oduc s, 74 (5), pp. 1100-
1105.
Published: Ap il 22, 2011
Copy igh 2011 Ame ican Chemical Socie y and
Ame ican Socie y o Pha macognosy 1100 dx.doi.o g/10.1021/np200036 |J. Na . P od. 2011, 74, 1100–1105
ARTICLE
pubs.acs.o g/jnp
A chazolid A-15-O-β-D-glucopy anoside and iso-A chazolid B: Po en
V-ATPase Inhibi o y Polyke ides om he Myxobac e ia Cys obac e
iolaceus and A changium gephy a
Nicole Ho s mann,
‡
Sebas ian Essig,
†
S enja Bockelmann,
§
Helmu Wieczo ek,
§
Ma kus Huss,
§
Flo enz Sasse,
||
and Di k Menche*
,†,‡
†
Ins i u €u O ganische Chemie, Rup ech -Ka ls-Uni e si €a Heidelbe g, Im Neuenheime Feld 270, D-69120 Heidelbe g, Ge many
‡
Helmhol z-Zen um €u In ek ions o schung, Medizinische Chemie and
)
Chemische Biologie, Inhoffens asse 7,
D-38124 B aunschweig, Ge many
§
Fachbe eich Biologie/Chemie, Ab eilung Tie physiologie, Uni e si €a Osnab €uck, D-49069 Osnab €uck, Ge many
b
SSuppo ing In o ma ion
Vacuola ype ATPases (V-ATPases) a e a amily o he e o-
mul ime ic ATP-dependen ion pumps ha ene gize ans-
po p ocesses, in pa icula o p o ons ac oss memb anes
by hyd olysis o ATP.
13
In acellula V-ATPases a e in ol ed
in a ious cellula p ocesses, including ecep o -media ed
endocy osis,
4
in acellula memb ane affic, p ocessing o p o-
ho mones, deg ada ion o p o eins, and elease o neu o ans-
mi e s.
5
Fu he mo e, plasma memb ane V-ATPases ha e c i-
ical unc ions in diffe en physiological p ocesses such as u ina y
acidifica ion,
6,7
bone eso p ion,
8
and spe m ma u a ion.
9
V-AT-
Pases a e associa ed wi h a ious human diseases, including
os eopo osis, ubula acidosis, umo me as asis, in ec ion by
influenza and o he i uses, and bac e ial in ec ions by an h ax o
diph he ia
10
and ha e an inc easingly eme ging po en ial as d ug
a ge s.
1113
This ende s he de elopmen and molecula unde -
s anding o po en V-ATPase inhibi o s impo an esea ch goals.
In he la e 1990s, he polyke ide mac olide a chazolids A (1)
and B (2) we e epo ed om he myxobac e ium A changium
gephy a by he g oups o H€ofle and Reichenbach.
14,15
Wi h
ac i i ies in he low nanomola egion in i o and in i o, hey
a e among he mos po en V-ATPase inhibi o s known.
14,16,17
On a molecula le el, hey selec i ely a ge V-ATPases, which
adds o hei a ac i eness o u he de elopmen . A chazolid
A(1) has been shown o bind o he V
O
subuni c in a
nonco alen manne .
17
Mul iple copies o subuni c o m a ing,
he s uc u e o which was analyzed by X- ay c ys allog aphy a a
esolu ion o 2.1 Å in a bac e ial homologue,
18
and subsequen ly
he s uc u e o he comple e euka yo ic V-ATPase enzyme was
de e mined by c yo-elec on mic oscopy wi h a esolu ion o
16.5 Å.
19
Ve y ecen ly, he binding si e in his subuni has been
analyzed in mo e de ail by c oss-linking expe imen s o an
a chazolid A de i a i e in combina ion wi h si e-di ec ed mu a-
genesis s udies o he p o ein, demons a ing ha a chazolid A
(1) binds o he equa o ial egion o he c- ing.
