Structure Assignment, Total Synthesis, and Evaluation of the Phosphatase Modulating Activity of Glucolipsin A

Article abstract of DOI:10.1021/jo035079f

The previously unknown stereostructure of glucolipsin A (1), a complex glycolipid endowed with glucokinase-activating properties, was unambiguously elucidated as (2R,2′R,3S,3′S) by comparison of its spectroscopic and analytical data with those of all conc

Full text of DOI:10.1021/jo035079f

Str u ctu r e Assign m en t, Tota l Syn th esis, a n d Eva lu a tion of th e
P h osp h a ta se Mod u la tin g Activity of Glu colip sin A
Alois Fu¨rstner,*,† J uliana Ruiz-Caro,† Heino Prinz,‡ and Herbert Waldmann‡
Max-Planck-Institut fu¨r Kohlenforschung, Kaiser-Wilhelm-Platz 1, D-45470 Mu¨lheim/ Ruhr, Germany,
and Max-Planck-Institut fu¨r Molekulare Physiologie, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany
fuerstner@mpi-muelheim.mpg.de
Received J uly 25, 2003
The previously unknown stereostructure of glucolipsin A (1), a complex glycolipid endowed with
glucokinase-activating properties, was unambiguously elucidated as (2R,2′R,3S,3′S) by comparison
of its spectroscopic and analytical data with those of all conceivable C2-symmetric stereoisomers.
This set of macrodiolides was prepared by a sequence comprising auxiliary guided aldol reactions,
glycosidation of the resulting â-hydroxy acid derivatives with trichloroacetimidate 7, followed by
hydrolytic cleavage of the auxiliaries used. The hydroxy acids thus formed were subjected to a
macrodilactonization reaction mediated by 2-chloro-1,3-dimethylimidazolinium chloride (22) as the
activating agent; this transformation is highly productive only in the presence of admixed potassium
cations which likely serve as templates to preorganize two substrate molecules in a favorable head-
to-tail arrangement. Glucolipsin and analogues were subjected to enzymatic assays that revealed
that glycoconjugates of this type effectively inhibit the activity of the dual specific phosphatase
Cdc25A with IC50 values in the low micromolar range, while being hardly active against the tyrosine
phosphatase PTP1B in vitro. This activity profile was compared to that of other glycolipids previously
prepared in this laboratory, including cycloviracin B1 (2), caloporoside (38), woodrosin I (39),
sophorolipid lactone (40), and tricolorin G (41).
In tr od u ction
Glucokinase catalyzes the phosphorylation of glucose
to glucose-6-phosphate which, in turn, plays a central role
in the entire carbohydrate metabolism.1 It is well estab-
lished that this enzyme is allosterically inhibited by long-
chain fatty acid- or stearoyl-CoA esters. Small molecules
that either competitively bind to the fatty acid-CoA
cofactor site or sequester these negative effectors would
result in an upregulation of glucokinase activity and as
such might ultimately lead to complementary drugs for
therapeutic intervention in diabetes.2
During a search for natural products able to relieve
the inhibitory effects of stearoyl-CoA on glucokinase, a
team at Bristol-Myers Squibb discovered the structurally
rather unusual glycoconjugate glucolipsin A (1) and a
homologue thereof. These macrocyclic dilactones pro-
duced by Streptomyces purpurogeniscleroticus and No-
cardia vaccinii strains exhibit RC50 values of 4.6-5.4 µM
in the pertinent bioassays.3 While spectroscopic investi-
gations unraveled the symmetric structure of 1 and
showed the presence of two â-glucose entities within its
† Max-Planck-Institut fu¨r Kohlenforschung.
‡ Max-Planck-Institut fu¨r Molekulare Physiologie.
(1) Nelson, D. L.; Cox, M. M. Lehninger Biochemie, 3rd ed.;
Springer: Berlin, Germany, 2001.
macrocyclic core, the absolute stereochemistry of the
chiral centers (*) at the periphery remained elusive. It
is tempting to speculate that glucolipsin might have the
same configuration at the branching sites as cycloviracin
B1 (2), a constitutionally related glycoconjugate endowed
(2) See the following for a leading reference and literature cited
therein: Grimsby, J .; Sarabu, R.; Corbett, W. L.; Haynes, N.-E.;
Bizzarro, F. T.; Coffey, J . W.; Guertin, K. R.; Hilliard, D. W.; Kester,
R. F.; Mahaney, P. E.; Marcus, L.; Qi, L.; Spence, C. L.; Tegni, J .;
Magnuson, M. A.; Chu, C. A.; Dvorozniak, M. T.; Matschinsky, F. M.;
Grippo, J . F. Science 2003, 301, 370.
10.1021/jo035079f CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/23/2003
J . Org. Chem. 2004, 69, 459-467
459

Fu¨rstner et al.
SCHEME 1. Retr osyn th etic An a lysis of
with antiviral properties.4 It has recently been shown by
total synthesis that compound 2 is (3R,3′R) configured.5
The notion that 1 and 2 might share the same stereo-
chemical pattern is corroborated by a seemingly consis-
tent set of NMR data,5 although possible effects of the
adjacent methyl branch in 1 on the observed shifts leave
some ambiguity in this regard. Since this methyl group
is absent in 2, no conclusions concerning the stereochem-
istry of the additional chiral centers at C-2/C-2′ in
glucolipsin can be drawn and even their relative config-
uration remains unknown.
Glu colip sin A
Outlined below is an unambiguous assignment of the
stereostructure of glucolipsin A by comparison of the
published data with those of all conceivable C2-sym-
metrical dimers of this type. As part of a long-term project
on bioactive glycoconjugates,5-9 we prepared this set of
compounds by a highly integrated synthesis route based
on a template-directed cyclodimerization reaction as the
key step. In contrast to previous assumptions, however,
this study revealed that the fatty acid moiety of glu-
colipsin A comprises a syn-aldol with a (3S)-configured
stereocenter, opposite the (3R)-configured motif found in
cycloviracin 2. Finally, synthetic 1 and congeners were
subjected to a more detailed screening program that
revealed an unusual profile as modulators of various
phosphatases, allowing for the stimulation as well as the
inhibition of enzymatic activity in a dose-dependent
manner.
tion of the entire set of possible stereoisomers as required
for the unambiguous structure elucidation of 1.
