Biosynthesis of isoprenoids. A rapid method for the preparation of isotope-labeled 4-diphosphocytidyl-2C-methyl-D-erythritol

Article abstract of DOI:10.1021/ja001385o

4-Diphosphocytidyl-2C-methyl-D-erythritol serves as an intermediate in the nonmevalonate pathway of isoprenoid biosynthesis. The compound has been prepared in millimole quantity by a sequence of one-pot reactions using ¹³C-labeled pyruvate and dihydroxyacetone phosphate or ¹³C-labeled glucose as starting materials and recombinant enzymes of the nonmevalonate isoprenoid pathway as catalysts. The method has been used for the preparation of various 4-diphosphocytidyl-2C-methyl-D-erythritol isotopomers in high yield.

Full text of DOI:10.1021/ja001385o

VOLUME 122, NUMBER 40
OCTOBER 11, 2000
© Copyright 2000 by the
American Chemical Society
Biosynthesis of Isoprenoids. A Rapid Method for the Preparation of
Isotope-Labeled 4-Diphosphocytidyl-2C-methyl-D-erythritol
Felix Rohdich,† Christoph A. Schuhr,† Stefan Hecht,† Stefan Herz,†
Juraithip Wungsintaweekul,‡ Wolfgang Eisenreich,† Meinhart H. Zenk,‡ and
Adelbert Bacher†*
Contribution from the Lehrstuhl fu¨r Organische Chemie und Biochemie, Technische UniVersita¨t Mu¨nchen,
Lichtenbergstrasse 4, D-85747 Garching, Federal Republic of Germany, and Biozentrum-Pharmazie,
UniVersita¨t Halle, Weinbergweg 22, D-06099 Halle/Saale, Federal Republic of Germany
ReceiVed April 20, 2000
Abstract: 4-Diphosphocytidyl-2C-methyl-D-erythritol serves as an intermediate in the nonmevalonate pathway
of isoprenoid biosynthesis. The compound has been prepared in millimole quantity by a sequence of one-pot
reactions using 13C-labeled pyruvate and dihydroxyacetone phosphate or 13C-labeled glucose as starting materials
and recombinant enzymes of the nonmevalonate isoprenoid pathway as catalysts. The method has been used
for the preparation of various 4-diphosphocytidyl-2C-methyl-D-erythritol isotopomers in high yield.
Introduction
Escherichia coli which could not be explained via the meva-
lonate pathway (refs 6-8; for reviews, see also refs 9-11). The
data suggested that pyruvate and a triose serve as substrates of
an alternative pathway. Arigoni and co-workers also showed
that exogenous 1-deoxy-D-xylulose is utilized efficiently for
terpenoid biosynthesis via this alternative pathway,7 and that
plants use both isoprenoid pathways for the biosynthesis of
different terpenoids.12
Several enzymes of the nonmevalonate isoprenoid pathway
have recently been obtained from microorganisms and plants.
1-Deoxy-D-xylulose 5-phosphate synthase, specified by the dxs
gene, catalyzes the formation of 1-deoxy-D-xylulose 5-phosphate
Terpenes are one of the largest groups of natural products,
and include numerous medically important compounds such as
cholesterol, vitamin A, carotenoids, and paclitaxel (Taxol).1 In
animals, fungi, and archaea, the universal terpene precursors,
isopentenyl pyrophosphate and dimethylallyl pyrophosphate, are
biosynthesized via the mevalonate pathway, which has been
studied in considerable detail.2-5
More recently, the research groups of Rohmer and Arigoni
independently observed 13C-labeling patterns in terpenoids of
certain bacteria such as Rhodopseudomonas palustris and
* Address correspondence to this author. Phone: +49-89-289-13360.
Fax: +49-89-289-13363. E-mail: adelbert.bacher@ch.tum.de.
† Technische Universita¨t Mu¨nchen.
(6) Flesch, G.; Rohmer, M. Eur. J. Biochem. 1988, 175, 405-411.
(7) Broers, S. T. J. Ph.D Thesis Nr. 10978, ETH Zu¨rich, Switzerland,
1994.
(8) Rohmer, M.; Knani, M.; Simonin, P.; Sutter, B.; Sahm, H. Biochem.
J. 1993, 295, 517-524.
(9) Eisenreich, W.; Schwarz, M.; Cartayrade, A.; Arigoni, D.; Zenk, M.;
Bacher, A. Chem. Biol. 1998, 5, R221-R233.
