Complete stereochemistry of neamphamide A and absolute configuration of the β-methoxytyrosine residue in papuamide B

Article abstract of DOI:10.1021/jo0508853

The absolute stereochemistry of the three unresolved structural components in neamphamide A (1) was determined to be (R)-β-methoxy-L-tyrosine, (2R,3A,4S)-4-amino-7-guanidino-2,3-dihydroxy-heptanoic acid, and (2R,3R,4R)-3-hydroxy-2,4,6-trimethylheptanoic acid. Stereochemical assignments were made by chemical degradation of 1, derivatization of the resulting products, and then spectroscopic and chromatographic comparison of the derivatives with synthetically prepared standards. Using the same analytical protocol developed for 1, the β-methoxytyrosine residue in papuamide B (2) was found to be (R)-β-methoxy-D-tyrosine. This represents a rare example of divergent stereochemistry in an unusual amino acid residue that is present in two closely related classes of peptides.

Full text of DOI:10.1021/jo0508853

Complete Stereochemistry of Neamphamide A and Absolute
Configuration of the â-Methoxytyrosine Residue in Papuamide B
Naoya Oku,† Ravi Krishnamoorthy,‡ Alan G. Benson,‡ Robert L. Ferguson,‡ Mark A. Lipton,‡
Lawrence R. Phillips,§ Kirk R. Gustafson,*,† and James B. McMahon†
Molecular Targets Development Program, Center for Cancer Research, National Cancer Institute,
Building 1052, Room 121, Frederick, Maryland 21702-1201, Department of Chemistry,
Purdue University, West Lafayette, Indiana 47907-2084, and Biological Testing Branch,
Developmental Therapeutics Program, National Cancer Institute, Building 1047, Room 5,
Frederick, Maryland 21701-1201
manuscripts@ncifcrf.gov
Received May 2, 2005
The absolute stereochemistry of the three unresolved structural components in neamphamide A
(1) was determined to be (R)-â-methoxy-L-tyrosine, (2R,3R,4S)-4-amino-7-guanidino-2,3-dihydroxy-
heptanoic acid, and (2R,3R,4R)-3-hydroxy-2,4,6-trimethylheptanoic acid. Stereochemical assign-
ments were made by chemical degradation of 1, derivatization of the resulting products, and then
spectroscopic and chromatographic comparison of the derivatives with synthetically prepared
standards. Using the same analytical protocol developed for 1, the â-methoxytyrosine residue in
papuamide B (2) was found to be (R)-â-methoxy-D-tyrosine. This represents a rare example of
divergent stereochemistry in an unusual amino acid residue that is present in two closely related
classes of peptides.
Introduction
antifungal, cytotoxic, and sodium ionophore properties.4
Distinguishing structural characteristics of this family
of peptides include a preponderance of unusual amino
acid residues and unique N-terminal polyketide derived
moieties. The novel structural features and diverse
biological effects of these peptidic metabolites have
generated considerable interest among synthetic chem-
A series of potent HIV inhibitory cyclic depsipeptides
have recently been described from a number of marine
sponges. Peptides in this family include neamphamide
A (1) from Neamphius huxleyi,1 the callipeltins from
Callipelta sp. and Latrunculia sp.,2 and the papuamides
from Theonella sponges.3 In addition to antiviral activity,
these compounds have been reported to exhibit potent
(2) (a) 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. (b) D’Auria, M. V.; Zampella, A.; Paloma, L. G.; Minale, L.;
Debitus, C.; Roussakis, C.; Le Bert, V. Tetrahedron 1996, 52, 9589-
9596. (c) Zampella, A.; Randazzo, A.; Borbone, N.; Luciani, S.; Trevisi,
L.; Debitus, C.; D’Auria, M. V. Tetrahedron Lett. 2002, 43, 6163-6166.
(3) Ford, P. W.; Gustafson, K. R.; McKee, T. C.; Shigematsu, N.;
Maurizi, L. K.; Pannell, L. K.; Williams, D. E.; de Silva, E. D.; Lassota,
P.; Allen, T. M.; Van Soest, R.; Andersen, R. J.; Boyd, M. R. J. Am.
Chem. Soc. 1999, 121, 5899-5909.
* To whom correspondence should be addressed. Tel: (301) 846-
5391. Fax: (301) 846-6919.
† Molecular Targets Development Program, CCR, NCI.
‡ Purdue University.
§ Biological Testing Branch, DTP, NCI.
(1) Oku, N.; Gustafson, K. R.; Cartner, L. K.; Wilson, J. A.;
Shigematsu, N.; Hess, S.; Pannell, L. K.; Boyd, M. R.; McMahon, J. B.
J. Nat. Prod. 2004, 67, 1407-1411.
10.1021/jo0508853 CCC: $30.25 © 2005 American Chemical Society
Published on Web 07/26/2005
6842
J. Org. Chem. 2005, 70, 6842-6847