P r ep a r a tion of th e Bu ild in g Block s. To meet this
interim goal in a reliable and efficient way, recourse was
taken to established auxiliary-based aldol methodology
(Scheme 2).10 The required aldehyde 4 was prepared from
commercially available 12-bromo-1-dodecanol (3)11 by
reaction with isobutylmagnesium bromide in the pres-
Resu lts a n d Discu ssion
Str a tegy. On the basis of our previous experiences
with sugar-based dilactones,5 it was assumed that the
specific array of oxygen atoms in the core region of
glucolipsin A endows this compound with some degree
of ionophoric character. This property can be exploited
as a key design feature of an expeditious synthesis route,
which allows the assembly of the target via a template-
directed macrodilactonization reaction of a rather simple
glycosylated aldol derivative A preorganized around a
suitable metal cation. As can be seen from Scheme 1, this
plan results in a considerable retrosynthetic simplifica-
tion and should therefore be well suited for the prepara-
12
ence of Li2CuCl4 and subsequent PCC oxidation of the
resulting alcohol. As expected, reaction of aldehyde 4 with
the boron enolate derived from 5 and n-Bu2BOTf under
standard conditions delivered the (2R,3S)-configured syn-
aldol derivative 6 in essentially diastereomerically pure
form (de 99%) after purification of the crude product by
flash chromatography.13
The subsequent glycosidation reaction, however, turned
out to be far from routine.14 In fact, the conversion of
â-hydroxy carbonyl compounds to the corresponding
â-glycosides constitutes a formidable challenge for which
no general solution yet exists.15 This is mainly caused
by the poor glycosyl acceptor properties of aldol deriva-
tives and by their insufficient chemical stability under
the required acidic reaction conditions. In line with this,
(3) Qian-Cutrone, J .; Ueki, T.; Huang, S.; Mookhtiar, K. A.; Ezekiel,
R.; Kalinowski, S. S.; Brown, K. S.; Golik, J .; Lowe, S.; Pirnik, D. M.;
Hugill, R.; Veitch, J . A.; Klohr, S. E.; Whitney, J . L.; Manly, S. P. J .
Antibiot. 1999, 52, 245.
(4) (a) Tsunakawa, M.; Komiyama, N.; Tenmyo, O.; Tomita, K.;
Kawano, K.; Kotake, C.; Konishi, M.; Oki, T. J . Antibiot. 1992, 45, 1467.
(b) Tsunakawa, M.; Kotake, C.; Yamasaki, T.; Moriyama, T.; Konishi,
M.; Oki, T. J . Antibiot. 1992, 45, 1472.
(5) (a) Fu¨rstner, A.; Albert, M.; Mlynarski, J .; Matheu, M.; DeClercq,
E. J . Am. Chem. Soc. 2003, 125, 13132. (b) Fu¨rstner, A.; Albert, M.;
Mlynarski, J .; Matheu, M. J . Am. Chem. Soc. 2002, 124, 1168. (c)
Fu¨rstner, A.; Mlynarski, J .; Albert, M. J . Am. Chem. Soc. 2002, 124,
10274.
(6) (a) Fu¨rstner, A.; Mu¨ller T. J . Am. Chem. Soc. 1999, 121, 7814.
(b) Fu¨rstner, A.; Mu¨ller, T. J . Org. Chem. 1998, 63, 424. (c) Lehmann,
C. W.; Fu¨rstner, A.; Mu¨ller, T. Z. Kristallogr. 2000, 215, 114.
(7) Fu¨rstner, A.; Radkowski, K.; Grabowski, J .; Wirtz, C.; Mynott,
R. J . Org. Chem. 2000, 65, 8758.
(10) (a) Evans, D. A.; Nelson, J . V.; Taber, T. R. Top. Stereochem.
1982, 13, 1. (b) Heathcock, C. H. In Asymmetric Synthesis; Morrison,
J . D., Ed.; Academic Press: New York, 1984; Vol. 3, pp 111-212. (c)
Cowden, C. J .; Paterson, I. Org. React. 1997, 51, 1.
(11) This product can be prepared on a large scale from 1,12-
dodecandiol and HBr in toluene according to the following: Chong, J .
M.; Heuft, M. A.; Rabbat, P. J . Org. Chem. 2000, 65, 5837.
(12) Nunomoto, S.; Kawakami, Y.; Yamashita, Y. J . Org. Chem.
1983, 48, 1912.
(8) (a) Fu¨rstner, A.; J eanjean, F.; Razon, P. Angew. Chem. 2002,
114, 2203; Angew. Chem., Int. Ed. 2002, 41, 2097. (b) Fu¨rstner, A.;
J eanjean, F.; Razon, P.; Wirtz, C.; Mynott, R. Chem. Eur. J . 2003, 9,
307. (c) Fu¨rstner, A.; J eanjean, F.; Razon, P.; Wirtz, C.; Mynott, R.
Chem. Eur. J . 2003, 9, 320.
(9) (a) Fu¨rstner, A.; Konetzki, I. J . Org. Chem. 1998, 63, 3072. (b)
Fu¨rstner, A.; Konetzki, I. Tetrahedron 1996, 52, 15071. (c) Fu¨rstner,
A.; Konetzki, I. Tetrahedron Lett. 1998, 39, 5721.
(13) (a) Evans, D. A.; Ennis, M. D.; Mathre, D. J . J . Am. Chem. Soc.
1982, 104, 1737. (b) Evans, D. A.; Bartroli, J .; Shih, T. L. J . Am. Chem.
Soc. 1981, 103, 2127.
(14) General reviews see: (a) Paulsen, H. Angew. Chem. 1982, 94,
184; Angew. Chem., Int. Ed. Engl. 1982, 21, 155. (b) Schmidt, R. R. In
Comprehensive Organic Synthesis; Trost, B. M., Fleming, I., Eds.;
Pergamon: Oxford, UK, 1991; Vol. 6, pp 33-61. (c) Toshima, K.;
Tatsuta, K. Chem. Rev. 1993, 93, 1503.
460 J . Org. Chem., Vol. 69, No. 2, 2004

Phosphatase Modulating Activity of Glucolipsin A
Mukaiyama conditions (SnCl2, AgClO4, MeCN)20,21 es-
sentially met with failure.
SCHEME 2
Saponification of product 8 with aqueous LiOH in THF
in the presence of H2O222 furnished hydroxy acid 9 as the
required substrate for the cyclodimerization step by
concomitant cleavage of the oxazolidinone and the re-
sidual acetate. The corresponding (2S,3R)-configured
analogue 10 was obtained following the same reaction
sequence employing the antipodal auxiliary (cf. Support-
ing Information).
In an attempt to probe whether the presence of the
auxiliary in 6 or its enantiomer 14 is responsible for the
low yield in the glycosidation reaction (e.g. by sequester-
ing the Lewis-acidic promoter), the corresponding methyl
ester 15 derived from 14 was reacted with donor 7 under
similar conditions (Scheme 3). Although the yield of the
desired glycoside 16 was raised to 60%, the significantly
lower â:R ratio of 2.5:1 renders this detour unrewarding
overall.