(10) Rohmer, M. Nat. Prod. Rep. 1999, 16, 565-574.
(11) Rohmer, M. ComprehensiVe Natural Products Chemistry; Barton,
D., Nakanishi, K., Eds.; Pergamon: Oxford, 1999; Vol. 2, pp 45-68.
(12) Schwarz, M. K.; Arigoni, D. ComprehensiVe Natural Products
Chemistry; Barton, D., Nakanishi, K., Eds.; Pergamon: Oxford, 1999; Vol.
2, pp 367-400.
‡ Universita¨t Halle.
(1) Saccettini, J. C.; Poulter, C. D. Science 1997, 277, 1788-1789.
(2) Qureshi, N.; Porter, J. W. Biosynthesis of Isoprenoid Compounds;
Porter, J. W., Spurgeon, S. L., Eds.; John Wiley: New York, 1981; Vol. 1,
pp 47-94.
(3) Bloch, K. Steroids 1992, 57, 378-382.
(4) Bach, T. J. Lipids 1995, 30, 191-202.
(5) Bochar, D. A.; Friesen, J. A.; Stauffacher, C. V.; Rodwell, V. W.
ComprehensiVe Natural Product Chemistry; Barton, D., Nakanishi, K., Eds.;
Pergamon: Oxford, 1999; Vol. 2, pp 15-44.
10.1021/ja001385o CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/23/2000

9572 J. Am. Chem. Soc., Vol. 122, No. 40, 2000
Rohdich et al.
(7) from pyruvate (6) and D-glyceraldehyde 3-phosphate (5).13-16
The enzyme product is converted into 2C-methyl-D-erythritol
4-phosphate (8) by a reductoisomerase specified by the dxr gene
of Escherichia coli.17 The branched polyol 8 is converted into
4-diphosphocytidyl-2C-methyl-D-erythritol (9) by the catalytic
action of the 4-diphosphocytidyl-2C-methyl-D-erythritol syn-
thase specified by the ispD (ygbP) gene of E. coli.18
Scheme 1
Isotope-labeled precursors were crucial in the relatively short
history of the investigation of the alternative terpenoid pathway
which involved the reassessment of numerous erroneous claims
on the universality of the mevalonate pathway. The possibility
to prepare 9 in isotope-labeled form in a very high overall yield
should benefit future studies in the area. More specifically, 13C-
labeled 9 will be helpful for in vivo and in vitro studies
identifying further downstream metabolites as well as the
mechanisms of the novel nonmevalonate pathway enzymes.
Results
This paper describes the use of recombinant enzymes of the
nonmevalonate isoprenoid pathway for the efficient preparation
of 13C- and 14C-labeled 4-diphosphocytidyl-2C-methyl-D-eryth-
ritol (9) in millimole quantity. The synthesis can be performed
as a one-pot reaction using D-glyceraldehyde 3-phosphate (5)
and pyruvate (6) as starting materials (Scheme 1). Using the
methods described below, a wide variety of isotopomers of 9
could also be prepared from commercially available starting
materials such as glucose (1), respectively, pyruvate (6) labeled
with 13C or 14C (Table 1).
Unlabeled dihydroxyacetone phosphate (4) was obtained from
dimeric dihydroxyacetone phosphate ethyl hemiacetal (3)19 and
was further converted into unlabeled 5 by the catalytic action
of triose phosphate isomerase. Isotope-labeled samples of 5 were
obtained from isotopomers of 1 using enzymes of the glycolytic
pathway which are commercially available. Crude 5 from these
respective starting materials could be converted enzymatically
into 9 without prior isolation.
The enzyme-assisted synthetic reactions can be monitored
conveniently in real time by 13C NMR when 13C-labeled starting
materials are used, since the label enhances both the sensitivity
and the selectivity of NMR diagnosis. Figure 1 shows the
relevant sections of 13C NMR spectra from an experiment for
the preparation of [1,2,2-methyl,3,4-13C5]-9 from [U-13C6]-1 and
[2,3-13C2]-6. Spectrum A was obtained at the start of the enzyme
reaction, and spectrum B was recorded prior to the addition of
1-deoxy-D-xylulose 5-phosphate reductoisomerase. In Figure 1B,
signals reflecting the 13C-labeled starting materials ([U-13C6]-
glucose and [2,3-13C2]pyruvate) have virtually disappeared, and
the spectrum is dominated by the signals of [U-13C5]1-deoxy-
D-xylulose 5-phosphate (7). The resulting [U-13C5]-7 can be
further converted into [1,2,2-methyl,3,4-13C5]-9, without prior
isolation, by the sequential addition of 1-deoxy-D-xylulose
(13) Sprenger, G. A.; Scho¨rken, U.; Wiegert, T.; Grolle, S.; deGraaf, A.
