Total synthesis and comparative evaluation of luzopeptin A - C and quinoxapeptin A - C

Article abstract of DOI:10.1021/ja993019e

Full details of the total syntheses of luzopeptin A - C and quinoxapeptin A - C, C₂-symmetric cyclic depsidecapeptides bearing two pendant heterocyclic chromophores, are disclosed and serve to establish the quinoxapeptin relative and absolute configuration. Key elements of the approach include the late-stage introduction of the chromophore and penultimate L-Htp acylation permitting the divergent synthesis of the luzopeptins, quinoxapeptins, and structural analogues from a common advanced intermediate. Symmetrical pentadepsipeptide coupling and macrocyclization of the 32-membered ring conducted at the single secondary amide site provided the common cyclic decadepsipeptide. The convergent preparation of the required pentadepsipeptide with installation of the labile ester in the final coupling was achieved under surprisingly effective racemization-free conditions. The quinoxapeptins were shown to bind to DNA by high-affinity bisintercalation analogous to sandramycin and the luzopeptins. Significant similarities in the DNA binding of sandramycin and luzopeptin A were observed, and these compounds proved distinguishable from the quinoxapeptins, indicating that the structural alterations in the chromophore impact the affinity and selectivity more than the changes in the decadepsipeptide. The luzopeptins proved to be more potent cytotoxic agents than the corresponding quinoxapeptin, but the quinoxapeptins proved to be more potent inhibitors of HIV-1 reverse transcriptase. In addition, a well-defined potency order was observed in the cytotoxic assays (A > B > C) in which the distinctions were extraordinarily large, with the removal of each L-Htp acyl substituent resulting in a 100-1000-fold reduction in potency. An equally well-defined but reverse potency order was observed in HIV-1 reverse transcriptase inhibition (C > B > A). Thus, the non-naturally occurring synthetic precursor 6 (quinoxapeptin C) was found to exhibit the most potent HIV-1 reverse transcriptase inhibition in the series and to lack a dose-limiting in vitro cytotoxic activity, making it the most attractive member of the series examined.

Full text of DOI:10.1021/ja993019e

J. Am. Chem. Soc. 1999, 121, 11375-11383
11375
Total Synthesis and Comparative Evaluation of Luzopeptin A-C and
Quinoxapeptin A-C
Dale L. Boger,* Mark W. Ledeboer, Masaharu Kume, Mark Searcey, and Qing Jin
Contribution from the Department of Chemistry and The Skaggs Institute for Chemical Biology,
The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
ReceiVed August 19, 1999
Abstract: Full details of the total syntheses of luzopeptin A-C and quinoxapeptin A-C, C2-symmetric cyclic
depsidecapeptides bearing two pendant heterocyclic chromophores, are disclosed and serve to establish the
quinoxapeptin relative and absolute configuration. Key elements of the approach include the late-stage
introduction of the chromophore and penultimate L-Htp acylation permitting the divergent synthesis of the
luzopeptins, quinoxapeptins, and structural analogues from a common advanced intermediate. Symmetrical
pentadepsipeptide coupling and macrocyclization of the 32-membered ring conducted at the single secondary
amide site provided the common cyclic decadepsipeptide. The convergent preparation of the required
pentadepsipeptide with installation of the labile ester in the final coupling was achieved under surprisingly
effective racemization-free conditions. The quinoxapeptins were shown to bind to DNA by high-affinity
bisintercalation analogous to sandramycin and the luzopeptins. Significant similarities in the DNA binding of
sandramycin and luzopeptin A were observed, and these compounds proved distinguishable from the
quinoxapeptins, indicating that the structural alterations in the chromophore impact the affinity and selectivity
more than the changes in the decadepsipeptide. The luzopeptins proved to be more potent cytotoxic agents
than the corresponding quinoxapeptin, but the quinoxapeptins proved to be more potent inhibitors of HIV-1
reverse transcriptase. In addition, a well-defined potency order was observed in the cytotoxic assays (A > B
> C) in which the distinctions were extraordinarily large, with the removal of each L-Htp acyl substituent
resulting in a 100-1000-fold reduction in potency. An equally well-defined but reverse potency order was
observed in HIV-1 reverse transcriptase inhibition (C > B > A). Thus, the non-naturally occurring synthetic
precursor 6 (quinoxapeptin C) was found to exhibit the most potent HIV-1 reverse transcriptase inhibition in
the series and to lack a dose-limiting in vitro cytotoxic activity, making it the most attractive member of the
series examined.
The luzopeptins (1-3) and quinoxapeptins (4-6) are closely
related members of a growing class of naturally occurring C2-
symmetric cyclic decadepsipeptides which also include quinal-
dopeptin and sandramycin (6)1-4 that bind to DNA by bisin-
tercalation (Figure 1). The luzopeptins were first isolated from
Actinomadura luzonensis,5 and their structures were established
through the single-crystal X-ray structure determination of
luzopeptin A by Clardy6 and recently confirmed by total
synthesis.7 In addition to their potent cytotoxic activity (A > B
. C) and antitumor activity,5,8-10 the luzopeptins have been
shown to inhibit HIV reverse transcriptase (C > B > A).4,11
Quinoxapeptins A and B, which were isolated more recently
(1) Toda, S.; Sugawara, K.; Nishiyama, Y.; Ohbayashi, M.; Ohkusa, N.;
Yamamoto, H.; Konishi, M.; Oki, T. J. Antibiot. 1990, 43, 796.
(2) Matson, J. A.; Bush, J. A. J. Antibiot. 1989, 42, 1763. Matson, J. A.;
Colson, K. L.; Belofsky, G. N.; Bleiberg, B. B. J. Antibiot. 1993, 46, 162.
(3) Total synthesis of sandramycin: Boger, D. L.; Chen, J.-H. J. Am.
Chem. Soc. 1993, 115, 11624. Boger, D. L.; Chen, J.-H.; Saionz, K. W. J.
Am. Chem. Soc. 1996, 118, 1629.
from a norcardioform actinomycete of indeterminant morphol-
ogy obtained from a bark disc of Betula papyrifera,12 were first
identified at Merck while screening for HIV reverse transcriptase
(RT) inhibition. Both the luzopeptins and quinoxapeptins were
shown to inhibit single and double mutants responsible for the
emerging clinical resistance to RT inhibitors, and luzopeptin C
was found to be capable of suppressing HIV replication in
infected MT-4 cells at noncytotoxic concentrations.11,12 At the
time of their disclosure, only the two-dimensional structure of
the quinoxapeptins had been established. Because of their close
(7) Luzopeptins A-C: Boger, D. L.; Ledeboer, M. W.; Kume, M. J.
Am. Chem. Soc. 1999, 121, 1098. Quinoxapeptins A-C: Boger, D. L.;
Ledeboer, M. W.; Kume, M.; Jin, Q. Angew. Chem., Int. Ed. 1999, 38,
2424.
(8) Huang, C.-H.; Mong, S.; Crooke, S. T. Biochemistry 1980, 19, 5537.
Huang, C.-H.; Prestayko, A. W.; Crooke, S. T. Biochemistry 1982, 21, 3704.
Huang, C.-H.; Mirabelli, C. K.; Mong, S.; Crooke, S. T. Cancer Res. 1983,
43, 2718. Huang, C.-H.; Crooke, S. T. Cancer Res. 1985, 45, 3768.
(9) Rose, W. C.; Schurig, J. E.; Huftalen, J. B.; Bradner, W. T. Cancer
Res. 1983, 43, 1504. Huang, C.-H.; Crooke, S. T. Anti-Cancer Drug Des.
1986, 1, 87.
(10) Boger, D. L.; Chen, J.-H. Bioorg. Med. Chem. Lett. 1997, 7, 919.
(11) Take, Y.; Inouye, Y.; Nakamura, S.; Allaudeen, H. S.; Kubo, A. J.
Antibiot. 1989, 42, 107. Inouye, Y.; Take, Y.; Nakamura, S.; Nakashima,
H.; Yamamoto, N.; Kawaguchi, H. J. Antibiot. 1987, 40, 100.
(12) Lingham, R. B.; Hsu, A. H. M.; O’Brien, J. A.; Sigmund, J. M.;
Sanchez, M.; Gagliardi, M. M.; Heimbuch, B. K.; Genilloud, O.; Martin,
I.; Diez, M. T.; Hirsch, C. F.; Zink, D. L.; Liesch, J. M.; Koch, G. E.;
Gartner, S. E.; Garrity, G. M.; Tsou, N. N.; Salituro, G. M. J. Antibiot.
1996, 49, 253.
(4) Boger, D. L.; Chen, J.-H.; Saionz, K. W.; Jin, Q. Bioorg. Med. Chem.
1998, 6, 85. Boger, D. L.; Saionz, K. W. Bioorg. Med. Chem. 1999, 7,
315.
(5) Konishi, M.; Ohkuma, H.; Sakai, F.; Tsuno, T.; Koshiyama, H.; Naito,
T.; Kawaguchi, H. J. Antibiot. 1981, 34, 148. Ohkuma, H.; Sakai, F.;
Nishiyama, Y.; Ohbayashi, M.; Imanishi, H.; Konishi, M.; Miyaki, T.;
Koshiyama, H.; Kawaguchi, H. J. Antibiot. 1980, 33, 1087. Tomita, K.;
Hoshino, Y.; Sasahira, T.; Kawaguchi, H. J. Antibiot. 1980, 33, 1098.