Access to the anti-aldol series was secured by the
method developed by Abiko and Masamune employing a
norephedrine-derived auxiliary (Scheme 4).23 Specifically,
enolization of 17 with dicyclohexylboryl triflate and Et3N
followed by addition of aldehyde 4 at low temperature
gave the desired anti-aldol derivative 18 in good yield
and excellent diastereoselectivity (de > 99% after flash
chromatography). Subsequent reaction with trichloro-
acetimidate 7 in the presence of TMSOTf as the catalyst
furnished glycoside 19 in a respectable yield of 74% as a
5:1 mixture of anomers. The cleavage of the auxiliary in
19, however, could only be effected with (n-Bu)4NOH in
the effective yields obtained in the glycosidation of
compound 6 remained rather low despite considerable
experimentation with various glucosyl donors and dif-
ferent promoter systems.16 Best results were obtained
with use of an excess (3 equiv) of trichloroacetimidate 75
activated in situ by TMSOTf (20 mol %) in MeCN.17
Under these conditions, product 8 was obtained in 45%
isolated yield; the undesired R-anomer (â:R ) 5.2:1) was
removed by routine flash chromatography. The use of
other donors, including the phenyl thioglycoside 11,18 the
anomeric sulfoxide 12,19 or the glycosyl fluoride 13 under
(15) For representative examples illustrating this notion see: (a)
Evans, D. A.; Kaldor, S. W.; J ones, T. K.; Clardy, J .; Stout, T. J . J .
Am. Chem. Soc. 1990, 112, 7001. (b) Paterson, I.; McLeod, M. D.
Tetrahedron Lett. 1995, 36, 9065. (c) Toshima, K.; Misawa, M.; Ohta,
K.; Tatsuta, K.; Kinoshita, M. Tetrahedron Lett. 1989, 30, 6417. (d)
Mitchell, S. A.; Pratt, M. R.; Hruby, V. J .; Polt, R. J . Org. Chem. 2001,
66, 2327.
(16) Promotors screened include the following: TMSOTf, TBSOTf,
triflic acid, BF3‚Et2O, SnCl4, Cl3CCHO. Although TBSOTf has previ-
ously been recommended as a superior promotor for glycosidation
reactions of sensitive substrates, we did not observe any improvement
in our case upon replacing TMSOTf by TBSOTf; cf.: (a) Marzabadi,
C. H.; Franck, R. W. Tetrahedron 2000, 56, 8385. (b) Roush, W. R.;
Narayan, S. Org. Lett. 1999, 1, 899. (c) For the use of Cl3CCHO as a
particularly mild promotor for sensitive substrates see: Schmidt, R.
R.; Gaden, H.; J atzke, H. Tetrahedron Lett. 1990, 31, 327.
(17) (a) Schmidt, R. R. Angew. Chem. 1986, 98, 213; Angew. Chem.,
Int. Ed. Engl. 1986, 25, 212. (b) Schmidt, R. R.; Kinzy, W. Adv.
Carbohydr. Chem. Biochem. 1994, 50, 21.
(19) (a) Kahne, D.; Walker, S.; Cheng, Y.; Engen, D. V. J . Am. Chem.
Soc. 1989, 111, 6881. (b) Sildersleeve, J .; Pascal, R. A.; Kahne, D. J .
Am. Chem. Soc. 1998, 120, 5961.
(20) Mukaiyama, T.; Murai, Y.; Shoda, S. Chem. Lett. 1981, 431.
(21) For a recent successful application of a glycosyl fluoride to the
formation of 2-deoxy-â-glycosides from â-hydroxy ketones and
a
detailed discussion see: Blanchard, N.; Roush, W. R. Org. Lett. 2003,
5, 81.
(22) Gage, J . R.; Evans, D. A. Org. Synth. 1990, 68, 83.
(23) (a) Abiko, A.; Liu, J .-F.; Masamune, S. J . Org. Chem. 1996, 61,
2590. (b) Abiko, A.; Liu, J .-F.; Masamune, S. J . Am. Chem. Soc. 1997,
119, 2586. (c) Inoue, T.; Liu, J .-F.; Buske, D. C.; Abiko, A. J . Org. Chem.
2002, 67, 5250. (d) Abiko, A. Org. Synth. 2002, 79, 116.
(18) (a) Nicolaou, K. C.; Ueno, H. In Preparative Carbohydrate
Chemistry; Hanessian, S., Ed.; Marcel Dekker: New York, 1997; pp
313-338. (b) Crich, D.; Sun, S. J . Am. Chem. Soc. 1998, 120, 435.
J . Org. Chem, Vol. 69, No. 2, 2004 461

Fu¨rstner et al.
SCHEME 3
SCHEME 5
aldol step, access to the diastereomeric anti-aldol deriva-
tive 21 was secured (cf. Supporting Information).
Cyclod im er iza tion Rea ction s a n d Com p letion of
th e Tota l Syn th esis. With the complete set of stereo-
isomeric hydroxy acids in hand, the envisaged template
directed cyclization reactions were investigated. In line
with our expectations,5 smooth macrodilactonization of
substrate 9 was achieved when the reaction was per-
formed with 2-chloro-1,3-dimethylimidazolinium chloride
(22) as the activating agent25,26 in the presence of
admixed potassium cations derived from KH used as the
base to deprotonate the starting material (Scheme 5).
Under these conditions, the cyclic dimer 23 was obtained
in 54% isolated yield.
SCHEME 4
As can be seen from Table 1, all other isomeric hydroxy
acids behaved similarly well and afforded the corre-
sponding C2-symmetrical dimers in equally satisfactory
yields. HPLC analyses of the crude reaction mixtures
showed the striking preference for the formation of the
macrodilactones over other conceivable products (cf. entry
1, footnote b), whereas in the absence of potassium
cations an almost statistical mixture of the cyclic mono-
mer, the desired cyclic dimer, and higher oligomers are
formed.5 Together with the fact that the reaction rate is
considerably accelerated by the admixed cations, this
result lends credence to the notion that the macrodilac-
tonization reactions of 9 and analogues proceed in a
template-directed manner in which the potassium cations
help to preorganize the substrate in a suitable head-to-
tail arrangement (cf. Scheme 1).27,28
combination with H2O2,24 whereas LiOH failed completely
in this particular case. By following the same sequence
of reactions using ent-17 as the reagent in the crucial
(24) Hasegawa, T.; Yamamoto, H. Synlett 1998, 882.
(25) (a) Isobe, T.; Ishikawa, T. J . Org. Chem. 1999, 64, 5832. (b)
Isobe, T.; Ishikawa, T. J . Org. Chem. 1999, 64, 6984. (c) Isobe, T.;
Ishikawa, T. J . Org. Chem. 1999, 64, 6989. (d) See also: Garcia, D.
M.; Yamada, H.; Hatakeyama, S.; Nishizawa, M. Tetrahedron Lett.
1994, 35, 3325. (e) Corey, E. J .; Gin, D. Y.; Kania, R. S. J . Am. Chem.
Soc. 1996, 118, 9202. (f) Akaji, K.; Kurijama, N.; Kiso, Y. J . Org. Chem.
1996, 61, 3350.
(26) For an application of this reagent in organometallic chemistry
see: Fu¨rstner, A.; Seidel, G.; Kremzow, D.; Lehmann, C. W. Organo-
metallics 2003, 22, 907.
(27) Fu¨rstner, A. In Templated Organic Synthesis; Diederich, F.,
Stang, P. J ., Eds.; Wiley-VCH: Weinheim, Germany, 2000; pp 249-
273.
