Practical synthesis of hydroxamate-derived siderophore components by an indirect oxidation method and syntheses of a DIG-siderophore conjugate and a biotin-siderophore conjugate

Article abstract of DOI:10.1021/jo990769y

A practical large-scale synthesis of hydroxamate-derived siderophore components (30 and 40) that utilizes an efficient indirect oxidation method is described and applied to the syntheses of nonradioactive labeled siderophores. Oxidation of imines derived from L-ornithine (17) and its tripeptide (19) afforded oxaziridines that were isomerized to stable nitrones (16 and 18). Acid-catalyzed hydrolysis of nitrones provided hydroxylamines that were converted to the desired hydroxamic acids (30 and 40) suitable for constructing siderophore-drug conjugates (2). The entire synthetic sequence required no chromatographic separation. DIG- and biotin-labeled ferrichrome analogues designed to detect and isolate ferrichrome receptors in various microbes were also synthesized.

Full text of DOI:10.1021/jo990769y

J . Org. Chem. 1999, 64, 7451-7458
7451
P r a ctica l Syn th esis of Hyd r oxa m a te-Der ived Sid er op h or e
Com p on en ts by a n In d ir ect Oxid a tion Meth od a n d Syn th eses of a
DIG-Sid er op h or e Con ju ga te a n d a Biotin -Sid er op h or e Con ju ga te
Yun-Ming Lin and Marvin J . Miller*
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556
Received May 10, 1999
A practical large-scale synthesis of hydroxamate-derived siderophore components (30 and 40) that
utilizes an efficient indirect oxidation method is described and applied to the syntheses of
nonradioactive labeled siderophores. Oxidation of imines derived from L-ornithine (17) and its
tripeptide (19) afforded oxaziridines that were isomerized to stable nitrones (16 and 18). Acid-
catalyzed hydrolysis of nitrones provided hydroxylamines that were converted to the desired
hydroxamic acids (30 and 40) suitable for constructing siderophore-drug conjugates (2). The entire
synthetic sequence required no chromatographic separation. DIG- and biotin-labeled ferrichrome
analogues designed to detect and isolate ferrichrome receptors in various microbes were also
synthesized.
In tr od u ction
siderophore-drug conjugates. They all contain the sid-
erophore component derived from the tripeptide of N5-
hydroxy-N5-acetyl-L-ornithine, the common iron chelator
in most hydroxamate-derived siderophores. The cytotoxic
unit is connected to the iron chelator via a serine spacer.
We have been engaged in the synthesis of siderophore-
drug conjugates (2) for biological evaluation.5 In a previ-
ous study, we found that a siderophore component
derived from the tripeptide of N5-hydroxy-N5-acetyl-L-
ornithine was recognized by certain microbes and actively
transported inside the cell. This was determined by the
growth-promoting ability of the siderophore for S. flexneri
SA 240.6 Our current strategy for active drug transport
focuses on the connection of drugs to siderophores. These
siderophore-drug conjugates (2) could be recognized by
microbes and transported inside the cell by the iron
uptake system. Once inside the cell, the drug could be
released and deliver an effective dosage to the microbes.
This strategy would overcome the cell permeability
problem and thus has great potential for the development
of a new generation of antibacterial and antifungal
agents.
Siderophores are secreted by organisms as microbial
iron transport agents to solubilize iron(III) under iron-
deficient conditions.1 Hydroxamate-based siderophores,
such as albomycins (1, Figure 1), contain ligands derived
from the tripeptide of N5-hydroxy-N5-acetyl-L-ornithine.
These compounds form strong, selective complexes with
Fe(III). The siderophore-iron complex is recognized by
outer membrane receptors and actively transported into
the cell. Because iron is a limiting nutrient at the onset
of microbial infection, siderophores are essential for
microbial pathogenicity.2
Effective drug delivery is becoming increasingly im-
portant because of the emergence of multidrug-resistant
pathogens. Bacteria have developed the ability to limit
the permeability of their cell walls, to make proteins that
pump drugs rapidly out of their cells, to acquire enzymes
to deactivate drugs, and to alter the drugs' targets.3 Lack
of effective drug delivery to the target cell due to cell wall
permeability is partially responsible for current drug
resistance.
A unique way to combat the cell permeability problem
is to use the cell’s natural mechanism for nutrient
assimilation to smuggle drugs into cells. This “Trojan
Horse” approach in drug delivery is being rigorously
studied in our laboratory.4 Siderophore-drug conjugates
(2, Figure 1) are composed of iron chelators, chemical
linkers, and biologically active components (“drug”).
Albomycins, antibiotics produced by Streptomyces griseus
and Streptomyces substropicus, are examples of natural
(4) (a) Miller, M. J .; Malouin, F. Acc. Chem. Res. 1993, 26, 241. (b)
Miller, M. J .; Malouin, F.; Dolence, E. K.; Gasparski, C. M.; Ghosh,
M.; Guzzo, P. R.; Lotz, B. T.; McKee, J . A.; Minnick, A. A.; Teng, M.
Iron Transport-Mediated Drug Delivery. In Recent Advances in the
Chemistry of Antiinfective Agents; Bently, P. H., Ponsford, R., Eds.;
Special Publication No. 119; Royal Society of Chemistry: Royal Society
of Chemistry, Cambridge, 1993; Chapter 10. (c) Miller, M. J .; Malouin,
F. Siderophore-Mediated Drug Delivery. The Design, Synthesis and
Study of Siderophore-Antibiotic and Antifungal Conjugates. In The
Development of Iron Chelators for Clinical Use; Bergeron, R. J .,
Brittenham, G. M., Eds.; CRC Press: Boca Raton, FL, 1994; Chapter
13.
* To whom correspondence should be addressed. Tel: (219) 631-
7571. Fax: (219) 631-6652. E-mail: Marvin.J .Miller.2@nd.edu.
(1) (a) Neilands, J . B. J . Biol. Chem. 1995, 270, 26723. (b) Koster,
W.; Braun, V. J . Biol. Chem. 1990, 265, 21407.
(2) (a) Wooldridge, K., G., Williams, P. H. FEMS Microbiol. Rev.
1993, 12, 325. (b) Neilands, J . B. Biochem. Biophys. 1993, 302, 1.
(3) (a) Connell, N. D.; Nikaido, H. Membrane Permeability and
Transport in Mycobacterium tuberculosis. In Tuberculosis: Pathogen-
esis, Protection, and Control; Bloom, B. R., Ed.; ASM Press: Wash-
ington, DC, 1994; Chapter 22. (b) Liu, J .; Rosenberg, E. Y.; Nikaido,
H. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 11254.
