Enterobactin and enantioenterobactin

Full text of DOI:10.1021/jo9520194

3548
J . Org. Chem. 1996, 61, 3548-3550
Sch em e 1
En ter oba ctin a n d En a n tioen ter oba ctin
Eric R. Marinez, Eric K. Salmassian,
Thomas T. Lau, and Carlos G. Gutierrez*
Department of Chemistry and Biochemistry,
California State University, Los Angeles, California 90032
Received November 15, 1995
In response to biochemical requirements for iron, and
the great insolubility of environmental ferric ion, micro-
organisms secrete siderophores (from the Greek: sideros
) iron, pherein ) to carry), small molecules capable of
high affinity binding and transport of Fe(III). Entero-
bactin (1),1 produced by E. coli and many other Gram-
negative bacteria,2 exhibits the largest binding constant
(log KML) 49) for ferric ion of any natural substance.3 It
is composed of three L-serine residues linked head to tail
into a trilactone platform to which are attached three
pendant N-(2,3-dihydroxybenzoyl) groups. The catechol
oxygens define the hexadentate binding site for Fe(III).
Although many synthetic analogs have been prepared,
none exhibit greater ferric ion complex stability constants
than enterobactin. Enantioenterobactin (2) is the syn-
thetic antipode prepared from D-serine.4
than catechol. We present here an efficient synthesis of
enterobactin and enantioenterobactin.
The reactions outlined in Scheme 1 use D-serine for
preparation of enantioenterobactin (2); the reactions with
L-serine produce enterobactin (1) from intermediates
enantiomeric to 3-5. The triserine lactone nucleus 5a
has been prepared generally following the approach
developed by Shanzer and Libman,7 which involves
â-lactonization of N-tritylserine (3a )8,9 and its subsequent
trimerization to the macrocyclic lactone with the aid of
an organotin template.10 This strategy is attractive
because of its directness and economy of steps relative
to other syntheses,4,11 yet it includes some low yield
transformations, the lactonization of N-tritylserine (3a )
with diisopropylcarbodiimide in the presence of 4-(di-
methylamino)pyridine (DMAP) to 4a (23-26% yields)
and trimerization of the N-tritylserine â-lactone (4a ) to
macrocyclic triserine lactone (5a ) using 2,2-dibutyl-2,1,3-
dioxastannolane as a template, a remarkable transfor-
mation even at 20-23% yield. We have significantly
increased the yields of these reactions using methodology
which also allows for the preparation of considerable
quantities of the triserine nucleus 5a . The overall yield
of enantioenterobactin or enterobactin from commercial
starting materials has been increased 10-fold.
Enterobactin is a particularly intriguing ligand and
binds Fe(III) more strongly than other tricatechol ligands.
The triserine lactone backbone appears to be a strongly
contributing factor in the ligand’s efficacy, allowing
considerable preorganization of the free ligand and
relatively strain-free binding of the ferric ion in the
complex.5
Enterobactin and enantioenterobactin have been of
interest for structural studies5 and as probes for elucida-
tion of details of the biological processes which recognize
and transport ferric enterobactin into the bacterial cell
and subsequently make iron available for cellular bio-
chemistry.6 In addition, the enterobactin triserine lac-
tone backbone is itself attractive as a platform for the
preparation of hybrid synthetic ligands which incorporate
the serine trilactone nucleus with binding units other
Resu lts a n d Discu ssion
* To whom correspondence should be addressed. Tel.: (213) 343-
2356. Fax: (213) 343-6411. E-mail: cgutier@calstatela.edu.
(1) Pollack, J . R.; Nielands, J . B. Biochem. Biophys. Res. Commun.
1970, 38, 989. O’Brien, I. G.; Gibson, F. Biochem. Biophys. Acta 1970,
215, 393.
The formation of serine â-lactones appears to be very
sensitive to the identity of the serine N-protecting group.
(2) Rutz, J . M.; Abdullah, T.; Singh, S. P.; Kalve, V. I.; Klebba, P.
E. J . Bacteriol. 1991, 173, 5964.
(3) Loomis, L. D.; Raymond, K. N. Inorg. Chem. 1991, 30, 906.
(4) Rastetter, W. H.; Erickson, T. J .; Venuti, M. C. J . Org Chem.
1980, 45, 5012. Rastetter, W. H.; Erickson, T. J .; Venuti, M. C. J . Org.
Chem. 1981, 46, 3579.
(5) Karpishin, T. B.; Dewey, T. M.; Raymond, K. N. J . Am. Chem.
