Total synthesis and stereochemical revision of (+)-aeruginosin 298-A.

Article abstract of DOI:10.1021/ol006759x

[structure:see text] Novel routes toward both enantiomers of the bicyclic proline surrogate 2-carboxy-6-hydroxyoctahydroindole, i.e., Choi, were developed on the basis of the oxidative cyclization of L-tyrosine. Synthesis of the proposed sequence of (+)-a

Full text of DOI:10.1021/ol006759x

ORGANIC
LETTERS
2000
Vol. 2, No. 26
4213-4216
Total Synthesis and Stereochemical
Revision of (+)-Aeruginosin 298-A
Peter Wipf* and Joey-Lee Methot
Department of Chemistry, UniVersity of Pittsburgh, Pittsburgh, PennsylVania 15260
pwipf+@pitt.edu
Received October 20, 2000
ABSTRACT
Novel routes toward both enantiomers of the bicyclic proline surrogate 2-carboxy-6-hydroxyoctahydroindole, i.e., Choi, were developed on the
basis of the oxidative cyclization of L-tyrosine. Synthesis of the proposed sequence of (+)-aeruginosin 298-A did not provide the natural
product. Incorporation of a D-leucine residue, in contrast, led to the total synthesis of this thrombin inhibitor.
The discovery of novel anticoagulant agents for the treatment
of thrombosis continues to receive significant attention.1
Thrombin, a key enzyme in the blood coagulation cascade,
catalyzes the conversion of fibrinogen to fibrin which then
polymerizes to form a haemostatic plug.2 Current therapeutic
agents, such as heparins and coumarins, require careful
monitoring of the patient to avoid excessive haemorrhage
and are not orally active.3
In 1998, Tulinsky and co-workers reported an X-ray
crystallographic structure of the ternary complex of 1 bound
to hirugen-thrombin.6 Surprisingly, the binding mode closely
resembled that of D-Phe-Pro-Arg chloromethyl ketone with
the L-Leu residue occupying the D-S3 subsite. Their work
also confirmed the absolute stereochemistry of the novel
hydroindole core 2 (L-Choi). Conformationally restricted
In 1994, Murakami and co-workers isolated the thrombin
inhibitor (IC50 of 0.5 µM) aeruginosin 298-A (1) from the
blue-green freshwater algae Microcystis aeruginosa.4 At the
time, only the leucine stereochemistry was assigned by chiral
GC analysis of the acid hydrolysate. The configurations of
the hydroxyphenyllactic acid and argininol fragments were
later established by Marfey analysis.5
proline derivatives such as 2 project peptide chains into
defined regions of space, promoting specific turns in peptide
folding and conferring a bioactive conformation.7 Further-
more, the hydroxyl group in 2 can participate in hydrogen
bonding and increase water solubility or can be functionalized
(e.g., as a sulfate) as found for certain aeruginosins.
(1) Vacca, J. Annual Reports in Medicinal Chemistry; Bristol, J. A., Ed.;
Academic Press: San Diego, 1998; Vol. 33, pp 81-90.
(2) Buchanan, M. R.; Brister, S. J.; Ofosu, F. A. Thrombin: Its Key
Role in Thromogenesis: Implications For Its Inhibition Clinically; CRC
Press: Boca Raton, FL, 1995.
Our approach toward 2 highlights the utility of our tyrosine
oxidation-rearrangement methodology to prepare function-
(3) Das, J.; Kimball, S. D. Bioorg. Med. Chem. Lett. 1995, 3, 999-
(5) Ishida, K.; Okita, Y.; Matsuda, H.; Okino, T.; Murakami, M.
Tetrahedron 1999, 55, 10971-10988.
1007.
(4) Murakami, M.; Okita, Y.; Matsuda, H.; Okino, T.; Yamaguchi, K.
Tetrahedron Lett. 1994, 35, 3129-3132. Aeruginosin 298-A exhibits modest
selectivity against trypsin (IC50 of 1.7 µM) but does not inhibit papain,
elastase, chymotrypsin, or plasmin. One dozen aeruginosins have since been
isolated with thrombin IC50 values ranging from 0.04 to 15 µM, all sharing
the same hydroindole core 2.5
(6) Steiner, J. L. R.; Murakami, M.; Tulinsky, A. J. Am. Chem. Soc.
