Solid-phase synthesis and configurational reassigment of callipeltin E. Implications for the structures of callipeltins A and B

Article abstract of DOI:10.1021/jo060351h

Two possible isomers of the natural product callipeltin E (1, 5) were synthesized by using an Fmoc-based solid-phase strategy in 7 steps, in 20% and 26% overall yields, respectively. The ¹H NMR spectrum of synthetic 5 correlated closely with that of the natural product, whereas that of 1 did not, providing confirmation of the configurational reassignment of the N-terminal residue of callipeltin E as D-allothreonine. This result strongly implies that the corresponding residue in the closely related cyclic depsipeptides callipeltins A and B should also be considered a D-allothreonine residue.

Full text of DOI:10.1021/jo060351h

Solid-Phase Synthesis and Configurational Reassigment of
Callipeltin E. Implications for the Structures of Callipeltins A and B
Selc¸uk C¸ alimsiz, AÄ ngel I. Morales Ramos, and Mark A. Lipton*
Department of Chemistry and Cancer Center, Purdue UniVersity, 560 OVal DriVe,
West Lafayette, Indiana 47907-2084
lipton@purdue.edu
ReceiVed February 20, 2006
Two possible isomers of the natural product callipeltin E (1, 5) were synthesized by using an Fmoc-
based solid-phase strategy in 7 steps, in 20% and 26% overall yields, respectively. The 1H NMR spectrum
of synthetic 5 correlated closely with that of the natural product, whereas that of 1 did not, providing
confirmation of the configurational reassignment of the N-terminal residue of callipeltin E as
D-allothreonine. This result strongly implies that the corresponding residue in the closely related cyclic
depsipeptides callipeltins A and B should also be considered a D-allothreonine residue.
Introduction
stants, it was suggested by Bifulco et al. that both of the
threonine residues in callipeltin A have the D-allo configuration,5
consistent with what was independently found in the structurally
related cyclic depsipeptide neamphamide A (4, Figure 1).6 Since
the configuration of the threonine residue in callipeltin E
corresponds to the revised D-allothreonine in callipeltin A, the
revision of configuration of 2 calls into question the original
configurational assignment of callipeltin E. To resolve this
question and definitively establish the configuration of callipeltin
E, we decided to synthesize the two possible isomers of
callipeltin E: the originally reported structure (1) and its
D-allothreonine isomer (5). Herein we report the efficient solid-
phase syntheses of 1 and 5, and their spectral correlation with
the natural product.
Callipeltin E (1, Figure 1), an acyclic hexapeptide member
of the callipeltin family, was isolated from the marine sponge
Latrunculia sp. by D’Auria and co-workers in 2002.1 1 was
shown to be a truncated, open-chain derivative of callipeltins
A and B (2 and 3, Figure 1), previously isolated cyclic
depsipeptides that possess anti-HIV and antifungal activity and
cytotoxicity against several human carcinoma cell lines.2,3 Due
to the acid-sensitive nature of the â-OMeTyr residue present in
1-3, its configuration was only determined recently as the
2R,3R isomer by D’Auria and co-workers using chemical
degradation of callipeltin A and derivatization of the resulting
amino acids.4 By extension, the â-OMeTyr residue present in
1 is now believed to be the 2R,3R isomer. Recently, by
employing quantum mechanical calculation of coupling con-
Results and Discussion
* Address correspondence to this author. Phone: +765-494-0132. Fax: +765-
494-0239.
Our approach to the synthesis of callipeltin E employed an
Fmoc-based, solid-phase strategy. It was envisaged that, due to
(1) Zampella, A.; Randazzo, A.; Borbone, N.; Luciani, S.; Trevisi, L.;
Debitus, U.; D’Auria, M. V. Tetrahedron Lett. 2002, 43, 6163-6166.
(2) Zampella, A.; D’Auria, M. V.; Paloma, L. G.; Casapullo, A.; Minale,
L.; Debitus, C.; Henin, Y. J. Am. Chem. Soc. 1996, 118, 6202-6209.