(5) (a) Diarra, M. S.; Lavoie, M. C.; J acques, M.; Darwish, I.; Dolence,
E. K.; Dolence, J . A.; Ghosh, A.; Ghosh, M.; Miller, M. J .; Malouin, F.
Antimicrob. Agents Chemother. 1996, 40, 2610. (b) Dolence, E. K.;
Minnick, A. A.; Miller, M. J . J . Med. Chem. 1990, 33, 461. (c) Ghosh,
A.; Miller, M. J . J . Org. Chem. 1993, 58, 7652. (d) McKee, J . A.;
Sharma, S. K.; Miller, M. J . Bioconjugate Chem. 1991, 2, 281. (e)
Ramurthy, S.; Miller, M. J . J . Org. Chem. 1996, 61, 4120. (f) Ghosh,
M.; Miller, M. J . J . Org. Chem. 1994, 59, 1020. (g) Dolence, E. K.;
Minnick, A. A.; Lin, C.-E.; Miller, M. J . J . Med. Chem. 1991, 34, 968.
(6) Dolence, E. K.; Lin, C.-E.; Miller, M. J . J . Med. Chem. 1991, 34,
956.
10.1021/jo990769y CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/15/1999

7452 J . Org. Chem., Vol. 64, No. 20, 1999
Lin and Miller
F igu r e 1. Albomycins and siderophore-drug conjugates.
Sch em e 1
Sch em e 2
11 was therefore limited by the generation and utilization
of DMD solution in higher concentration. Benzoyl peroxide-
mediated oxidation of ω amino group of protected lysine
was employed in Bergeron’s total synthesis of nanno-
chelin A.11 This oxidation protocol was recently improved
by Phanstiel.12
Hydroxamic acids are also essential iron(III) chelators
for hydroxamate-derived siderophores. Thus, efficient
and rapid access to large quantities of hydroxamates are
highly desirable not only for the syntheses of a sidero-
phore-drug conjugates library, but also for the syn-
theses of natural siderophore analogues such as myco-
bactin analogues.7 Our goal was to develop a practical
synthesis in which primary amines would be converted
to the corresponding hydroxylamines and hydroxamic
acids.
Hydroxamates were synthesized previously in this
laboratory by N-alkylation of simple O-substituted hy-
droxamic acid 3 with a variety of alkylating agents.
Alternatively, formation of oxime 7 from aldehyde 6 and
O-substituted hydroxylamine followed by subsequent
reduction and in situ acylation produced the suitably
protected hydroxamic acid derivatives 8 (Scheme 1).8
These methods allowed us to synthesize several sidero-
phores and many analogues.9
Most recently, hydroxylamines and hydroxamic acids
were synthesized by direct oxidation of lysine and orni-
thine using dimethyldioxarine (DMD) (Scheme 2) that
led to the efficient total synthesis of mycobactins and
analogues.10 Oxidation of lysine methyl ester (9) with
DMD provided nitrone 10 that was converted to hydrox-
amic acid (11) in two steps. Practical large-scale synthesis
of hydroxamate-derived siderophore components such as
In 1978, Naegeli and Keller-Schierlein reported an
elegant total synthesis of D-ferrichrome by an indirect
oxidation method (Scheme 3).13 Their indirect oxidation
featured the conversion of primary amines to imines
followed by oxidation of imines to oxaziridines. Acid-
catalyzed hydrolysis of oxaziridines afforded hydroxy-
lamines which led to the successful synthesis of D-fer-
richrome. This indirect oxidation is an attractive approach
for the synthesis of hydroxamate-derived siderophore
components from amino acids and peptides since more
than one amino group could be oxidized simultaneously.
Furthermore, it should be possible to scale-up this
oxidation process. We therefore adopted this indirect
oxidation strategy and have found that alternate genera-
tion of intermediate nitrone derivatives provides distinct
advantages for large scale, practical syntheses of es-
sential hydroxylamine and hydroxamate components of
siderophores.
Herein, we would like to report an efficient scalable
synthesis of hydroxylamines and hydroxamic acids start-
ing from N2-protected L-ornithine (17) and its tri-
peptide (19) through nitrone intermediates (16 and 18)
by an indirect oxidation method (Scheme 4). The
syntheses of biotin-siderophore conjugate (20, Figure 2)
and DIG-siderophore conjugate (21, Figure 2) suitable
for detecting receptor proteins (vida infra) are also
described.
(7) Xu, Y.; Miller, M. J . J . Org. Chem. 1998, 63, 4314.
(8) Miller, M. J . Chem. Rev. 1989, 89, 1563
(9) (a) Lee, B. H.; Miller, M. J . J . Org. Chem. 1983, 48, 24. (b) Lee,
B. H.; Miller, M. J .; Prody, C. A.; Neilands, J . B J . Med. Chem. 1985,
28, 317. (c) Lee, B. H.; Miller, M. J .; Prody, C. A.; Neilands, J . B.J .
Med. Chem. 1985, 28, 323. (d) Lee, B. H.; Gerfen, G. J .; Miller, M. J .
J . Org. Chem. 1984, 49, 2418. (e) Maurer, P. J .; Miller, M. J . J . Am.
Chem. Soc. 1982, 104, 3096. (f) Maurer, P. J .; Miller, M. J . J . Am.
Chem. Soc. 1983, 105, 240. (g) Maurer, P. J .; Miller, M. J . J . Org. Chem.
1981, 46, 2835.
(11) (a) Bergeron, R. J .; Phanstiel IV, O. J . Org. Chem. 1992, 57,
7140. (b) Milewska, M. J .; Chimiak, A. Synthesis 1990, 233.
(12) (a) Wang, Q. X.; King, J .; Phanstiel IV, O. J . Org. Chem. 1997,
62, 8104. (b) Phanstiel, IV, O.; Wang, Q. X.; Powell, D. H.; Ospina, M.
P.; Leeson, B. A. J . Org. Chem. 1999, 64, 803.
(10) (a) Hu, J .; Miller, M. J . J . Org. Chem. 1994, 59, 4858. (b) Hu,
J .; Miller, M. J . J . Am. Chem. Soc. 1997, 119, 3462.
(13) Naegeli, H.-U.; Keller-Schierlein, W. Helv. Chim. Acta 1978,
61, 2088.

Synthesis of Hydroxamate-Derived SiderophoreComponents
J . Org. Chem., Vol. 64, No. 20, 1999 7453
Sch em e 3
Figu r e 2. DIG-siderophore conjugate and biotin-ferrichrome
analogue.
Sch em e 5
Sch em e 4
fective when 17 was treated with 1 equiv of potassium
hydroxide in the presence of 3 Å molecular sieves.14
Oxidation of the resulting imine with m-CPBA under
basic conditions proceeded rapidly and gave oxaziridine
27. Oxaziridine 27 was hydrolyzed to provide the corre-
sponding hydroxylamine 28 under conditions similar to
those described in the model study.