A.; Taylor, S. V.; Begley, T. P.; Bringer-Meyer, S.; Sahm, H. Proc. Natl.
Acad. Sci. U.S.A. 1997, 94, 12857-12862.
Figure 1. Parts of 13C NMR spectra of a mixture containing [U-13C6]-
1 and [2,3-13C2]-6 prior to the addition of enzymes (A) and after
incubation with hexokinase, glucose 6-phosphate isomerase, fructose
6-phosphate kinase, aldolase, triose phosphate isomerase, and 1-deoxy-
D-xylulose 5-phosphate synthase (B). Signals arising from glucose (1),
pyruvate (6), and 1-deoxy-D-xylulose 5-phosphate (7) are indicated by
G, P, and D, respectively. T ) Tris hydrochloride.
(14) Lois, L. M.; Campos, N.; Putra, S. R.; Danielsen, K.; Rohmer, M.;
Boronat, A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 2105-2110.
(15) Lange, B. M.; Wildung, M. R.; McCaskill, D.; Croteau, R. Proc.
Natl. Acad. Sci. U.S.A. 1998, 95, 2100-2104.
(16) Kuzuyama, T.; Takagi, M.; Takahashi, S.; Seto, H. J. Bacteriol.
2000, 182, 891-897.
(17) Takahashi, S.; Kuzuyama, T.; Watanabe, H.; Seto, H. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 9879-9884.
(18) Rohdich, F.; Wungsintaweekul, J.; Fellermeier, M.; Sagner, S.; Herz,
S.; Kis, K.; Eisenreich, W.; Bacher, A.; Zenk, M. H. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 11758-11763.
5-phosphate reductoisomerase and 4-diphosphocytidyl-2C-meth-
yl-D-erythritol synthase, together with their respective cosub-
strates.
(19) Mizuno, M.; Masuda, S.; Takemaru, K.; Hosono, S.; Sato, T.;
Takeuchi, M.; Kobayashi, Y. Microbiology 1996, 142, 3103-3111.
The overall yield of [1,2,2-methyl,3,4-13C5]-9 was 60% based

Biosynthesis of Isoprenoids
J. Am. Chem. Soc., Vol. 122, No. 40, 2000 9573
Table 1. Preparation of 13C-Labeled
4-Diphosphocytidyl-2C-methyl-D-erythritol (9) from Pyruvate (6),
Glucose (1), and Glyceraldehyde 3-Phosphate (5)
The enzymes used in this study are either commercially available
(hexokinase, glucose 6-phosphate isomerase, fructose 6-phos-
phate kinase, aldolase, and glucose dehydrogenase) or can be
prepared by recombinant DNA techniques.13,14,16,17
C2 precursor
C3 precursor
product
[2,3-13C2]-6
[2,3-13C2]-6
6
[U-13C6]-1
5
[1,2-methyl,3,4-13C5]-9
[2,2-methyl-13C2]-9
[1,3,4-13C1]-9
One limitation for the scale-up of the procedure is the
necessity to purify the final product by chromatographic
procedures. The purification of 1 mmol of 9 using semiprepara-
tive HPLC columns (2 × 25 cm) requires about 15 h. The
efficient purification of larger batches would be possible using
preparative scale HPLC columns.
[1,2,3-13C1]-1a)
a Equimolar mixture of [1-13C1]-, [2-13C1]-, and [3-13C1]-1.
Table 2. 13C NMR Data of 13C-Labeled 9 Using D2O as Solvent
[2,2-
[1,2,2-
The method can be scaled down to very small volumes to
prepare radiolabeled samples of high specific activity. Thus,
radioactive labels can be introduced into 9 virtually without
dilution. As an example, 7.4 µmol of [2-14C]-9 have been
prepared from [2-14C]pyruvate at a specific activity of 15.8 mCi
mmol-1 with a yield of 47%.