(3) D’Auria, M. V.; Zampella, A.; Paloma, L. G.; Minale, L.; Debitus,
C.; Roussakis, C.; LeBert, V. Tetrahedron 1996, 52, 9589-9596.
(4) Zampella, A.; D’Orsi, R.; Sepe, V.; Casapullo, A.; Monti, M. C.;
D’Auria, M. V. Org. Lett. 2005, 7, 3585-3588.
(5) Bassarello, C.; Zampella, A.; Monti, M. C.; Gomez-Paloma, L.;
D’Auria, M. V.; Riccio, R.; Bifulco, G. Eur. J. Org. Chem. 2006, 604-
609.
(6) Oku, N.; Krishnamoorthy, R.; Benson, A. G.; Ferguson, R. L.; Lipton,
M. A.; Phillips, L. R.; Gustafson, K. R.; McMahon, J. B. J. Org. Chem.
2005, 70, 6842-6847.
10.1021/jo060351h CCC: $33.50 © 2006 American Chemical Society
Published on Web 07/21/2006
J. Org. Chem. 2006, 71, 6351-6356
6351

C¸ alimsiz et al
FIGURE 1. Two possible isomers of callipeltin E (1 and 5), callipeltin A (2), callipeltin B (3), and neamphamide A (4).
the acid-labile nature of the (2R,3R)-â-methoxytyrosine (â-
MeOTyr) residue, the coupling conditions, cleavage from the
resin, and removal of protecting groups would be performed
under either mildly acidic or nonacidic conditions. Our initial
strategy followed closely the synthetic plan we had developed
for callipeltins A and B. Thus, anchoring the side chain of the
N-methylglutamine residue to the Sieber amide resin (10)7 was
initially explored rather than a linear C to N strategy. Addition-
ally, the protecting groups used in the synthesis were chosen
for their facile removal by hydrogenolysis.
The initial retrosynthetic analysis for the synthesis of cal-
lipeltin E is shown in Scheme 1. It was envisaged that both
isomers 1 and 5 could be obtained from pentapeptide 6 via
coupling of either D-allo or L-threonine, followed by resin
cleavage and hydrogenolytic deprotection. Pentapeptide 6 would
be obtained by sequential N to C coupling of N-methylalanine
to the resin-bound dipeptide 7, followed by standard C to N
coupling of the leucine and D-arginine residues. 7 could be
derived in turn from the regioselective ring opening of anhydride
8 with deprotected Sieber Resin 10, followed by N to C coupling
of â-methoxytyrosine 9.
Cyclic anhydride 8 was synthesized from Fmoc-Glu(OtBu)
in 88% overall yield (Scheme 2) by acid deprotection followed
by treatment with neat acetic anhydride, giving 8. N-Methyl-
alanine benzyl ester (11) was prepared in 67% overall yield
from Boc-N-methylalanine by esterification followed by Boc
deprotection.
The peptide coupling conditions were screened by employing
a model system in which L-tyrosine was used in lieu of the
â-methoxytyrosine residue. All reactions were monitored by
cleavage of a small aliquot of resin and analysis of the crude
cleavage product by reverse-phase HPLC and MALDI-MS.
Thus, Fmoc deprotection of the Sieber resin linker followed by
the regioselective addition of cyclic anhydride 8 provided resin-
bound Fmoc-MeGln (12). Activation of 12 with PyBOP,
followed by introduction of Tyr(OBn)-OAllyl‚HCl provided
dipeptide 13 without any evidence of epimerization as indicated
by HPLC (Scheme 3).