(6) Arnold, E.; Clardy, J. J. Am. Chem. Soc. 1981, 103, 1243. Konishi,
M.; Ohkuma, H.; Sakai, F.; Tsuno, T.; Koshiyama, H.; Naito, T.; Kawaguchi,
H. J. Am. Chem. Soc. 1981, 103, 1241.
10.1021/ja993019e CCC: $18.00 © 1999 American Chemical Society
Published on Web 11/24/1999

11376 J. Am. Chem. Soc., Vol. 121, No. 49, 1999
Boger et al.
Scheme 1
our strategy allows for a divergent synthesis of the luzopeptins,
quinoxapeptins, and structural analogues from a common
intermediate. Key elements of the synthesis include late-stage
introduction of the chromophore and late-stage L-Htp acylation,
symmetrical pentadepsipeptide coupling, and macrocyclization
of the 32-membered decadepsipeptide conducted at the single
secondary amide site. The convergent assemblage of the
pentadepsipeptide with installment of the potentially labile ester
linkage in the final coupling reaction is achieved surprisingly
effectively under racemization-free conditions (Figure 1).
The quinoxapeptins were shown to bind to DNA by high-
affinity bisintercalation analogous to sandramycin and the
luzopeptins. Significant similarities in the DNA binding selec-
tivity of sandramycin and luzopeptin A were observed, and these
compounds proved distinguishable from the quinoxapeptins,
indicating that the structural alterations in the chromophore
impact the selectivity more than the changes in the deca-
depsipeptide.
Comparisons of the biological activities of the natural
products and key analogues revealed important trends including
the observation that 6, which has not yet been identified as a
natural product and was dubbed quinoxapeptin C in analogy
with luzopeptin C, exhibits the most potent HIV-1 RT inhibition
in the series and lacks a dose-limiting in vitro cytotoxic activity,
making it the most attractive member of the series examined to
date.
Pentadepsipeptide Synthesis. The N-methyl-â-hydroxyvali-
nol required for incorporation into the pentadepsipeptide 20 was
prepared as summarized in Scheme 1. Sharpless epoxidation
of 3-methyl-2-buten-l-ol with L-(+)-DIPT provided known (2S)-
epoxide 8.20 Treament of 8 with methyl isocyanate gave the
corresponding carbamate 9 (94%). Subsequent base-catalyzed
epoxide opening generated 10, which smoothly rearranged to
the more stable cyclic carbamate 11 (>25:1) under the reaction
conditions (5.0 equiv of NaH, THF, 25 °C, 24-72 h, 66-
85%).21 Protection of the primary alcohol as its THP ether (99%)
was followed by hydrolysis of the carbamate (KOH, (CH2-
OH)2-H2O, 150 °C, 25 h, 92-94%) to provide the desired
amine 13. Coupling of 13 with BOC-Gly-Sar-OH mediated by
EDCI-HOAt22 and subsequent acid-catalyzed removal of the
Figure 1. Structures of the luzopeptins, quinoxapeptins, and sandra-
mycin.
structural relationship, the relative and absolute configuration
of the quinoxapeptin cyclic decadepsipeptide could be safely
assumed to be the same as those of the luzopeptins (established
by X-ray), but that of the L-Htp acyl substituent was unknown.
Thus, the total synthesis of the quinoxapeptins confirmed the
relative and absolute configuration of the cyclic decadepsipep-
tide and established that of the 2-methylcyclopropanecarboxylic
acid L-Htp acyl substituent.
Despite the potent biological activity of these natural products,
there have been only a limited number of reported efforts toward
the synthesis13-16 of members of this family of DNA bisinter-
calators.3,4,8,17-19 Herein, we provide the full details7 of the total
synthesis of luzopeptin A-C, quinoxapeptin A and B, and
several key analogues from a common intermediate which
unambiguously establish the relative and absolute stereochem-
istry of the quinoxapeptin acyl substituent, 2-methylcyclopro-
panecarboxylic acid, of the L-(4S)-hydroxy-2,3,4,5-tetrahydro-
pyridazine-3-carboxylic acid (L-Htp) subunit. Since the luzopeptins
and quinoxapeptins contain the identical cyclic decadepsipeptide
core while differing only in the structure of the pendant
chromophore and in the acyl substituent of the L-Htp subunit,
(13) Olsen, R. K.; Apparao, S.; Bhat, K. L. J. Org. Chem. 1986, 51,
3079.
(14) Hughes, P.; Clardy, J. J. Org. Chem. 1989, 54, 3260. Greck, C.;
Bischoff, L.; Genet, J. P. Tetrahedron: Asymmetry 1995, 6, 1989.
(15) Ciufolini, M. A.; Swaminathan, S. Tetrahedron Lett. 1989, 30, 3027.
Ciufolini, M. A.; Xi, N. J. Chem. Soc., Chem. Commun. 1994, 1867. Xi,
N.; Ciufolini, M. A. Tetrahedron Lett. 1995, 36, 6595. Ciufolini, M.; Xi,
N. J: Org. Chem. 1997, 62, 2320. Xi, N.; Alemany, L. B.; Ciufolini, M. A.
J. Am. Chem. Soc. 1998, 120, 80.
(16) Boger, D. L.; Schu¨le, G. J. Org. Chem. 1998, 63, 6421.
(17) Fox, K. R.; Davies, H.; Adams, G. R.; Portugal, J.; Waring, M. J.
Nucleic Acids Res. 1988, 16, 2489. Fox, K. R.; Woolley, C. Biochem.
Pharmacol. 1990, 39, 941.
(18) Searle, M. S.; Hall, J. G.; Wakelin, L. P. G. Biochem. J. 1988, 256,
271. Searle, M. S.; Hall, J. G.; Denny, W. A.; Wakelin, L. P. G. Biochem.
J. 1989, 259, 433.
(19) Zhang, X.; Patel, D. J. Biochemistry 1991, 30, 4026. Leroy, J. L.;
Gao, X.; Misra, V.; Gueron, M.; Patel, D. J. Biochemistry 1992, 31, 1407.
(20) Gao, Y.; Hanson, R. M.; Klunder, J. M.; Ko, S. Y.; Masamune, H.;
Sharpless, K. B. J. Am. Chem. Soc. 1987, 109, 5765.
(21) Roush, W. R.; Adam, M. A. J. Org. Chem. 1985, 50, 3752. The
crystallinity of 11 provided the opportunity to ensure pure material free of
the isomer 10 was utilized.

Luzopeptin A-C and Quinoxapeptin A-C
J. Am. Chem. Soc., Vol. 121, No. 49, 1999 11377
THP group provided 16 (72% overall from 13). Oxidation of
the primary alcohol of 16 to the carboxylic acid was most
reliably achieved with RuO2-NaIO4 (87%)23 and was followed
by BOC/FMOC exchange of the amine protecting group to
provide 18 (79% from 17).24
Scheme 2
Esterification of 18 with the fully functionalized dipeptide
1916 incorporating an acyclic precursor to the L-Htp subunit was
achieved with DCC-DMAP (3 equiv/2 equiv, CH2Cl2, -20 to
0 °C, 17 h, 73%)3,7,25 in a surprisingly effective reaction. It was
found that addition of increasing amounts of DMAP not only
suppresses the racemization of the N-methyl-L-â-hydroxyvaline
residue but also improves the overall reaction conversion (Table
1 in Scheme 1). Alternative coupling methods provided
significantly lower conversions, and near complete racemization
was observed when the reaction was run in the absence of
DMAP.
Scheme 3
To accurately quantify the extent of racemization, carboxylic
acid 18 was prepared by an altenative route to ensure its
enantiomeric purity. Thus, benzylation of 17 (2 equiv of BnBr,
1.2 equiv of NaHCO3, DMF, 23 °C, 20 h, 84%) allowed for
purificaton of the corresponding benzyl ester on a semiprepara-
tive Diacel Chiracel OD column in order to remove any minor
enantiomer and provided S-23 in enantiomerically pure form
(eq 1).26 Hydrogenolysis of S-23 (10% Pd-C, CH3OH, 23 °C,
1 h, quantitative) regenerated 17 free from any contaminate
enantiomer, which was converted to 18 as previously described.
When this material was used in the esterification reaction,
pentadepsipetide 20 was obtained as a single isomer without
any detectable diastereomeric contaminants, thereby obviating
the need for chiral chromatography purification, and diastereo-
merically pure material was used in the subsequent steps.27
With the 20 in hand, we examined the L-Htp ring-forming
reaction which involves BOC deprotection, acetal cleavage, and
imine cyclization (Scheme 2). Thus, treatment of 19 with TFA-
H2O (9/1, 23 °C, 2 h, 86%)15,16 provided 24 in excellent yield
and was accompanied by complete TBS deprotection. Likewise,
20 was smoothly converted to 25 under identical conditions
(TFA-H2O 9/1, 23 °C, 2.5 h, 68%). Importantly, both 24 and
25 proved stable to standard isolation, purification (SiO2), and
characterization techniques.