462 J . Org. Chem., Vol. 69, No. 2, 2004

Phosphatase Modulating Activity of Glucolipsin A
TABLE 1. Resu lts of th e Ma cr od ila cton iza tion
Rea ction s Med ia ted by Im id a zolin iu m Ch lor id e 22 in
CH2Cl2 (0.05 M) in th e P r esen ce of P ota ssiu m Ca tion s a s
th e Tem p la tes
TABLE 2. Com p a r ison of Releva n t Sign a ls of th e 13C
NMR Sp ectr a (MeOH-d 4) of Glu colip sin A (1) w ith Th ose
of th e Syn th etic P r od u ctsa
glucolipsin
A (1)
24
26
28
30
position
(2R,3S) (2S,3R) (2R,3R) (2S,3S)
1
2
3
4
1′
2′
3′
4′
5′
6′
175.7
46.6
78.9
13.4
104.5
75.6
78.0
72.4
75.4
66.3
175.8
46.7
78.9
176.0
45.9
77.9
175.5
45.8
83.1
9.3
106.7
75.2*
78.0
72.6
75.1*
66.1
175.8
41.7
76.8
8.4
100.0
75.1*
78.1
72.4
75.4*
66.9
13.3
13.4
104.5
75.6*
78.0
72.4
75.5*
66.3
102.0
75.1*
78.2
72.4
75.6*
65.9
a
Signals marked with an asterisk may be interchanged;
arbitrary numbering scheme as shown.
subjected the stereoisomeric dilactones as well as selected
key intermediates formed en route to 1 to biological
screens established in our laboratories.30 In particular,
compounds 24, 26, 28, and 30 were investigated as
possible inhibitors of protein phosphatases.
a
Isolated yield of analytically pure compound unless stated
b
otherwise. The product distribution in the crude mixture accord-
ing to HPLC was as follows: cyclic monomer (11%), cyclic dimer
25 (87%), higher oligomers (3%). c [R]20 -19.4 (c 0.65, MeOH).
D
[R]20D -23.7 (c 0.75, CH2Cl2). e HPLC yield. f [R]20D -2.1 (c 0.65,
d
g
MeOH). Products formed upon hydrogenolytic cleavage of the
Reversible phosphorylation of proteins is among the
most important regulatory biological processes. It is vital
to innumerable biological phenomena and involved in the
establishment of diseases ranging from diabetes over
immunological disorders to the development of cancer.
While the phosphorylating kinases are established drug
targets, their natural antagonists, i.e., the dephospho-
rylating phosphatases, have only recently moved into the
focus of medicinal chemistry research, and new types of
phosphatase inhibitors are in urgent demand.
The dual-specificity phosphatase Cdc25A and the ty-
rosine phosphatase PTP1B were chosen as representative
enzymes for screening. The family of Cdc25 protein
phosphatases is critically involved in cell cycle control.31
Their physiological substrates are cyclin-dependent ki-
nases which trigger key transitions in the process of
eukaryotic cellular division. Their oncogenic properties
together with the fact that Cdc25A and B are overex-
pressed in many human tumors render these isoenzymes
molecular targets of utmost interest in the quest for
anticancer drugs.30-33 On the other hand, PTP1B is a key
negative regulator of insulin-receptor activity, and PTP1B
inhibitors are expected to enhance insulin sensitivity and
act as effective therapeutics for the treatment of type II
diabetes, insulin resistance, and obesity.34
benzyl ether protecting groups.
Hydrogenolytic cleavage of the benzyl ether groups in
these products proceeded quantitatively in all cases with
Pd(OH)2 as the precatalyst. The spectroscopic data of the
unprotected macrodiolides thus obtained were compared
with those of glucolipsin A (1) reported in the literature.3
Particularly diagnostic are the shifts of the anomeric
carbon atom and the signals of the aldol region. As can
be seen from Tables 2 and 3, it is the (2R,2′R,3S,3′S)-
isomer 24 that shows an excellent match with the natural
product. The characteristic pattern signature of its 1H
NMR spectrum is superimposable to that of authentic
glucolipsin A depicted in ref 3. While these comparisons
rigorously establish the syn arrangement of the aldol
entity, it is surprising to find that the absolute configu-
ration of C-3/C-3′ in 1 is opposite that one found in the
otherwise closely related glycoconjugate cycloviracin B1
(2).5
P h osp h a ta se Activity-Mod u la tin g P r op er ties of
Glycolip id s. Given the interesting biological properties
of glucolipsin A, cycloviracin B1, and related glycolipids
and light of the fact that natural products frequently
inhibit enzymes with significantly differing activity29 we
The results of the screen for phosphatase-inhibiting
activity are shown in Table 4. All four isomeric dilactones
(28) Attempts to monitor the complexation of K+ by macrodiolide
hosts via NMR titration experiments were inconclusive. However, it
is known that the association constants between alkali metal cations
and sugar-based crown ethers are usually rather low; see the following
for a leading reference and literature cited therein: Shizuma, M.;
Kadoya, Y.; Takai, Y.; Imamura, H.; Yamada, H.; Takeda, T.; Arakawa,
R.; Takahashi, S.; Sawada, M. J . Org. Chem. 2002, 67, 4795.
(29) Breinbauer, R.; Vetter, I. R.; Waldmann, H. Angew. Chem., Int.
Ed. 2002, 41, 2878.
(30) For a recent study see: Fu¨rstner, A.; Feyen, F.; Prinz, H.;
Waldmann, H. Angew. Chem. 2003, 115, 5519; Angew. Chem., Int. Ed.
2003, 42, 5361.
(31) Reviews: (a) Galaktinov, K.; Lee, A. K.; Eckstein, J .; Draetta,
G.; Meckler, J .; Loda, M.; Beach, D. Science 1995, 269, 1575. (b)
Draetta, G.; Eckstein, J . Biochim. Biophys. Acta 1997, 1332, M53. (c)
Nilsson, I.; Hoffmann, I. Prog. Cell Cycle Res. 2000, 4, 107.
J . Org. Chem, Vol. 69, No. 2, 2004 463

Fu¨rstner et al.