Complete Stereochemistry of Neamphamide A
istry groups. As a result, syntheses of structural subunits
such as 3,4-dimethylglutamine (3,4-diMeGln),5 4-amido-
7-guanidino-2,3-dihydroxyheptanoic acid (3, Agdha),6 and
3-hydroxy-2,4,6-trimethylheptanoic acid (4, Htmha)7 have
been described. These efforts provided essential building
blocks for use in total synthesis of the parent peptides,
and they also helped to unambiguously define the abso-
lute stereochemistry of several key stereogenic centers.
However, synthetic efforts to prepare the intact natural
products have been hampered by unresolved questions
concerning the stereochemistry of the â-methoxytyrosine
(5, âOMeTyr) residues, which occur throughout this
family of peptides. While stereoselective syntheses of all
four diastereomers of 5 have been reported,8 the absolute
stereochemistry of this residue in the natural peptides
was never established. Decomposition of 5 during acid
hydrolysis of the parent peptide prevented the successful
application of standard techniques such as Marfey’s
analysis.9 In the original report of neamphamide A (1),
the chirality of the Agdha, Htmha, and âOMeTyr resi-
dues was left undefined. Described herein is the first
complete stereochemical assignment of neamphamide A
(1) and a general method for determining the absolute
stereochemistry of âOMeTyr (5) residues in other pep-
tides. This technique was also used to unambiguously
define the stereochemistry of the same residue in pap-
uamide B (2), and the results of these analyses should
help focus the synthetic efforts underway to prepare the
parent peptides.
Results and Discussion
Our prior studies had established the absolute stere-
ochemistry of nine amino acid residues of neamphamide
A (1) as D-Arg, L-Leu, D- and L-Asn, L-NMeGln, L-
homoproline, D-allo-Thr (two residues), and (3S,4R)-3,4-
diMe-L-Gln. In addition, C4 of the Agdha (3) moiety was
assigned a S-configuration by chemical degradation
involving diol-cleavage, oxidative work up, and acid
hydrolysis, followed by Marfey’s analysis of the resulting
arginine subunit.1 In the current study, a synthetic
standard of 3 with defined stereochemistry was available
for comparative purposes,6 but direct application of
Marfey’s method to the peptide hydrolysate to determine
the stereochemistry at C2 and C3 proved unsuitable. The
L-FDAA derivative of 3 had poor retention on reversed-
phase chromatography packings and unacceptable peak
broadening in the LC-MS analysis. Accordingly, the
γ-amino acid 3 was isolated from the hydrolysate of 1
and converted into cyclic derivatives appropriate for
NMR-based configurational analysis at C2, C3, and C4.
Compound 1 was hydrolyzed in 6 N HCl (107 °C, 18.5
h), defatted with EtOAc, and the resulting aqueous
hydrolysate was purified by anion- and then cation-
exchange chromatography to give a mixture of 3 and Arg.
While dissolved in CD3OD for NMR studies, compound
3 was gradually converted to the trideuteriomethyl ester
derivative 6. This transformation was evidenced from 1H
resonances at δ 4.10 (H3 of 3) and 4.37 (H2 of 3), which
diminished in intensity, while resonances at δ 3.97 (H3
of 6) and 4.35 (H4 of 6) emerged and grew with time
(Supporting Information). Positive ion ESI-MS data
obtained before and after the NMR experiments showed
the appearance of a dominant ion peak at m/z 25210 and
(4) Trevisi, L.; Bova, S.; Cargnelli, G.; Danieli-Betto, D.; Floreani,
M.; Germinario, E.; D’Auria, M. V.; Luciani, S. Biochem. Biophys. Res.
Commun. 2000, 279, 219-222. (b) Trevisi, L.; Cargnelli, G.; Ceolotto,
G.; Papparella, I.; Semplicini, A.; Zampella, A.; D’Auria, M. V.; Luciani,
S. Biochem. Pharmacol. 2004, 68, 1331-1338.
(5) (a) Liang, B.; Carroll, P. J.; Joullie, M. M. Org. Lett. 2000, 2,
4157-4160. (b) Okamoto, N.; Hara, O.; Makino, K.; Hamada, Y.
Tetrahedron: Asymmetry 2001, 12, 1353-1358. (c) Acevedo, C. M.;
Kogut, E. F.; Lipton, M. A. Tetrahedron 2001, 57, 6353-6359.
(6) Thoen, J. C.; Morales-Ramos, AÄ . I.; Lipton, M. A. Org. Lett. 2002,
4, 4455-4458.
(7) (a) Guerlavais, V.; Carroll, P. J.; Joullie´, M. M. Tetrahedron:
Asymmetry 2002, 13, 675-680. (b) Zampella, A.; Sorgente, M.; D’Auria,
M. V. Tetrahedron: Asymmetry 2002, 13, 681-685. (c) Zampella, A.;
D’Auria, M. V. Tetrahedron: Asymmetry 2002, 13, 1237-1239. (d)
Turk, J. A.; Visbal, G. S.; Lipton, M. A. J. Org. Chem. 2003, 68, 7841-
7844.
(8) (a) Okamoto, N.; Hara, O.; Makino, K.; Hamada, Y. J. Org. Chem.
2002, 67, 9210-9215. (b) Hansen, D. B.; Wan, X.; Carroll, P. J.; Joullie´,
M. M. J. Org. Chem. 2005, 70, 3120-3126.
(9) Marphy, P. Carlsberg Res. Commun. 1984, 49, 591-596.
J. Org. Chem, Vol. 70, No. 17, 2005 6843