Soc. 1993, 115, 1842. Karpishin, T. B.; Raymond, K. N. Angew. Chem.
1992, 104, 486.
(6) Raymond, K. N. Pure Appl. Chem. 1994, 66, 773 and references
therein.
(7) (a) Shanzer, A.; Libman, J . J . Chem. Soc., Chem. Commun. 1983,
846. (b) Shanzer, A.; Libman, J .; Lifson, S.; Felder, C. E. J . Am. Chem.
Soc. 1986, 108, 7609.
(8) Guttman, St.; Boissonnas, R. A. Helv. Chim. Acta 1958, 41, 1852.
Guttman, St. Helv. Chim. Acta 1962, 45, 2622.
(9) A superior preparation of N-tritylamino acids is presented by:
Barlos, K.; Papaioannou, D.; Theodoropolous, D. J . Org. Chem. 1982,
47, 1324.
(10) Shanzer, A.; Libman, J .; Frolow, F. J . Am. Chem. Soc. 1981,
103, 7339.
(11) Corey, E. J .; Bhattaracharyya, S. Tetrahedron Lett. 1977, 3919.
S0022-3263(95)02019-6 CCC: $12.00 © 1996 American Chemical Society

Notes
J . Org. Chem., Vol. 61, No. 10, 1996 3549
The carbodiimide/DMAP procedure converts N-tritylserine
to â-lactone 4a in modest yield, yet fails or leads to
extremely low yields of lactone when the protecting group
is t-Boc or Cbz.7a Vederas has reported efficient synthe-
ses of â-lactones under Mitsunobu conditions,12 and this
methodology is useful for the conversion of N-t-Boc- and
Cbz-protected serines 3c and 3d to lactones 4c and 4d .
On the other hand, N-tritylserine 3a does not cyclize to
the lactone under these Mitsunobu conditions.
A more efficient synthesis of enantioenterobactin (or
enterobactin) would avoid or reduce the number of
protection/deprotection steps: that is, the reaction se-
quence 3b f 4b f 5b f 2, where the N-protecting group
in 3b is the benzyl-protected catechol which appears in
the final enantioenterobactin product. Unfortunately we
have been unsuccessful in converting N-[2,3-bis(benzyl-
oxy)benzoyl]-D-serine (3b) to the â-lactone 4b under a
host of variations of either the carbodiimide or Mitsunobu
procedures. The predominant reaction course here is the
decarboxylation of 3b to produce the enamine.12e
A slight modification of the Vederas12b procedures has
allowed us to make N-t-Boc â-lactone 4c in high yield
(84%) from commercial N-t-Boc-D-serine (3c). These
reactions are amenable to scale up (the lactonization 3c
f 4c can routinely be run on 8-9 g of starting material
to recover product in 81-84% yield). This compares very
favorably with other methods, which produce the â-lac-
tones in low yields which further decrease as the amount
of material to be cyclized is increased.
The organotin-mediated trimerization of serine â-lac-
tones also exhibits a sensitivity to the identity of the
N-protecting group. Attempts at trimerization of N-t-
Boc â-lactone 4c to the triserine lactone 5c were unsuc-
cessful, a particularly discouraging result in that 4c can
be produced in exceptionally high yield. It may be that
the urethane carbonyl in this protecting group is too good
a ligand for tin, in as much as none of the template effects
of the stannylene acetal were observed. Likewise, we
have been unsuccessful in oligomerizing â-lactones 4d
bearing the N-(benzyloxycarbonyl) protecting group.
Since N-tritylserine â-lactone (4a ) has been trimerized
by Shanzer,7 an interchange of N-protecting groups was
done in good yield (80%) by removal of the t-Boc group
in 4c with triflouroacetic acid and toluenesulfonic acid,12b
followed by tritylation of the intermediate â-serine tosy-
late salt (4e) to produce 4a .
mediated cyclic oligomerization of â-lactones appears to
be thermodynamically controlled, with the trimer as the
more stable product: after 24 h the reaction mixture
consisted largely of the tetrolide and pentolide, while
after 110 h the major product was the triolide 5a . With
the improvements in these two key transformations, the
overall yield for the conversion of commercial N-t-Boc-
D-serine to the serine trilactone 5a was 54%.
The triserine lactone platform 5a was converted into
enantioenterobactin by detritylation to trisammonium-
trilactone salt 5f with dry HCl, and formation of the
hexabenzylenantioenterobactin 5b (79% yield) by reac-
tion with 2,3-bis(benzyloxy)benzoyl chloride. Hydro-
genolysis on Pd-C produced enantioenterobactin (2).