During a scale-up reaction, however, a substantial
amount (ca. 50%) of nitrone 16 was isolated as a white
solid from the reaction mixture. We, therefore, decided
to intentionally isomerize the unstable oxaziridine 27 to
nitrone 16. Thus, isomerization of crude oxaziridine 27
with TFA in methylene chloride afforded nitrone 16 in
80% overall yield from 17 after recrystallization. Al-
though this isomerization added one step to the synthe-
sis, it offered the important advantage of a stable
protected hydroxylamine equivalent that was easily
crystallized. This indirect oxidation process has been
carried out on 30 g of N2-Cbz-L-ornithine 17 to afford 33
g of nitrone 16. In contrast to hydroxylamine 28, which
is susceptible to decomposition, oxidation, and dispro-
portionation reactions,15 nitrone 16 can be stored at room
temperature indefinitely without any decomposition.
Resu lts a n d Discu ssion
To examine the feasibility and practical aspects of this
indirect oxidation approach, we conducted model studies
on 6-aminocaproic acid. As shown in Scheme 5, imine
formation of 6-aminocaproic acid (22) with benzaldehyde
was effective when refluxed in toluene with removal of
water. The resulting imine 23 was oxidized with m-
chloroperoxybenzoic acid (m-CPBA) to give oxaziridine
24 in 81% overall yield.
Oxaziridine 24 was hydrolyzed to hydroxylamine 25
under acid-catalyzed conditions. Benzaldehyde byproduct
and other organic impurities were readily removed by
extraction. Concentration of the aqueous solution thus
provided pure hydroxylamine 25. Subsequent acetylation
of 25 with acetyl chloride afforded hydroxamic acid 26
in 52% overall yield from oxaziridine 24. It is noteworthy
that the entire process required only one column chro-
matographic purification in the last step and it did not
require the protection of the carboxyl group.
Indirect oxidation of N2-Cbz-L-ornithine 17 was then
attempted. Although the benzaldehyde-derived imine was
not formed in refluxing toluene, it was obtained in
quantitative yield under basic conditions. As shown in
Scheme 6, imine formation with benzaldehyde was ef-
(14) Zhang, L.; Finn, J . M. J . Org. Chem. 1995, 60, 5719.
(15) Ottenheijm, H. C. J .; Herscheid, J . D. M. Chem. Rev. 1986, 86,
697.

7454 J . Org. Chem., Vol. 64, No. 20, 1999
Lin and Miller
Sch em e 6
Sch em e 7
Nitrone 16 is an advanced intermediate in preparing
exochelin MN16 and analogues.
Acid-catalyzed hydrolysis of nitrone 16 afforded hy-
droxylamine 28 in 84% yield after removal of benzalde-
hyde by extraction. The acetylation of hydroxylamine 28
was then attempted. Under standard acetylation condi-
tions (e.g, acetyl chloride and base in acetonitrile), the
free carboxylic group was readily activated and led to a
mixture of products due to an intramolecular cyclization.
Thus, other reaction conditions were examined in order
to effect the selective acetylation of the hydroxylamine
functional group without the need for protection of the
carboxylic acid. At pH 4, the hydroxylamine functional
group is nucleophilic and acetic anhydride is activated
by acid catalysis. This would lead to acceleration of the
acetylation reaction of hydroxylamine. The activation of
the carboxyl functionality of 28 would be minimized.
Treatment of hydroxylamine 28 with acetic anhydride
at pH 4 resulted in selective N,O-diacetylation and
provided hydroxamate 29 in 95% yield.
Hydroxamate 29 is hydrophobic and, therefore, easily
extracted from the reaction mixture in pure form. The
new acetylation conditions avoided the last-step column
chromatograhic separation that was required in the
model system. Finally, treatment of hydroxamate 29 with
Hunig’s base in methanol afforded desired hydroxamic
acid 30 as the corresponding salt in quantitative yield.
It is noteworthy that this indirect oxidation process does
not require any chromatographic purification or carboxyl
protection.
phore components of albomycins by this indirect oxida-
tion procedure, an appropriately protected ornithine
tripeptide was required. A partially protected tripeptide
35 was synthesized under active ester-mediated coupling
conditions18 from N2-Cbz-N5-Boc-L-ornithine (33) and N5-
Boc-L-ornithine in an iterative approach. Thus, the
N-hydroxysuccinimide (NHS) active ester of 33 was
synthesized by treating 33 and NHS with dicyclohexyl-
carbodiimide (DCC) in THF. The resulting active ester
was aminolyzed with N5-Boc-L-ornithine to give dipeptide
34 in quantitative yield. Under the same coupling condi-
tions, dipeptide 34 was converted to the partially pro-
tected tripeptide 35 in quantitative yield. The side-chain
protecting groups were cleanly removed using 70% TFA
in acetic acid19 to give 36 in 82% yield (Scheme 8).
When treated with 3 equiv of benzaldehyde and 4 equiv
of KOH, triamine 36 was converted to the corresponding
triimine, which was oxidized to afford trioxaziridine 37
(Scheme 9). Crude oxaziridine 37 was isomerized to
nitrone 18 in 58% overall yield from tripeptide 36.
Nitrone 18 was readily purified by recrystallization.
Acid-catalyzed hydrolysis of nitrone 18 gave trihy-
droxylamine 38 in 97% yield. Acetylation of 38 with acetic
anhydride under the previously described buffered condi-
tions gave fully acetylated product 39 in 93% yield. The
O-acetyl groups were removed to afford the desired
siderophore component 40 as the diisopropylethylamine
salt in quantitative yield. Again, column chromatographic
separation was not required in the entire indirect oxida-
tion process. Large-scale indirect oxidation of ornithine
tripeptide 36 (ca. 12 g) provided gram quantities of 18
A fully unprotected nitrone (32) was obtained through
this indirect oxidation-isomerization protocol in 47%
total yield from N2-Boc-L-ornithine (31) (Scheme 7). This
should allow the preparation of fully unprotected amino
acid-derived hydroxylamines that are of significant bio-
logical interest.17
We next focused our attention on the corresponding
ornithine tripeptide. To synthesize the desired sidero-
(16) Sharman, G. J .; Williams, D. H.; Ewing, D. F.; Ratledge, C.
Chem. Biol. 1995, 2, 553.
(17) (a) Wittman, M. D.; Halcomb, R. L.; Danishefsky, S. J . J . Org.
Chem. 1990, 55, 1981. (b) Gould, S. J .; J u, S. J . Am. Chem. Soc. 1989,
111, 2329.
(18) Bodanszky, M. “Principles of Peptide Synthesis,” 2nd Ed.,
Springer-Verlag: New York, 1993.
(19) Klausner, Y. S.; Bodanszky, M. Bioorg. Chem. 1973, 2, 354.

Synthesis of Hydroxamate-Derived SiderophoreComponents
Sch em e 8
J . Org. Chem., Vol. 64, No. 20, 1999 7455
Sch em e 9
(ca. 6 g) suitable for further elaboration and construction
of siderophore-drug conjugate libraries that will be
reported in due course.