Oku et al.
SCHEME 1a
a Reagents and conditions: (a) 6 N HCl, 107 °C, 18.5 h; (b) solvent partitioning, 6 N HCl/EtOAc; (c) CD3OD, 65 h; (d) PPTS, 2,2-
dimethoxypropane, DMF, 45 °C (30 min) f 60 °C (1.5 h); (e) 2 N HCl in MeOH, 60 °C, 15.5 h; (f) H2O, reflux, 60 h. bData reproduced from
ref 13.
complete loss of the original peak at m/z 235 (Supporting
Information), which supported the conversion of 3 to the
methyl ester 6. Preparation of the acetonide of 6 with
2,2-dimethoxypropane and PPTS in DMF exclusively
yielded the 2,3-O,O-isopropylidene derivative 7.11 In 1D
gNOESY experiments, selective irradiation of one of the
acetonide methyl signals at δ 1.36 excited the two
oxymethine protons at δ 4.65 (H2) and δ 4.74 (H3). No
NOE enhancement was observed with these protons upon
irradiation of the other methyl group at δ 1.40, thus
indicating H2 and H3 were on the same face of the
dioxolane ring. To correlate the erythro-diol moiety at
C2-C3 with the S-configuration of neighboring C4, 7 was
treated with 2 N HCl to recover 3, which was then
refluxed in H2O for 60 h to achieve partial conversion
into Agdha γ-lactam (8, 63% yield).12 The coupling
constituent. Careful interpretation of the 1H, gCOSY, and
constants observed between protons on the lactam ring
gHSQC spectra of the EtOAc extract allowed assignment
of 8 were JH2,H3 ) 4.7-4.9 Hz and JH3,H4 ) 3.6 Hz. This
of the NMR signals from 4, and these were compared
combination of couplings was compared to the reported
with published data for four synthetic diastereomers of
values for four similarly functionalized synthetic γ-lac-
this fatty acid7d including (2R,3R,4R)-, (2S,3R,4R)-,
tams (Scheme 1),13 and they were only compatible with
1
(2S,3R,4S)-, and (2R,3R,4S)-4. H NMR chemical shifts
the (2R,3R,4S)-isomer. Further support for this stereo-
chemical assignment was provided by mutual NOE
interactions between H2 (δ 4.31) and H4 (δ 3.52) in a 1D
for 4 showed the greatest similarity to those for the
(2R,3R,4R)-isomer, which indicated that the hydroxy acid
recovered from the hydrolysis of 1 had to have either
gNOESY experiment. Thus, the configuration of 3 in the
(2R,3R,4R) or (2S,3S,4S)-stereochemistry. To establish
peptide 1 was determined to be (2R,3R,4S), which is
the absolute stereochemistry of these chiral centers, 4
consistent with the stereochemistry previously assigned
from 1 was condensed with (S)-(+)-phenylglycine methyl
ester (PGME),14 and the product was subjected to LC-
MS analysis. To prepare appropriate chromatographic
standards, synthetic (2R,3R,4R)-4 was reacted either
with (S)-(+)- or (R)-(-)-PGME to obtain (2R,3R,4R)-4 (S)-
(+)-PGME amide and (2R,3R,4R)-4 (R)-(-)-PGME amide.
The latter derivative is chromatographically equivalent
to the (S)-(+)-PGME amide of enantiomeric (2S,3S,4S)-4
when chromatographed on an achiral support. The
reversed-phase HPLC retention time for the PGME
to the Agdha residue in callipeltin A (9).2
The EtOAc extract of the acid hydrolysate of neam-
phamide A (1) contained free Htmha (4) as the major
(10) In ref 1a, the authors reported a molecular ion of Agdha at m/z
252 and attributed the increase of 17 mu to the formation of an NH3
adduct. Although it was not mentioned if CD3OD was used in NMR
experiments prior to the mass measurement, it is possible that the
mass observed was the molecular ion of 6.
(11) Meyers, A. I.; Lawson, J. P. Tetrahedron Lett. 1982, 23, 4883-
4886.
(12) Hardy, P. M. Synthesis 1978, 290-291.
(13) Huang, Y.; Dalton, D.; Carroll, P. J. J. Org. Chem. 1997, 62,
372-376.
(14) Nagai, N.; Kusumi, T. Tetrahedron Lett. 1995, 36, 1853-1856.
6844 J. Org. Chem., Vol. 70, No. 17, 2005