Palladium-catalyzed deprotection of the allyl ester in 13
(Scheme 4) was followed by activation of the resin-bound
carboxylic acid and coupling with N-methylalanine benzyl ester
(11). Fmoc removal followed by peptide coupling of Fmoc-
leucine yielded resin-bound tetrapeptide 14. Attempts to couple
Nω-nitro-NR-Fmoc-D-arginine to 15 produced a single product
14 amu lighter than the expected pentapeptide 6. Similar results
were observed when Nω-Nω-bis(benzyloxycarbonyl)-NR-Fmoc-
D-arginine or Nω-Nω-bis(allyoxycarbonyl)-NR-Fmoc-D-arginine
1
were coupled to 14. H NMR comparison of the unexpected
product and callipeltin E showed a loss of the N-methylalanine
N-methyl group, suggesting the formation of pentapeptide 15.
To confirm the site of demethylation, authentic 15 was
synthesized by using Fmoc-alanine in place of Fmoc-N-
methylalanine. The product of this synthesis was the expected
one with no loss of the glutamine N-methyl group.
Changing the addition sequence by performing the C to N
couplings first and adding the N-methylalanine residue last again
afforded the demethylated product 15. At this stage, it was
concluded that demethylation resulted when the N-methylalanine
and D-arginine were both present in the growing peptide. It was
proposed that the anchoring point of peptide 6 to the solid
support might have an effect on the conformation of the peptide,
promoting a pseudocyclic conformation containing a strong
(7) Sieber, P. Tetrahedron Lett. 1987, 28, 2107-2110.
6352 J. Org. Chem., Vol. 71, No. 17, 2006

Synthesis and Configurational Reassigment of Callipeltin E
SCHEME 1. Initial Retrosynthetic Analysis
SCHEME 2
peptides from resins with use of acidic conditions can result in
acid-promoted demethylation.8 On the basis of these results, our
synthetic strategy was revised.
On the basis of the proposed demethylation process, it was
decided to change the anchoring point of the solid support to
the peptide backbone. It was envisaged that placing a bulky
linker on the carboxylate terminus of the N-methylalanine might
hinder its demethylation. By employing this strategy, not only
the pseudocyclic conformation of the hexapeptide backbone
would be avoided, but also only C to N couplings would be
needed. For these reasons the acid-labile 2-chlorotrityl chloride
resin (16)9 was chosen.
The required residue Fmoc-(2R,3R)-â-methoxytyrosine (19)
was synthesized from the previously reported (2R,3R)-â-
methoxytyrosine allyl ester (17)6 in two steps in 56% overall
yield (Scheme 5). Also, Fmoc-NR-methylglutamine (22) was
prepared from commercially available Nδ-trityl-NR-Fmoc-
SCHEME 3
glutamine (20) via the oxazolidinone intermediate 21 employing
10,11
Freidinger’s procedure
in 82% yield (Scheme 5).
In the synthesis of 1, each peptide coupling was complete in
30 min, using 1.5 equiv of HOAt/HATU and 1.5 equiv of the
Fmoc-protected amino acid in the presence of 3 equiv of Hunig’s
base. 2-Chlorotrityl chloride resin (16) activation, followed by
coupling with Fmoc-N-methylalanine, 19, 22, Fmoc-leucine,
Nω,Nω-bis(benzyloxycarbonyl)-NR-Fmoc-D-arginine, and Fmoc-
D-allothreonine, respectively, afforded the resin-bound hexapep-
tide 23 in 71% purity as judged by HPLC analysis (Scheme 6).
(8) Teixido, M.; Albericio, F.; Giralt, E. J. Pept. Res. 2005, 65, 153-
166.
(9) Barlos, K.; Chatzi, O.; Dimitrios, G.; Stavropoulos, G. Int. J. Pept.
Protein Res. 1991, 38, 513-520.
hydrogen bond between the protected guanidine and the alanine
carbonyl (Figure 2). This hydrogen bond might weaken the
methyl-nitrogen bond, making the CH3 a labile electrophile.
It has been previously reported that cleavage of N-methylated
(10) Freidinger, R. M.; Hinkle, J. S.; Perlow, D. S.; Arison, B. H. J.
Org. Chem. 1983, 48, 77-81.