Synthetic procedures for the preparation of intermediates of
the nonmevalonate pathway were reported in the literature for
1-deoxy-D-xylulose 5-phosphate (7)21,22 and 2C-methyl-d-eryth-
ritol 4-phosphate (8).23,24 The chemical synthesis of 8 involves
14 steps23 and can be used for the preparation of the [1-3H]-
isotopomer with high specific activity, but the introduction of
isotopes at other positions would not be achieved easily. In
contrast, a wide variety of isotopomers carrying stable or
radioactive labels in the methylerythritol moiety of 9 can be
obtained with the method reported in this paper.
methyl-13C2]-9 [1,3,4-13C1]-9 methyl,3,4-13C5]-9
δ
13C
position ppm JCH Hz JCC Hz JCH Hz JCP Hz JCH Hz JCC Hz JCP Hz
1
2
66.25
73.75
141
142 40
n.d.a
127 38
135 n.d.
135 39
40
40
2-methyl 18.13 127
3
4
73.28
66.86
n.d.
n.d.
7
6
n.d.
5
a n.d., not determined due to signal overlapping.
Experimental Section
Materials. ATP, CTP, NADPH, and TPP were purchased from
Sigma (Deisenhofen, Germany). [1-13C1]-, [2-13C1]-, [3-13C1]-, and
[U-13C6]glucose were obtained from Omicron, South Bend, IN. [2,3-
13C2]Pyruvate was purchased from Isotec, Miamisburg, OH. [2-14C]-
Pyruvate was obtained from NEN, Boston, MA.
Proteins. Hexokinase from yeast (E.C.2.7.1.1), glucose 6-phosphate
isomerase from yeast (1.1.1.49), fructose 6-phosphate kinase from rabbit
muscle (E.C.2.7.1.11), aldolase from rabbit muscle (E.C.5.3.1.1),
glucose dehydrogenase from Bacillus megaterium (E.C.1.1.1.47), triose
phosphate isomerase from rabbit muscle (E.C.5.3.1.1), and inorganic
pyrophosphatase from E. coli (E.C.3.6.1.1.) were purchased from Sigma
(Deisenhofen, Germany). Recombinant 4-diphosphocytidyl-2C-methyl-
D-erythritol synthase from E. coli was prepared by a published
procedures.18 The preparation of recombinant 1-deoxy-D-xylulose
5-phosphate synthase from B. subtilis and recombinant 1-deoxy-D-
xylulose 5-phosphate reductoisomerase from E. coli will be published
elsewere.
General Procedures. NMR spectra were obtained using a Bruker
AVANCE DRX 500 spectrometer (Karlsruhe, Germany). Chemical
shifts were referenced to external trimethylsilylpropane sulfonate or
85% ortho-phosphoric acid, respectively. UV/VIS spectra were deter-
mined with an Ultrospec 2000 spectrometer (Amersham Pharmacia
Biotech, Heidelberg, Germany). ESI mass spectra were recorded on a
LCQ Finnigan mass spectrometer (San Jose, CA) in the negative ion
detection mode. Optical rotation was measured on a Perkin-Elmer 241
MC polarimeter (Weiterstadt, Germany).
Crude [U-13C5]1-Deoxy-D-xylulose 5-Phosphate. A reaction mix-
ture containing 120 mM Tris hydrochloride, pH 8.0, 6 mM MgCl2, 31
mM ATP, 1.6 mM thiamine pyrophosphate, 15 mM [U-13C6]glucose,
30 mM [2,3-13C2]sodium pyruvate, 174 U of hexokinase, 106 U of
glucose 6-phosphate isomerase, 48 U of fructose 6-phosphate kinase,
26 U of aldolase, 284 U of triose phosphate isomerase, and 2.8 mg
Figure 2. 13C NMR signals of [1,2,2-methyl,3,4-13C5]-9 (A), [1,3,4-
13C1]-9 (B), and [2,2-methyl-13C2]-9 (C). 13C13C and 13C31P coupling
constants are given in Table 2.
on 13C-labeled 1. [2,2-Methyl-13C2]-9 and [1,3,4-13C1]-9 were
prepared from [2,3-13C2]-6 and [1,2,3-13C1]-1, respectively, in
similar yields. The purity of the compounds was assessed by
NMR (Table 2, Figure 2) and mass spectrometry.