20
While he
a chazolids we e o iginally isola ed om A changium gephy a,
mo e ecen epo s ha e indica ed ha he myxobac e ium
Cys obac e iolaceus p oduces a g ea e di e si y o a chazolids,
and wo o he de i a i es, a chazolid C (3)
21
and a chazolid D
(4), wi h glucosides a C-7 (3and 4) and an addi ional hyd oxyl
g oup (4),
22
ha e been isola ed om his myxobac e ium.
While he a chazolids we e o iginally epo ed as plana
s uc u es, hei absolu e and ela i e s e eochemis y was de e -
mined in 2006 by high-field NMR s udies in combina ion wi h
Recei ed: Janua y 12, 2011
ABSTRACT: Two s uc u ally no el analogues o he mac olides
a chazolids A and B, a chazolid A-15-O-β-D-glucopy anoside
(a chazolid E, 5) and iso-a chazolid B (a chazolid F, 6), we e isola ed
om he myxobac e ium Cys obac e iolaceus and A changium gephy a,
espec i ely. Mac olac one 5 ep esen s he fi s 15-O-glycoside o he
a chazolids. iso-A chazolid B (6) inco po a es a C-3 alkene and
p esen s he fi s cons i u ional isome epo ed o his na u al
p oduc class. The s uc u es o hese polyke ides we e de e mined
by spec oscopic analysis, in pa icula by HMBC, HMQC, and
ROESY NMR in es iga ions and by chemical deg ada ion. iso-A ch-
azolid B (6) demons a ed ex emely high an ip oli e a i e and V-AT-
Pase inhibi o y effec s, wi h IC
50
alues in he picomola ange, while
only mode a e ac i i y was obse ed o glycoside 5.iso-A chazolid B
p esen s he mos po en a chazolid known.
1101 dx.doi.o g/10.1021/np200036 |J. Na . P od. 2011, 74, 1100–1105
Jou nal o Na u al P oduc s ARTICLE
molecula modeling and de i a iza ion
23
and subsequen ly con-
fi med by o al syn heses o a chazolids A (1)
24,25
and B (2).
26
So
a only a limi ed numbe o SAR s udies on syn he ic
27
and
na u al analogues
21,22
ha e been epo ed; his will be c i ical in
ad ancing hese mac olide an ibio ics and may help in de i ing a
be e molecula unde s anding o he unc ion o he V-ATPase
enzyme.
28
He ein, we desc ibe he isola ion, s uc u e elucida ion,
and biological e alua ion o wo s uc u ally no el a chazolid
de i a i es, a chazolid A-15-O-β-D-glucopy anoside (a chazolid
E, 5)andiso-a chazolid B (a chazolid F, 6), om he myxobac e -
ium Cys obac e iolaceus and A changium gephy a, espec i ely.
’RESULTS AND DISCUSSION
In he cou se o p e ious s udies
27
he myxobac e ium Cys o-
bac e iolaceus s ain Cb i105
22
has been iden ified as an
efficien sou ce o s uc u ally no el a chazolid de i a i es, while
A changium gephy a s ain A 3548
16
was a mo e p oduc i e
sou ce o a chazolids A (1) and B (2). In o de o s udy mino
na u al a chazolid de i a i es, each o hese o ganisms was
cul i a ed in 350 L bio eac o s wi h 300 L o medium in he
p esence o Ambe li e XAD-16 (1%) o adso p ion. In a simila
ashion, he adso be esin and cell mass we e ha es ed a e 10
days by cen i uga ion and ex ac ed wi h ace one o gi e a c ude
ex ac . A de ailed HPLC-MS analysis o hese c ude mix u es in
combina ion wi h he cha ac e is ic UV is spec a o he
a chazolids (maximum 238/239 nm) sugges ed he p esence o
a leas one no el de i a i e in bo h e men a ion b o hs. Con-
secu i e pu ifica ion by gel ch oma og aphy (Sephadex LH-20),
MPLC, and finally e e sed-phase HPLC ga e he no el me a-
boli es a chazolid A-15-O-β-D-glucopy anoside (a chazolid F, 5,
1.0 mg yield; appa en yield <0.0035 mg/L) om Cys obac e
iolaceus s ain Cb i105 and iso-a chazolid B (a chazolid E, 6,
1.5 mg yield; appa en yield <0.005 mg/L) om A changium
gephy a s ain A 3548.