(11) Aurelio, L.; Box, J. S.; Brownlee, R. T. C.; Hughes, A. B.; Sleebs,
M. M. J. Org. Chem. 2003, 68, 2652-2667.
J. Org. Chem, Vol. 71, No. 17, 2006 6353

C¸ alimsiz et al
SCHEME 4
No demethylation of the MeAla was observed after coupling
of the Fmoc-arginine residue to the growing peptide, supporting
our hypothesis about the cause of demethylation. Removal of
the Fmoc group and cleavage of the hexapeptide from the resin,
followed by reverse-phase HPLC purification, yielded a partially
deprotected hexapeptide that was fully deprotected by hydro-
genation in 2-propanol.12 Final purification by reverse-phase
HPLC afforded 1 in 20% overall yield. The second isomer 5
was prepared in the same manner, using Fmoc-D-allothreonine
in place of Fmoc-threonine, in 26% overall yield.
1
Visual comparison of the H NMR spectra of callipeltin E,
1, and 5 revealed several important differences. In particular,
there were no significant differences between the spectra of
callipeltin E and 5 in the region from 3.8 to 4.5 ppm, whereas
the spectrum of 1 was substantially different in that region
(Figure 313). This distinction is especially noteworthy since the
signals for the N-terminal Thr/D-alloThr residue lie within that
region of the 1H NMR. On the basis of these results we conclude
that 5 is identical with callipeltin E, thereby confirming that
D-allothreonine is the N-terminal residue.14
Conclusion
In summary, a reliable, solid-phase synthesis for both isomers
of callipeltin E was developed. Callipeltin E was synthesized
in 7 steps in 26% overall yield. The H NMR of synthetic 5
FIGURE 2. Proposed conformation of 6 leading to acid-promoted
demethylation.
1
correlated closely with that of the natural product, confirming
the reassignment of the configuration of the N-terminal residue
in callipeltin E as D-allothreonine. This finding also reinforces
the recent configurational reassignment of the corresponding
residue in callipeltin A and, by extension, in callipeltin B.
Combined with the recent confirmation of the configurational
assignment of the AGDHE residue in callipeltin A,15 we now
believe that the structures of 2 and 3 have been firmly
established pending their confirmation by total synthesis.
SCHEME 5
(12) Hydrogenation in methanol and ethanol afforded N-methylated and
N-ethylated byproducts, respectively.
(13) NMR spectrum obtained from Prof. M. V. D’Auria and reproduced
with permission.
1
(14) Tabulated H NMR signals of callipeltin E; 1 and 5 are compared
in Table 1 in the Supporting Information.
(15) Cranfill, D. C.; Morales-Ramos, A. I.; Lipton, M. A. Org. Lett. 2005,
7, 5881-5883.
6354 J. Org. Chem., Vol. 71, No. 17, 2006

Synthesis and Configurational Reassigment of Callipeltin E
1
FIGURE 3. Comparison of the 3.8-4.5 ppm region of the H NMR spectra of (a) callipeltin E, (b) 1, and (c) 5.
SCHEME 6
was bubbled through the reaction for an additional 5 min. The
Experimental Section
solvents were removed by filtration and the resin washed sequen-
tially with the following solvents: 9:1 MeOH-DIEA (2×), DMF
(3×), i-PrOH (2×), DMF, i-PrOH, CH2Cl2, and Et2O.
All reactions were monitored by cleaving the growing peptide
from a small quantity of resin and analysis of the crude cleavage
product by reverse-phase HPLC and MALDI-TOF mass spectrom-
etry.
To the washed resin was added CH2Cl2 (0.35 mL) and a 25%
piperidine/DMF solution (0.35 mL), and N2 was bubbled through
the reaction for 10 min. The solvents were removed by filtration
and the resin was treated again with a 25% piperidine/DMF solution
(0.7 mL) for 15 min. The solvent was removed by filtration and
the resin washed with DMF (3×), i-PrOH (2×), and DMF (2×).