1998, 120, 597-598.
(7) See, for example: Zhang, R.; Brownewell, F.; Madalengoita, J. S. J.
Am. Chem. Soc. 1998, 120, 3894-3902. Blanco, M. J.; Paleo M. R.; Penide,
C.; Sardina, F. J. J. Org. Chem. 1999, 64, 8786-8793. Tam, J. P.; Miao,
Z. J. Am. Chem. Soc. 1999, 121, 9013-9022.
10.1021/ol006759x CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/28/2000

facial selectivity. Dienone 5 can be selectively reduced to
6; however, further reduction again proceeded with little
selectivity. Substituent effects may control which face of an
olefin is adsorbed on the metal catalyst.10 Accordingly, we
speculated that varying the distance between the olefin and
the ester could alter the selectivity for cis-hydrogenation.
Elimination of the mesylate derived from 4 with activated
zinc11 gave 7 in 94% yield. In contrast to 6, catalytic
hydrogenation of the â,γ-enone 7 proceeded with excellent
(25:1) facial selectivity leading to the cis-fused hydroindole
8 in 92% yield.12
The ketone was reduced to either the equatorial alcohol
(9; 87%) with NaBH4 or the axial alcohol with L-Selectride
(10; 69%, 9:1).13 Stereochemical assignments were made on
the basis of comparison with the natural product and related
work.9 TBS-protection of 10 followed by epimerization of
Figure 1. Diastereoselective cyclooxidation of Cbz-L-tyrosine: an
entry to the aeruginosin core segment.
14
the hindered methyl ester with LiNEt2 in 10% HMPA/
THF afforded the fully protected unnatural amino acid D-3
with up to 12:1 selectivity at C(2).
The synthesis of natural aeruginosin 298-A by this route
would require starting with costly D-tyrosine. Consequently
we considered an alternative pathway (Scheme 2) to the
natural L-Choi configuration L-3.
alized hydroindole amino acids (Figure 1).8 Oxidation of
L-Cbz-tyrosine with PhI(OAc)2 followed by exposure to basic
methanol furnishes 4 in >98:2 diastereoselectivity. Recent
efforts have demonstrated the versatility of 4 for the synthesis
of various natural products.9
We had hoped to access the cis-fused ring system in 2 by
dehydration of the tertiary alcohol of 4, followed by
hydrogenation (Scheme 1). Treatment of 4 with POCl3 in
pyridine gave the kinetic elimination product 5. Unfortu-
nately, catalytic hydrogenation of this diene with various
catalysts (Pt/C, Pd/C, PtO2, Rh(PPh3)3Cl) and solvents
(MeOH, EtOH, THF, AcOH/EtOH) proceeded with little
Scheme 2. Synthesis of the L-Choi Corea
Scheme 1. Initial Approach to the Choi Corea
a Reagents and conditions: (a) Bz2O, DMAP, pyr., CH2Cl2, 50
°C, 90%; (b) NaHCO3, DMSO, 90 °C; 12 (78%); (c) activated Zn
dust, AcOH/THF, 65 °C, 75%; (d) SmI2, AcOH/THF, 65 °C, 5
min, 63%; (e) H2, 5% PtO2, 10% AcOH/EtOH, 0 °C, 95%; (f)
NaBH4, MeOH, 0 °C; (g) L-Selectride, THF, -78 °C; (h) TBSOTf,
imidazole, CH2Cl2, 87%.
The syn-diastereomer of 4 was accessed through a
thermodynamic equilibration of the benzoyl-protected15
(8) Wipf, P.; Kim, Y. Tetrahedron Lett. 1992, 33, 5477-5480.
(9) Wipf, P.; Kim, Y.; Goldstein, D. M. J. Am. Chem. Soc. 1995, 117,
11106-11112. Goldstein, D. M.; Wipf, P. Tetrahedron Lett. 1996, 37, 739-
742. Wipf, P.; Li, W. J. Org. Chem. 1999, 64, 4576-4577. Wipf, P.;
Mareska, D. A. Tetrahedron Lett. 2000, 41, 4723-4727.