TABLE 3. Com p a r ison of Releva n t Sign a ls [δ (m u ltip licity, J (Hz))] of th e 1H NMR Sp ectr a (MeOH-d 4) of Glu colip sin A
(1) w ith Th ose of th e Syn th etic P r od u ctsa
position
glucolipsin A
2.63 (m)
24 (2R,3S)
26 (2S,3R)
28 (2R,3R)
30 (2S,3S)
2
3
4
1′
2′
3′
4′
5′
6a′
6b′
2.63 (quint, 7.2)
4.05 (dt, 7.2, 5.0)
1.31 (d, 7.0)
4.36 (d, 7.6)
3.20 (dd, 9.1, 7.7)
3.35 (t, 9.0)
3.15 (dd, 9.7, 8.8)
3.46 (t, 9.8)
3.85 (dd, 11.4, 9.9)
4.56 (bd, 10.3)
2.60 (quint, 7.0)
4.11 (q, 5.9)
1.22 (d, 7.0)
4.31 (d, 7.7)
3.17 (dd, 9.1, 7.7)
3.34 (t, 9.0)
3.16 (dd, 9.8, 8.9)
3.36 (td, 9.0, 1.7)
3.69 (dd, 11.5, 9.2)
4.73 (dd, 11.5, 1.7)
3.28 (qd, 6.8, 4.3)
4.02-4.05 (m)
1.06 (d, 7.0)
3.10 (qd, 6.8, 4.1)
4.27-4.31 (m)
1.12 (d, 6.8)
4.37 (d, 7.7)
3.20 (dd, 9.2, 7.7)
3.37 (t, 9.0)
3.15 (dd, 9.6, 9.0)
3.55 (td, 9.8, 1.0)
3.84 (dd, 11.4, 9.8)
4.49 (dd, 11.4, 1.0)
4.05 (m)
1.31 (d, 7.0)
4.36 (d, 7.7)
3.20 (dd, 9.0, 7.8)
3.35 (dd, 9.8, 9.1)
3.15 (t, 9.8)
3.46 (t, 9.9)
3.85 (t, 9.9)
4.57 (d, 10.5)
4.37 (d, 7.8)
3.21 (dd, 9.1, 7.9)
3.36 (dd, 9.0, 7.8)
3.14 (dd, 9.2, 7.3)
3.52 (td, 9.8, 1.6)
4.11 (t, 11.4)
4.32 (dd, 11.4, 1.6)
a
Arbitrary numbering scheme as shown at the top of Table 2.
TABLE 4. In h ibition of Cd c25A a n d P TP 1B by Glu colip sin a n d An a logu es (n .i.: less th a n 50% in h ibition a t 50 µM)
inhibit Cdc25A in the low micromolar range but they do
not affect the activity of PTP1B. The IC50 values for the
four diastereomers are identical within the error limit,
indicating that the configuration of the stereocenters in
the core region is not important for Cdc25A inhibition.
The monomeric â-hydroxyacids 31, 32, and 33, which
(33) (a) For comprehensive literature coverage see: Lyon, M. A.;
Ducruet, A. P.; Wipf, P.; J azo, J . S. Nature Rev. Drug Disc. 2002, 1,
961. (b) For a representative study see: Brohm, D.; Philippe, N.;
Metzger, S.; Bhargava, A.; Mu¨ller, O.; Lieb, F.; Waldmann, H. J . Am.
Chem. Soc. 2002, 124, 13171.
(34) (a) Zhang, Z.-Y. Curr. Op. Chem. Biol. 2001, 5, 416. (b) J ohnson,
T. O.; Ermolieff, J .; J irousek, M. R. Nature Rev. Drug Disc. 2002, 1,
696.
(32) See: (a) Cangi, M. G.; Cukor, B.; Soung, P.; Signoretti, S.;
Moreira, G.; Ranashinge, M.; Cady, B.; Pagano M.; Loda, M. J . Clin.
Invest. 2000, 106, 753. (b) Dixon, D.; Moyana, T.; King, M. J . Exp. Cell
Res. 1998, 240, 236. (c) Gasparotto, D.; Maestro, R.; Piccinin, S.;
Vukosavljigevic, T.; Barman, L.; Sulfaro, S.; Boiocchi, M. Cancer Res.
1997, 57, 2366. (d) Wu, W.; Fan, Y. H.; Kemp, B. L.; Walsh, G.; Mao,
L. Cancer Res. 1998, 58, 4082.
464 J . Org. Chem., Vol. 69, No. 2, 2004

Phosphatase Modulating Activity of Glucolipsin A
TABLE 5. In h ibition of Cd c25A a n d P TP 1B by Differ en t
Typ es of Glycolip id s (n .i.: less th a n 50% in h ibition a t 50
µM)
F IGURE 1. Glycolipids that did not inhibit Cdc25A and
PTP1B.
activity and tricolorin G (41) was devoid of any noticeable
effects in our assays.
Remarkably, in the course of the inhibition studies an
unexpected phosphatase-stimulating activity was re-
corded for some of the compounds investigated. Thus,
glycolipids 28 (but not 24, 26, and 30), 31-33, 35, and
39-41 stimulate the activity of PTP1B at concentrations
of 1-5 µM. Such an effect was not observed for the
phosphatase Cdc25. We have not investigated this effect
in detail; however, involvement of allosteric binding could
be a possible explanation.
were obtained from carboxylic acids 9, 21, and 10 by
removal of the protecting groups, inhibit both Cdc25A
and PTP1B. The isomeric compound 34, however, devi-
ates from this pattern as it is only a weak Cdc25A
inhibitor and has no effect on the activity of the tyrosine
phosphatase.
To obtain deeper insights into possible structure/
activity relationships, other glycolipids obtained in previ-
ous synthesis projects were investigated. The results are
compiled in Figure 1 and Table 5. Compounds 35-37
were obtained in the context of studies leading to the total
synthesis of cycloviracin B1 (2).5 None of these turned out
to be an inhibitor of Cdc25A or PTP1B. Notably, these
compounds differ from the ones shown in Table 4 by only
one stereogenic center missing in their â-hydroxyester
segments and by the presence of at least one polar
headgroup at the lipidic appendices; otherwise the struc-
tures are very similar to the glucolipsin series. Table 5
summarizes the results for the phospholipase inhibitor
caloporoside (38),9 woodrosin I (39),8 sophorolipid lactone
(40),7 and tricolorin G (41).6a Caloporoside proved to be
a strong inhibitor of both Cdc25A and even more potent
against PTP1B. Woodrosin is a good Cdc25 inhibitor
whereas compound 40 has only very weak inhibiting
Given the amphiphilic character of the glycolipids
investigated, the formation of micelles might also be a
factor that influences the enzyme activity-modulating
properties of this compound class.35 However, the finding
that structurally closely related compounds exhibit fairly
differing inhibitory potency and that the observed stimu-
lation is enzyme dependent (no effect on Cdc25A) and
differs substantially even for compounds with very
similar structure (compare compounds 28 with com-
pounds 24, 26, and 30) argue against a major influence
of such a physical effect.
(35) Aggregate formation has for instance been found to influence
the inhibitory activity of certain protein kinase inhibitors: McGovern,
S. L.; Shoichet, B. K. J . Med. Chem. 2003, 46, 1478.
J . Org. Chem, Vol. 69, No. 2, 2004 465

Fu¨rstner et al.