Cyclic Decadepsipeptide Formation and Completion of the
Total Synthesis of Luzopeptin A-C. Linear decadepsipeptide
formation was accomplished by independent deprotection of the
amine and carboxylic acid of 20. Selective removal of the benzyl
ester of 20 (H2, 10% Pd-C, 76-78%) conducted at 10-12 °C
in order to minimize the slow but competitive loss of the FMOC
protecting group provided 22, while FMOC deprotection of 20
(Et2NH-CH3CN, ca. 100%) supplied the complementary
coupling partner 21. Coupling of 21 and 22 was mediated by
EDCI-HOAt (CH2Cl2, 0 °C, 2 h, 64%), providing 26, and
proceeded smoothly in the absence of added bases (Scheme 3).
Addition of bases (i-Pr2NEt or sym-collidine) and the use of
DMF as a solvent substantially lowered the overall conversion
and may reflect the substrate and product sensitivity to
â-elimination or retro-aldol reactions. The FMOC group and
the benzyl ester were cleaved in a single operation by transfer
hydrogenolysis (25% aqueous HCO2NH4, 10% Pd-C, EtOH-
H2O, 98%)29 to give crude amino acid 27 which was im-
(22) EDCI ) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride; HOAt ) 1-hydroxy-7-azabenzotriazole; DCC ) 1,3-dicyclohexyl-
carbodiimide; DMAP ) 4-dimethylaminopyridine; HOBt ) 1-hydoxyben-
zotriazole; SES ) 2-(trimethylsilyl)ethylsulfonyl.
(23) Carlsen, P. H. J.; Katsuki, T.; Martin, V. S.; Sharpless, K. B. J.
Org. Chem. 1981, 46, 3936. A related sequence enlisting KMnO4 as the
oxidant on a luzopeptin dipeptide has been reported15 and failed to provide
17 in our preliminary efforts.
(24) Carpino, L. A.; Han, G. Y. J. Org. Chem. 1972, 22, 3404.
(25) Hassner, A.; Alexanian, V. Tetrahedron Lett. 1978, 19, 4475.
(26) Analysis of the starting ester revealed that 23 existed as a mixture
(94:6) of enatiomers. The major (S)-enantiomer of 23 was chromatographi-
cally purified on a semipreparative Diacel Chiracel OD column (10 µm, 2
× 25 cm, 15% i-PrOH-hexane, 7.0 mL/min flow rate). The relative ratio
of enatiomers was determined on an analytical Chiracel OD column (10
µm, 0.46 × 25 cm, 10% i-PrOH-hexane, 1.0 mL/min flow rate). The efluent
was monitored at 235 nm, and the enantiomers eluted with a retention time
of 9.98 (major S-23) and 11.48 min (minor R-23), respectively (R ) 1.15).
(27) The epimers of 20 were chromatographically separated, and their
relative ratio was determined on a semipreparative Diacel Chiracel OD
column (10 µm, 2 × 25 cm, 50% i-PrOH-hexane, 7.0 mL/min flow rate).
The effluent was monitored at 265 nm, and the diastereomers eluted with
a retention time of 26.3 (20) and 32.3 min (epi-20), respectively (R ) 1.23).
(28) Characterization of these three products may be found in the
Supporting Information.

11378 J. Am. Chem. Soc., Vol. 121, No. 49, 1999
Boger et al.
mediately subjected to macrocyclization (EDCI-HOAt 5.0
equiv/5.5 equiv, CH2Cl2, 0 °C, 2 h, 63% overall from 23),
providing the 32-membered cyclic decadepsipeptide 28. This
closure was unusually good given the large ring size being
formed, and alternative reagents including DPPA (0%) and
HATU-HOAt (ca. 20%) were less effective at promoting the
macrocyclization. The well-defined 1H NMR spectrum indicated
that 28 adopts a single symmetrical rigid solution conformation
and that the material was free of contaminate diastereomers.
Attempts at installing the two L-Htp units by treatment of 28
with TFA-H2O mixtures met with limited success. While some
of the desired diol (12-42%)28 could be generated, its formation
was accompanied by variable amounts of the mono-TBS product
(10-28%)28 and an elimination product (37-65%)28 derived
from the loss of one of the L-Htp hydroxyl groups. However,
this could be avoided by running the reaction under anhydrous
conditions. Thus, treatment of 28 with TFA-CH2Cl2 in the
presence of anisole (1/1/0.4, 0 °C for 2 h, 0 to 23 °C, 1 h, 68%)
cleanly provided the fully functionalized cyclic decadepsipeptide
29 incorporating the L-Htp subunits without competitive OTBS
cleavage. The alternative order of first closing the L-Htp subunit
and then conducting the macrocyclization was also briefly
examined in order to establish whether this more conforma-
tionally restricted substrate might close more readily. Thus,
simultaneous benzyl ester and FMOC deprotection of 26,
treatment of the resulting 27 with 10% H2O-TFA to promote
L-Htp ring closure, and subsequent macrocyclization effected
by treatment with EDCI-HOAt provided a mixture of 29 and
the corresponding mono OTBS derivative (ca. 1:1) in variable
conversions (20-55%). Although this was not carefully exam-
ined, this did not appear to present any advantage over the
approach detailed in Scheme 3.
Completion of the synthesis required SES deprotection
followed by incorporation of the appropriate chromophore.
Removal of the NSES group proved challenging, and treatment
of 28 or 29 with Bu4NF or CsF30 under a variety of conditions
((BOC2O)3 led to deprotection of the OTBS groups without
removal of the NSES. Under more forcing conditions, depro-
tection attempts led to substrate degradation, presumably as a
result of the basic nature of the reagents. In our search for an
alternative and acidic set of conditions for NSES cleavage, we
found that N-SES-Phe-OCH3 could be deprotected upon treat-
ment with anhydrous HF (neat, 0 °C, 1 h, 65%), while 70%
HF-pyridine complex (25% in THF, or neat, 0 to 23 °C) was
ineffective (eq 2). Gratifyingly, exposure of 29 to anhydrous
subsequent mild basic hydrolysis of the phenolic acetates
provided luzopeptin A (50%) and smaller amounts of luzopeptin
B (20%) identical in all respects to natural material.1,5,33
Total Synthesis of Quinoxapeptin A-C and Establishment
of the Absolute Stereochemistry. Since the absolute stereo-
chemistry of the L-Htp acyl substituent was unknown, comple-
tion of the total syhthesis of quinoxapeptin A required incor-
poration of quinoxaline chromophore 33 and subsequent acylation
with both (S,S)-34 and (R,R)-34.34 Comparison of the spectro-
scopic data with those reported for authentic material would
establish the absolute stereochemistry. Thus, treatment of 29
with anhydrous HF provided 30 which was immediately coupled
with 6-methoxyquinoxaline-2-carboxylic acid (33)35 to provide
6 (65% overall from 29), which was named quinoxapeptin C
in analogy with the luzopeptins (Scheme 4). Quinoxapeptin C
(6) was acylated with (S,S)-34 (80%) and (R,R)-34 (74%),
providing 4 and 35, respectively. From each of the acylation
reactions a small amount of the mono-acylated product 36 (18%)
or 37 (16%) was isolated. Comparison of the spectroscopic data
established that 4 derived from (S,S)-34 was identical to the
spectroscopic data for quinoxapeptin A12 and could be distin-
guished from 35 derived from (R,R)-34. Although the 1H NMR
spectra of 4 and 35 were nearly identical, the assignment rested
on the distinguishable chemical shifts of the cyclopropane (δ
0.94/0.62 versus 0.87/0.72) and the Gly R-protons (δ 4.02 versus
3.98). The rigid conformation of the cyclic decadepsipeptide
places the L-Htp acyl substituent above the Gly R-center, thereby
perturbing the chemical shift of the cyclopropane protons and
of one of the two Gly R-protons. In addition, chemical shifts
for the Ser/Gly amide protons were also found to differ slightly
(δ 9.00/8.95 versus 8.97/8.86). Acetylation of 36, bearing the
(S,S)-2-methylcyclopropanecarboxylic acid substituent, with
Ac2O (62%) provided quinoxapeptin B (5) which was identical
to the natural material,12 confirming the absolute stereochemistry
assignment of the cyclic decadepsipeptide and the L-Htp acyl
substituent. The bisacetate 38 was also prepared by acetylation
of 6 (Ac2O, 84%) to provide a direct comparison analogue to
luzopeptin A differing only in the chromophore, and it may
constitute an as yet unidentified member of the quinoxapeptin
family of natural products.
DNA Binding Studies. Structural studies of luzopeptin A18,19
and sandramycin3 have confirmed their bisintercalation inter-
action with duplex DNA spanning two base pairs and shown
that it requires two amide bond trans-to-cis isomerizations to
allow the chromophores to adopt the appropriate distance and
parallel orientation for binding. Both have been shown to bind
to DNA with extremely high affinities (107 M-1), and their
binding significantly retards the mobility of DNA under non-
denaturing electrophoretic conditions. The high affinity of
sandramycin and a series of synthetic analogues has been
demonstrated by surface plasmon resonance studies to be derived
from the stability of the adduct formed between the duplex and
HF (neat, 2-3 mL, 100 µL of anisole/5-10 mg, 0 °C, 1-1.5
h) led to deprotection of both NSES and OTBS groups,
providing 30 which could be converted to the corresponding
NBOC derivative (31, 33, equiv of BOC2O, 170 equiv of
NaHCO3, THF, 23 °C, 48 h, 69%) for identification purposes.