(10) Rylander, P. N. Hydrogenation Methods; Academic Press: London,
1985; pp 29-52.
a Reagents and conditions: (a) POCl3, pyr., 89%; (b) H2, 5%
Rh/Al2O3, MeOH, quant.; (c) Ms2O, DMAP, pyr., CH2Cl2, -30
°C to 0 °C; activated Zn dust, AcOH/THF, 94%; (d) H2, 1% PtO2,
10% AcOH/EtOH, 0 °C, 92%; (e) NaBH4, MeOH, 0 °C; (f)
L-Selectride, THF, -78 °C; (g) TBSOTf, pyr., CH2Cl2, 94%; (h)
LiNEt2, 10% HMPA/THF, -78 °C; t-BuOH; 69%.
(11) Knochel, P.; Yeh, M. C. P.; Berk, S. C.; Talbert, J. J. Org. Chem.
1988, 53, 2392-2394.
4214
Org. Lett., Vol. 2, No. 26, 2000

alcohol 11 to 12 via a retro-Michael-Michael addition
sequence at 90 °C in basic DMSO.16 While a modest 1.6:1
ratio of 12:11 was obtained after 1 h, chromatographic
separation and recycling of recovered 11 provided an overall
yield of 78% for 12. MM2-level calculations suggest that
diastereomer 12 is preferred by 0.3 kcal/mol.
Scheme 4. Synthesis of the D-Hpla-L-Leu Segmenta
Benzoate 12 required elevated temperatures (65 °C) to
provide 13 in good yield using either zinc or SmI2.17
Interestingly, SmI2 reduction of 12 at room temperature
rendered only the saturated ketobenzoate. Hydrogenation
followed by L-Selectride reduction (86%, 3.8:1) and TBS-
protection afforded the L-enantiomer of 3. The minor
reduction product 15 could be recycled back to 14 by Ley
oxidation (83%).18 Finally the alcohol was protected as a
TBS silyl ether (87%).19
With an efficient synthesis of the core accomplished, we
turned to the preparation of the argininol (Argol) and
hydroxyphenyl lactic acid (Hpla) fragments. The former was
synthesized in six steps from L-arginine (Scheme 3). Selective
a Reagents and conditions: (a) t-BuLi, CuBr‚SMe2, THF, -78
°C to -45 °C; I, BF3‚OEt2, -45 °C to -20 °C, 83%; (b) TBSOTf,
imidazole, CH2Cl2; (c) H2, 10% Pd/C, EtOAc, 92%; (d) DMP,
CH2Cl2, 84%; (e) NaClO2, NaH2PO4, 2-methyl-2-butene, t-BuOH/
H2O, 93%; (f) L-Leu-OBn, DEPC, iPr2NEt, CH2Cl2, 73%; (g) H2,
Pd/C, EtOH, 93%.
Standard protective group manipulations followed by Dess-
Martin21 and NaClO2 oxidations led to Hpla 21. Finally,
DEPC-mediated coupling to L-Leu-OBn and hydrogenolysis
provided the desired segment LLeu-22.
Scheme 3. Synthesis of the Argol Fragmenta
For the assembly of the tetrapeptide and subsequent
deprotection, the Cbz group of L-3 was exchanged for an
Alloc group (Scheme 5). Subsequent saponification and
pentafluorophenyl ester-mediated coupling to argol 18
provided dipeptide 23 in 69% yield. Alloc deprotection
Leu
followed by DEPBT22-mediated segment coupling to L
-
a Reagents and conditions: (a) AllocCl, aq. NaOH, 79%; (b)
CbzCl, aq. NaOH, THF, 63%; (c) IBCF, NMM, DMF, -20 °C;
NaBH4, H2O, 72%; (d) TBSOTf, imidazole, CH2Cl2, 81%; (e)
CbzCl, DMAP, K2CO3, DMF, 86%; (f) Bu3SnH, Pd(PPh3)4, AcOH,
THF, 88%.