No specific adaptations are required for the preparation of
different isotopomers since a wide variety of 13C-labeled
pyruvate and glucose isotopomers are commercially available.
(20) Effenberger, F.; Straub, A. Tetrahedron Lett. 1987, 28, 1641.
(21) Thiel, R.; Adam, K.-P. Tetrahedron Lett. 1999, 40, 5307-5308.
(22) Proteau, P. J.; Woo, Y.-H.; Williamson, R. T.; Phaosiri, C. Org.
Lett. 1999, 1, 921-923.
(23) Kis, K.; Wungsintaweekul, J.; Eisenreich, W.; Zenk, M. H.; Bacher,
A. J. Org. Chem. 2000, 65, 587-592.
(24) Koppisch, A. T.; Blagg, B. S. J.; Poulter, C. D. Org. Lett. 2000, 2,
215-217.

9574 J. Am. Chem. Soc., Vol. 122, No. 40, 2000
Rohdich et al.
(6.2 U) of 1-deoxy-D-xylulose 5-phosphate synthase in a total volume
of 122 mL was incubated at 37 °C. The pH was maintained at 8.0 by
the addition of 1 M NaOH. The formation of 1-deoxy-D-xylulose
5-phosphate was monitored by 13C NMR and was found to be virtually
complete after 22 h.
Pure 9 was obtained as a white powder with an overall yield of about
60%. Analytical data of 9 were as follows: ꢀ268 ) 7410 M-1 cm-1
(pH 7.0); [R]589, ) 9.5° (c 0.3 g 100 mL-1 in H2O). Mass spectrom-
etry: 9 (unlabeled), m/e 520.4; [1,2,2-methyl,3,4-13C5]-9, m/z 525.6;
[2,methyl-13C2]-9, m/z 522.6; [1,3,4-13C1]-9, m/z 521.4.
Crude [3,4,5-13C1]1-Deoxy-D-xylulose 5-Phosphate. [3,4,5-13C1]-
7 was prepared as described above with an equimolar mixture of
[1-13C1]-, [2-13C1]-, [3-13C1]glucose and unlabeled pyruvate as starting
materials.
1H, 13C, and 31P NMR data agree with previously published results.18
More specifically, the presence of a cytidyl moiety was gleaned from
1
the H NMR data. The 31P NMR spectrum displayed two doublets at
-7.2 and -7.8 ppm with 31P31P couplings of 20 Hz reflecting the
diphospho moiety. The 13C NMR data and the coupling patterns of
[2,2-methyl-13C2]-, [1,2,2-methyl,3,4-13C5]-, and [1,3,4-13C1]-9 are sum-
marized in Table 2.
Crude [1,2-13C2]1-Deoxy-D-xylulose 5-Phosphate. Dimeric dihy-
droxyacetone phosphate ethyl hemiacetal (barium salt, 1.9 g, 3.2 mmol)
was treated with 19 mL of Dowex 50 WX8 resin (H+-form) in 44 mL
of water at 65 °C for 4 h. The resin was filtered off, and the pH of the
solution was adjusted to 8 by the addition of 1 M NaOH.19 The solution
was added to 70 mL of a solution containing 150 mM Tris hydrochlo-
ride, pH 8.0, 57 mM sodium [2,3-13C2]pyruvate, 10 mM MgCl2, and
2.5 mM thiamine pyrophosphate. Triose phosphate isomerase (17 000
U) was added, and the solution was incubated for 105 min at 37 °C.
1-Deoxy-D-xylulose 5-phosphate synthase (3.3 mg, 7.4 U) was added.
The reaction was found to be virtually complete after 8 h.
[2,2-methyl-13C2]-, [1,2,2-methyl,3,4-13C5]-, and [1,3,4-13C1]4-
Diphosphocytidyl-2C-methyl-D-erythritol. Crude [U-13C5]-, [3,4,5-
13C1]-, or [1,2-13C2]-7 obtained by either of the two procedures described
above can be used for the following steps without purification. Glucose
(0.66 g, 3.7 mmol), 46 U of glucose dehydrogenase, 51.5 mg of NADP+
(0.5 mmol), and 4 mM MgCl2 were added to a solution containing
approximately 3.7 mmol of 7. The mixture was incubated for 5 min at
37 °C, and a solution (1.4 mL) containing 2.6 mg (18 U) of 1-deoxy-
D-xylulose 5-phosphate reductoisomerase was added. The formation
of 13C-labeled 8 was monitored by 13C NMR and was found to be
virtually complete after 18 h.
