Expanding the pyrimidine diphosphosugar repertoire: The chemoenzymatic synthesis of amino- and acetamidoglucopyranosyl derivatives

Article abstract of DOI:10.1002/1521-3773(20010417)40:8<1502::AID-ANIE1502>3.0.CO;2-K

The exploitation of a unique thymidylyltransferase (E_p) allows the rapid syntheses of thymidine and uridine 5′-(aminodeoxy-α-D-hexopyranosyl diphosphates), 5′(acetamidodeoxy-α-D-hexopyranosyl diphosphates), and even 5′-(aminodideoxy-α-D-hexopyr

Full text of DOI:10.1002/1521-3773(20010417)40:8<1502::AID-ANIE1502>3.0.CO;2-K

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[1] a) P. J. Fagan, J. C. Calabrese, B. Malone, Science 1991, 252, 1160 ± 1161;
b) A. L. Balch, J. W. Lee, B. C. Noll, M. M. Olmstead, Inorg. Chem.
1993, 32, 3577 ± 3578; c) R. E. Douthwaite, M. L. H. Green, A. H. H.
Stephens, J. F. C. Turner, J. Chem. Soc. Chem. Commun. 1993, 1522 ±
1523; d) J. T. Park, J.-J. Cho, H. Song, J. Chem. Soc. Chem. Commun.
1995, 15 ± 16; e) H.-F. Hsu, Y. Du, T. E. Albrecht-Schmitt, S. R. Wilson,
J. R. Shapley, Organometallics 1998, 17, 1756 ± 1761; f) A. L. Balch,
M. M. Olmstead, Chem. Rev. 1998, 98, 2123 ± 2165.
(49%). Compound 2a was shown to be stable towards
transformation into 2b under similar irradiation conditions,
and this implies the photochemical route selectively forms 2b
from 1. Compounds 2a and 2b were not interconvertible upon
prolonged heating at 808C, that is, no intermetal movement of
the isocyanide ligand occurs in these clusters.
In conclusion, we have demonstrated the first transforma-
tion of the bonding mode of C60 from m3-h2,h2,h2 (p) to m3-
h1,h2,h1 (s) on an Os3 framework induced by an external
ligand (1 !2a ‡ 2b). We have shown that the selective
formation of isomer 2b can be accomplished by UV
irradiation. Efforts are currently underway to understand
the thermal and photochemical conversion pathways of the p
and s C60 ± metal interactions. Reactivity studies and selective
functionalization of the C60 ligand of 2a and 2b are also in
progress.
[2] a) M. Sawamura, H. Iikura, E. Nakamura, J. Am. Chem. Soc. 1996, 118,
12850 ± 12851; b) H. Iikura, S. Mori, M. Sawamura, E. Nakamura, J.
Org. Chem. 1997, 62, 7912 ± 7913.
Â
[3] a) M. Rasinkangas, T. T. Pakkanen, T. A. Pakkanen, M. Ahlgren, J.
Rouvinen, J. Am. Chem. Soc. 1993, 115, 4901; b) I. J. Mavunkal, Y. Chi,
S.-M. Peng, G.-H. Lee, Organometallics 1995, 14, 4454 ± 4456; c) A. N.
Chernega, M. L. H. Green, J. Haggitt, A. H. H. Stephens, J. Chem. Soc.
Dalton Trans. 1998, 755 ± 767; d) K. Lee, C. H. Lee, H. Song, J. T. Park,
H. Y. Chang, M.-G. Choi, Angew. Chem. 2000, 112, 1871 ± 1874; Angew.
Chem. Int. Ed. 2000, 39, 1801 ± 1804.
[4] a) H.-F. Hsu, J. R. Shapley, J. Am. Chem. Soc. 1996, 118, 9192 ± 9193;
b) K. Lee, H.-F. Hsu, J. R. Shapley, Organometallics 1997, 16, 3876 ±
3877; c) K. Lee, J. R. Shapley, Organometallics 1998, 17, 3020 ± 3026;
d) J. T. Park, H. Song, J.-J. Cho, M.-K. Chung, J.-H. Lee, I.-H. Suh,
Organometallics 1998, 17, 227 ± 236; e) H. Song, K. Lee, J. T. Park, M.-
G. Choi, Organometallics 1998, 17, 4477 ± 4483; f) H. Song, K. Lee, J. T.
Park, M.-G. Choi, J. Organomet. Chem. 2000, 599, 49 ± 56.