Alternatively, coupling of 30 with 3-hydroxy-6-methoxyquino-
line-2-carboxylic acid31,32 (80% overall from 29) without
deliberate protection of the chromophore phenol provided
luzopeptin C (3) identical in all respects (1H NMR, 13C NMR,
IR, MS, [R]D) with natural material.1,5 Peracetylation and
(33) Altering the length of time exposed to the hydrolysis conditions
alters the relative amounts of 1-3 obtained.
(34) Arai, I.; Mori, A.; Yamamoto, H. J. Am. Chem. Soc. 1985, 107,
8254. Mori, A.; Arai, I.; Yamamoto, H. Tetrahedron 1986, 42, 6447. The
acid chlorides were prepared from the corresponding carboxylic acids (0.9
equiv of (COCl)2, catalytic DMF, CH2Cl2, 0 °C for 10-15 min, 25 °C for
1-2 h) and used directly. For the carboxylic acid precursor to 1R,2R-34:
[R]23 -74.4 (c 0.29, EtOH), lit. [R]24 -71.9 (c 1.00, EtOH). For the
D
D
carboxylic acid precursor to 1S,2S-34: [R]23 +75.6 (c 0.25, EtOH). The
D
(29) Anwer, M. K.; Spatola, A. F. Synthesis 1980, 929.
(30) Weinreb, S. M.; Demko, D. M.; Lessen, T. A. Tetrahedron Lett.
1986, 27, 2099.
13C NMR of 4 and 5 proved diagnostic of a trans- versus cis-2-
methylcyclopropanecarboxylic acid ester (δ 18 versus 12 for CH3), limiting
the possibilities to 1R,2R-34 or 1S,2S-34.
(31) Prepared from methyl 2-benzyloxy-6-methoxyquinoline-2-carboxy-
late32 by sequential treatment with H2, 10% Pd-C, EtOH, 23 °C, 3 h (97%)
and LiOH, THF/CH3OH/H2O 3/1/1, 23 °C, 10 h (70%).
(32) Boger, D. L.; Chen, J.-H. J. Org. Chem. 1995, 60, 7369.
(35) Dumaitre, B. A.; Dodic, N.; Daugan, A. C. M.; Pianetti, P. M. C.
PCT Int. Appl. WO9401408; Chem. Abstr. 1995, 122, 31342e. A detailed
procedure for the preparation of 33 is provided in the Supporting
Information.

Luzopeptin A-C and Quinoxapeptin A-C
J. Am. Chem. Soc., Vol. 121, No. 49, 1999 11379
Table 2. Comparison of DNA Binding Properties
compound
KB, M-1 (107)a (-)-unwindingb (+)-windingc
sandramycin
luzopeptin A
luzopeptin B
luzopeptin C
quinoxapeptin A
quinoxapeptin B
quinoxapeptin C
quin diacetate 38
37
3.4 (10)
1.38 (5)
0.022
0.044
0.22
0.22
0.044-0.11
0.22
0.22
0.044-0.11
0.044-0.11
0.022
0.044-0.11
0.044-0.11
0.033
0.044-0.11
0.044-0.11
0.044-0.11
1.51 (4)
1.04 (4)
0.43 (3.5)
0.044-0.11
>0.22
>0.22
>0.22
0.22 (3.5)
35
a Apparent binding constant, calf thymus DNA. The base pair/agent
ratio at saturated high-affinity binding is given in parentheses. b Agent/
base pair ratio required to unwind negatively supercoiled ΦX174 DNA
(form I to form II gel mobility, 1% agarose gel). c Agent/base pair ratio
required to induce complete rewinding or postive supercoiling of
ΦX174 DNA (form II to form I gel mobility, 1% agarose gel).
base pair ratios of 0.044 for sandramycin, 0.044-0.11 for
luzopeptin C and quinoxapeptin C, and 0.22 for luzopeptins A
and B and quinoxapeptins A and B. These results suggest that
sandramycin, luzopeptin C, and quinoxapeptin C bind to DNA
with either a higher unwinding angle, a greater stability, or a
slower off-rate than the other luzopeptins and quinoxapeptins,
demonstrating that a small change in the structure has a
significant effect on the DNA binding characteristics.
Further evidence of the effect of small perturbations in the
depsipeptide structure on DNA binding is seen with quinox-
apeptin diacetate 38, the quinoxapeptin A monoester containing
the R,R-stereochemistry in the cyclopropane ring (37), and the
corresponding diester 35. Each of these unnatural agents
unwound ΦX174 DNA at agent/base pair ratios of 0.044-0.11
but failed to show any rewinding at agent/base pair ratios of
0.22. This suggests that the unnatural agents bind with a smaller
unwinding angle, with lower stability, or with faster off-rates
than the natural products.
DNA Binding Affinity. Apparent absolute binding constants
and apparent binding site sizes were obtained by measurement
of fluorescence quenching upon titration with calf thymus (CT)
DNA. The excitation and emission spectra for the luzopeptins
and the quinoxapeptins were determined in aqueous buffer (Tris-
HCl, pH 7.4, 75 mM NaCl). The quinoxapeptins, which have
the quinoxaline chromophore, exhibited intense fluorescence in
solution with enhanced excitation (360 nm) and emission (460
nm) maxima when compared with the luzopeptins (excitation
340 nm, emission 520 nm) and sandramycin (excitation 360
nm, emission 530 nm) at similar concentrations. This greatly
facilitated the measurement of fluorescence quenching in this
series and allowed measurements to be carried out at initial agent
concentrations of 1-10 µM. For the titration, small aliquots of
CT-DNA (320 µM in base pairs) were added to 2 mL of a
solution of the agent (10 µM) in Tris-HCl (pH 7.4), 75 mM
NaCl buffer. Additions were carried out at 15-min intervals to
allow equilibration. The titration was deconvoluted by Scatchard
analysis using the equation rb/c ) Kn - Krb, where rb is the
number of agent molecules bound per DNA nucleotide phos-
phate, c is the free drug concentration, K is the apparent binding
constant, and n is the number of agent binding sites per
nucleotide phosphate. A plot of rb/c versus rb gives the
association constant (slope) and the apparent binding site size
(x-intercept) for the agents (see Table 2).
Figure 2. Agarose gel electrophoresis. (A) Lanes 1 and 7, untreated
supercoiled ΦX174 DNA, 95% form I and 5% form II; lanes 2-6,
quinoxapeptin A-treated ΦX174 DNA; lanes 8-12, quinoxapeptin
B-treated ΦX174 DNA. The [agent]-to-[base pair] ratios were 0.022
(lanes 2 and 8), 0.033 (lanes 3 and 9), 0.044 (lanes 4 and 10), 0.11
(lanes 5 and 11), and 0.22 (lanes 6 and 12). (B) Lanes 1 and 7, untreated
supercoiled ΦX174 DNA, 95% form I and 5% form II; lanes 2-6,
quinoxapeptin C-treated ΦX174 DNA; lanes 8-12, R,R-quinoxapeptin
A analogue (35)-treated ΦX174 DNA. The [agent]-to-[base pair] ratios
were 0.022 (lane 2 and 8), 0.033 (lanes 3 and 9), 0.044 (lanes 4 and
10), 0.11 (lanes 5 and 11), and 0.22 (lanes 6 and 12).
the agent, with dissociation rate constants on the order of 10-5
s-l (t1/2 ) 19 h).4
The sequence selectivity of sandramycin and a series of
synthetic analogues has been studied by fluorescence and surface
plasmon resonance binding studies. Using both techniques,
sandramycin was shown to display a preference for 5′-purine-
pyrimidine sequences, with the highest affinity for 5′-CATG.
This sequence has a narrow minor groove and the lowest
stability, and it allows the formation of H-bonds between two
glycine amides (NH) and thymidine C2 carbonyls which were
detected in the NMR-derived structures. However, the differ-
ences in sequence selectivity are relatively small, and attempts
to footprint both the luzopeptins and sandramycin have met with
limited success.3,17 Details of the sequence selectivites of the
luzopeptins remain unexplored, while studies of the DNA
binding interactions of the quinoxapeptins have not been
disclosed.
Bifunctional Intercalation. Confirmation that the luzopeptins
and quinoxapeptins bind to DNA with intercalation was derived
from their ability to induce the unwinding of negatively
supercoiled DNA. This was established by their ability to
gradually decrease the agarose gel electrophoresis mobility of
supercoiled ΦX174 (unwinding) at increasing concentrations
followed by a return to normal mobility (rewinding) at even
higher agent concentrations. Similar types of changes have been
reported for sandramycin3 and luzopeptin A.8 Under the
conditions employed, both sandramycin and luzopeptin C
completely unwound ΦX174 DNA at a 0.022 agent/base pair
ratio (Figure 2 and Table 2), and quinoxapeptin C unwound
ΦX174 DNA at a 0.033 agent/base pair ratio. Luzopeptins A
and B and quinoxapeptins A and B completely unwound ΦX174
DNA at an even higher agent/base pair ratio, 0.044-0.011.
