Archazolid A-15-O-β-D-glucopyranoside and iso-archazolid B: potent V-ATPase inhibitory polyketides from the myxobacteria Cystobacter violaceus and Archangium gephyra.
Abstract
Two structurally novel analogues of the macrolides archazolids A and B, archazolid A-15-O-β-D-glucopyranoside (archazolid E, 5) and iso-archazolid B (archazolid F, 6), were isolated from the myxobacterium Cystobacter violaceus and Archangium gephyra, respectively. Macrolactone 5 represents the first 15-O-glycoside of the archazolids. iso-Archazolid B (6) incorporates a C-3 alkene and presents the first constitutional isomer reported for this natural product class. The structures of these polyketides were determined by spectroscopic analysis, in particular by HMBC, HMQC, and ROESY NMR investigations and by chemical degradation. iso-Archazolid B (6) demonstrated extremely high antiproliferative and V-ATPase inhibitory effects, with IC(50) values in the picomolar range, while only moderate activity was observed for glycoside 5. iso-Archazolid B presents the most potent archazolid known.
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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.
13
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.
1113
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 CH
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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