[5] a) S. Zhang, T. L. Brown, Y. Du, J. R. Shapley, J. Am. Chem. Soc. 1993,
115, 6705 ± 6709; b) S. Ballenweg, R. Gleiter, W. Krätschmer, Tetrahe-
dron Lett. 1993, 34, 3737 ± 3740; c) Y.-H. Zhu, L.-C. Song, Q.-M. Hu, C.-
M. Li, Org. Lett. 1999, 1, 1693 ± 1695.
Experimental Section
Details on the synthesis as well as full spectroscopic characterization of 1,
2a, and 2b are given in the Supporting Information. For the X-ray structure
analyses, data were collected on a CCD diffractometer with MoKa radiation
(l ˆ 0.71073 Š) by using
w scans. Crystallographic data (excluding
structure factors) for the structures reported in this paper have been
deposited with the Cambridge Crystallographic Data Centre as supple-
mentary publication no. CCDC-151706 (1), CCDC-151707 (2a), and
CCDC-151708 (2b). Copies of the data can be obtained free of charge
on application to CCDC, 12 Union Road, Cambridge CB21EZ, UK (fax:
(‡44)1223-336-033; e-mail: deposit@ccdc.cam.ac.uk).
[6] A. V. Rivera, G. M. Sheldrick, M. B. Hursthouse, Acta Crystallogr. Sect.
B 1978, 34, 1985 ± 1988.
2a: Analysis calcd for C85H14N2O8S2Os3 (2a ´ CS2): C 55.92, H 0.77, N 1.53, S
3.51; found: C 55.50, H 0.68, N 1.29, S 3.57; IR (C6H12): nÄ ˆ 2079 (s), 2068
1
1
(s), 2019 (s), 1986 cm (s) (CO); nÄ ˆ 2189 (w), 1634 cm (vw) (CN);
1H NMR (400 MHz, CS2/CDCl3, 298 K): d ˆ 7.49 ± 6.98 (m, 10H; Ph), 5.50
(d, 1H, JH,H ˆ 13 Hz; CH2), 4.95 (brs, 2H; CH2), 4.87 (d, 1H, JH,H ˆ 13 Hz;
CH2); 13C NMR (carbonyl region, 100 MHz, C6H4Cl2/C6D5CD3, 298 K):
d ˆ 179.96, 179.00, 178.32, 176.74, 175.74, 174.55, 173.39, 168.95; MS
Expanding the Pyrimidine Diphosphosugar
Repertoire: The Chemoenzymatic Synthesis of
Amino- and Acetamidoglucopyranosyl
Derivatives**
‡
‡
(FAB ): m/z: 1754 [M ].
Jiqing Jiang, John B. Biggins, and Jon S. Thorson*
X-ray data for 2a: Brown crystals were obtained by slow diffusion of
hexane into a solution of 2a in CS2 at room temperature. A crystal of
dimensions 0.12 Â 0.14 Â 0.42 mm was used for data collection:
C84H14N2O8Os3 ´ CS2, Mr ˆ 1825.7; monoclinic, space group P21/c, Z ˆ 4,
1calcd ˆ 2.105 gcm 3, a ˆ 19.4334(2), b ˆ 10.6922(2), c ˆ 29.0892(2) Š, b ˆ
107.6158, Vˆ 5760.9(1) Š3. The structure was solved by direct methods
and refined by full-matrix least-squares analysis to give R ˆ 0.0448 and
Rw ˆ 0.0695 (based on F 2) for 878 variables and 11490 observed reflections
with I > 2s(I) and 1.47 < q < 26.23. Data collection at Tˆ 293(2) K.