Complete rewinding of the supercoiled DNA occurred at agent/
Analogous to previous studies, luzopeptin A3 was found to
exhibit a high affinity for duplex DNA (1.4 × 107 M-1) with a
saturating stoichiometry of 1:5 agent/base pairs, and this affinity
is slightly less than that observed for sandramycin. Quinox-

11380 J. Am. Chem. Soc., Vol. 121, No. 49, 1999
Table 3. Comparative Oligonucleotide Binding Properties
Boger et al.
5′-GCATGC
5′-GCGCGC
5′-GCTAGC
5′-GCCGGC
sandramrnycin
luzopeptin A
KB (107 M-1
∆G° (kcal mol-1
KB (107 M-1
∆G° (kcal mol-1
KB (107 M-1
∆G° (kcal mol-1
KB (107 M-1
∆G° (kcal mol-1
)
23.0
-11.4
28.4
14.5
-11.1
12.7
8.5
-10.8
10.3
8.5
-10.8
14.3
)
)
)
)
)
-11.5
-11.0
-10.9
-11.1
quinoxapeptin A
quinoxapeptin C
)
1.65
1.00
2.70
1.02
-9.8
1.12
-9.6
-9.5
0.68
-9.3
-10.1
1.95
-9.9
-9.5
1.00
-9.5
)
apeptin A, constituting only a change of chromophore, has
approximately the same affinity for duplex DNA as luzopeptin
A. Removal of the cyclopropyl esters to generate quinoxapeptin
B (minus one cyclopropyl ester) and quinoxapeptin C (minus
both esters) causes an incremental decrease in affinity (A, 1.51
× 107 M-1; B, 1.04 × 107 M-1; C, 0.43 × 107 M-1). This is in
contrast to the luzopeptins, which have been demonstrated to
increase in DNA binding affinity with removal of the acetate
esters.8 In our studies, solubility of luzopeptin B and C prevented
confirmation of this result. The lower DNA binding affinity of
quinoxapeptin C for duplex DNA, which was also consistently
observed in the oligonucleotide studies (see below), suggests
that the enhanced ability of this agent to unwind supercoiled
DNA arises from something other than a greater binding affinity.
DNA Binding Selectivity. Preliminary studies of the DNA
binding selectivity of luzopeptin A and quinoxapeptins A and
C were conducted by measuring their absolute binding constants
with the deoxynucleotides 5′-d(GCXXGC)2, where XX ) AT,
GC, TA, and CG (Table 3). For each agent the characteristic
fluorescence excitation and emission spectra were recorded in
10 mM Tris-HCl, 75 mM NaCl (pH 7.4) buffer. The addition
of the deoxyoligonucleotides caused a marked quenching of
fluorescence of the agents, with quenching ranging from 50 to
96%. To minimize fluorescence decrease due to dissolution or
photobleaching, the solutions were stirred in 4-mL cuvettes in
the dark with the minimum exposure to the excitation beam
necessary to obtain a reading. The titrations were carried out
with a 15-min equilibration time after deoxynucleotide addition.
Scatchard plots of luzopeptin A binding to the deoxyoligo-
nucleotides exhibited a downward convex curvature which, as
in the case of sandramycin, we have interpreted to indicate a
high-affinity bisintercalation and a lower affinity binding
potentially involving monointercalation. Using the model de-
scribed by Feldman36 which assumes one ligand with two
binding sites, we were able to deconvolute the curves according
to the equation
Figure 3. (a) Fluorescence quenching of luzopeptin A (excitation at
338 nm and emission at 530 nm in 10 mM Tris-HCl (pH 7.4) and 75
mM NaCl buffer solution) with increasing 5′-d(GCGCGC)2 concentra-
tions. (b) Scatchard plot of fluorescence quenching of luzopeptin A
with 5′-d(GCGCGC)2 (nonlinear fit). (c) Fluorescence quenching of
quinoxapeptin A (excitation at 360 nm and emission at 460 nm in 10
mM Tris-HCl (pH 7.4) and 75 mM NaCl buffer solution) with
increasing 5′-d(GCATGC)2 concentrations. (d) Scatchard plot of
fluorescence quenching of luzopeptin A with 5′-d(GCATGC)2 (non-
linear fit).
such that both a linear, single-site analysis and a Feldman two-
site analysis both gave almost identical values for the high-
affinity binding constant. An example of the analysis using the
latter approach is given in Figure 3. Interestingly, quinoxapeptin
A has a significantly lower binding constant for the deoxyoli-
gonucleotides than luzopeptin A. Comparisons between the
binding constants of quinoxapeptin A and quinoxapeptin C
remain qualitatively similar to those observed with duplex CT-
DNA in that the latter compound consistently displays a lower
DNA binding affinity than the former. Both were substantially
less effective than luzopeptin A and sandramycin. In addition,
the binding constants for quinoxapeptin A and C proved to be
remarkably similar across the oligonucleotide series and, unlike
those of luzopeptin A, showed a small preference for two of
the sequences. Both agents bind with slightly higher affinity to
5′-AT (0.1-0.3 kcal mol-1) and 5′-TA (0.5-0.7 kcal mol-1),
but the differences are relatively small. Notably, it is now the
reverse 5′-TA vs 5′-AT that binds most tightly, and these
differences stem from alterations in the chromophore.
rb/c ) 1/2(K1(n1 - rb) + K2(n2 - rb) +
(K (n - r ) - K (n - r ))2 + 4K K n n
x
1
1
b
2
2
b
1 2 1 2
where K1 and K2 represent the association constants for high-
and low-affinity binding, and n1 and n2 represent the number
of bound agents per duplex for the separate binding events. A
typical plot for luzopeptin A is shown in Figure 3. Similar to
sandramycin, luzopeptin A shows a marked affinity for 5′-
GCATGC compared with the three other deoxyoligonucleotides.
The distinctions are small but indicate a preference, like
sandramycin, for the 5′-AT intercalation site. There is little, if
any, discrimination by this agent for the other three sequences.
Quinoxapeptin A and quinoxapeptin C differed in their
binding to the oligonucleotides. The Scatchard plots for both
agents were only very slightly curved and approached linearity
Consequently, while there are substantial similarities in DNA
binding properties of the agents in this class, bisintercalation
spanning two base pairs, there are also significant differences
in the stability, affinity, and selectivity of binding. These
differences, which are derived from relatively small structural
(36) Feldman, H. A. Anal. Biochem. 1972, 48, 317.

Luzopeptin A-C and Quinoxapeptin A-C
Table 4. Biological Activity
J. Am. Chem. Soc., Vol. 121, No. 49, 1999 11381
(4R)-4-(Hydroxymethyl)-3,5,5-trimethyl-2-oxazolidinone (11). A
stirred suspension of NaH (2.24 g, 97.5 mmol, 5.9 equiv) in THF (40
mL) at 25 °C was treated dropwise with a solution of 9 (2.63 g, 16.5
mmol, 1 equiv) in THF (15 mL) and stirred for 36 h. The mixture was
quenched dropwise with saturated aqueous NH4Cl (20 mL) and
extracted with CH2Cl2 (4 × 60 mL). The combined organic phases
were dried (NaSO4), filtered, and concentrated. Flash chromatography
(SiO2, 66% EtOAc-hexane) provided a mixture (>20:1) of isomers
(1.63 g, 10.3 mmol, 62%, typically 62-85%) which was further purified
by recrystallization (EtOAc-hexane).
(4R)-3,5,5-Trimethyl-4-1(tetrahydro-2H-pyran-2-yl)oxy]methyl]-
2-oxazolidinone (12). A solution of 11 (4.40 g, 27.6 mmol, 1 equiv)
and 3,4-dihydro-2H-pyran (3.3 mL, 36 mmol, 1.3 equiv) in CH2Cl2
(75 mL) at 25 °C was treated with pyridinium p-toluenesulfonate (PPTs,
350 mg, 1.39 mmol, 0.05 equiv) and stirred for 24 h. The solution was
poured into half-saturated aqueous NaCl (100 mL) and extracted with
CH2Cl2 (4 × 75 mL). The combined organic phase was dried (MgSO4),
filtered, and concentrated. Flash chromatography (SiO2, 50% EtOAc-
hexane) provided 12 (6.55 g, 27.0 mmol, 99%).
HIV-1 RTa
(µM)
L1210b
(nM)
HCT116c
(nM)
compound
1
2
3
4
5
luzopeptin A
luzopeptin B
luzopeptin C
quinoxapeptin A
quinoxapeptin B
quinoxapeptin C
6
3
0.008
0.3
30
>100
1
30
>100
0.4
0.6
0.9
0.3
0.7
0.9
0.8
2
0.3
2
>100
2
7
6
>100
6
>100
ndd
0.007
35
37
38
>100
7
sandramycin
0.001
a HIV-l reverse transcriptase inhibition. b L1210 mouse leukemia
cytotoxic assay. c Human colon carcinoma assay. d Not determined.
changes, may account for the well-defined trends observed in
their biological properties discussed below.