Hydrogenolysis of 40 provided fully deprotected sid-
erophore component 41 in 88% yield (Scheme 10). Treat-
ment of 41 with commercially available biotin active ester
and digoxigenin (DIG) active ester afforded the desired
biotin-ferrichrome analogue (20) and DIG-siderophore
conjugate (21) without the need for protection of the
constituent hydroxamic and carboxylic acid.
These two siderophore conjugates were designed to
detect the presence of receptor proteins in new microbial
strains in order to target specific microbial species in the
“Trojan Horse” active drug delivery approach. Binding
of the siderophore-iron complex in 20 or in 21 to receptor
proteins should immobilize the receptors on an avidin or
a DIG antibody coated stationary phase. Affinity column
chromatography is anticipated to separate ligand bound
protein from other proteins. The utilization and bio-
logical testing of these two conjugates is currently
underway.
In conclusion, a practical and efficient synthesis of
hydroxylamines and hydroxamate siderophore compo-
nents derived from amino acids has been achieved
through an indirect oxidation method. It provides a large
laboratory-scale synthetic protocol for preparation of
hydroxylamines and hydroxamates through stable ni-
trone intermediates with no need of column chromato-
graphic purification. In addition, a pH 4 buffered solution
was found to facilitate the acetylation of hydroxylamines
selectively in the presence of free carboxylates. Large
quantities of siderophore component 40 are now readily
available for the preparation of siderophore-drug con-
jugate libraries for biological evaluation.

7456 J . Org. Chem., Vol. 64, No. 20, 1999
Lin and Miller
N2-Ben zyloxyca r b on yl-N5-(3-p h en yloxa zir id in yl)-L-
or n ith in e (27). To a solution of KOH (6.6 g, 117.8 mmol) in
methanol (250 mL) at room temperature was added 17 (30.0
g, 112.8 mmol), followed by benzaldehyde (12 mL, 118.2 mmol)
and 3 Å molecular sieves. The reaction mixture was stirred at
room temperature for 16 h. The molecular sieves were filtered
off and washed with methanol. The filtrate was concentrated
to give 43.3 g (98%) of imine as a white foam: 1H NMR (300
MHz, CD3OD) δ 8.35 (s, 1H), 7.74 (m, 2H), 7.48-7.30 (m, 8H),
5.10 (m, 2H), 4.12 (m, 1H), 3.65 (m, 2H), 1.94-1.70 (m, 4H);
13C NMR (75 MHz, CD3OD) δ 179.4, 164.4, 158.2, 138.4, 137.1,
132.0, 129.7, 129.4, 129.3, 128.9, 128.8, 67.3, 61.9, 57.5, 31.9,
Sch em e 10
28.0; IR (neat) 3285, 1708, 1692 cm-1
.
To a solution of imine (43.3 g, 110.4 mmol) in CH3OH (200
mL) at 0 °C was added a solution of m-CPBA (25.1 g, 124.0
mmol) in CH3OH (80 mL) over 1 h. The reaction was stirred
at 0 °C for an additional h. The resulting precipitate was
filtered off and washed with CH3OH. The filtrate was concen-
trated at room temperature to give a white solid, which was
partitioned between H2O (250 mL) and EtOAc (250 mL). The
pH of the mixture was adjusted to 2 with 1 N HCl. The layers
were separated, and the aqueous layer was extracted with
EtOAc. The combined extracts were washed with brine, dried
(Na2SO4), filtered, and concentrated to give crude oxaziridine
27 as a white solid, which was used without further purifica-
tion. An analytical sample was obtained as a mixture of
diastereomers by trituration from diethyl ether and hexanes:
1H NMR (300 MHz, CDCl3) δ 7.38-7.33 (m, 10H), 6.75 (br,
1H), 5.57 (d, J ) 8.1 Hz, 1H), 5.10 (s, 2H), 4.51 (s, 1H), 4.41
(m, 1H), 3.16 (m, 1H), 2.62 (m, 1H), 2.06-1.84 (m, 4H); 13C
NMR (75 MHz, CDCl3) δ 176.1, 156.2, 136.1, 134.3, 130.1,
128.52, 128.48, 128.2, 128.1, 127.6, 80.5, 80.3, 67.1, 61.2, 53.7,
Exp er im en ta l Section
Gen er a l Meth od s. All reactions were carried out under N2
unless otherwise stated. Instruments and general methods
used have been described earlier.20 Benzaldehyde was freshly
distilled before use. Technical-grade m-CPBA (ACROS) was
dehydrated over P2O5 in a desiccator under vaccum until mp
> 90 °C before use.
2-(5′-Ca r boxyl)-3-p h en yloxa zir id in e (24). To a suspen-
sion of 6-aminocaproic acid 22 (2.6 g, 20 mmol) in toluene (40
mL), was added benzaldehyde (2.0 mL, 20 mmol). The mixture
was refluxed under N2, using a Dean-Stark trap (3 Å
molecular sieves) for 12 h. The volatiles were removed to give
4.0 g of imine 23 as a bright yellow syrup, which was used
without further purification.
To a solution of imine 23 (4.0 g) in CH2Cl2 (20 mL) at 0 °C
was added a solution of m-CPBA (4.1 g, 20.7 mmol) in CH2Cl2
(50 mL) dropwise. The reaction mixture was stirred at 0 °C
for 5 h. The resulting m-CPBA precipitate was filtered off and
washed with CH2Cl2. The filtrate was washed with Na2S2O3
and brine, dried (Na2SO4), filtered, and concentrated to give
3.5 g (81% in two steps) of 24 as a yellow solid, which was
used without further purification. An analytical sample was
obtained by flash column chromatographic separation on silica
gel (2:1 hexanes/EtOAc) as a white solid: mp 71-72 °C. 1H
NMR (300 MHz, CDCl3) δ 7.39 (m, 5H), 4.49 (s, 1H), 3.03 (dt,
J ) 12.3, 4.5 Hz, 1H), 2.73 (dt, J ) 12.3, 6.9 Hz, 1H), 2.37 (t,
J ) 7.5 Hz, 2H), 1.75-1.68 (m, 4H), 1.51-1.49 (m, 2H); 13C
NMR (75 MHz, CDCl3) δ 179.9, 134.6, 130.0, 128.4, 127.5, 80.4,
61.8, 33.8, 27.6, 26.7, 24.4; IR (neat) 3050, 1690, 1465, 1410
cm-1; HRFABMS calcd for C13H18NO3 (MH+) 236.1287, found
236.1263.
30.1, 29.9, 24.0. IR (neat) 3206, 1713, 1683, 1413 cm-1
;
HRFABMS Calcd for C20H23N2O5 (MH+) 371.1607, found
371.1610.