An extensive body of in vivo genetic evidence indicates that
the glycosyltransferases involved in secondary metabolism are
extremely promiscuous with respect to their nucleotide
diphosphosugar (NDP-sugar) donor.[1] Yet, in vitro experi-
ments in this area are limited to only a few examples, partly
because of the lack of the required NDP-sugar substrates for
2b: Analysis calcd for C84H14N2O8Os3: C 57.66, H 0.81, N 1.60; found: C
56.76, H 0.61, N 1.22; IR (C6H12): nÄ ˆ 2085 (vs), 2052 (s), 2026 (vs), 2015 (w),
1
1
[*] Prof. Dr. J. S. Thorson, Dr. J. Jiang, J. B. Biggins
Laboratory for Biosynthetic Chemistry
Memorial Sloan ± Kettering Cancer Center
1275 York Avenue, Box 309, New York, NY 10021 (USA)
Fax : (‡1)212-717-3066
1992 (w), 1982 (w), 1968 cm (m) (CO); nÄ ˆ 2185 (w), 1629 cm (vw)
(CN); 1H NMR (400 MHz, CS2/CDCl3, 298 K): d ˆ 7.49 ± 7.19 (m, 10H;
Ph), 5.66 (d, 1H, JH,H ˆ 13 Hz; CH2), 5.48 (d, 1H, JH,H ˆ 16 Hz; CH2), 5.41
(d, 1H, JH,H ˆ 16 Hz; CH2), 4.88 (d, 1H, JH,H ˆ 13 Hz; CH2); 13C NMR
(carbonyl region, 100 MHz, C6H4Cl2/C6D5CD3, 223 K): d ˆ 181.9, 176.9,
‡
‡
E-mail: jthorson@sbnmr1.ski.mskcc.org
and
176.1, 175.8, 175.5, 174.5, 173.2, 169.2; MS (FAB ): m/z 1754 [M ].
X-ray crystal data for 2b: Brownish black crystals were obtained by slow
diffusion of methanol into a solution of 2b in toluene at room temperature.
A crystal of dimensions 0.41 Â 0.29 Â 0.11 mm was used for data collection:
The Sloan ± Kettering Division
Joan and Sanford I. Weill Graduate School of Medical Sciences
Cornell University
C84H14N2O8Os3, Mr ˆ 1749.6; monoclinic, space group P21/c, Z ˆ 4, 1calcd
ˆ
3
[**] This contribution was supported by the National Institutes of Health
(GM58196 and CA84374), a Cancer Center Support Grant (CA-
08748), and a grant from the Special Projects Committee of the
Society of Memorial Sloan ± Kettering Cancer Center. J.S.T. is an
Alfred P. Sloan Research Fellow and a Rita Allen Foundation Scholar.
2.024 gcm
,
a ˆ 19.9376(8), b ˆ 23.0770(9), c ˆ 12.8318(5) Š, b ˆ
103.462(1)8, Vˆ 5741.7(4) Š3. The structure was solved by direct methods
and refined by full-matrix least-squares analysis to give R ˆ 0.0779 and
Rw ˆ 0.1995 (based on F 2) for 851 variables and 8192 observed reflections
with I > 2s(I) and 1.37 < q < 23.34. Data collection at Tˆ 193(2) K.
Supporting information for this article is available on the WWW under
http://www.angewandte.com or from the author.
Received: November 10, 2000 [Z16079]
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these experiments.[2] Thus, the reliance of these unique
glycosyltransferases on pyrimidine diphosphosugars (uri-
dine (UDP)/thymidine (dTDP)) has revitalized interest in
methods to expand the repertoire of UDP/dTDP-sugars.[3]
We recently reported that Salmonella enterica LT2 a-d-
glucopyranosyl phosphate thymidylyltransferase (Ep),[4] which
catalyzes the reaction shown in Equation (1), can convert a
phosphate series and their corresponding acetamidodeoxy
analogues provides insight into the ability of the active site of
Ep to accommodate additional steric bulk.