δ 7.45-7.27 (m, 5H), 5.69 (d, 1H, J ) 7.3 Hz), 4.80 (quint, 1H,
J ) 6.6 Hz), 3.98-3.94 (m, 1H), 3.79 (qd, 1H, J ) 7.0, 2.7 Hz),
3.50-3.00 (br s, 1H, -OH), 1.52 (non., 1H, J ) 6.6 Hz), 1.65-
1.13 (m, 24H), 1.24 (d, 3H, J ) 7.1 Hz), 0.90 (d, 3H, J ) 6.6
Hz), 0.87 (d, 6H, J ) 6.6 Hz); 13C NMR (100 MHz, CDCl3) δ
177.4, 152.6, 133.2, 128.8, 128.7, 125.6, 78.9, 71.6, 54.7, 42.2,
39.0, 33.9, 29.9, 29.7, 29.6, 27.9, 27.4, 26.0, 22.6, 14.3, 10.2;
IR (KBr) 3445, 2922, 2851, 1797, 1688, 766, 698 cm-1; MS (EI)
m/z (rel intensity) 473 ([M+], 1), 367 (16), 262 (14), 233 (100),
178 (11), 134 (15), 118 (27), 116 (11), 107 (37), 95 (11), 82 (18),
70 (14), 67 (10), 57 (50), 56 (12), 55 (18), 41 (16); HRMS calcd
for C29H47NO4 [M+] 473.350509, found 473.350808. Anal. Calcd
for C29H47NO4: C, 73.53; H, 10.00; N, 2.96. Found: C, 73.02;
H, 10.08; N, 2.91.
The results concerning the phosphatase-inhibiting
activity of the glycolipids compiled in Tables 4 and 5
should be of general interest to this particular area of
medicinal chemistry and chemical biology since glycolip-
ids have not been identified as phosphatase inhibitors
so far. Moreover, the observation that selectivity can be
achieved between a dual specificity phosphatase and a
tyrosine phosphatase even with the small set of com-
pounds investigated suggests that further detailed study
of this new class of inhibitors is worthwhile.
Exp er im en ta l Section
Com p ou n d 8. TMSOTf (5 µL, 0.027 mmol) was added to a
solution of alcohol 6 (64 mg, 0.13 mmol) and trichloroacetimi-
date 7 (256 mg, 0.40 mmol)5 in CH3CN (6.7 mL) at -30 °C
and stirring was continued for 2 h at that temperature. After
neutralization with triethylamine and evaporation of the
solvent, the residue was purified by flash chromatography
(hexane:EtOAc 20/1 f 9/1) to yield glycoside 8 (48 mg, 38%,
pure â-isomer) as a colorless syrup. [R]20D + 10 (c 5.11, CDCl3);
1H NMR (400 MHz, CDCl3) δ 7.43-7.00 (m, 20H), 5.34 (d, 1H,
J ) 7.1 Hz), 4.91 (d, 1H, J ) 11.3 Hz), 4.88 (d, 1H, J ) 11.0
Hz), 4.75 (d, 1H, J ) 10.9 Hz), 4.73 (d, 1H, J ) 11.0 Hz), 4.65
(d, 1H, J ) 11.2 Hz), 4.49 (d, 1H, J ) 10.9 Hz), 4.46 (d, 1H, J
) 7.7 Hz), 4.43-4.41 (m, 1H), 4.27 (br d, 1H, J ) 11.6 Hz),
4.10 (dd, 1H, J ) 11.6, 4.8 Hz), 3.96 (quint, 1H, J ) 6.6 Hz),
3.99-3.89 (m, 1H), 3.60 (tm, 1H, J ) 8.8 Hz), 3.45-3.39 (m,
2H), 3.35 (dd, 1H, J ) 9.0, 7.7 Hz), 1.95 (s, 3H), 1.69-1.05 (m,
27H), 1.44 (non., 1H, J ) 6.6 Hz), 0.78 (d, 6H, J ) 6.6 Hz),
0.73 (d, 3H, J ) 6.6 Hz); 13C NMR (100 MHz, CDCl3) δ 175.0,
170.6, 152.7, 138.6, 138.5, 137.8, 133.2, 128.7, 128.6, 128.4,
128.3 (2x), 128.0, 127.9, 127.7, 127.6, 127.5, 125.6, 103.0, 85.0,
82.5, 80.3, 78.7, 77.8, 75.6, 75.0, 74.9, 72.7, 63.3, 55.0, 42.1,
39.0, 33.8, 30.0, 29.9, 29.7, 29.6, 27.9, 27.4, 25.4, 22.6, 20.8,
14.1, 12.7; IR (film) 3031, 2925, 2853, 1780, 1743, 1703, 1235,
1196, 1090, 1072, 1029, 736, 699 cm-1; MS (EI) m/z (rel
intensity) 474 (3), 456 (33), 412 (6), 253 (11), 240 (8), 181 (7),
160 (8), 134 (12), 97 (5), 92 (10), 91 (100), 81 (5), 69 (7), 55 (7),
43 (16); HRMS (ESIpos) calcd for C58H77NO10 [(M + NH4)+]
965.589122, found 965.58939. Anal. Calcd for C58H77NO10: C,
73.47; H, 8.18; N, 1.48. Found: C, 73.44; H, 8.24; N, 1.46. A
second fraction was collected that contained the corresponding
R-anomer (9 mg, 7%).
14-Meth ylp en ta d eca n -1-ol. To a stirred solution of 12-
bromo-1-dodecanol (3) (10.2 g, 38 mmol) and Li2CuCl4 (0.1 M
in THF, 11.5 mL) in THF (19 mL) was slowly added a solution
of isobutylmagnesium bromide (2 M in Et2O, 47.5 mL, 95
mmol) at 0 °C under Ar. Once the addition was complete,
stirring was continued for 45 min at 0 °C before the mixture
was allowed to reach ambient temperature. The reaction was
then quenched with aq HOAc (20% w/w), the aqueous layer
was repeatedly extracted with Et2O, and the combined organic
phases were washed with aq NaHCO3 (5 mol %) and brine,
then dried over Na2SO4 before the solvent was evaporated.
Flash chromatography of the crude product (hexane/EtOAc
20/1 f 10/1) afforded 14-methylpentadecan-1-ol as a white
1
solid (9.1 g, 98%). H NMR (400 MHz, CDCl3) δ 3.64 (t, 2H, J
) 6.7 Hz), 1.98 (br s, 1H, -OH), 1.57 (quint, 2H, J ) 6.7 Hz),
1.54 (non., 1H, J ) 6.6 Hz), 1.37-1.13 (m, 22H), 0.87 (d, 6H,
J ) 6.6 Hz); 13C NMR (100 MHz, CDCl3) δ 62.9, 39.0, 32.7,
29.9, 29.7, 29.6, 29.4, 27.9, 27.4, 25.7, 22.6; IR (film) 3157, 2918,
2849, 1471, 1363, 1052, 718 cm-1; MS (EI) m/z (rel intensity)
224 (25), 196 (15), 168 (14), 82 (35), 69 (80), 68 (31), 67 (10),
57 (99), 56 (100), 55 (69), 43 (75), 41 (44); HRMS calcd for
C16H34O [(M + H)+] 243.268790, found 243.268820. Anal. Calcd
for C16H34O: C, 79.27; H, 14.14. Found: C, 79.18; H, 14.02.