Nitr on e 16. To crude oxaziridine 27 at room temperature
was added TFA (50 mL), followed by CH2Cl2 (50 mL). The
reaction mixture was stirred at room temperature for 1 h. The
volatiles were removed, and the resulting slurry was dissolved
in EtOAc (200 mL). Hexanes (400 mL) and benzaldehyde (6
mL) were added, and the reaction mixture was stirred at room
temperature for 12 h. The mixture was cooled to 0 °C, and
the resulting solid was filtered off, washed with EtOAc, and
dried to give 37.1 g of nitrone 16 as an off-white solid. The
i
crude product was recrystallized from PrOH/EtOAc/hexanes
to give 33.4 g (80% yield from 17) of 16 as a fluffy white solid:
1
mp 150.5-151 °C; H NMR (500 MHz, DMSO-d6) δ 8.24 (m,
2H), 7.85 (s, 1H), 7.68 (d, J ) 8.5 Hz, 1H), 7.42-7.29 (m, 8H),
5.03 (s, 2H), 4.03 (m, 1H), 3.91 (t, J ) 7.0 Hz, 2H), 1.91 (m,
2H), 1.76 (m, 1H), 1.65 (m, 1H); 13C NMR (125 MHz, DMSO-
d6) δ 173.8, 156.4, 137.1, 133.8, 131.1, 130.0, 128.5, 128.1,
128.0, 127.8, 65.6, 53.6, 27.9, 24.2; IR (KBr) 3340, 1717, 1696
cm-1; HRFABMS calcd for C20H23N2O5 (MH+) 371.1607, found
371.1615.
N6-Hyd r oxy-6-a m in oca p r oic Acid (25). A solution of
oxaziridine 24 (470 mg, 2.0 mmol) in CH2Cl2 (30.0 mL) was
treated with TFA/H2O (10.0 mL, v/v, 4/1) at room temperature.
The reaction mixture was stirred for 4 h, H2O (20 mL) was
added, and the layers were separated. The aqueous layer was
washed with CH2Cl2 and concentrated to yield 300 mg of 25
as a yellow oil: 1H NMR (300 MHz, CD3CN) δ 5.65 (br, 3H),
3.16 (t, J ) 7.5 Hz, 2H), 2.28 (t, J ) 7.2 Hz, 2H), 1.68-1.56
(m, 4H), 1.41-1.39 (m, 2H); 13C NMR (75 MHz, CD3CN) δ
175.2, 52.0, 33.8, 26.4, 24.9, 23.8.
N2-Ben zyloxyca r bon yl-N5-h yd r oxy-L-or n ith in e Hyd r o-
ch lor id e (28). A mixture of nitrone 16 (3.7 g, 0.01 mol),
hexanes (20 mL), 0.5 N HCl (40 mL), and TFA (10 mL) was
heated at 60 °C for 15 min. The volatiles were removed to give
a light yellow oil. To this residue were added CH2Cl2 (40 mL)
and 1 N HCl (60 mL). The mixture was heated at 40 °C briefly
to dissolve the residue and allowed to stir at room temperature
for 40 min. The organic layer was separated, and the aqueous
layer was washed with CH2Cl2 and hexanes. The aqueous layer
was concentrated to give 2.68 g (84%) of 28 as a white foam.
No further recrystallization was attempted due to the instabil-
ity of hydroxylamine: 1H NMR (300 MHz, CD3CN) δ 7.34 (m,
5H), 6.14 (br, 1H), 5.08 (AB, J ) 12.6 Hz, 2H), 4.27 (m, 1H),
3.54 (m, 2H), 2.07-1.71 (m, 4H); 13C NMR (75 MHz, CD3CN)
δ 166.7, 157.9, 137.8, 129.4, 129.0, 128.8, 67.5, 52.0, 51.3, 28.1,
20.9; IR (KBr) 3331, 1739, 1687 cm-1; HRFABMS calcd for
N6-Acetyl-N6-h yd r oxy-6-a m in oca p r oic Acid (26). To a
mixture of hydroxylamine 25 (300 mg, 2.0 mmol) and NaHCO3
(420 mg, 5.0 mmol) in CH3CN (20.0 mL) at room temperature
was added CH3COCl (0.16 mL, 2.26 mmol). The reaction
mixture was stirred at room temperature for 12 h. The solid
was filtered off and washed with methanol. The filtrate was
concentrated and purified by flash column chromatography on
silica gel (EtOAc to 1:2 MeOH/EtOAc), yielding 200 mg (52%
from 24) of 26 as a white foam: 1H NMR (300 MHz, CD3OD)
δ 3.59 (t, J ) 6.9 Hz, 2H), 2.29 (t, J ) 7.5 Hz, 2H), 2.09 (s,
3H), 1.63 (m, 4H), 1.34 (m, 2H); 13C NMR (75 MHz, CD3OD) δ
177.7, 173.5, 48.6, 34.8, 27.4, 27.2, 25.7, 20.2; IR (neat) 3189,
1683, 1624 cm-1; HRFABMS calcd for C8H16NO4 (MH+)
190.1079, found 190.1082.
C
13H19N2O5 (MH+) 283.1294, found 283.1311.
N2-Ben zyloxyca r b on yl-N5-a cet yl-N5-O-a cet yl-L-or n i-
th in e (29). To a solution of 28 (2.64 g, 8.30 mmol) in KOAc/
HOAc buffer (pH 4, [KOAc] ) 0.05 M, 80 mL), was added
KHCO3 (0.83 g). Acetic anhydride (14 mL, 150 mmol) was
(20) Hu, J .; Miller, M. J . J . Am. Chem. Soc. 1997, 119, 3462.

Synthesis of Hydroxamate-Derived SiderophoreComponents
J . Org. Chem., Vol. 64, No. 20, 1999 7457
added dropwise at room temperature. The pH of the solution
was maintained at 4 by adding solid potassium bicarbonate
during this process. The reaction mixture was stirred at room
temperature for 4.5 h. The volatiles were removed to give an
oily residue. To this residue were added 0.5 N HCl (200 mL)
and EtOAc (150 mL). The organic layer was separated, and
the aqueous layer was extracted with EtOAc. The combined
organic layers were washed with brine, dried (Na2SO4),
filtered, and concentrated to give 2.9 g (95%) of 29 as a
colorless oil: 1H NMR (300 MHz, DMSO-d6) δ 12.20 (br, 1H),
7.59 (d, J ) 7.8 Hz, 1H), 7.35-7.28 (m, 5H), 5.02 (s, 2H), 3.93
(m, 1H), 3.58 (m, 2H), 2.18 (s, 3H), 1.90 (s, 3H), 1.70-1.55 (m,
4H); 13C NMR (125 MHz,CD3OD) δ 175.7, 173.4, 170.4, 158.8,
138.4, 129.6, 129.1, 128.9, 67.7, 55.0, 48.2, 29.9, 24.6, 20.3, 18.3;
IR (neat) 3329, 1796, 1715, 1652 cm-1; HRFABMS calcd for
ornithine provided 53 g (quant) of 35 as a white foam, which
was used without further purification. An analytical sample
was obtained by flash column chromatographic separation on
silica gel (2.5% HOAc/EtOAc) as a white solid: mp 88-90 °C;
1H NMR (500 MHz, DMSO-d6/D2O, (95%) 60 °C) δ 7.29 (m,
5H), 4.98 (s, 2H), 4.23 (dd, J ) 5.0, 8.5 Hz, 1H), 4.14 (dd, J )
5.0, 8.5 Hz, 1H), 3.95 (dd, J ) 5.0, 8.5 Hz, 1H), 2.88 (m, 6H),
1.69-1.35 (m, 12H), 1.32 (s, 27H); 13C NMR (125 MHz, DMSO-
d6/D2O) δ 174.2, 173.1, 172.6, 157.0, 156.9, 137.5, 129.3, 128.8,
128.4, 79.0, 65.5, 55.2, 52.9, 52.5, 40.0, 29.8, 28.9, 26.6, 26.4;
IR (neat) 3320 (br), 1710, 1695, 1665, 1660, 1525 cm-1
;
HRFABMS calcd for C38H62N6O12Na (M + Na)+ 817.4323,
found 817.4347.