Only two of the aminodeoxy-a-d-glucopyranosyl phos-
phates examined, 2-amino-2-deoxy-a-d-glucopyranosyl phos-
phate (26; see Figure 1) and 2-acetamido-2-deoxy-a-d-gluco-
pyranosyl phosphate (27; Figure 1), were commercially avail-
able. The syntheses of the
remaining analogues di-
verged from the key inter-
mediates 8, 13, and 19
(Scheme 1). 1-Ethylthio-
b-d-pyranosides 8 and 19
were derived from the
previously reported glyco-
sides 6[5] and 17,[6] respec-
tively, whereas 13 was syn-
thesized from the previ-
OH
O
HO
HO
HO
O
O
N
O
O
HO
HO
HO
O
HN
HN
O
P
–O
O
–O
P
O
O–
HO
O
P
O
P
–O
O
N
P
O
O
P
O
2
–O
O
O
O
O
–O
O–
O
O
O–
(1)
OH
OH
Ep
PPi
3
1
ously reported 12,[7] in a manner strategically similar to the
synthesis of deoxy-a-d-glucopyranosyl phosphate.[3] Specifi-
cally, this effective strategy invoked a protection scheme to
selectively expose the position of substitution, followed by
activation (with TsCl or Tf2O; Ts ˆ para-toluenesulfonyl,
Tf ˆ trifluoromethanesulfonyl) and SN2 displacement by so-
dium azide. From the divergent point (8, 13, and 19), an
efficient and selective reduction of the azide group with
SnCl2, followed by acetylation, gave the desired 1-ethylthio-b-
d-pyranoside precursors 9, 14, and 20. Finally, 9, 14, and 20
wide array of a-d-hexopyranosyl phosphates into their
corresponding dTDP- and UDP-nucleotide sugars.[3] Herein
we expand this methodology to include glycosides that are
common to biological systems, namely aminodeoxy-a-d-
hexopyranosyl phosphates and acetamidodeoxy-a-d-hexopyr-
anosyl phosphates. A general chemoenzymatic method to
rapidly generate these reagents is presented. This method is
significant because it provides a substrate set for developing
in vitro glycosylation systems. Furthermore, a direct compar-
ison between a series of aminodeoxy-a-d-glucopyranosyl
R
N3
NH
NHAc
N3
N3
O
O
O
O
O
a)
b)
c)
BnO
BnO
HO
HO
AcO
AcO
BnO
BnO
AcO
AcO
SEt
SEt
SEt
SEt
SEt
O
HO
BnO
AcO
BnO
OAc
AcO
O P
–O
O–
4 R = H
5 R = Ac
6
7
8
9
HO
OBn
OBn
OBn
OH
R
O
O
O
O
c)
c)
d)
N3
BnO
AcHN
BnO
NH
HO
SEt
SEt
BnO
O
HO
BnO
BnO
BnO
O P
–O
O–
12
13
14
10 R = H
11 R = Ac
BnO
AcO
AcO
OH
BnO
O
O
O
O
a)
b)
O
BnO
N3
AcO
N3
AcO
N3
HO
NH
R
BnO
AcHN
SEt
SEt
O
BnO
AcO
AcO
HO
OAc
BnO
O P
–O
O–
15 R = H
16 R = Ac
17
18
19
20
AcO
AcO
AcO
AcO
OH
BzO
O
O
O
O
O
a)
e)
d)
N3
BzO
H2N
HO
SEt
SEt
SEt
O
AcO
AcO
BnO
BnO
OAc
HO
O P
–O
O–
21
23
24
25
22
Scheme 1. An overview of the key steps in the described syntheses of analogues of Ep substrates. The box highlights the point from which the aminodeoxy-a-
d-glucose phosphate series and the acetamidodeoxy-a-d-glucose phosphate series diverge. Reaction conditions: a) Me3SiSEt, ZnI2 (84.2% overall yield);
b) 1) MeONa, 2) NaH, BnBr (77.3% average overall yield, two steps); c) 1) SnCl2, PhSH, Et3N, 2) Ac2O, py (84.0% average overall yield, two steps);
d) 1) Tf2O, py, 2) NaN3 (87.7% average overall yield, two steps); e) 1) NaOMe, 2) CH3C(OCH3)2CH3, TsOH, 3) NaH, BnBr, 4) HCl/MeOH, 5) BzCl,
DMAP, Et3N (87.3% average overall yield, five steps); final steps (not shown): 1) phosphorylation, 2) reductive deprotection, 3) cation exchange to give the
‡
Na salt (44.4% average overall yield, three steps). Bn ˆ benzyl, Bz ˆ benzoyl, DMAP ˆ 4-dimethylaminopyridine, py ˆ pyridine.
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were phosphorylated by the reaction with dibenzyl phosphate
as previously described,[3] and reductively deprotected to form
5, 11, and 16, respectively. The same procedure also led to the
conversion of azides 8, 13, and 19 into the desired amines 4,
10, and 15, respectively. An aminodideoxy sugar, 4-amino-4,6-
dideoxy-a-d-glucopyranosyl phosphate (21), was also synthe-
sized from peracetylated d-fucose (22) by using a similar
strategy (Scheme 1).