Exp er im en ta l Section
Melting points were recorded on a digital capillary melting
point apparatus and are reported uncorrected. NMR spectra
were recorded at either 300 or 400 MHz for 1H and at 75 or 100
MHz for 13C. J values are given in Hz. Solvents were dried by
standard methods and used just after distillation. All reactions
were run under a dry nitrogen or argon atmosphere and were
stirred magnetically, except as noted. Separations were done
by flash chromatography or by centrifugally accelerated radial
thin layer chromatography on silica gel. Glassware was dried
in a 140 °C oven for a minimum of 4 h. The following entries
describe the largest scale at which we have run these reactions.
N-(t-Boc)-D-Ser in e â-La cton e (4c). A solution of 12.560 g
(48.00 mmol) of dry triphenylphosphine (in vacuo 72 h over P2O5)
in 300 mL of anhydrous acetonitrile was cooled to -65 °C (dry
ice/m-xylene) and stirred 15 min with a mechanical stirrer.
Dimethyl azodicarboxylate (DMAD)15 (5.28 mL, 48.0 mmol) was
added dropwise over 10 min. After 20 min a solution of N-(t-
Boc)-D-serine (8.800 g, 42.88 mmol) in 100 mL of anhydrous
acetonitrile was added dropwise to the reaction mixture over
30 min. The reaction mixture was stirred for an additional 1.5
h at -65 °C, allowed to slowly warm to room temperature, and
subsequently evaporated in vacuo. The residue was chromato-
graphed on silica gel with 85:15 hexanes/ethyl acetate, followed
by 70:30 hexanes/ethyl acetate to separate 6.760 g (84%) of 4c
as white crystals: mp 118.9-119.7 °C (lit.12a,e for L enantiomer
mp 119.5-120.5 °C); IR (CH2Cl2 cast) 3356, 1835, 1678, 1529,
1289, 1104 cm-1 1H NMR (300 MHz, CDCl3) δ 5.59, (d, 1 H),
;
5.10 (m, 1 H), 4.45 (m, 2 H), 1.46 (s, 9 H); 13C NMR (CD2Cl2) δ
170.0, 155.1, 81.5, 66.6, 59.9, 28.2.
D-3-Am in o-2-oxet a n on e p -Tolu en esu lfon ic Acid Sa lt
(4e).12e A mixture of N-(tert-butoxycarbonyl)-D-serine â-lactone
(4c) (6.650 g, 35.5 mmol) and anhydrous p-toluenesulfonic acid
(6.541 g, 38.0 mmol, dried 3 days in vacuo over P2O5) was cooled
in an ice bath for 15 min. Anhydrous trifluoroacetic acid (80
mL) was added over 10 min (stirring was initiated when
possible). The pale yellow solution was stirred at 0 °C for 15
min, followed by removal of the trifluoroacetic acid at the rotary
evaporator, maintaining the temperature below 30 °C. The
syrup was further evaporated at the vacuum pump (∼0.2 mm)
for 1 h. In a glovebag, the resulting solid was triturated with
anhydrous diethyl ether, filtered and washed with ether, and
then dried under reduced pressure (0.2 mm) overnight to give
8.941 g (97%) of salt 4e: 1H NMR (300 MHz, DMF-d7) δ 7.64-
7.7 (d, 2 H), 7.15 (d, 2 H, J ) 8), 5.54 (dd, 1 H, J ) 4.6, 6.5), 4.74
(m, 1 H), 4.66 (m, 1 H), 2.3 (s, 3 H).
N-Tr ityl-D-ser in e â-La cton e (4a ). A solution of 9.240 g
(35.7 mmol) of â-lactone tosylate salt 4e in 100 mL of dry CH2-
Cl2 was cooled to 0 °C, followed by the addition of 19.9 mL of
freshly distilled Et3N over 5 min. A solution of 14.88 g (53.6
mmol) of trityl chloride and 50 mL of CH2Cl2 was added over 15
min. The mixture was reacted for an additional 30 min at 0 °C
and then warmed to room temperature and stirred 6 days. The
reaction mixture was then passed through a 2 in silica gel plug
The cyclic trimerization of 4a to 5a using 2,2-dibutyl-
2,1,3-dioxastannolane as a template in refluxing chloro-
form or carbon tetrachloride produces the tritylserine
trilactone 5a in 20-23% yield, along with higher cyclic
oligomers, as reported earlier.7 Roelens has observed
that a mixture of the stannylene acetal and dibutyltin
dichloride increases the conversion of propiolactone to the
corresponding triolide from 25% to 54% relative to
stannylene acetal alone.13 Yet we saw no corresponding
increase in the production of 5a when 4a was subjected
to these reaction conditions. However, we did observe a
remarkable increase in the production of macrocyclic
trimer 5a (81% yield) when 4a was oligomerized in
refluxing xylene for an extended time.14 Organotin-
(12) (a) Arnold, L. D.; Kalantar, T. H.; Vederas, J . C. J . Am. Chem.