Tr ip ep tid e 36. A solution of tripeptide 35 (30.0 g, 38 mmol)
in TFA/HOAc (35 mL/15 mL) was stirred at room temperature
for 1 h. The volatiles were removed, and the resulting residue
was dissolved in H2O (200 mL). The aqueous solution was
washed with EtOAc, concentrated, and azeotroped with tolu-
ene to give 26 g (82%) of 36 as a glassy white foam: 1H NMR
(300 MHz, D2O) δ 7.27 (m, 5H), 4.97 (s, 2H), 4.25 (m, 2H),
4.04 (br, 1H), 2.88 (m, br, 6H), 1.64 (m, br, 12H); 13C NMR (75
MHz, D2O) δ 173.7, 173.4, 172.4, 156.9, 135.5, 128.0, 127.6,
126.8, 66.3, 53.7, 52.5, 51.4, 38.1, 27.4, 27.1, 26.6, 22.5, 22.4;
IR (neat) 3265, 1676 cm-1; HRFABMS calcd for C23H39N6O6
(MH+) 495.2931, found 495.2924.
C
17H23N2O7 (MH+) 367.1505, found 367.1495.
N2-Ben zyloxyca r b on yl-N5-a cet yl-N5-h yd r oxy-L-or n i-
th in e (30). A solution of hydroxamate 29 (2.60 g, 7.10 mmol)
in a diisopropylethylamine methanol solution (6%, 30 mL) was
stirred at room temperature for 12 h. The volatiles were
removed to give 3.22 g (quant) of 30 as a light yellow oil: 1H
NMR(500 MHz, CD3OD) δ 7.36-7.28 (m, 5H), 5.06 (s, 2H),
4.03 (m, 1H), 3.70 (hept, J ) 6.5 Hz, 2H), 3.60 (m, 2H), 3.19
(q, J ) 7.5 Hz, 2H), 2.08 (s, 3H), 1.82-1.66 (m, 4H) 1.35 (d, J
) 6.5 Hz, 12H), 1.35 (t, J ) 7.5 Hz, 3H); 13C NMR (125 MHz,
CD3OD) δ 178.9, 173.7, 158.2, 138.6, 129.6, 129.0, 128.9, 67.5,
57.5, 55.8, 48.6, 43.8, 31.5, 24.3, 20.4, 18.1, 13.3; IR (neat) 3396,
Nitr on e Tr ip ep tid e 18. To a solution of KOH (3.5 g, 62.5
mmol) in methanol (200 mL) at room temperature was added
tripeptide 36 (12.0 g, 14.4 mmol), followed by benzaldehyde
(4.8 mL, 47.3 mmol) and 3 Å molecular sieves. The reaction
mixture was stirred at room temperature for 17 h. The
molecular sieves were filtered off and washed with methanol.
The filtrate was concentrated to give 17.7 g of imine as a white
foam. To a solution of imine in methanol (200 mL) at 0 °C
was added a solution of m-CPBA (9.6 g, 47.4 mmol) in
methanol (50 mL) over 1 h. The reaction was stirred at 0 °C
for an additional h. The reaction mixture was concentrated at
room temperature to a small volume (ca. 50 mL). The resulting
mixture was partitioned between H2O (250 mL) and EtOAc
(250 mL). The pH of this mixture was adjusted to 2 with 1 N
HCl. The layers were separated, and the aqueous layer was
extracted with EtOAc. The combined organic extracts were
washed with brine, dried (Na2SO4), filtered, and concentrated
to give 37 as a white solid, which was used without further
purification.To crude oxaziridine tripeptide 37 at room tem-
perature was added TFA (30 mL), followed by CH2Cl2 (30 mL).
The reaction mixture was stirred at room temperature for 1
h. The volatiles were removed, and the resulting residue was
dissolved in EtOAc (100 mL). Hexanes (200 mL) and benzal-
dehyde (3 mL) were added. The mixture was then stirred at
room temperature for 12 h. The reaction mixture was then
concentrated to a small volume (ca 100 mL), and the resulting
white solid was filtered off and rinsed with EtOAc. The filtrate
was concentrated to give 12.65 g of crude nitrone 18 as a tan
foam.The crude nitrone was recrystallized from CH3CN/EtOAc
/hexanes to give 6.72 g (58% total yield from 36) of 18 as a tan
solid: mp 83-85 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.24 (m,
7H), 8.13 (d, J ) 7.8 Hz, 1H), 7.84-7.83 (m, 3H), 7.52 (d, J )
8.1 Hz, 1H), 7.40-7.26 (m, 14H), 4.99 (m, 2H), 4.36 (m, 1H),
4.21 (m, 1H), 4.09 (m, 1H), 3.89 (m, 6H), 1.87-1.56 (m, 12H);
13C NMR (75 MHz, DMSO-d6) δ 173.2, 171.5, 171.3, 156.0,
137.0, 133.4, 133.3, 133.28, 131.05, 131.03, 129.8, 128.3, 128.0,
127.8, 127.7, 79.2, 65.7, 65.68, 65.5, 65.46, 54.2, 51.9, 51.7, 29.1,
29.0, 28.0, 24.0, 23.9, 23.8; IR (KBr) 3412, 3294, 1710, 1676,
1661, 1637 cm-1; HRFABMS calcd for C44H51N6O9 (MH+)
807.3718, found 807.3737.
3265, 1715, 1610 cm-1
.