In a separate vial, HATU (20 mg, 0.052 mmol), HOAt (7 mg, 0.052
mmol), and Fmoc-(2R,3R)-â-methoxytyrosine (19) (27 mg, 0.052
mmol) were dissolved in DMF (0.4 mL). DIEA (27 µL, 0.16 mmol)
was added and the mixture was stirred for 1.5 min and added to
the resin. N2 was bubbled through the reaction for 30 min, the
solvent was removed by filtration, and the resin was washed with
DMF (3×), i-PrOH (2×), DMF (2×), i-PrOH, CH2Cl2, and Et2O.
To the washed resin was added CH2Cl2 (0.35 mL) and a 25%
piperidine/DMF solution (0.35 mL), and N2 was bubbled through
Callipeltin E (D-Allothreonine Isomer, 5). 2-Chlorotrityl
chloride resin (16, 50 mg, 100-200 mesh, 1% DVB, 1.2 mmol/g)
was washed with dry DMF and dry CH2Cl2 (3×) under a positive
N2 atmosphere. In a flame-dried vial, SOCl2 (6.5 µL, 0.090 mmol)
was dissolved in CH2Cl2 (0.5 mL). The resulting solution was
transferred to the resin and the mixture was agitated by bubbling
N2 through it for 1 h. The solvent was removed by filtration and
the resin was washed with DMF (2×) and CH2Cl2 (3×). In another
flame -dried vial, Fmoc-N-methylalanine (12 mg, 0.037 mmol) and
DIEA (25.6 µL, 0.147 mmol) were dissolved in CH2Cl2 (0.5 mL)
and added to the preactivated resin. N2 was bubbled through the
reaction for 1.5 h, after which the excess sites on the resin were
quenched with a solution of 9:1 MeOH-DIEA (0.5 mL) and N2
J. Org. Chem, Vol. 71, No. 17, 2006 6355

C¸ alimsiz et al
the reaction for 10 min. The solvents were removed by filtration
and the resin was treated again with a 25% piperidine/DMF solution
(0.7 mL) for 15 min. The solvent was removed by filtration and
the resin washed with DMF (3×), i-PrOH (2×), and DMF (2×).
In a separate vial, HATU (20 mg, 0.052 mmol), HOAt (7 mg, 0.052
mmol), and Fmoc-NR-methylglutamine (22) (20 mg, 0.052 mmol)
were dissolved in 0.4 mL of DMF. DIEA (30 µL, 0.173 mmol)
was added and the mixture stirred for 1.5 min and added to the
resin. N2 was bubbled through the reaction for 30 min, the solvent
was removed by filtration, and the resin was washed with DMF
(3×), i-PrOH (2×), DMF (2×), i-PrOH, CH2Cl2, and Et2O.
To the washed resin was added CH2Cl2 (0.35 mL) and a 25%
piperidine/DMF solution (0.35 mL), and N2 was bubbled through
the reaction for 10 min. The solvents were removed by filtration
and the resin was treated again with a 25% piperidine/DMF solution
(0.7 mL) for 15 min. The solvent was removed by filtration and
the resin washed with DMF (3×), i-PrOH (2×), and DMF (2×).
In a separate vial, HATU (20 mg, 0.052 mmol), HOAt (7 mg, 0.052
mmol), and Fmoc-leucine (18 mg, 0.052 mmol) were dissolved in
0.4 mL of DMF. DIEA (30 µL, 0.173 mmol) was added and the
mixture was stirred for 1.5 min and added to the resin. N2 was
bubbled through the reaction for 30 min, the solvent was removed
by filtration, and the resin was washed with DMF (3×), i-PrOH
(2×), DMF (2×), i-PrOH, CH2Cl2, and Et2O.
In a 250-mL round-bottom flask 5 (4 mg) was dissolved in
AcOH-iPrOH (2.0 mL) and 10% Pd(OH)2-C (∼1 mg) was added.