Soc. 1985, 107, 7105. (b) Arnold, L. D.; Drover, J . C. G; Vederas, J . C.
J . Am. Chem. Soc. 1987, 109, 4649. (c) Arnold, L. D.; May, R. G.;
Vederas, J . C. J . Am. Chem. Soc. 1988, 110, 2237. (d) Pansare, S. V.;
Arnold, L. D.; Vederas, J . C. Org. Synth. 1991, 70, 10. (e) Ramer, S.
E.; Moore, R. N.; Vederas, J . C. Can. J . Chem. Soc. 1986, 64, 706.
(13) Roelens, S. J . Chem. Soc., Chem. Commun. 1990, 58.
(14) Plattner, D. A.; Brunner, A.; Dobler, M.; Mu¨ller, H.-M.; Petter,
W.; Zbinden, P.; Seebach, D. Helv. Chim. Acta 1993, 76, 2004.
(15) The more readily commercially available diethyl azodicarboxy-
late (DEAD) can be used in place of DMAD, although chromatograpic
separation of the product is generally easier with the latter.

3550 J . Org. Chem., Vol. 61, No. 10, 1996
Notes
with CH2Cl2. The solvent was removed at the rotary evaporator
and the residue recrystallized from hexanes to give 7.271 g of
4a . The mother liquor was evaporated and the residue flash
chromatographed with 92:8 hexanes/ethyl acetate to give an
additional 2.229 g of 4a (combined yield 81%): mp 192-194 °C
[lit.7 mp 193-195 °C for L-enantiomer]; IR (CH2Cl2 cast) 3324,
3063, 1814, 1594, 1487, 1446, 1214, 1125 cm-1; 1H NMR (CDCl3)
δ 7.43 (m, 9H), 7.28 (m, 6H), 4.61 (m, J ) 4.7, 5.6, 11.5 Hz, 1H),
3.55 (pseudo t, J ) 5.6 Hz, 1H), 3.12 (q, J ) 5.6, 4.7 Hz, 1H),
2.69 (d, J ) 11.5 Hz, 1H); 13C NMR (CDCl3) δ 172.0, 145.0, 128.4,
128.3, 127.0, 70.8, 70.6, 64.5.
triethylamine (1.5 × 1.236 mmol) in 1 mL of THF were added
simultaneously dropwise over 10 min. The mixture was stirred
at 0 °C an additional 15 min, at rt for 30 min, and then passed
through a 1 in. plug of silica gel with 50 mL of CH2Cl2. The
solvent was removed at reduced pressure and the residue
separated by centrifugally accelerated radial thin layer chro-
matography. Elution with 2:1 CH2Cl2/hexanes yielded 70 mg
of unreacted triserine lactone 5a . Elution with 1:4 EtOAc/CH2-
Cl2 provided 126 mg of hexabenzylenantioenterobactin (7) (51%
conversion; 79% yield accounting for recovered starting material)
1
of as a foam:4 IR (CHCl3) 1755, 1655 cm-1; H NMR (CDCl3) δ
Tr is(N-tr itylser in e) Tr ila cton e (5a ). N-Trityl-D-serine
â-lactone (4a ) (1.000 g, 3.04 mmol) and 90 mg (0.3 mmol) 2,2-
dibutyl-2-stanna-1,3-dioxolane16 were refluxed in 15 mL of dry
m-xylene for 110 h. After being cooled to room temperature,
the reaction mixture was filtered through a 2 in. plug of silica
gel with 300 mL of CH2Cl2. The solvent was removed at the
rotary evaporator and the residue separated (in two ca. half-
gram batches) by centrifugally accelerated radial thin layer
chromatography with 2:1 CH2Cl2:hexanes to give 0.806 g (81%)
of 5a which was identical to that prepared along the lines of
Shanzer:7 mp > 260 °C; IR (Nujol) 1745, 1600 cm-1; 1H NMR
(CD2Cl2) δ 7.4-7.6 (m, 18 H), 7.2-7.35 (m, 27 H), 3.90 (pseudo
t, J ) 10.8 Hz, 3H), 3.44 (q, J ) 10.8, 4.5 Hz, 3H), 3.30 (m, J )
10.8, 4.5, 10.8 Hz, 3H), 2.58 (d, J ) 10.4 Hz, 3H); 13C NMR (CD2-
Cl2) δ 172.6, 145.8, 128.9, 128.3, 127.0, 71.4, 66.6, 55.1. Anal.