To evaluate the synthetic utility of our thymidylyltransfer-
ase,[8] Ep, a-d-glucopyranosyl phosphate, Mg2‡, nucleotide
triphosphate (NTP), and inorganic pyrophosphatase[9] were
incubated at 378C for 30 min, and the extent of product
formation was determined by HPLC (Figure 1).[10] For each
assay, confirmation of the product was based on high-
resolution mass spectrometry (HR-MS) of the HPLC-isolated
products, and in some cases also on HPLC coelution with
commercially available standards.[11] Control reactions
showed that no product formation was observed in the
absence of Ep, glucopyranosyl phosphate, Mg2‡, or NTP. The
fundamental goal of this work was to assess the utility of Ep in
simplifying the synthesis of nucleotide sugar pools. Figure 1
clearly illustrates that Ep is advantageous for this task: of the
nine substrate analogues tested, seven with thymidine tri-
phosphate (dTTP) and four with uridine triphosphate (UTP)
provide appreciable amounts of product (more than 50%
conversion) under the conditions described.
A
comparison of the aminodeoxy-a-d-glucopyranosyl
phosphate/dTTP assay results (Figure 1, 4, 10, 15 and 26)
with the native reaction of Ep (Figure 1, 3/dTTP) reveals that
the position of the amino substituent has absolutely no effect
on product formation, and, with the exception of 4, a similar
phenomenon is observed in the presence of UTP. The
divergence of 4 from this trend is consistent with our previous
observations of a UTP-dependent Ep ªadverse cooperationº
in the presence of certain hexopyranosyl phosphates,[3]
perhaps a result of allosteric activation by dTTP.[12] An
evaluation of the acetamidodeoxy-a-d-glucopyranosyl phos-
phate/dTTP assays (Figure 1, 5, 11, 16, and 27), in comparison
to their non-acetylated counterparts (Figure 1, 4, 10, 15, and
26, respectively), revealed that a bulky N-acetyl group at C2
or C3 (16 and 27) is tolerated, whereas
the identical substituent at C4 or C6 (5
and 11) results in a complete loss of
activity. Given that these effects most
likely derive from unfavorable steric
interactions, it follows that the active
site of Ep is able to accommodate
additional C2/C3 bulk, whereas steric
interactions limit the allowed C4/C6
substitutions. To our surprise, product
formation from 5/UTP was eight times
that from 5/dTTP. This is the first
example that contradicts the typical
adverse UTP-dependent effect on
yields, as illustrated by 4 and 16 in
the present study. Finally, a compar-
ison of aminodideoxy-a-d-glucopyra-
nosyl phosphate (21)[13] with 10 reveals
that C6 deoxygenation does not affect
dTTP-dependent Ep catalysis, but
greatly diminishes UTP-dependent
conversion (Figure 1). Given that in-
dependent deoxygenation at C6[3] or
amino substitution at C4 (10) have no
effect on product yield (in the context
of our assay conditions), data from
independent substitutions may not be
reliable in predicting the effects of
multiple substitutions on product
yield.
In conclusion, the presented work
further substantiates the promiscuous
nature of Ep and the advantages of
exploiting this unique characteristic to
synthesize an incredible array of val-
uable nucleotide diphosphosugar re-
agents. Thus, these studies will broadly
impact efforts to understand and ex-
ploit the biosynthesis of glycosylated
Figure 1. The Ep-catalyzed conversion of ªunnaturalº substrates. Percent conversion ˆ [AP/(AP
‡
AT)]  100 (AP ˆ the NDP-sugar product peak integration, AT ˆ the NTP peak integration). The
composition of all the products was confirmed by HR-MS.
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bioactive natural products.[1] Moreover, this work suggests
that Ep can accept greater C2/C3 steric bulk in the substrate
(relative to 3), which opens the door to even more imaginative
substitutions at these positions. Efforts are in progress to
further expand the scope of this methodology.
tion of the calicheamicin aryltetrasaccharide. See: a) J. S. Thorson, B.