The close simila i y o he
1
H NMR,
13
C NMR, and wo-
dimensional NMR spec a o hese wo no el me aboli es
sugges ed co e s uc u es closely ela ed o hose o he pa en
na u al p oduc s a chazolids A (1) (Table 1) and B (2). Toge he
wi h he
13
C NMR spec a and HRMS analysis o he no el
me aboli e 5, indica ing he molecula o mula o be C
48
H
72
-
N
2
O
12
S, co esponding o 162 mass uni s (i.e., C
6
H
10
O
5
) highe
han a chazolid A (1), he da a sugges his mac olide o be a
hexose o a chazolid A (1). The O-glycosidic na u e o his com-
pound also became e iden om a double a δ4.22 (7.7 Hz),
a ibu ed o an anome ic p o on (1000-H). The signals o 2000-H
o 5000-H we e esol ed in CD
3
OD and assigned o glucose. The
13
C NMR da a also displayed he expec ed numbe o ca bons
and chemical shi s o glucose. The posi ion o he glucose uni
was u he confi med by ROESY expe imen s, which showed
in e ac ions o H-1000 wi h H-14, and by long- ange CH
co ela ion om 1000-H o C-15 and om H-15 o C-1000. The e-
o e, he suga uni is si ua ed a posi ion C-15, and he new
glycoside has he cons i u ion shown in Figu e 1. I s s uc u e was
u he confi med by TOCSY expe imen s and HMBC and
HMQC in e ac ions. Fu he p oo o i s cons i u ion was
ob ained by hyd olysis and GC-/HPLC compa ison wi h an
au hen ic sample o glucose. Fo he new glycoside, we sugges
he name a chazolid E (5).
The
1
H NMR spec um o he no el me aboli e 6 om
A changium gephy a likewise showed cha ac e is ic
1
H NMR
pa e ns o he a chazolid co e (Table 1). Howe e , in con as o
a chazolid A (1), a me hyl g oup a C-2 was missing, which
oge he wi h HRMS da a (molecula o mula C
41
H
60
N
2
O
7
S)
sugges ed 6 o be an isome o a chazolid B (2). C ucial in
deducing he s uc u e o he no el me aboli e was a double o
H-4 in he olefinic egion [δ6.21 ppm (J= 15.4 Hz)], one
double o a iple o H-3 [δ5.81 ppm (J= 15.4, 7.3 Hz)], and
he appea ance o he H-2 p o ons in he alipha ic egion [δ3.17,
d(J= 15.4, 7.3 Hz)]. Consequen ly, his no el de i a i e bea s a
double bond be ween C-3 and C-4 as shown, ins ead o an alkene
be ween C-2 and C-3. The E-configu a ion o he double bond
was assigned on he basis o he size o he coupling cons an
(15.4 Hz) and NOE da a, as shown in Figu e 2. The s uc u e o
his no el mac olide was confi med by HMBC co ela ion as well
as HSQC da a.
29
P e ious eeding s udies wi h
13
C-en iched ace a e and
me hionine o A changium gephy a ha e shown ha he mac o-
cyclic co e o a chazolid A, wi h he excep ion o C-23, is de i ed
om hese building blocks.