The system was flushed and evacuated three times with argon, and
then flushed with H2 twice and maintained under a positive pressure
of H2. The reaction proceeded for 140 min, with the balloon being
refilled with hydrogen hourly. The catalyst was removed by
filtration and the solution was concentrated under reduced pressure.
This procedure was repeated three times until all of 5 was
deprotected.
The combined crude product (14 mg) was purified with reverse-
phase HPLC (C8, 5-40% gradient of CH3CN-H2O over 50 min,
retention time 33 min), yielding 5 (7.7 mg, 26%) as a colorless
1
foam. H NMR (500 MHz, CD3OD) δ 7.19 (d, 2H, J ) 8.5 Hz),
6.78 (d, 2H, J ) 8.5 Hz), 5.22 (t, d, 1H, J ) 7, 2 Hz), 5.11 (q, 1H,
J ) 7.3 Hz), 4.83 (overlapped, 1H), 4.74 (d, d, 1H, J ) 10.4, 3.8
Hz), 4.39 (d, d, 1H, J ) 7.9, 5.7 Hz), 4.32 (d, 1H, J ) 8.9 Hz),
4.13 (m, 1H), 3.89 (d, 1H, J ) 5.5 Hz), 3.2 (m, 2H), 3.13 (s, 3H),
3.08 (s, 3H), 2.86 (s, 3H), 1.99 (overlapped, 2H), 1.98, 1.72
(overlapped, 2H), 1.97, 1.65 (overlapped, 2H), 1.63 (overlapped,
2H), 1.6 (overlapped, 2H), 1.4 (d, 3H, J ) 6.9 Hz), 1.28 (d, 2H, J
) 6.3 Hz), 0.95 (d, 3H, J ) 6.7 Hz), 0.93 (d, 3H, J ) 6.7 Hz). 13
C
NMR (125 MHz, CD3OD) δ 129.1, 129, 114.8, 83.9, 65.8, 57.9
56.3, 55.7, 53.2, 52.8, 48.4, 40.5, 39.6, 31.5, 30.1, 28.5, 24.9, 24.6,
22.2, 17.4,13.3. HRMS (ESI) calcd for C36H60N10O11 (H+) 809.4521,
found 809.4535.
To the washed resin was added CH2Cl2 (0.35 mL) and a 25%
piperidine/DMF solution (0.35 mL), and N2 was bubbled through
the reaction for 10 min. The solvents were removed by filtration
and the resin was treated again with a 25% piperidine/DMF solution
(0.7 mL) for 15 min. The solvent was removed by filtration and
the resin was washed with DMF (3×), i-PrOH (2×), and DMF
(2×). In a separate vial, HATU (20 mg, 0.052 mmol), HOAt
(7 mg, 0.052 mmol), and Nω-Nω-bis(benzyloxycarbonyl)-NR-Fmoc-
D-arginine (34.8 mg, 0.052 mmol) were dissolved in 0.4 mL of
DMF. DIEA (30 µL, 0.173 mmol) was added and the mixture was
stirred for 1.5 min and added to the resin. N2 was bubbled through
the reaction for 30 min, the solvent was removed by filtration, and
the resin was washed with DMF (3×), i-PrOH (2×), DMF (2×),
i-PrOH, CH2Cl2, and Et2O.
To the washed resin was added a 2% DBU/DMF solution (0.4
mL), and N2 was bubbled through the reaction for 20 min. The
solvents were removed by filtration and the resin was bubbled again
with a 2% DBU/DMF solution (0.4 mL) for 20 min. The solvent
was removed by filtration and the resin washed with DMF (3×),
i-PrOH (2×), and DMF (2×). In a separate vial, HATU (20 mg,
0.052 mmol), HOAt (7 mg, 0.052 mmol), and Fmoc-D-allothreonine
(18 mg, 0.052 mmol) were dissolved in 0.4 mL of DMF. DIEA
(30 µL, 0.173 mmol) was added and the mixture was stirred for
1.5 min and added to the resin. N2 was bubbled through the reaction
for 30 min, the solvent was removed by filtration, and the resin
was washed with DMF (3×), i-PrOH (2×), DMF (2×), i-PrOH,
CH2Cl2, and Et2O.