Shen, R. E. Whitwam, W. Liu, Y. Li, J. Ahlert, Bioorg. Chem. 1999, 27,
172 ± 188; b) R. E. Whitwam, J. Ahlert, T. R. Holman, M. Ruppen,
J. S. Thorson, J. Am. Chem. Soc. 2000, 122, 1556 ± 1557; c) J. B. Biggins,
J. R. Prudent, D. J. Marshall, M. Ruppen, J. S. Thorson, Proc. Natl.
Acad. Sci. USA 2000, 97, 13537 ± 13542; d) J. S. Thorson, E. L. Sievers,
J. Ahlert, E. Shepard, R. E. Whitwam, K. C. Onwueme, M. Ruppen,
Curr. Pharm. Des. 2000, 6, 1841 ± 1879).
Received: November 24, 2000
Revised: January 10, 2001 [Z16173]
Note added in proof: The three-dimensional structure of Ep and the
structure-based engineering of Ep to expand the methodology reported
here have recently been reported (W. A. Barton, J. Lesniak, J. B. Biggins,
P. D. Jeffrey, J. Jiang, K. R. Rajashankar, J. S. Thorson, D. B. Nikolov, Nat.
Struct. Biol. 2001, in press).
[1] J. S. Thorson, T. J. Hosted, Jr., J. Jiang, J. B. Biggins, J. Ahlert, M.
Ruppen, Curr. Org. Chem. 2001, 5, 89 ± 111, and references therein.
[2] P. J. Solenberg, P. Matsushima, D. R. Stack, S. C. Wilkie, R. C.
Thompson, R. H. Baltz, Chem. Biol. 1997, 4, 195 ± 202.
[3] J. Jiang, J. B. Biggins, J. S. Thorson, J. Am. Chem. Soc. 2000, 122,
6803 ± 6804; for recent reviews see: K. M. Koeller, C.-H. Wong, Nat.
Biotechnol. 2000, 18, 835 ± 841; J. M. Elhalabi, K. G. Rice, Curr. Med.
Chem. 1999, 6, 93 ± 116; M. M. Palcic, Curr. Opin. Biotechnol. 1999, 10,
616 ± 624; H. J. M. Gijsen, L. Qiao, W. Fitz, C.-H. Wong, Chem. Rev.
1996, 96, 443 ± 473; C.-H. Wong, Pure Appl. Chem. 1995, 67, 1609 ±
1616; C.-H Wong, R. L. Halcomb, Y. Ichikawa, T. Kajimoto, Angew.
Chem. 1995, 107, 569 ± 593; Angew. Chem. Int. Ed. Engl. 1995, 34,
521 ± 546; Y. Ichikawa, G. C. Look, C.-H. Wong, Anal. Biochem. 1992,
202, 215 ± 238.
[4] This enzyme (E.C. 2.7.7.24) is also known as dTDP-glucose synthase,
dTDP-glucose pyrophosphorylase, thymidine diphosphoglucose py-
rophosphorylase, and thymidine diphosphate glucose pyrophosphor-
ylase. The name ªEpº historically derives from the original pyrophos-
phorylase designation.
[Ru(N2)(PiPr3)(ꢀN2Me2S2ꢁ)]: Coordination of
Molecular N2 to Metal Thiolate Cores under
Mild Conditions**
Dieter Sellmann,* Barbara Hautsch, Annette Rösler,
and Frank W. Heinemann
Dedicated to Professor Ernst-Gottfried Jäger
on the occasion of his 65th birthday
X-ray crystallography has revealed the structure of FeMo
nitrogenase and its FeMo cofactors, however, the molecular
mechanism of biological N2 fixation has remained as un-
known as low-molecular weight compounds catalyzing the
reduction of N2 under mild and biologically compatible
conditions.[1] These conditions rule out the use of alkali
metals or comparably strong reductants at any stage in the
design of a nonenzymatic chemical system for modeling the
biological N2 reduction. This includes the first stage, the
synthesis of N2 complexes.
All mechanisms postulated for biological N2 fixation
consider the coordination of N2 to the metal sulfur core of
the Fe7MoS9 cofactors as the first key step.[1] However, metal
sulfur complexes that bind N2 under mild conditions are
unknown, in spite of numerous intensive efforts.[2] There are
only 12 N2 complexes with sulfur coligands,[3] only two of
which could be prepared directly from molecular N2.[3a,f] Their
preparation, however, required strong reductants or precur-
sors prepared by use of strong reductants. None of these
complexes meets the severe constraints with regard to mild
conditions.
[5] V. Maunier, P. Boullanger, D. Lafont, Y. Chevalier, Carbohydr. Res.
1997, 299, 49 ± 57.