23,30
As shown in Figu e 3 o
a chazolid B, he 5E,9Z,11Z,13Eolefinic double bonds a e wi hin
hese ace a e o p opiona e agmen s and no be ween hese
building blocks, as commonly obse ed in polyke ide biosyn h-
esis ( iz., 18Eand 20Ealkenes). This may sugges a flexible
dehyd a ase domain o mo e likely an isome iza ion p ocess. The
occu ence o he double-bond isome s a chazolid B (2) and iso-
a chazolid B (6) may also o igina e om a mig a ion o he 2,3-
double bond (a chazolid B) o he unusual 3,4-posi ion in iso-
a chazolid B (6) du ing he biosyn hesis o hese mac olides.
Fo biological e alua ion o he analogues, hei inhibi o y
effec on he g ow h o he mammalian mu ine issue cell line
L-929 was e alua ed, in di ec compa ison wi h a chazolids A, B,
C, and D (Table 2). A chazolid A-15-O-β-D-glucopy anoside
1102 dx.doi.o g/10.1021/np200036 |J. Na . P od. 2011, 74, 1100–1105
Jou nal o Na u al P oduc s ARTICLE
Table 1. NMR Da a o A chazolid A (1), A chazolid A-15-O-β-D-glucopy anoside (a chazolid E, 5) and iso-A chazolid B
(a chazolid F, 6) in CD
3
OD
a
a chazolid A (1)
a chazolid A-15-O-β-D-glucopy anoside
(a chazolid E, 5)
iso-a chazolid B
(a chazolid F, 6)
posi ion δ
C
δ
H
mul . (Jin Hz) δ
C
δ
H
mul . (Jin Hz) δ
C
δ
H
mul . (Jin Hz)
1 168.3 168.5 172.5
2 129.7 123.0 39.6 3.17d (7.3)
3 142.1 6.82 ddd (7.9, 7.5, 1.3) 142.3 6.81 (7.5) 121.7 5.81 d (15.4, 7.3)
4 40.6 2.91 dd (15.0, 7.5) 3.03 dd (15.0, 7.5) 40.9 2.93 dd (14.3, 7.0)/ 3.04 dd
(14.7, 8.1)
139.7 6.21 d (15.4)
5 136.9 137.5 134.8
6 130.4 5.21 dd (9.5, 1.2) 130.8 5.21 d (9.2) 134.8 5.33 d (8.8)
7 73.6 4.03 dd (9.4, 9.3) 73.9 4.04 (9.5) 73.3 4.13 (9.2)
8 41.6 2.30 ddq (9.5, 9.8, 7.0) 41.7 2.32 m 41.5 2.36 ddq (9.5, 9.5, 6.6)
9 132.7 5.27 d (9.6) 132.6 5.29 d (9.5) 132.3 5.26 d (9.9)
10 134.8 135.0 134.6
11 130.8 5.81 d (1.0) 131.2 5.80 s 130.6 5.77 s
12 133.6 133.5 133.1
13 129.3 6.56 dd (15.5, 0.9) 131.3 6.73 d (14.3) 129.2 6.56 d (15.8)
14 133.6 5.79 dd (15.5, 6.0) 130.9 5.73 dd (16.1, 6.2) 133.4 5.83 m
b
15 75.5 4.31 dd (6.4, 3.2) 80.6 4.60 m 75.8 4.28 b s
16 44.5 1.80 ddq (9.0, 7.0, 3.2) 44.0 1.75 m 43.8 1.76 m
17 89.8 3.40 d (9.0) 89.0 3.47 d (6.6) 89.9 3.34 d (9.3)
18 135.7 136.2 135.8
19 129.9 5.87 dd (10.9, 1.2) 130.1 5.87 d (10.6) 130.0 5.81 d (11.1)
20 127.6 6.17 dd (15.5, 10.9) 127.8 6.16 dd (13.0, 12.3) 127.3 6.22 dd (15.0, 10.9)