Nitr on e 32. A crude oxaziridine was prepared from N2-Boc-
L-ornithine 31 (10.0 g, 43.1 mmol) by the indirect oxidation
procedure as described above. It was then isomerized by adding
TFA (60.0 mL), followed by CH2Cl2 (60 mL) at room temper-
ature. The resulting red solution was allowed to stir at room
temperature for 1 h. The volatiles were removed, and the
residue was dissolved in EtOAc (150 mL). Then a THF solution
of Hunig’s base was added until the pH of the mixture was
8-9. The mixture was cooled at 0 °C for 1 h, and the resulting
precipitate was filtered off and dried to give 7.36 g (47% overall
yield from 31) of 32 as a yellow solid. An analytical sample
was obtained by recrystallization from H2O/CH3CN: mp 207-
207.5 °C dec; 1H NMR (300 MHz, D2O) δ 8.18 (m, 2H), 7.96 (s,
1H), 7.60-7.54 (m, 3H), 4.06 (m, 2H), 3.77 (m, 1H), 1.95 (m,
4H); 13C NMR (75 MHz, DMSO-d6/D2O (95%)) δ 175.3, 142.5,
133.9, 131.5, 130.5, 130.4, 66.6, 55.7, 28.8, 24.2; IR (KBr) 3436,
1626, 1591 cm-1; HRFABMS calcd for C12H16N2O3 (MH+)
237.1239, found 237.1231.
Dip ep tid e 34. To a solution of 33 (24.0 g, 65 mmol) and
NHS (8.0 g, 69 mmol) in THF (150 mL) at 0 °C was added a
solution of DCC (13.6 g, 66 mmol) in THF (100 mL). The reac-
tion was stirred to room temperature for 10 h. The resulting
DCU was filtered off and washed with THF. The THF filtrate
was used as the active ester solution in the following reaction.
To a solution of N5-Boc-L-ornithine (18.0 g, 77 mmol) and
NaHCO3 (14 g, 170 mmol) in H2O/THF (400 mL/300 mL) at
room temperature was added the active ester obtained from
above. The reaction mixture was stirred at room temperature
for 6 h. The volatiles were evaporated, and the aqueous residue
was diluted with EtOAc (500 mL) and acidified to pH 2 with
10% citric acid. The layers were separated, and the aqueous
layer was extracted with EtOAc. The combined extracts were
washed with brine, dried (Na2SO4), filtered, and concentrated
to give 38 g (quant) of 34 as a white foam, which was used
without further purification. An analytical sample was ob-
tained by flash column chromatographic separation on silica
gel (2.5% HOAc/EtOAc) as a white solid: mp 58-60 °C; 1H
NMR (500 MHz, DMSO-d6/D2O, (95%) 60 °C) δ 7.29 (m, 5H),
4.98 (s, 2H), 4.15 (m, 1H), 3.99 (dd, J ) 5.5, 9.0 Hz, 1H), 2.88
(t, J ) 6.5 Hz, 4H), 1.69-1.35 (m, 8H), 1.32 (s, 18H); 13C NMR
(125 MHz, DMSO-d6/D2O (95%)) δ 173.5, 172.1, 156.0, 155.7,
137.1, 128.4, 127.8, 127.7, 77.5, 65.4, 54.2, 51.7, 39.8, 29.4, 28.5,
28.3, 26.1; IR (neat) 3320 (br), 1710, 1700, 1690, 1665, 1520
cm-1; HRFABMS calcd for C28H44N4O9Na (M + Na)+ 603.3006,
found 603.3005.
N2-Z-N5-H yd r oxyl-L-or n it h yl-N5-h yd r oxyl-L-or n it h yl-
N5-h yd r oxyl-L-or n ith in e Hyd r och lor id e (38). To a solution
of nitrone 18 (644 mg, 0.80 mmol) in TFA/H2O (9 mL/1 mL)
at room temperature was added CH2Cl2 (10 mL). The solution
was stirred at 40-50 °C for 15 min. The volatiles were removed
to give a light yellow oily residue. To this residue were added
CH2Cl2 (20 mL) and 1 N HCl (30 mL). The mixture was stirred
at room temperature for 1 h, and the layers were separated.
The aqueous layer was washed with CH2Cl2 and hexanes and
concentrated to give 503 mg (97%) of 38 as a light yellow foam.
Tr ip ep tid e 35. Analogous active ester-mediated coupling
(DCC, NHS) of dipeptide 34 (38 g, 65.5 mmol) with N5-Boc-L-

7458 J . Org. Chem., Vol. 64, No. 20, 1999
Lin and Miller
Further recrystallization was not attempted due to the insta-
bility of hydroxylamine: 1H NMR (300 MHz, D2O/DMSO-d6)
δ 7.24 (m, 5H), 4.95 (s, 2H), 4.18 (m 2H), 4.00 (m 1H), 3.12 (m
6H), 1.64 (m, 12H); 13C NMR (75 MHz, D2O/DMSO-d6) δ 176.0,
175.5, 174.6, 159.2, 137.8, 130.4, 130.0, 129.2, 68.6, 55.9, 54.7,
53.7, 51.5, 29.6, 29.4, 29.0, 21.2, 21.1, 21.0; IR (KBr) 3441,
methylcarbonyl-ꢀ-aminocaproic acid N-hydroxysuccinimide es-
ter (Boehringer, 5 mg, 0.0076 mmol). The reaction mixture was
stirred at room temperature for 12 h. CH3OH was added, and
the white solid was filtered off. The filtrate was concentrated
and separated by reversed-phase column chromatography (C18,
2:1 H2O/CH3OH to 1:1 H2O/CH3OH, 5% of HOAc was added
in the eluent) to give 7 mg (85%) of 21 as an oil: 1H NMR
(600 MHz, CD3OD) δ 7.69 (t, J ) 6.0 Hz, 1H), 5.90 (s, 1H),
4.92 (m, 2H), 4.42 (m, 1H), 4.35 (m, 2H), 3.88 (AB, J ) 15 Hz,
2H), 3.70 (m, 3H), 3.62-3.55 (m, 4H), 3.39 (dd, J ) 11.4, 4.8
Hz, 1H), 3.33 (dd, J ) 6.0, J ) 9.6 Hz, 1H), 3.25 (m, 2H), 2.25
(t, J ) 7.2 Hz, 2H), 2.14 (m, 1H), 2.10 (s, 3H), 2.10 (s, 3H),
2.09 (s, 3H), 1.90-1.87 (m, 4H), 1.75-1.60 (m, 26H), 1.44 (m,
1H), 1.34-1.26 (m, 5H), 0.97 (s, 3H), 0.78 (s, 3H); 13C NMR
(150 MHz, CD3OD) δ 179.0, 177.8, 176.8, 175.7, 174.9, 174.5,
174.3, 174.2, 173.6, 118.2, 87.3, 77.6, 76.1, 76.0, 68.8, 57.8, 54.8,
54.5, 54.1, 48.9, 48.8, 48.7, 47.5, 42.7, 40.3, 38.5, 37.1, 36.7,
34.1, 34.0, 31.8, 31.75, 31.3, 30.8, 30.7, 30.5, 30.4, 28.8, 28.2,
28.0, 27.0, 26.0, 24.8, 24.8, 24.7, 24.6, 23.2, 21.2, 20.8, 20.8,
3265, 1705, 1661 cm-1
.