22 proceeded in 63% yield to give LLeu-24. The tetrapeptide
Scheme 5. Synthesis of the Proposed Structure of Aeruginosin
298-Aa
Alloc and Cbz protection followed by in situ NaBH4
reduction of the mixed anhydride20 formed with IBCF gave
primary alcohol 17 (72%). Standard protective group ma-
nipulations provided segment 18.
The key step in the synthesis of the Hpla fragment was a
BF3‚OEt2-catalyzed organocuprate addition to (R)-benzyl-
glycidol (I), providing adduct 19 in 83% yield (Scheme 4).
(12) The relative stereochemistry of 8 was confirmed by comparison of
the observed 1H NMR coupling constants (shown below) to those calculated
from an AM-1 minimized conformation (calculated coupling constants: Jab
) 9.3, Jac ) 4.9, Jad ) 11.3 Hz).
(13) Mitsunobu reaction of 9 with BzOH gave the axial benzoate in
modest yield (67%) along with elimination product.
(14) Use of bulkier bases (e.g., LDA/HMPA) did not provide the epimer
but instead a complex mixture, including the Claisen condensation dimer.
(15) Heating the unprotected alcohol 4 in basic DMSO led to rapid
decomposition.
a Reagents and conditions: (a) H2, Pd/C, EtOH; AllocCl, pyr.,
91%; (b) LiOH, THF/H2O, 40 °C; 18, iPr2NEt, FDPP, CH2Cl2, 69%;
1
(c) Bu3SnH, Pd(PPh3)4, AcOH, CH2Cl2; (d)
iPr2NEt, CH2Cl2, 63%; (e) HF (aq); H2, Pd/C, EtOH, 42%.
L
Leu-22, DEPBT,
(16) Diastereomers 11 and 12 can be differentiated by their H(C-2) H
NMR coupling constants; J11 ) 9.1, 2.4 Hz (dd); J12 ) 8.5 Hz (t).
(17) Molander, G. A. Org. React. 1994, 46, 211-367.
Org. Lett., Vol. 2, No. 26, 2000
4215

was treated with HF(aq) for 2 h, neutralized with NaOH(aq),
and extracted into CH2Cl2/EtOAc. Finally, hydrogenolysis
cleaved the Cbz protective groups (42%; two steps). How-
ever, this product was spectroscopically distinctively different
from natural aeruginosin 298-A; apparently it represented a
diastereomer.23
Upon reinspection of the configuration of other members
of the aeruginosin family, we began to suspect that the
stereochemical assignment of the leucine residue might be
incorrect.24 Thus, we also prepared the D-leucine analogue
of aeruginosin 298-A using the same strategy (Scheme 6).
stereochemistry of (+)-aeruginosin 298-A. A direct com-
parison between synthetic and natural samples is unfortu-
nately not possible, since the natural product is no longer
available.
In conclusion, a concise synthesis of the novel bicyclic
amino acid 2 from L-tyrosine has been developed that can
readily be adapted to the preparation of analogues and
peptidomimetic scaffolds. The total synthesis of (+)-aerugi-
nosin 298-A demonstrates the potentially biomimetic incor-
poration of an oxidative cyclization product of L-tyrosine
into the highly functionalized backbone of this potent
thrombin inhibitor. Key steps of the total synthesis include
the efficient construction of other nonproteinogenic building
blocks in aeruginosin 298-A as well as an extensive
optimization of coupling strategies.
Scheme 6. Synthesis of D-Leu-aeruginosin 298-Aa
The preparation of LLeu- as well as DLeu-aeruginosin 298-A
compels one to a reassignment of the configuration of the
natural product. As a consequence, the binding mode of
aeruginosin 298-A to thrombin is as expected,27 with a
D-leucine occupying the D-S3 binding site of the enzyme.