21 135.1 5.63 dd (15.2, 7.0) 134.8 5.59 dd (15.2, 6.8) 135.2 5.59 dd (15.4, 7.3)
22 42.0 3.10 ddq (7.5, 7.0, 4.0) 42.0 3.12 dd (10.1, 6.1) 41.5 3.06 qdd (7.2, 6.6, 5.8)
23 77.6 5.97 d (4.1) 77.8 6.02 d (3.3) 72.3 5.85 dd (6.6, 1.8)
Me-2 12.6 1.91 d (1.2) 12.8 1.93 s
Me-5 16.8 1.73 d (1.2) 16.8 1.75 d (1.1) 13.3 1.87 d (0.7)
Me-8 17.7 0.84 d (7.0) 17.9 0.85 d (6.6) 17.8 0.87 d (6.6)
Me-10 24.7 1.80 b s 24.9 1.80 s 24.8 1.82 s
Me-12 19.9 1.93 d (1.2) 20.0 1.95 d (1.1) 20.1 1.95 d (1.1)
Me-16 12.6 0.74 d (7.0) 12.9 0.78 d (7.0) 12.8 0.73 d (7.3)
OMe-17 56.2 3.16 s 56.2 3.18 s 56.0 3.16 s
Me-18 13.0 1.64 d (1.2) 12.9 1.64 s 12.2 1.64 s
Me-22 17.6 1.15 d (7.0) 17.5 1.22 d (7.0) 17.2 1.07 d (7.0)
1073.3 6.04 dd (9.1, 4.5) 73.5 6.05 dd (9.2, 4.8) 73.2 6.03 dd (9.2, 4.4)
20173.9 174.1 174.2
30156.1 156.3 155.4
40116.7 7.21 s 116.6 7.18 s 117.5 7.29 s
5046.0 1.92 m 46.1 1.86 m/1.93 m 45.9 1.91 m/1.82 m
6025.8 1.77 m 26.0 1.81 m 24.8 1.78 m
7023.4 1.01 d (6.0) 23.5 1.04 d (6.6) 23.2 1.02 d (7.0)
8022.4 1.02 d (6.0) 22.5 1.05 d (6.6) 23.2 1.03 d (6.6)
100 158.2 158.4 158.3
200 27.5 2.75 s 27.7 2.76 s 27.4 2.75 s
1000 102.0 4.27 d (7.7)
2000 75.3 3.28 ddd (12.1, 8.8, 5.5)
3000 78.1 3.39 m
4000 77.9 3.22 dd (7.2, 5.0, 2.3)
5000 71.9 3.39 m
6000 62.9 3.75 dd (11.7, 5.5)/3.89 dd (11.7, 2.6)
a
Reco ded a 600 MHz (
1
H) and 150 MHz (
13
C).
b
The coupling cons an s o H-14 could be deduced in d
6
-ace one: δ5.79 ppm, dd, (J= 16.1, 4.1 Hz).
1103 dx.doi.o g/10.1021/np200036 |J. Na . P od. 2011, 74, 1100–1105
Jou nal o Na u al P oduc s ARTICLE
demons a ed an ip oli e a i e ac i i ies wi h submic omola
concen a ions (IC
50
= 0.51 μM). I was less po en han he
pa en na u al p oduc a chazolid A (1), bu h ee imes mo e
ac i e han he co esponding 7-O-β-glucosyla ed a chazolid (3)
(IC
50
= 1.6 μM).
27
This may sugges ha he 15-OH migh no
be as impo an o binding compa ed wi h he hyd oxyl a C-7;
his is in ag eemen wi h p e ious da a ob ained o a 15-oxo
de i a i e.
27
Toge he wi h p e ious da a hese esul s sugges
he agmen be ween C-7 and C-15 o be pa o he pha ma-
copho e egion o hese mac olide an ibio ics. iso-A chazolid B
(6), in con as , demons a ed ex emely po en an ip oli e a i e
ac i i y, wi h an IC
50
alue in he subnanomola ange, which is
a ound 10 imes mo e ac i e han a chazolids A (1) and B (2).