Callipeltin E (L-Threonine Isomer, 1). 1 was prepared in the
same manner as 5. The product was cleaved from the resin by
treatment with a 2% TFA/CH2Cl2 solution for 15 min, followed
by removal of the resin. Evaporation of solvent under reduced
pressure afforded crude product (40 mg), which was purified with
reverse-phase HPLC (C8, 40-60% gradient of CH3CN-H2O over
60 min, retention time 29.5 min), yielding protected 1 (15.1 mg)
as a colorless, glassy solid. After hydrogenation (4 mg at a time)
the combined crude product was purified with reverse-phase HPLC
(C8, 5-40% gradient of CH3CN-H2O over 50 min, retention time
32.5 min), yielding 1 (5.8 mg, 20%) as a colorless foam. 1H NMR
(500 MHz, CD3OD) δ 7.24 (d, 2H, J ) 8.5 Hz), 6.81 (d, 2H, J )
8.5 Hz), 5.23 (q, 1H, J ) 9.2 Hz), 5.13 (q, 1H, J ) 7.2 Hz), 4.9
(overlapped, 1H), 4.74-4.8 (m, 1H), 4.45 (d, d, 1H, J ) 7.8, 6.6
Hz), 4.38 (d, 1H, J ) 9.1 Hz), 4.08 (p, 1H, J ) 6.5 Hz), 3.78 (d,
1H, J ) 6.5 Hz), 3.24 (m, 2H), 3.16 (s, 3H), 3.14 (s, 3H), 2.87 (s,
3H), 1.99 (overlapped, 2H), 1.98, 1.72 (overlapped, 2H), 1.97, 1.65
(overlapped, 2H), 1.63 (overlapped, 2H), 1.6 (overlapped, 2H), 1.44
(d, d, 3H, J ) 7.26, 1.77 Hz), 1.31 (d, 2H, J ) 6.3 Hz), 1.01 (d,
3H, J ) 5.7 Hz), 0.93 (d, 3H, J ) 3.6 Hz). 13C NMR (125 MHz,
CD3OD) δ 129.1, 129, 128.9, 114.8, 83.8, 65.8, 58.7, 55.7, 55.4,
52.9, 52.7, 48.4, 40.5, 39.6, 31.5, 31.2, 28, 25, 24.6, 22.2, 18.9,
13.3. HRMS (ESI) calcd for C36H60N10O11 (H+) 809.4521, found
809.4532.
To 13 was added a 2% DBU/DMF solution (0.4 mL), and N2
was bubbled through the reaction for 20 min. The solvents were
removed by filtration and the resin was bubbled again with a 2%
DBU/DMF solution (0.4 mL) for 20 min. The solvent was removed
by filtration and the resin was washed with DMF (3×), i-PrOH
(2×), DMF (2×), i-PrOH, CH2Cl2, and Et2O. The product was
cleaved from the resin by treatment with a 2% TFA/CH2Cl2 solution
for 15 min, followed by removal of the resin by filtration.
Evaporation of the solvent under reduced pressure afforded 47 mg
of crude product. The crude product was purified with reverse-
phase HPLC (C8, 40-60% gradient of CH3CN-H2O over 60 min,
retention time 29.5 min), yielding protected 5 (13.6 mg) as a
colorless, glassy solid.
Acknowledgment. We gratefully acknowledge the assistance
of Prof. M. V. D’Auria for her provision of NMR spectra, a
manuscript preprint, and helpful discussions. We thank Dr.
Huaping Mo for assistance with the variable-temperature
experiments. We also thank the National Institutes of Health
(AI-50888) for support of this work.
Supporting Information Available: Full experimental details
and tabulated NMR spectra. This material is available free of charge
via the Internet at http://pubs.acs.org.
JO060351H
6356 J. Org. Chem., Vol. 71, No. 17, 2006