N2-Z-N5-Acetyl-N5-O-a cetyl-L-or n ith yl-N5-a cetyl-N5-O-
a cetyl-L-or n ith yl-N5-a cetyl-N5-O-a cetyl-L-or n ith in e (39).
Acetylation of 38 (456 mg, 0.701 mmol) under the previously
described pH 4 buffer conditions provided 520 mg (93%) of 39
as an oil. 1H NMR (300 MHz,CD3OD) δ 7.34 (m, 5H), 5.08 (m,
2H), 4.39 (m, 2H), 4.14 (m, 1H), 3.71-3.69 (m, 6H), 2.19 (s,
br, 9H), 1.99 (br, 9H), 1.68 (m, 12H); 13C NMR (75 MHz, CD3-
OD) δ 174.8, 174.6, 173.8, 170.4, 158.5, 138.3, 129.6, 129.2,
129.0, 67.8, 55.9, 54.0, 53.2, 48.6, 30.4, 29.8, 24.6, 20.4, 18.4;
IR (KBr) 3328, 1796, 1718, 1655 cm-1; HRFABMS calcd for
C
35H51N6O15 (MH+): 795.3412, found: 795.3429.
N2-Z-N5-Acet yl-N5-h yd r oxyl-L-or n it h yl-N5-a cet yl-N5-
10.4; IR (neat) 3292 (br), 1734, 1720, 1646, 1638, 1617 cm-1
;
h y d r o x y l-L -o r n it h y l-N 5 -a c e t y l-N 5 -h y d r o x y l-L -o r n i-
th in e, DIP EA sa lt (40). Treatment of 39 (478 mg, 0.602
mmol) with 6% Hunig’s base in methanol (20 mL) at room
temperature for 12 h provided 474 mg (99%) of 40 as a light
yellow oil. 1H NMR (300 MHz, CD3OD) δ 7.33 (m, 5H), 5.07
(s, 2H), 4.36 (m, 1H), 4.18 (m, 2H), 3.69 (hept, J ) 6.6 Hz,
2H), 3.58 (m, 6H), 3.19 (q, J ) 7.5 Hz, 2H), 2.11 (s, 3H), 2.09
(s, 3H), 2.07 (s, 3H), 1.66 (m, 12H), 1.36-1.29 (m, 15H); 13C
NMR (75 MHz, CD3OD) δ 178.1, 175.0, 174.0, 173.9, 173.7,
173.1, 158.5, 138.3, 129.6, 129.1, 129.0, 129.0, 67.8, 56.1, 56.0,
55.7, 54.8, 48.4, 43.8, 31.2, 30.6, 30.4, 24.5, 24.4, 24.2, 20.6,
HRFABMS calcd for C52H83N7O17Na (M + Na)+ 1100.5743,
found 1100.5732.
Biotin Con ju ga te (20). To a solution of 41 (30.0 mg, 0.056
mmol) and NaHCO3 (5.0 mg, 0.059 mmol) in THF/H2O (1:1, 4
mL) at room temperature was added D-biotinonyl-ꢀ-amino-
caproic acid N-hydroxysuccinimide ester (Calbiochem, 31.0 mg,
0.056 mmol) The reaction mixture was stirred at room tem-
perature for 48 h. TFA was added, and the volatiles were
removed. The residue was purified by reversed-phase column
chromatography (C18, 4:1 H2O/CH3OH to 2:1 H2O/CH3OH,
eluent containing 10% HOAc) to give 8 mg (16%) of 20 as a
colorless oil: 1H NMR (300 MHz, D2O) δ 4.61 (dd, J ) 7.8, 4.8
Hz, 1H), 4.20 (dd, J ) 7.8, 4.5 Hz, 1H), 4.36-4.30 (m, 3H),
3.65 (br, 6H), 3.33 (m, 1H), 3.17 (t, J ) 6.6 Hz, 2H), 3.00 (dd,
J ) 12.9, 4.8 Hz, 1H), 2.30 (t, J ) 8.1 Hz, 2H), 2.24 (t, J ) 7.2
Hz, 2H), 2.14 (m, 6H), 2.08 (s, 3H), 1.70-1.32 (m, 24H); 13C
NMR (125 MHz, D2O) δ 176. 9, 176.7, 176.5, 175.6, 173.8,
173.2, 169.2, 165.2, 62.0, 60.2, 55.3, 53.2, 52.8, 50.9, 47.1, 39.6,
39.0, 35.4, 35.1, 27.9, 27.8, 27.6, 25.5, 25.1, 24.8, 22.4. 22.2,
20.4, 19.2; IR (neat) 3281, 3089, 1685, 1642 (br) cm-1; HR-
FABMS calcd for C37H63N9O13SNa (M + Na)+ 895.4086, found
895.4095.
20.4, 19.5, 18.2, 13.4; IR (KBr) 3403, 3278, 1715, 1630 cm-1
;
HRFABMS calcd for C29H45N6O12 (MH+): 669.3095, found:
669.3095. The free acid was obtained by passing 40 through a
reverse phase column (C18, 2:1 H2O/CH3OH, 10% of HOAc was
added in the eluent). HPLC analysis indicated 99.5% purity
of the free acid.21
Sid er op h or e Com p on en t (41). A mixture of 40 (137 mg,
0.172 mmol) and Pd/C (10%, 15 mg) in methanol/water (20
mL/5 mL) was stirred under an atmosphere of hydrogen for 1
h. The catalyst was filtered off and washed with methanol.
The filtrate was concentrated to give 100 mg (88%) of 41 as a
colorless oil: 1H NMR (300 MHz, CD3OD) δ 4.21 (m, 1H), 3.97
(m, 1H), 3.74 (m, 1H), 3.56-3.47 (m, 8H), 3.00 (q, J ) 7.5 Hz,
2H), 1.94-1.92 (m, 9H), 1.57-1.46 (m 12H), 1.15-1.08 (m,
15H); HRFABMS calcd for C21H39N6O10 (MH+) 535.2728, found
535.2706.
Ack n ow led gm en t. We gratefully acknowledge fi-
nancial support by the National Institutes of Health
(GM 25845 and AI 30988) and Maureen Metcalf for
assistance with the manuscript.
DIG Con ju ga te (21). To a mixture of 41 (20.0 mg, 0.030
mmol) and NaHCO3 (10.0 mg, 0.12 mmol) in DMF (1 mL) at
room temperature was added a solution of digoxigenin-3-O-
1
Su p p or tin g In for m a tion Ava ila ble: Copies of H NMR
and 13C NMR spectra for compounds 16, 18, 20, 21, 24-30,
32, 34-36, and 38-41. This material is available free of charge
via the Internet at http://pubs.acs.org.
(21) HPLC analysis was carried out using a Nova-Pak C18 3.9 ×
150 mm column (WAT 086344). A CH3CN/H2O gradient (10% to 90%,
with 0.1% TFA added to both solvents) was used as the mobile phase
with a flow rate of 1.0 mL/min.
J O990769Y