No ably, i is he mos po en a chazolid known. iso-A chazolid B
(6) was also e alua ed o inhibi ion o pu ified V-ATPase
holoenzyme om he midgu o he obacco ho nwo m Manduca
sex a. Likewise, i demons a ed ex emely high po ency in his in
i o es , compa able o ha o a chazolids A (1) and B (2).
These da a indica e ha a ce ain deg ee o s uc u al flexibili y in
he C-1 o C-4 egion is possible while e aining he biological
po ency. The obse a ion ha a chazolid F is equipo en o
a chazolids A and B in he enzyme assay bu has a much mo e
p onounced an ip oli e a i e ac i i y is no ewo hy and may sug-
ges a diffe en biological a ailabili y and/o possibly also ano he
biological a ge . No ably, he solu ion con o ma ion o a ch-
azolid A (1)
23,31
in his egion closely esembles ha o an
E-configu ed C-3 alkene, as p esen in iso-a chazolid B (6), which
may indica e ha he solu ion con o ma ion o his s uc u al
domain also co ela es o he bioac i e con o ma ion.
Because o he ex emely po en cy o oxic ac i i y o a ch-
azolid F agains cell line L-929, i was u he e alua ed agains a
ange o o he cance cell lines. As shown in Table 3, a chazolid F
showed ex eme po ency in all cases, wi h IC
50
alues in he
picomola ange. Fu he mo e, a ce ain deg ee o selec i i y was
obse ed o specific cell lines, which migh be impo an o he
u u e de elopmen o a chazolids as chemo he apeu ic agen s.
’EXPERIMENTAL SECTION
Gene al Expe imen al P ocedu es. Op ical o a ions we e
de e mined on a Pe kin-Elme 241 ins umen . UV spec a we e
eco ded on a Shimadzu UV-2102 PC scanning spec ome e . IR spec a
we e measu ed wi h a Nicole 20DXB FT-IR spec ome e . NMR
spec a we e eco ded in CD
3
OD and CO(CD
3
)
2
on a B uke DMX-
600 and a B uke WM-400 spec ome e . ESIþand DCI mass spec a
( eac an gas ammonia) we e ob ained on a B uke ICR APEX-QE
spec ome e ; high- esolu ion da a we e acqui ed using peak ma ching
(M/DM = 10 000). Pu e compounds we e cha ac e ized by analy ical
HPLC on a Nucleosil C
18
(column: 125 2 mm, 5 μm, low a e:
0.3 mL/min), wi h diode a ay de ec ion. P epa a i e HPLC was ca ied
Figu e 3. Biosyn he ic o igin o a chazolid B (2) and iso-a chazolid B
(6), in analogy wi h he biosyn hesis o a chazolid A (C-me hyl g oups
may o igina e om me hionine o me hyl-malonyl-CoA).
23
Figu e 1. A chazolid A-15-O-β-D-glucopy anoside (a chazolid E, 5):
numbe ing o a oms (a) as well as HMBC (single a ows) and ROESY
in e ac ions (double a ows) (b).
Figu e 2. HMBC (single a ows) and ROESY in e ac ions (double
a ows) o he C-1 o C-7 subuni o 6.
Table 2. Inhibi ion o he V
1
/V
o
Holoenzyme Ac i i y and
Cy o oxici y o he A chazolids
enzyme inhibi ion
V
1
/V
o
holoenzyme:
IC
50
[nmol/mg enzyme]
a
g ow h inhibi ion
L-929: IC
50
[nM]
g
a chazolid A (1) 0.6
b
0.81
a chazolid B (2) 0.6
c
1.1
a chazolid C (3) 210
d
1600
d
a chazolid D (4) 1200
e
330
e
a chazolid E (5) n.d.
510
a chazolid F (6) 0.7 0.11
a
The specific enzyme ac i i y o he con ols wi hou inhibi o s was
app oxima ely 1.5 μmol mg
1
min
1
.
b
0.8 nmol/mg enzyme acco ds
o 10 nM.
c
Value aken om e 17.
d
Value aken om e 22.
e
Value
aken om e 21.
n.d.: no de e mined.
g
O igin o he mammalian cell
line: Mu ine connec i e issue DSM ACC 2.