Biological Properties. The biological comparisons conducted
with the synthetic samples are summarized in Table 4. The
luzopeptins proved to be more potent cytotoxic agents than the
corresponding quinoxapeptin, but the quinoxapeptins proved to
be more potent inhibitors of HIV-1 reverse transcriptase. The
quinoxapeptin derivative 35 possessing the unnatural (R,R)-2-
methylcyclopropanecarboxylic acid substituent proved to be only
slightly less potent than natural quinoxapeptin A (4) in both
the HIV-RT and cytotoxic assays. In addition, the quinoxapep-
tins displayed activity trends analogous to those observed with
the luzopeptins, with the important exception that the RT
inhibition was more potent and the cytotoxic activity less potent,
enhancing the selective RT inhibition observed with the
quinoxapeptins. The comparison of the quinoxapeptin diacetate
derivative 38 with luzopeptin A (1) is instructive in this regard,
where 38 was nearly 10-fold more potent against HIV-1 RT
and 1000 times less potent in the L1210 cytotoxic assay. The
HIV-1 RT inhibition follows the trend of quinoxapeptin C >
A analogous to the luzopeptin C > B > A potency, with the
L-Htp-free alcohols being the most active agents in each series.
The reverse potency order was observed in the cytotoxic assays
with quinoxapeptin A . C and luzopeptin A > B . C, with
the L-Htp-free alcohols being inactive. The distinctions here were
extraordinarily large given the small structural differences with
the removal of each L-Htp acyl substituent resulting in a 100-
1000-fold reduction in potency. From comparisons that also
include the more potent sandramycin (7) which lacks a
comparable L-Htp substituent altogether, it appears as if sub-
stituents at this location diminish the cytotoxic potency and the
presence of a free alcohol greatly reduces the potency. Thus,
the synthetic precursor 6 (quinoxapeptin C), which has not yet
been disclosed as a natural product, exhibits the most potent
HIV-1 RT inhibition in the series and lacks a dose-limiting in
vitro cytotoxic activity, making it the most attractive member
of the series examined.
(2R,2′RS)-3-Methyl-2-(N-methylamino)-1-[(tetrahydro-2H-pyran-
2′-yl)oxy]-3-butanol (13). A solution of 12 (6.50 g, 26.7 mmol, 1 equiv)
and potassium hydroxide (7.50 g, 133 mmol, 5 equiv) in a mixture of
ethylene glycol and H2O (4:1, 55 mL) was warmed at reflux in a 150
°C bath for 20 h. The cooled solution was poured into half-saturated
aqueous NaCl (100 mL) and extracted with CH2Cl2 (4 × 100 mL).
The combined organic phase was dried (MgSO4), filtered, and
concentrated. Flash chromatography (SiO2, 3% Et3N-EtOAc) provided
13 (5.47 g, 25.1 mmol, 94%).
N-BOC-Gly-Sar-L-Me-Valinol(â-OH) (16). A solution of BOCGly-
SarOH (14, 562 mg, 2.28 mmol, 1.1 equiv), 13 (450 mg, 2.07 mmol,
1 equiv), and 2,6-lutidine (0.27 mL, 2.3 mmol, 1.1 equiv) in CH2Cl2-
DMF (4:1, 10 mL) at 0 °C was sequentially treated with HOAt (310
mg, 2.28 mmol, 1.1 equiv) and EDCI (593 mg, 3.11 mmol, 1.5 equiv).
The solution was stirred at 0 °C for 15 min, warmed to 25 °C, and
stirred for an additional 24 h. The solution was diluted with CH2Cl2
(30 mL), washed with saturated aqueous NaHCO3 (2 × 20 mL), 1 M
aqueous HCl (2 × 20 mL), and saturated aqueous NaCl (1 × 50 mL),
dried (MgSO4), filtered, and concentrated to provide crude 15 (843
mg) which was used directly in the next reaction without purification.
A solution of 15 (843 mg) in CH3OH (4 mL) at 25 °C was treated
with TsOH‚H2O (36 mg, 0.19 12 mmol, 0.1 equiv) and stirred for 2.5
h. The solution was concentrated, and flash chromatography (SiO2,
5-10% CH3OH-CH2Cl2 gradient) provided 16 (538 mg, 1.49 mmol,
72% overall from 13).
N-BOC-Gly-Sar-L-Me-Val(â-OH)-OH (17). From 16. A hetero-
geneous solution of 16 (3.30 g, 9.14 mmol, 1 equiv) in CCl4-CH3-
CN-H2O (2:2:3, 92 mL) at 25 °C was treated with NaIO4 (5.87 g,
27.4 mmol, 3 equiv) and RuO2‚H2O (36 mg, 0.27 mmol, 0.03 equiv)
and stirred for 24 h. The solution was poured into half-saturated aqueous
NaHCO3 (60 mL) and washed with CH2Cl2 (2 × 50 mL). The aqueous
phase was diluted with EtOAc (50 mL) and acidified to pH 2 with
concentrated HCl. The mixture was extracted with EtOAc (5 × 100
mL). The combined organic phase was dried (MgSO4), filtered, and
concentrated to provide 17 (3.00 g, 8.00 mmol, 88%).
From 23. A suspension of 23 (200 mg, 0.430 mmol) and 10% Pd-C
(20 mg) in CH3OH (20 mL) was stirred at 23 °C under H2 for 1 h.
Filtration and concentration in vacuo gave the acid S-17 (176 mg,
quantitative).
N-BOC-Gly-Sar-L-Me-Val(â-OH)-OBn (23). A solution of 17
(3.00 g, 8.00 mmol) and BnBr (1.90 mL, 16.0 mmol) in DMF at 25 °C
was treated with NaHCO3 (806 mg, 9.60 mmol). After 20 h, the solution
was poured into H2O (100 mL) and extracted with EtOAc (5 × 100
mL). The combined organic phase was washed with saturated aqueous
NaCl (100 mL), dried (MgSO4), filtered, and concentrated. Flash
chromatography (SiO2, EtOAc) provided 23 (3.11 g, 6.69 mmol, 84%).
The major (S)-enantiomer of 23 was chromatographically separated
on a semipreparative Diacel Chiracel OD column (10 µm, 2 × 25 cm,
15% i-PrOH-hexane; 7.0 mL/min flow rate). The relative ratio of
enantiomers was determined on an analytical Chiracel OD column (10
µm, 0.46 × 25 cm, 10% i-PrOH-hexane, 1.0 mL/min flow rate). The
effluent was monitored at 235 nm, and the enantiomers eluted with a
Experimental Section37
(2S)-1-[(N-Methylcarbamoyl)oxy]-3-methyl-2,3-epoxybutane (9).
A solution of 8 (4.65 g, 45.5 mmol, 1 equiv) and Et3N (12.6 mL, 91.0
mmol, 2 equiv) in CH2Cl2 at 25 °C was treated dropwise with methyl
isocyanate (4.0 mL, 68.4 mmol, 1.5 equiv) and stirred for 2 h. The
solution was quenched with H2O (5 mL), poured into saturated aqueous
NH4Cl (50 mL), and extracted with CH2Cl2 (3 × 50 mL). The combined
organic phase was dried (MgSO4), filtered, and concentrated. Flash
chromatography (SiO2, 5% EtOAc-CH2Cl2) provided 9 (6.77 g, 42.6
mmol, 94%).
(37) Full characterization data are provided in the Supporting Information.

11382 J. Am. Chem. Soc., Vol. 121, No. 49, 1999
Boger et al.
retention time of 9.98 (major S-23) and 11.48 min (minor R-23),
respectively (R ) 1.15).
(â-OH)]2 (Serine Hydroxyl) Dilactone (28). A solution of 26 (35.5
mg, 16.3 µmol) in degassed EtOH (1.4 mL) at 25 °C was treated with
10% Pd-C (18 mg) and 25% aqueous HCO2NH4 (142 µL). After 4 h,
additional HCO2NH4 (70 µL) was added, and the reaction was stirred
for an additional 2 h. The mixture was filtered and concentrated. The
residue was dissolved in EtOAc (30 mL) and washed with saturated
aqueous NaCl (2 × 3 mL). The aqueous layer was re-extracted with
EtOAc (2 × 20 mL). The combined organic phase was dried (Na2-
SO4), filtered, and concentrated in vacuo to yield 27 (32.0 mg) which
was used directly in the next reaction without further purification.
N-FMOC-Gly-Sar-L-Me-Val(â-OH)-OH (18). A sample of 17 (90
mg, 0.24 mmol) was treated with 4.0 M HCl in dioxane (2.0 mL), and
the solution was stirred at 25 °C for 30 min. The solution was
concentrated to provide a yellow solid. The solid was stirred with
anhydrous Et2O for 2 min, and the solvent was removed in vacuo. This
was repeated twice more. The solid was dissolved in 10% aqueous
Na2CO3 (0.76 mL), cooled to 0 °C, and treated with a solution of
FMOC-Cl (65 mg, 0.25 mmol, 1.05 equiv) in dioxane (0.65 mL). After
5 min, the solution was warmed to 25 °C and stirred for 8 h. The
solution was poured into H2O (10 mL) and washed with Et2O (3 × 5
mL). The aqueous phase was acidified to pH 1 with concentrated HCl
and extracted with CHCl3 (5 × 10 mL). The combined organic phase
was washed with saturated aqueous NaCl (10 mL), dried (Na2SO4),
filtered, and concentrated to provide 18 (95 mg, 0.19 mmol, 79%).