Calcd for C66H57N3O6: C, 80.22; H, 5.81; N, 4.25. Found: C,
80.43; H, 5.69; N, 4.19.
8.48 (d, 3H), 7.65 (m, 3H), 7.08-7.45 (m, 36H), 5.15 (half of AB
q, J ) 11 Hz, 3H), 5.11 (s, 6H), 5.04 (half of AB q, J ) 11 Hz,
3H), 4.91 (m, 3H), 4.09 (m, 6H). Anal. Calcd for C72H63N3O15
:
C, 71.45; H, 5.25; N, 3.47. Found: C, 71.27; H, 5.05; N, 3.31.
En a n tioen ter oba ctin (2). All glassware and the stirbar
were first washed in a KOH-2-propanol bath, rinsed with
distilled water, and then soaked in 1 M HCl for 24 h, rinsed,
and then washed in a RBS 3519 solution for 24 h, rinsed with
distilled water and then with deionized water, and then air dried
at room temperature. Ethanol (1.8 mL) was added dropwise to
a solution of 74 mg of hexabenzylenantioenterobactin (7) and
0.3 mL ethyl acetate, and the mixture became heterogeneous.
After addition of 34 mg of 10% Pd-C ethyl acetate was added
dropwise until the solution became clear. The mixture was then
hydrogenated (110 psi) for 24 h at room temperature. The
product mixture was gravity filtered through acetone-saturated
fluted filter paper containing also a plug of cotton and washed
with 30 mL of acetone. Evaporation of the solvent at 35 °C and
drying in vacuo gave 38 mg (100%) enantioenterobactin as a pale
tan glassy residue. Further evaporation in vacuo for 5 days
yielded a tan solid: mp 202-203 °C (lit.4,7,11 mp 202-203 °C);
1H NMR (acetone-d6) δ 8.6 (d, 3H), 7.3 (d, 3H), 7.0 (d, 3H), 6.78
(t, 3H), 5.15 (m, H), 4.78 (m, 6H), 3.17 (b, 5H); 13C NMR (DMSO-
d6) δ 169.4, 169.0, 148.5, 146.3, 118.9, 118.5, 118.5, 116.3, 51.6,
63.8; IR (KBr) 3700-2500, 1745, 1634, 1580, 1530, 1455, 1340,
Hexa ben zylen a n tioen ter oba ctin (7). HCl in dry ethanol18
(0.642 mL of a 1.133 N solution) was added to a solution of 200
mg (0.202 mmol) of tris(N-tritylserine) trilactone 5a and 1.5 mL
of dry ethanol and the mixture refluxed for 2 min in a preheated
oil bath. The mixture was cooled and the solvent removed at
reduced pressure. Under a steady stream of dry N2, the residue
was washed 4 × 1 mL of dry diethyl ether and the supernatant
removed with a Pasteur pipette equipped with a cotton filter at
the tip. The residue was suspended in 5.0 mL of dry THF and
cooled to 0 °C for 0.5 h. Solutions of 2,3-bis(benzyloxy)benzoyl
chloride17 (436 mg, 1.236 mmol) in 1 mL of THF and 0.26 mL of
1260, 1170 cm-1
.
Ack n ow led gm en t. We thank the National Insti-
tutes of Health (NIGMS-MBRS S06 GM08101) for
support of this work.
(16) Considine, W. J . J . Organomet. Chem. 1966, 5, 263.
(17) Schuda, P. F.; Botti, C. M.; Venuti, M. O.P.P.I. Briefs 1984, 16,
119-25.
(18) Anhydrous ethanolic HCl is conveniently prepared by adding
freshly distilled acetyl chloride (1.6 mL, 22.5 mmol) to 25 mL of
J O9520194
anhydrous ethanol, followed by titration with
solution, using 0.25% phenolphthalein in ethanol as indicator.
a
standard NaOH
(19) RSB-35 is a surface active agent marketed by Pierce Chemical
Co.