1104 dx.doi.o g/10.1021/np200036 |J. Na . P od. 2011, 74, 1100–1105
Jou nal o Na u al P oduc s ARTICLE
ou on an Agilen Technologies 1200 Se ies om Agilen wi h a
Nucleosil column (250 21 mm, 5 μm, low a e: 18 mL/min, de ec-
ion: UV abso p ion a 254 nm) om Mache y, Nagel & Co. Analy ical
TLC was pe o med wi h TLC aluminum shee s, silica gel Si 60 F
254
(Me ck), sol en : mix u es o e hylace a e/pe oleum e he , de ec ion:
UV abso p ion a 254 nm, da k blue spo s on s aining wi h ce ium-
(IV)sul a e-phosphomolybdic acid in sul u ic acid ollowed by cha ing.
Cul u e Condi ions, P oduc ion, and Isola ion o A chazolid A-15-
O-β-D-glucopy anoside (a chazolid E, 5). Fo isola ion o a chazolid E
(5) om Cys obac e iolaceus, a 300 L e men a ion ba ch o s ain Cb
i105, isola ed a he HZI, was g own in M7 medium in he p esence o
3 L o Ambe li e XAD-16 adso be esin a 30 °C.
17
A e ha es ing by
cen i uga ion, he mix u e o we cell mass and adso be esin was
ex ac ed wi h ace one (4 4 L) by s i ing, sedimen a ion, and
decan ing. E apo a ion ga e he c ude ex ac (13.7 g), which was
dissol ed in me hanol o u he pu i ica ion by gel ch oma og aphy on
Sephadex LH 20 (Fluka S einheim, sol en : me hanol, low a e: 7 mL/min).
Fu he pu i ica ion by medium-p essu e RP ch oma og aphy (sol en :
me hanol/wa e , 85:15, de ec ion 230 nm, low a e: 55 mL/min) and
HPLC (sol en : ace oni ile/wa e , 63:37, de ec ion 254 nm diode
a ay, low a e: 15 mL/min) ga e a chazolid E (5) (1.5 mg) oge he
wi h o he a chazolids as p e iously epo ed.
22
Cul u e Condi ions, P oduc ion, and Isola ion o iso-A chazolid B
(a chazolid F, 6). A changium gephy a, s ain A 3548, isola ed a he
HZI, was cul u ed o 10 days a 30 °C in a 350 L bio eac o wi h 300 L o
medium in he p esence o 3 L o Ambe li e XAD-16 adso be esin,
acco ding o he p e iously epo ed p ocedu e.
16
XAD-16 adso be
esin was ho oughly elu ed wi h ace one (4 4 L). The ace one was
e apo a ed in acuo ollowed by dis ibu ion o he esidue be ween
wa e and e hyl ace a e. The o ganic laye was sepa a ed and concen-
a ed in acuo. The esidue was dissol ed in me hanol, washed h ee
imes wi h hep ane, and il e ed h ough a silica plug. E apo a ion ga e
he c ude ex ac (26.1 g), which was dissol ed in me hanol o u he
pu i ica ion by gel ch oma og aphy on Sephadex LH 20 (Fluka S ein-
heim, sol en : me hanol, low a e: 7 mL/min). Fu he pu i ica ion by
medium-p essu e RP ch oma og aphy (sol en : me hanol/wa e , 8:2,
de ec ion 230 nm, low a e: 65 mL/min) and high-p essu e RP ch oma-
og aphy (sol en : ace oni ile/wa e , 65:35, de ec ion 230 nm diode
a ay, low a e: 15 mL/min) ga e a chazolid F (6) (1.0 mg) oge he
wi h o he a chazolids as p e iously epo ed.
16
Physicochemical P ope ies o A chazolid E (5): colo less oil.