Benzyl 2-[N2-BOC-N1-[N-SES-D-Ser(N-FMOC-Gly-Sar-L-Me-
Val(â-OH))]-hydrazinol-3-(tert-butyldimethylsilyloxy)-4-(1,3-dioxan-
2-yl)-(2S,3S)-butanoate (20). A solution of 18 (385 mg, 0.774 mmol)
and 19 (300 mg, 0.387 mmol) in CH2Cl2 (2.3 mL) was dried over
molecular sieves (4 Å) for 2 h. The solution was transferred to a reaction
vessel, and CH2Cl2 (1.5 mL) was added. The solution was cooled to
-25 °C, and DMAP (94.4 mg, 0.773 mmol) was added. After the
complete dissolution of DMAP, DCC (1.16 mL, 1.0 M in CH2Cl2) was
added. After 1 h, the reaction was warmed to 0 °C and stirred for 17
h. The reaction mixture was diluted with EtOAc (3.6 mL), the
precipitate was removed by filtration, and the solution was concentrated.
Flash chromatography (SiO2, 75% EtOAc-hexane) provided a colorless
foam (354 mg, 73%). The obtained product constituted a mixture of
the desired compound 20 (major component, 88-100%) and the
epimerized product epi-20 (minor component, 0-12%).
The epimers of 20 were chromatographically separated, and their
relative ratio was determined on a semipreparative Diacel Chiracel OD
column (10 µm, 2 × 25 cm, 50% i-PrOH-hexane, 7.0 mL/min flow
rate). The effluent was monitored at 265 nm, and the diastereomers
eluted with a retention time of 26.3 (20) and 32.3 min (epi-20),
respectively (R ) 1.23).
N-SES-D-Ser(O-(N-FMOC-Gly-Sar-L-Me-Val(â-OH)))-L-Htp-
OBn (25). A solution of 20 (8 mg, 4.32 limol) in 90% aqueous
trifluoroacetic acid (0.5 mL) at 25 °C was stirred for 2.5 h. The solution
was concentrated, diluted with EtOAc (10 mL), and washed with
saturated aqueous NaHCO3 (2 mL). The combined organic phase was
dried (MgSO4), filtered, and concentrated. PTLC (SiO2, 10% CH3OH-
CH2Cl2) provided 25 (5.2 mg, 68%).
A solution of EDCI (15.3 mg, 78.2 µmol) and HOAt (11.9 mg, 85.7
µmol) in CH2Cl2 (9.5 mL) at 0 °C was treated with a solution of 27
(32.0 mg) in CH2Cl2 (1.5 mL) dropwise over 1 h (25 µL/min), followed
by a CH2Cl2 (2 × 0.5 mL) rinse dropwise over 20 min (50 µL/min).
The solution was stirred at 0 °C for 16 h. The solution was diluted
with CH2Cl2 and successively washed with H2O and saturated aqueous
NaHCO3. The aqueous layer was re-extracted with EtOAc, and the
combined EtOAc phase was dried (Na2SO4), filtered, and concentrated.
Flash chromatography (SiO2, EtOAc-hexane-CH3CN, 5:4:1) provided
28 (19.1 mg, 63% overall).
[N-SES-D-Ser-L-Htp(OTBS)-Gly-Sar-L-Me-Val(â-OH)]2 (Serine
Hydroxyl) Dilactone (29). A solution of 28 (8 mg, 4.32 µmol) and
anisole (120 µL) in CH2Cl2 (0.3 mL) at 0 °C was treated with
trifluoroacetic acid (0.3 mL) and stirred for 2 h. The solution was
warmed to 25 °C and stirred for an additional 1 h. The solution was
concentrated, diluted with EtOAc (2.5 mL), and washed with saturated
aqueous NaHCO3 (2 mL). After re-extraction with EtOAc (4 × 2 mL),
the combined organic phase was dried (Na2SO4), filtered, and concen-
trated. Flash chromatography (SiO2, EtOAc-hexane-CH3CN, 5:4:1)
provided 29 (4.4 mg, 68%).
[N-BOC-D-Ser-L-Htp(OTBS)-GIy-Sar-L-Me-VaI(â-OH)]2 (Serine
Hydroxyl) Dilactone (31). A Teflon vessel charged with 29 (2.1 mg,
1.4 µmol) and anisole (5 drops) was treated (condensed) with 2-3 mL
of anhydrous HF at -78 °C. The solution was warmed to 0 °C and
stirred for an additional 75 min. The HF was removed at 0 °C under
a stream of N2 for 1 h. The residue was dissolved in H2O, filtered, and
concentrated in vacuo to provide 30 as a white residue. The residue
was suspended in THF (0.5 mL), treated with NaHCO3 (20 mg) and
BOC2O (10 mg), and stirred at 25 °C for 48 h. The solution was
concentrated, and PLTC provided 31 (1.1 mg, 0.96 µmol, 69%).
Luzopeptin C (3). In a Teflon vessel charged with 29 (2.0 mg, 1.3
µmol) and anisole (100 µL) was condensed 2-3 mL of anhydrous HF
at -78 °C. The solution was warmed to 0 °C and stirred for an
additional 90 min. The HF was removed at 0 °C under a stream of N2
for 1 h. The residue was dissolved in 1 M aqueous HCl and lyophilized
to provide 30 as the HCl salt which was used directly in the next
reaction.
Benzyl 2-{N2-BOC-N′-{N-SES-D-Ser[(2-(N2-BOC-N1-(N-SES-D-
Ser-(N-FMOC-Gly-Sar-L-Me-Val(â-OH)))-hydrazino)-3-(tert-
butyldimethylsilyloxy)-4-(1,3-dioxan-2-yl)-(2S,3S)-butanoyl)-Gly-
Sar-L-Me-Val(â-OH)]}-hydrazino}-3-(tert-butyldimethylsilyloxy)-
4-(1,3-dioxan-2-yl)-(2S,3S)-butanoate (26). A solution of 20 (60.0 mg,
47.8 µmol) in degassed CH3OH (6 mL) at 10-12 °C was treated with
10% Pd-C (60 mg) and stirred under H2 (balloon) for 3 h. The solution
was filtered, and the solvents were removed in vacuo. The residue was
dissolved in EtOAc (50 mL) and washed with 0.5% aqueous HCl (10
mL) and H2O (10 mL), dried (Na2SO4), filtered, and concentrated to
give crude acid 21 (42.3 mg) which was used directly in the next
reaction without further purification.
A solution of 30 in DMF (0.30 mL) at 0 °C was treated sequentially
with NaHCO3 (1.3 mg, 15 µmol, 12 equiv), HOBt (1.1 mg, 7.8, µmol,
6 equiv), and 6-methoxyquinoline-2-carboxylic acid (32, 1.4 mg, 6.5
µmol, 5 equiv). Once the solution was homogeneous, EDCI (1.3 mg,
6.5 tmol, 5 equiv) was added. The solution was stirred at 0 °C for 1 h,
warmed to 25 °C, and stirred for an additional 11 h. The solution was
concentrated in vacuo. PTLC (SiO2, 10% CH3OH-CH2Cl2) provided
luzopeptin C (3, 1.4 mg, 1.0 µmol, 80%).
Luzopeptin A (1) and Luzopeptin B (2). A solution of luzopeptin
C (3, 2.8 mg, 2.1 µmol) in Ac2O-pyridine (1:1, 240 µL) was stirred
at 25 °C for 14 h. The solution was poured into saturated aqueous
NaHCO3 (6 mL), extracted with CHCl3 (5 × 5 mL), dried (Na2SO4),
filtered, and concentrated to provide the crude product (3.1 mg, 2.1
µmol, 100%) as a crude yellow solid which was used directly in the
next reaction without further purification.
A solution of 20 (45.0 mg, 35.8 µmol) in CH3CN (0.6 mL) at 25 °C
was treated with Et2NH (0.3 mL) and stirred for 20 min. The solution
was diluted with CH3CN (2 mL) and the solvents were removed in
vacuo to give 22 which was used directly in the next reaction without
further purification.
A solution of acid 21, amine 22, EDCI (21.0 mg, 0.107 mmol), and
HOAt (14.90 mg, 0.107 mmol) in CH2Cl2 (0.5 mL) at 0 °C was stirred
for 2 h. The solution was concentrated, and the residue was dissolved
in EtOAc (50 mL). The organic phase was washed with 2% aqueous
HCl (10 mL), 1% aqueous NaHCO3 (10 mL), and saturated aqueous
NaCl (10 mL), dried (Na2SO4), filtered, and concentrated. Flash
chromatography (SiO2, 90% EtOAc-hexane) provided 26 (50.3 mg,
64% overall).
A solution of the product from above (3.1 mg, 2.1 µmol) in THF-
CH3OH (3:1, 0.80 mL) was treated with Na2CO3 (0.05 M in water,
0.20 mL) for 2 h. The solution was poured into saturated aqueous
NaHCO3 and extracted with CHCl3 (3 × 2 mL) and CH3CN (3 × 2
mL). The combined organic phase was dried (Na2SO4), filtered, and
concentrated. PTLC (SiO2, 8% CH3OH-CHCl3) provided luzopeptin
A (1, 1.3 mg, 0.91 µmol, 48%) and luzopeptin B (2, 0.4 mg, 0.3 µmol,
14%).