14-Meth ylp en ta d eca n a l (4). To a solution of 14-methyl-
pentadecan-1-ol (3.2 g, 13 mmol) in CH2Cl2 (150 mL) was
added PCC (5.6 g, 26 mmol). After the mixture had been
stirred for 2 h, it was filtered through a pad of Celite, the Celite
was carefully rinsed with CH2Cl2, the combined filtrates were
evaporated, and the residue was purified by flash chromato-
graphy (hexane:EtOAc 70/1) to yield product 4 as a white solid
(2.7 g, 86%). 1H NMR (400 MHz, CDCl3) δ 9.77 (t, 1H, J ) 1.9
Hz), 2.42 (td, 2H, J ) 7.3, 1.9 Hz), 1.65 (quint, 2H, J ) 7.3
Hz), 1.52 (non., 1H, J ) 6.6 Hz), 1.35-1.13 (m, 20H), 0.87 (d,
6H, J ) 6.6 Hz); 13C NMR (100 MHz, CDCl3) δ 202.9, 43.9,
39.1, 29.9, 29.7, 29.6, 29.4, 29.3, 29.2, 28.0, 27.4, 22.6; IR (film)
3200-2700, 2924, 2853, 2714, 1728, 1468, 1384, 1364, 1347,
1171, 718 cm-1; MS (EI) m/z (rel intensity) 240 ([M+], 3), 222
(14), 196 (7), 194 (7), 168 (4), 166 (6), 140 (6), 95 (39), 73 (3),
72 (6), 67 (34), 66 (9), 60 (3), 57 (94), 56 (51), 45 (9), 44 (22), 43
(100), 41 (63); HRMS calcd for C16H32O [M+] 240.245315, found
240.245396.
Hyd r oxy Acid 9. A solution of compound 8 (93 mg, 0.098
mmol) in THF/H2O (3:1, 2 mL) was treated with aq H2O2 (30%
w/w, 0.61 mL, 0.07 mmol) and LiOH‚H2O (9 mg, 0.19 mmol).
The mixture was stirred at 50-60 °C for 2 h. After treatment
with aq Na2SO3 (1.6 M, 2 mL), the aqueous layer was acidified
by addition of aq HCl (6 M) until pH 1 was reached. The
aqueous phase was repeatedly extracted with methyl tert-butyl
ether, the combined organic layers were dried (Na2SO4) and
evaporated, and the residue was purified by flash chromato-
graphy (hexane:EtOAc:HOAc, 8/2/0.01) to give product 9 as a
1
white solid (58 mg, 79%). [R]20 -3 (c 4.46, CHCl3); H NMR
D
(400 MHz, CDCl3) δ 7.33-7.24 (m, 15H), 6.00-5.00 (br s, 2H),
4.90 (d, 1H, J ) 11.1 Hz), 4.87 (d, 1H, J ) 11.0 Hz), 4.83 (d,
1H, J ) 11.0 Hz), 4.79 (d, 1H, J ) 11.0 Hz), 4.71 (d, 1H, J )
11.2 Hz), 4.62 (d, 1H, J ) 11.0 Hz), 4.55 (d, 1H, J ) 7.8 Hz),
4.07-4.02 (m, 1H), 3.84 (dd, 1H, J ) 11.9, 2.8 Hz), 3.68 (dd,
1H, J ) 11.9, 4.8 Hz), 3.66 (t, 1H, J ) 9.1 Hz), 3.53 (t, 1H, J
) 9.1 Hz), 3.39 (dd, 1H, J ) 9.1, 7.8 Hz), 3.39-3.33 (m, 1H),
2.67 (qd, 1H, J ) 7.1, 4.4 Hz), 1.51 (non., 1H, J ) 6.6 Hz),
1.65-1.12 (m, 24H), 1.22 (d, 3H, J ) 7.1 Hz), 0.86 (d, 6H, J )
6.6 Hz); 13C NMR (100 MHz, CDCl3) δ 179.0, 138.1-127.5,
102.5, 84.8, 82.1, 79.8, 77.8, 75.5, 75.0, 75.0, 62.2, 42.9, 39.1,
33.1, 30.0, 29.7, 29.6, 29.5, 28.0, 27.4, 25.7, 22.6, 11.2; IR (film)
Com p ou n d 6. To a stirred solution of (4R,5S)-4-methyl-5-
phenyl-3-propionyl-2-oxazolidinone (5) (900 mg, 3.86 mmol) in
CH2Cl2 (8.4 mL) at -5 °C were successively added n-Bu2BOTf
(1 M in CH2Cl2, 4.6 mL, 4.6 mmol) and triethylamine (0.68
mL, 5.0 mmol). The reaction mixture was stirred at -5 °C for
30 min before it was cooled to -78 °C. At that temperature, a
solution of aldehyde 4 (1 g, 4.3 mmol) in CH2Cl2 (10 mL) was
added dropwise, and the resulting mixture was stirred at -78
°C for 1 h and at room temperature for 15 min. The reaction
was then quenched with aqueous phosphate buffer (pH 7), the
aqueous layer was extracted with methyl tert-butyl ether (3×),
and the combined organic layers were washed with saturated
aqueous NH4Cl and dried over Na2SO4, before the solvent was
evaporated. The residue was purified by flash chromatography
(hexane:EtOAc 30/1 f 9/1) to give aldol 6 as a white solid (1.2
g, 63%). [R]20D + 14 (c 1.41, CDCl3); 1H NMR (400 MHz, CDCl3)
3500-2700, 2924, 2853, 1708, 1497, 1455, 1071, 734, 697 cm-1
;
HRMS (ESI-pos) calcd for C46H66O8 [(M + Na)+] 769.465538,
found 769.46428. Anal. Calcd for C46H66H8: C, 73.96; H, 8.91.
Found C, 73.83, H, 8.88.