CTP (2.0 g, 3.6 mmol) was added. The pH of the solution was
brought to 8.0 with 2 N NaOH. A solution (3 mL) containing
4-diphosphocytidyl-2C-methyl-D-erythritol synthase (2.1 mg, 51 U) was
added. The reaction mixture was incubated for 15 h and was then
lyophilized.
The crude lyophilized product (8 g) was dissolved in 80 mL of 100
mM ammonium formate containing 40% methanol. Aliquots of several
milliliters were placed on a HPLC column of Nucleosil SB10 (Macherey
& Nagel, Germany; 2 × 25 cm) which was developed with 100 mM
ammonium formate containing 40% methanol at a flow rate of 10 mL
min-1. The effluent was monitored photometrically (270 nm). The
retention volume of 9 was about 150 mL. Fractions were combined
and lyophilized. One millimole of product could be processed in about
25 runs requiring a total time of about 15 h.
[2-14C]4-Diphosphocytidyl-2C-methyl-D-erythriol. A reaction mix-
ture containing 150 mM Tris hydrochloride, 5 mM dithiothreitol, 21.7
mM dihydroxyacetone phosphate, 17 mM NADPH, 1.6 mM thiamine
pyrophosphate, 18.8 mM CTP, 15.8 mM [2-14C]pyruvate (15.8 mCi
mmol-1), 406 U of triose phosphate isomerase, 1.3 U of inorganic
pyrophosphatase, 170 µg (0.4 U) of 1-deoxy-D-xylulose 5-phosphate
synthase from B. subtilis, 70 µg (0.5 U) of 1-deoxy-D-xylulose
5-phosphate reductoisomerase from E. coli, and 100 µg (2.4 U) of
4-diphosphocytidyl-2C-methyl-D-erythritol synthase from E. coli in a
total volume of 1 mL was incubated at 37 °C. At intervals of 1 h,
aliquots (50 µL) of the reaction mixture were retrieved and applied to
a Multospher 120 RP 18-5 column (4.6 × 250 mm, CS-Chromatog-
raphie Service GmbH, Langerwehe, Germany) which had been
equilibrated with 10 mM tetra-n-butylammonium hydrogen sulfate, pH
6.0, at a flow rate of 0.75 mL min-1. The column was developed with
15 mL of 10 mM tetra-n-butylammonium hydrogen sulfate, pH 6.0,
followed by a linear gradient of 0-42% (v/v) methanol in 45 mL of
10 mM tetra-n-butylammonium hydrogen sulfate, pH 6.0. The effluent
was monitored using a radiodetector (Beta-RAM, Biostep GmbH,
Jahnsdorf, Germany). The retention volume of [2-14C]-9 was 29 mL.
After 8 h the reaction was stopped by passing the solution through a
Nanosep 10 K membrane (Pall Gelman, Rondorf, Germany). Crude
[2-14C]-9 was purified by semipreparative HPLC using a column of
Nucleosil SB5 (20 × 250 mm, CS-Chromatographie Service GmbH,
Langerwehe, Germany). The column was equilibrated and developed
with 100 mM ammonium formate as eluent at a flowrate of 5 mL min-1
.
The effluent was monitored using a radiodetector. The retention volume
of [2-14C]-9 was 600-650 mL. Fractions were combined and lyo-
philized. [2-14C]-9 (15.8 mCi mmol-1) (7.4 µmol) was obtained from
[2-14C]pyruvate with a yield of 47%.
Acknowledgment. This work was supported by the Deutsche
Forschungsgemeinschaft, the Fonds der Chemischen Industrie,
and the Hans-Fischer-Gesellschaft. We thank Angelika Werner
and Fritz Wendling for help with the preparation of the
manuscript.
The lyophilized powder (1.5 g) was dissolved in 15 mL of 10 mM
ammonium formate. Aliquots of 1 mL were placed on a semipreparative
HPLC column of Nucleosil 10 RP18 (Macherey & Nagel, Germany; 2
× 25 cm) which was developed with 10 mM ammonium formate at a
flow rate of 6 mL. The retention volume was 60 mL. Fractions were
combined and lyophilized repeatedly to remove ammonium formate.
JA001385O