This structural reassignment also explains the similarity28
between the binding modes of 298-A and 98-B29 (98-B has
a D-alloisoleucine residue at P3) to serine proteases.30
Acknowledgment. This work has been supported by the
National Institutes of Health (AI/GM-33506) and by a
graduate fellowship to J.M. from FCAR (Que´bec). We thank
Dr. H. Takahashi for preliminary experiments on the
thermodynamic equilibration of 11 to 12, Professor Mu-
1
rakami for providing H and 13C spectra of the natural
product, and Professor Tulinsky for helpful discussions
regarding the X-ray structural assignment.
a Reagents and conditions: (a) NH3Cl-D-Leu-OBn, DEPC,
iPr2NEt, CH2Cl2, 68%; (b) H2, Pd/C, EtOH, 95%; (c) Bu3SnH,
Pd(PPh3)4, AcOH, CH2Cl2; (d) DLeu-22, DEPBT, iPr2NEt, CH2Cl2,
59%; (e) HF (aq); H2, Pd/C, EtOH, 34%.
Supporting Information Available: Experimental pro-
cedures and spectroscopic data. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL006759X
Indeed, the 1H and 13C NMR data of DLeu-1 matched exactly
those reported for aeruginosin 298-A.25,26
(24) The 1H NMR data for 298-A (L-Leu) closely parallel those of 298-
B-major, 89-A, 89-B, and desulfated, dimethyl acetal-derivatized 89-A and
89-B (all have D-Leu) but are very different from 298-B-minor (L-Leu);
see Supporting Information.
While at this stage we cannot yet explain the discrepancy
between our assignment and the X-ray structure of (+)-
aeruginosin 298-A, we are confident that the revised structure
Leu-1, which matches all available spectroscopic data of the
(25) Marfey analysis of the acid hydrolysate of both LLeu- and DLeu-1
confirmed that the leucine residues were indeed in the L- and D-form,
respectively (procedure: Adamson, J. G.; Hoang, T.; Crivici, A.; Lajoie,
G. A. Anal. Biochem. 1992, 202, 210-214; see Supporting Information).
D
natural product, is indeed representative of the actual
(18) Griffith, W. P.; Ley, S. V. Aldrichimica Acta 1990, 23, 13-19.
(19) Cbz-Choi(TBS)-OMe (L-3) was converted into Ac-Choi-OMe, which
(26) [R]D:
(0.36, H2O).
L
Leu-1 -18 (0.25, H2O), DLeu-1 +16 (0.17, H2O), lit. +22
1
by H and 13C NMR and [R]D is identical to that prepared by Bonjoch et
(27) Based on the well-established binding mode of D-Phe-Pro-Arg
chloromethyl ketone (Bode, W.; Turk, D.; Karshikov, A. Protein Sci. 1992,
1, 426-471).
(28) The P1-P4 sequences of both 298-A and 98-B occupy the same
binding sites: the D-allo-Ile and D-Hpla of 98-B hydrogen bond to Gly216
and Gly219, respectively; the Leu and D-Hpla of 298-A also bind to Gly216
and Gly219, respectively.
(29) Sandler, B.; Murakami, M.; Clardy, J. J. Am. Chem. Soc. 1998,
120, 595-596.
(30) After submission of this manuscript, another synthesis of aeruginosin
298-A was published that is in agreement with our stereochemical
conclusions: Valls, N.; Lopez-Canet, M.; Vallribera, M.; Bonjoch, J. J.
Am. Chem. Soc. ASAP, Nov. 1, 2000 Web publication date.
al. (Bonjoch, J.; Catena, J.; Isabal, E.; Lopez-Canet, M.; Valls, N.
Tetrahedron: Asymmetry 1996, 7, 1899-1902).
(20) Rodriguez, M.; Llinares, M.; Doulut, S.; Heitz, A.; Martinez, J.
Tetrahedron Lett. 1991, 7, 923-926. Fresh NaBH4 was required to suppress
lactam formation.
(21) Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277-
7287. Ireland, R. E.; Liu, L. J. Org. Chem. 1993, 58, 2899.
(22) DEPBT: 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one.
Li, H.; Jiang, X.; Ye, Y.; Fan, C.; Romoff, T.; Goodman, M. Org. Lett.
1999, 1, 91.
(23) An alternative synthetic strategy whereby the Hpla-Leu side chain
was attached first gave identical material.
4216
Org. Lett., Vol. 2, No. 26, 2000