[R]
25D
37.89 (c0.57, MeOH); λ
max
(log ε) 239 nm (4.9); NMR
da a, see Table 1; HRMS (ESI) o C
48
H
72
N
2
O
12
NaS [M þNa]
þ
calcd
m/z923.4704, ound m/z923.4711.
Hyd olysis o A chazolid E. Glucose was de ec ed as he pe -TMS-
silyla ed me hyl-glycoside acco ding o he me hod o Chaplin.
32
Physicochemical p ope ies o a chazolid F (6): colo less oil;
[R]
25D
25.0 (c0.11, MeOH); λ
max
(log ε) 238 nm (4.9); IR ( ilm)
ν
max
2931, 2889, 2361, 1602, 1019 cm
1
; NMR da a, see Table 1;
HRMS (ESI) o C
41
H
60
N
2
O
7
NaS [M þNa]
þ
calcd 747.4018, ound
747.4012; HRMS o C
41
H
60
N
2
O
7
KS [M þK]
þ
calcd m/z763.3758,
ound m/z763.3752.
Cell Cul u e and G ow h Inhibi ion Assay. The L-929 mouse cell line
was om he Ge man Collec ion o Mic oo ganisms and Cell Cul u es
(DSMZ) and cul i a ed in DME medium (Gibco BRL) plus 10%
newbo n cal se um a 37 °Cand10%CO
2
in a mois a mosphe e.
G ow h inhibi ion was measu ed on mic o i e pla es. Aliquo s o 120 μLo
hesuspended cells (50000 mL
1
) we e added o 60 μL o a se ial dilu ion
o he inhibi o . A e 5 days, me abolic ac i i y pe well was de e mined
using he MTT assay.
33
The esul s we e ela ed o con ol wells, which
we e incuba ed wi h only he ehicle me hanol. These we e se o 100%.
V-ATPase Assays. V-ATPase was pu i ied acco ding o published
p ocedu es.
34
S anda d V-ATPase assays wi h a inal olume o 160 μL
and a pH o 8.1 consis ed o 3 μg o p o ein, 50 mM T is-MOPS, 3 mM
2-me cap oe hanol, 1 mM MgCl
2
, 20 mM KCl, 0.003% C
12
E
10
,20mM
NaCl, and 3 mM T is-HCl. A e 5 min o p eincuba ion a 30 °C wi h o
wi hou inhibi o s, 1 mM T is-ATP was added, and a e incuba ion o 2
min, he eac ion was s opped by placing he ube in liquid ni ogen. As a
con ol, he inhibi ion o he V-ATPase ac i i y by he es ablished
inhibi o a chazolid A was es ed in pa allel assays.
17
Ino ganic phos-
pha e p oduced in he assays o V-ATPase was measu ed acco ding o
he p o ocol o Wieczo ek e al.
35
(Table 2).
’ASSOCIATED CONTENT
b
SSuppo ing In o ma ion. Copies o NMR spec a o
a chazolid E (5) and a chazolid F (6). This ma e ial is a ailable
ee o cha ge ia he In e ne a h p://pubs.acs.o g.
’AUTHOR INFORMATION
Co esponding Au ho
*Phone: þ49 6221 546207. Fax: þþ49 6221 544205. E-mail:
di [email protected] g.de.
’ACKNOWLEDGMENT
This wo k was gene ously suppo ed by he Volkswagens i -
ung (Funding Ini ia i e: “In e play be ween Molecula
Con o ma ions and Biological Func ion”), he Fonds de Che-
mischen Indus ie (S ipendium o S.E.), and he “Wild-S i ung”.
We hank T. A nold, W.Collisi, and E. Pe sch o echnical suppo
and he Fe men a ion Se ice o he HZI o help wi h la ge-scale
e men a ion. Pa icula hanks a e also due o D . M. Nim z (HZI,
B aunschweig) o GC-MS analysis o glucose.
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50
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o a chazolid F
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