466 J . Org. Chem., Vol. 69, No. 2, 2004

Phosphatase Modulating Activity of Glucolipsin A
Com p ou n d 23. A solution of hydroxy acid 9 (40 mg, 0.053
mmol), 2-chloro-1,3-dimethylimidazolinium chloride (22) (22
mg, 0.13 mmol) and KH (5 mg, 0.11 mmol) in CH2Cl2 (1.1 mL)
was stirred for 1 h at 0 °C. 4-(Dimethylamino)pyridine (15 mg,
0.12 mmol) was then added and stirring was continued for 16
h. The mixture was then filtered through Celite, the filtrate
was evaporated, and the residue was purified by flash chro-
matography (hexane:EtOAc 20/1 f 10/1) to give cyclodimer
23 as a colorless syrup (21 mg, 54%). [R]20D +15 (c 0.7, CHCl3);
1H NMR (400 MHz, CDCl3) δ 7.35-7.16 (m, 15H), 4.95 (d, 1H,
J ) 11.0 Hz), 4.91 (d, 1H, J ) 10.9 Hz), 4.85 (d, 1H, J ) 10.9
Hz), 4.78 (d, 1H, J ) 11.0 Hz), 4.71 (d, 1H, J ) 10.9 Hz), 4.55
(d, 1H, J ) 10.9 Hz), 4.53 (d, 1H, J ) 7.7 Hz), 4.29 (d, 1H, J
) 10.6 Hz), 3.92-3.87 (m, 2H), 3.66 (t, 1H, J ) 8.9 Hz), 3.57
(tm, 1H, J ) 9.9 Hz), 3.41 (dd, 1H, J ) 9.0, 7.7 Hz), 3.29 (dd,
1H, J ) 9.9, 8.7 Hz), 2.71 (br quint, 1H, J ) 7.1 Hz), 1.51 (non.,
1H, J ) 6.7 Hz), 1.58-1.12 (m, 24H), 1.32 (d, 3H, J ) 6.9 Hz),
0.86 (d, 6H, J ) 6.6 Hz); 13C NMR (100 MHz, CDCl3) δ 173.7,
138.5-127.6, 103.7, 84.9, 82.6, 78.4, 75.7, 75.1, 72.9, 64.9, 45.7,
39.1, 30.2, 30.0, 29.8, 28.0, 27.5, 22.7, 14.4; IR (film) 2924, 2853,
1739, 1607, 1497, 1455, 1257, 1071, 750, 698 cm-1; MS (EI)
m/z (rel intensity) 1365 (<1), 513 (2), 279 (8), 253 (6), 240 (11),
193 (4), 181 (13), 109 (3), 97 (4), 91 (100), 81 (3), 57 (5), 55 (4),
43 (6), 41 (3); HRMS (ESI pos) calcd for C92H128O14 [(M +
NH4)+] 1474.964784, found 1474.96176.
1H, J ) 6.6 Hz), 1.31 (d, 3H, J ) 7.0 Hz), 1.58-1.11 (m, 24H),
0.87 (d, 6H, J ) 6.6 Hz); 13C NMR (150 MHz, CD3OD) δ 175.8,
104.5, 78.9, 78.0, 75.6, 75.5, 72.4, 66.3, 46.7, 40.3, 31.1 (2×),
31.0, 30.9 (2×), 30.8 (3×), 30.7, 29.1, 28.6, 25.2, 23.0, 13.3. For
a comparison of the 1H and 13C NMR data of this compound
with those of authentic glucolipsin A, see Tables 2 and 3.
En zym a tic Assa ys: (a ) P TP 1B In h ibition . PTP1B was
purchased from Calbiochem (human recombinant). The en-
zyme (0.001 U) was preincubated with the inhibitors in a
buffer (pH 7.2)36 containing HEPES (25 mM), EDTA (2.5 mM),
NaCl (50 mM), DTT (2 mM), and BSA (0.1%) for 15 min at
room temperature. Then p-NPP was added (end concentration
50 µM) and the read-out (405 nm) was recorded on a micro-
plate-reader at 37 °C continuously for 80 min. The reaction
rate was determined from the absorption difference between
30 and 60 min reaction time.
(b) Cd c25A In h ibition . The clone pET9d/His-Cdc25A was
expressed in the E. coli strain BL21-DE3 and purified in the
presence of 8 M urea. A 30-µg sample of the purified enzyme
was preincubated with the inhibitors in a pH 8.0 buffer
containing 50 mM Tris, 50 mM NaCl, and 2 mM DTE for 15
min at room temperature.37,38
Ack n ow led gm en t. Generous financial support by
the Deutsche Forschungsgemeinschaft (Leibniz award
to A.F.) and the Fonds der Chemischen Industrie is
gratefully acknowledged. We thank Dr. J . Qian-Cutrone,
Bristol-Myers Squibb, for providing additional copies of
the spectra of glucolipsin and some degradation prod-
ucts thereof. We are grateful to Ingrid Hofmann,
Heidelberg, for providing the Cdc25A clone.
Glu colip sin A (24). Pd(OH)2 (13 mg) was added to a
solution of cyclodimer 23 (21 mg, 0.014 mmol) in MeOH (5
mL) and the resulting suspension was stirred under H2 (1 atm)
for 3 d at ambient temperature. The catalyst was filtered off
through a short pad of Celite, the Celite was carefully rinsed
with MeOH, and the combined filtrates were evaporated giving
compound 24 as an analytically pure white solid (13 mg,
100%). [R]20D +9.5 (c 0.55, MeOH) [ref 3: [R]25D +11.39 (c 0.05,
1
MeOH)]; H NMR (600 MHz, pyridine-d5) δ 5.14 (d, 1H, J )
Su p p or tin g In for m a tion Ava ila ble: Full experimental
and analytical details for the preparation of the stereoisomers
of glucolipsin reported above and NMR spectra of all stereo-
isomeric macrodilactones in perbenzylated and deprotected
form. This material is available free of charge via the Internet
at http://pubs.acs.org.
11.2 Hz), 5.06 (d, 1H, J ) 7.7 Hz), 4.56-4.54 (m, 1H), 4.52 (t,
1H, J ) 10.6 Hz), 4.22 (td, 1H, J ) 8.8, 4.1 Hz), 4.18 (t, 1H, J
) 9.9 Hz), 4.06 (td, 1H, J ) 8.4, 4.4 Hz), 3.87 (td, 1H, J ) 9.4,
4.7 Hz), 3.01 (m, 1H), 2.14 (m, 1H), 1.97 (m, 1H), 1.78 (m, 2H),
1.66 (d, 3H, J ) 6.9 Hz), 1.49 (non., 1H, J ) 6.6 Hz), 1.40-
1.15 (m, 20H), 0.86 (d, 6H, J ) 6.6 Hz); 13C NMR (150 MHz,
pyridine-d5) δ 174.4, 105.1, 78.7, 78.6, 75.7, 75.2, 72.2, 66.4,
46.2, 39.3, 36.3, 30.7, 30.3, 30.2, 30.1, 28.2, 27.8, 24.2, 22.8,
J O035079F
1
14.4; H NMR (300 MHz, CD3OD) δ 4.56 (br d, 1H, J ) 10.3
(36) Hamaguchi, T.; Masuda, A.; Morino, T.; Osada, H. Chem. Biol.
1997, 4, 279.
(37) Blomberg I.; Hoffmann I. Mol. Cell. Biol. 1999, 19, 6183.
(38) Baratte, B.; Meijer, L.; Galaktionov, K.; Beach, D. Anticancer
Res. 1992, 12, 873.
Hz), 4.36 (d, 1H, J ) 7.6 Hz), 4.05 (dt, 1H, J ) 7.2, 5.0 Hz),
3.85 (dd, 1H, J ) 11.4, 9.9 Hz), 3.46 (t, 1H, J ) 9.8 Hz), 3.35
(t, 1H, J ) 9.0 Hz), 3.20 (dd, 1H, J ) 9.1, 7.7 Hz), 3.15 (dd,
1H, J ) 9.7, 8.8 Hz), 2.63 (quint, 1H, J ) 7.2 Hz), 1.52 (non.,
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