[6] W. A. Greenberg, E. S. Priestley, P. S. Sears, P. B. Alper, C. Rose-
nbohm, M. Hendrix, S.-C. Hung, C.-H. Wong, J. Am. Chem. Soc. 1999,
121, 6527 ± 6541.
[7] P. J. Garegg, I. Kvarnstrom, A. Niklasson, G. Niklasson, S. C. T.
Svensson, J. Carbohydr. Chem. 1993, 12, 933 ± 953.
[8] Ep was purified as described in ref. [3] from a rmlA expression strain
(L. Lindquist, R. Kaiser, P. R. Reeves, A. A. Lindberg, Eur. J.
Biochem. 1993, 211, 763 ± 770) and this homogeneous preparation
was utilized for the present study. The expression strain for this
enzyme was provided by Professor Hung-wen Liu (Medicinal
Chemistry, University of Texas, Austin).
[9] The inorganic pyrophosphatase was included to drive the reaction
forward. For examples, see: a) D. C. Crans, R. J. Kazlauskas, B. L.
Hirschbein, C.-H. Wong, O. Abril, G. M. Whitesides, Methods
Enzymol. 1987, 136, 263 ± 280; b) S. L. Haynie, G. M. Whitesides,
Appl. Biochem. Biotechnol. 1990, 23, 155 ± 170; c) Y. Ichikawa, R.
Wang, C.-H. Wong, Methods Enzymol. 1994, 247, 107 ± 124.
[10] The reaction of a mixture containing NTP (2.5 mm), sugar phosphate
(5.0 mm), MgCl2 (5.5 mm), and inorganic pyrophosphatase (10 U;
1 U ˆ the amount of protein needed to produce 1 mmolmin 1 of TDP-
d-glucose) in potassium phosphate buffer (pH 7.5, 50 mm, 50 mL) at
378C was initiated by the addition of Ep (3.52 U). The reaction was
incubated with slow agitation for 30 min at 378C, quenched with
MeOH (50 mL), centrifuged (5 min, 14000 Â g), and the supernatant
was stored at 208C until analysis by HPLC. Samples (30 mL) were
resolved on a Sphereclone 5 m SAX column (150 Â 4.6 mm) fitted with
a SecurityGuard cartridge (Phenomenex, Torrance, CA) by using a
linear gradient (potassium phosphate buffer, pH 5.0, 50 ± 200 mm,
1.5 mLmin 1, A275nm).
[11] HPLC product fractions from the assay described in ref. [10] were
lyophilized and submitted directly for HR-MS (fast-atom bombard-
ment (FAB)) analysis.
[12] Allosteric activation is common for the nucleotidylyltransferase
family. For examples, see: a) M. X. Wu, J. Preiss, Arch. Biochem.
Biophys. 1998, 358, 182 ± 188; b) D. A. Bulik, P. van Ophem, J. M.
Manning, Z. Shen, D. S. Newburg, E. L. Jarroll, J. Biol. Chem. 2000,
275, 14722 ± 14728. However, data is not yet available pertaining to
the allosteric effectors of Ep.
Our attempts to tackle this problem have focussed on sulfur
ligand complexes of iron and its congener ruthenium. They
[*] Prof. Dr. D. Sellmann, Dipl.-Chem. B. Hautsch,
Dipl.-Chem. A. Rösler, Dr. F. W. Heinemann
Institut für Anorganische Chemie
Universität Erlangen-Nürnberg
Egerlandstrasse 1, 91058 Erlangen (Germany)
Fax : (‡49)9131-852-7367
E-mail: sellmann@anorganik.chemie.uni-erlangen.de
[**] Transition Metal Complexes with Sulfur Ligands, Part 150. This work
was supported by the Deutsche Forschungsgemeinschaft and Fonds
der Chemischen Industrie. Part 149: D. Sellmann, F. Geipel, F. W.
2
Heinemann, Z. Anorg. Allg. Chem., in press. ꢁN2Me2S2 ꢂ ˆ 1,2-
[13] The product of this reaction, thymidine 5'-(4-amino-4,6-dideoxy-a-d-
glucopyranosyl diphosphate), is a critical intermediate in the forma-
ethanediamine-N,N'-dimethyl-N,N'-bis(2-benzenethiolate)(2 ).
Angew. Chem. Int. Ed. 2001, 40, No. 8
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