[2-{N2-BOC-N1-(N-SES-D-Ser)-hydrazino}-3-(tert-butyldimeth-
ylsilyloxy)-4-(1,3-dioxan-2-yl)-(2S,3S)-butanoyl-Gly-Sar-L-Me-Val-

Luzopeptin A-C and Quinoxapeptin A-C
J. Am. Chem. Soc., Vol. 121, No. 49, 1999 11383
Quinoxapeptin C (6). In a Teflon vessel charged with 29 (10.2 mg,
6.8 µmol) and anisole (100 µL) was condensed 2-3 mL of anhydrous
HF at -78 °C. The solution was warmed to 0 °C and stirred for an
additional 90 min. The HF was removed at 0 °C under a stream of N2
for 1 h. The residue was dissolved in 1 M aqueous HCl and lyophilized
to provide 30 as the HCl salt which was used directly in the next
reaction.
A solution of 30 in DMF (1.0 mL) at 0 °C was treated with NaHCO3
(8.6 mg, 102 µmol, 15 equiv), HOBt (4.6 mg, 34, µmol, 5 equiv), and
6-methoxyquinoxaline-2-carboxylic acid (32, 5.5 mg, 27 µmol, 4 equiv).
Once the solution was homogeneous, EDCI (5.2 mg, 27 µmol, 4 equiv)
was added. The solution was stirred at 0 °C for 1 h, warmed to 25 °C,
and stirred for an additional 5 h. The solution was poured into saturated
aqueous NaHCO3 (5 mL) and extracted with EtOAc (5 × 5 mL). The
combined organic phase was dried (Na2SO4), filtered and concentrated.
PTLC (SiO2, 8% CH3OH-CH2Cl2) provided 6 (5.8 mg, 4.4 µmol,
65%).
Quinoxapeptin A (4). A solution of (1S,2S)-methylcyclopropane-
carboxylic acid (5.6 mg, 56 µmol) in CH2Cl2 (40 µL) and DMF (0.0052
µmol) at 0 °C was treated with oxalyl chloride (6 µL, 67 µmol), stirred
at 0 °C for 20 min, warmed to 25 °C, and stirred for an additional 1.5
h. The solution was transferred dropwise (5-10 min) by syringe to a
solution of quinoxapeptin C (6, 1.0 mg, 0.76 µmol) in pyridine (50
µL) at 0 °C, and the resulting mixture was stirred for 1.5 h. The solution
was diluted with CH2Cl2 (2 mL) and washed with saturated aqueous
NaHCO3 (2 × 2 mL) and saturated aqueous NaCl (1 × 2 mL). After
re-extraction of the aqueous layer with CH2Cl2 (4 × 2 mL), the organic
layers were combined, dried (Na2SO4), and concentrated. PTLC
(alumina, 10 × 10 cm, 4% CH3OH-CH2Cl2) provided 4 (0.9 mg, 0.61
µmol, 80%) and the corresponding S,S-monoester 36 (0.2 mg, 0.14
µmol, 18%).
(1R,2R)-2-Methylcyclopropanecarboxylic Acid Diester of Quin-
oxapeptin C (35). A solution of (1R,2R)-methylcyclopropanecarboxylic
acid (8 mg, 80 µmol) in CH2Cl2 (40 µL) and DMF (0.0026 µmol) at
0 °C was treated with oxalyl chloride (6 µL, 67 µmol), stirred at 0 °C
for 10 min, warmed to 25 °C, and stirred for an additional 2 h. The
solution was transferred dropwise (5 min) by syringe to a solution of
quinoxapeptin C (6, 0.6 mg, 0.46 µmol) in pyridine (20 µL) at 0 °C,
and the resulting mixture was stirred for 1 h. The solution was diluted
with CH2Cl2 (2 mL) and washed with saturated aqueous NaHCO3 (2
× 2 mL) and saturated aqueous NaCl (1 × 2 mL). After re-extraction
of the above aqueous layer with CH2Cl2 (4 × 2 mL), the organic layers
were combined, dried (Na2SO4), filtered, and concentrated. PTLC
(alumina, 4% CH3OH-CH2Cl2) provided 35 (0.5 mg, 74%) and the
corresponding monoester 37 (0.1 mg, 16%).
mM Tris-HCl (pH 7.4), 75 mM NaCl. The reverse addition, of the
agent to the buffer, caused significant precipitation and gave lower
fluorescence intensities with a number of the agents. Type I calf thymus
DNA (Sigma) was dissolved in 10 mM Tris-HCl (pH 7.4), 75 mM
NaCl buffer to a concentration of 320 µM base pairs based on an
extinction coefficient of 12 824 M-l cm-1 at 260 nm. The purity was
checked by assuring that the absorbance ratio at 260:280 nm was greater
than 1.8. The concentration of the self-complementary deoxyoligo-
nucleotides 5′-(GCATGC)2, 5′-(GCGCGC)2, 5′-(GCTAGC)2, 5′-(GC-
CGGC)2 (Genbase Inc., San Diego, CA) was established as previously
described,4 and these were diluted to 320 µM base pairs in 10 mM
Tris-HCl (pH 7.4), 75 mM NaCl aqueous buffer.
The agent (20 µL, 1 mM in DMSO) was added to a clean, dry 4-mL
quartz cuvette containing a Teflon-coated stirrer bar. A further 20 µL
of DMSO was added, followed by 1960 µL of 10 mM Tris-HCl (pH
7.4), 75 mM NaCl aqueous buffer. After 5 min of stirring, an initial
fluorescence reading was taken with minimum exposure to the
excitation beam. At this point, aliquots of DNA solution (5-10 µL)
were added, and the solution was allowed to equilibrate for 15 min
before the subsequent readings were taken. The excitation and emission
wavelengths were 360 and 530 nm for sandramycin, 340 and 520 nm
for the luzopeptins, and 360 and 460 nm for the quinoxapeptins. The
results of the titrations were analyzed by Scatchard analysis. For CT-
DNA, the linear part of the Scatchard plot was used to determine high-
affinity binding constants. For the deoxynucleotides, a nonlinear fit of
the curve as described in the text was used to determine the high-
affinity binding constant.
DNA Unwinding. Due to the low solubility of the agents in water,
all agents were dissolved in DMSO as stock solutions, stored at -20
°C in the dark, and diluted to working concentrations in DMSO prior
to addition to the DNA. A buffer solution containing 0.25 µg of
supercoiled ΦX174 RF1 DNA (1.0 × 10-8 M) in 9 µL of 50 mM
Tris-HCl buffer solution (pH 8.0) was treated with 1 µL of agent in
DMSO. The control DNA was treated with 1 µL of DMSO. The agent-
to-base pair ratios were 0.011, 0.022, 0.033, 0.044, 0.11, and 0.22 for
sandramycin and 0.022, 0.033, 0.044, 0.11, and 0.22 for luzopeptin A,
B, and C and quinoxapeptin A, B, and C. The reactions were incubated
at 25 °C for 1 h and quenched with 4 µL of loading buffer (30%
aqueous glycerol, 0.25% bromophenol blue, 0.25% xylene cyanol FF).
Electrophoresis was conducted, on a 1% agarose gel at 50 V for 3 h.
The gel was stained with ethidium bromide and visualized using a UV
transilluminator, and the image was captured on an Eagle-Eye II
(Stratagene, La Jolla, CA).
Acknowledgment. We gratefully acknowledge the financial
support of the National Institutes of Health (CA41101) and the
Skaggs Institute for Chemical Biology, the award of a NIH
postdoctoral fellowship (F32 GM1876, M.W.L.), and the
sabbatical leave of Dr. M. Kume sponsored by Shiongi Co.,
Ltd. (1998-1999). We also gratefully acknowledge the prepara-
tion of substantial quantities 10 by Dr. G. Schule (ref 16) and
the development of the route to 16 by J.-H. Chen.
Quinoxapeptin B (5). A solution of 36 (0.4 mg, 0.76 µmol) in
Ac2O-pyridine (1:1, 480 µL) was stirred at 25 °C for 17 h. The solution
was diluted with CH2Cl2 (3 mL), poured into saturated aqueous
NaHCO3 (15 mL), extracted with CH2Cl2 (5 × 10 mL), dried (Na2-
SO4), filtered, and concentrated. PTLC (alumina, 4% EtOH-CH2Cl2
eluent) provided 5 (0.25 mg, 0.18 µmol, 62%).
Quinoxapeptin Diacetate (38). A solution of quinoxapeptin C (6,
1.0 mg, 0.76 µmol) in Ac2O-pyridine (1:1, 240 µL) was stirred at 25
°C for 15 h. The solution was diluted with CH2Cl2 (3 mL), poured into
saturated aqueous NaHCO3 (15 mL), extracted with CH2Cl2 (5 × 10
mL), dried (Na2SO4), filtered, and concentrated. PTLC (SiO2, 6% CH3-
OH-CH2Cl2) provided 38 (0.9 mg, 0.64 µmol, 84%).
Supporting Information Available: Full characterization
data on 1-6, 8, 9, 11-13, 16-18, 20, 25, 26, 28, 29, 31, and
35-38, and full details of the preparation of 33 (PDF). This
material is available free of charge via the Internet at
http://pubs.acs.org.
DNA Binding Studies. The analogues were dissolved in DMSO to
a concentration of 1 mM. A final concentration in the cuvette of 10
µM was achieved by adding 20 µL to the cuvette, diluting with DMSO
to 40 µL, and then adding 1960 µL of aqueous buffer containing 10
JA993019E