Complete Stereochemistry of Neamphamide A
SCHEME 2a
a Reagents and conditions: (a) BuLi, 4-BnPhCHO, THF, -30 °C, 3 h; (b) NaI, CH3I, THF, 0 °C, 3 h; (c) 0.25 N HCl, THF-MeCN, 25
°C, 16 h; (d) Ti(Oi-Pr)4, allyl alcohol, 25 °C, 24 h; (e) EtOAc-3 N HCl; (f) 0.2 N LiOH-MeOH, rt, 2 days; (g) H2, Pd(OH)2/C, MeOH-4.4%
HCOOH, rt, 4 h. *Obtained as hydrochloride salts.
SCHEME 3
amide prepared from the natural product was identical
to that for synthetic (S)-(+)-PGME amide, thus verifying
a (2R,3R,4R) configuration for the subunit 4 in 1, which
was the same as that established for callipeltin A (9).7c,d
The synthetic procedure we developed to obtain refer-
ence standards of the four diastereomers of âOMeTyr (5)
was based on the stereoselective synthesis of â-hydroxy-
tyrosine reported by Boger.15 An adaptation of Boger’s
procedure (Scheme 2), in which a partially diastereose-
lective addition of a metalated Scho¨llkopf reagent16 (10)
to 4-benzyloxybenzaldehyde formed the two key stereo-
genic centers, afforded roughly a 1:1 mixture of two
epimeric carbinols (11a,b). Chromatographic separation
afforded 11a and 11b in stereochemically pure form.
Spectral correlation with Boger’s published spectra15
permitted the assignment of configuration for each
isomer. Methylation of the hydroxyl group and hydrolysis
of the bis-lactim ether using mild acid afforded stere-
ochemically pure (S)- and (R)-O-benzyl-L-tyrosine methyl
esters (13a and 13b, respectively). Transesterification
with allyl alcohol mediated by titanium tetraisopropoxide
afforded the allyl esters 14a and 14b in 15% and 17%
overall yields, respectively, from 4-benzyloxybenzalde-
hyde. Repetition of this procedure using the enantiomeric
Scho¨llkopf reagent ent-10 afforded ent-14a and ent-14b
in stereochemically pure form and in comparable yields.
Treatment of the allyl esters 14a, 14b, ent-14a, and ent-
14b with 0.2 N LiOH and subsequent hydrogenolysis on
Pd(OH)2 gave (S)-âOMe-L-Tyr, (R)-âOMe-L-Tyr, (R)-âOMe-
D-Tyr, and (S)-âOMe-D-Tyr, respectively, in quantitative
yields.
In a stability test of 5 using the (S)-âOMe-D-Tyr as a
model, the amino acid tolerated 2 N HCl but partially
decomposed when kept in 6 N HCl for 1.5 h at room
temperature to give a burgundy-colored residue that was
insoluble in water but soluble in MeOH. We presumed
that acid-catalyzed decomposition of the âOMeTyr resi-
due might involve elimination of the â-methoxy group
followed by a nucleophilic attack to form condensation
products as illustrated in Scheme 3.
In an initial effort to avoid decomposition of this
residue during hydrolysis, 1 was fully protected with Cbz
and then hydrolyzed in alkaline media, for example, 1 N
or 2.5 N KOH-MeOH, to liberate N-Cbz-âOMeTyr or
N,O-bis(Cbz)-âOMeTyr. However, the reaction was in-
SCHEME 4a
a Reagents and conditions: (a) RuCl3, NaIO4, CCl4-MeCN-0.1
M sodium phosphate buffer ) 2:2:3, pH 7.6, rt, 16 h; (b) CbzCl,
1,4-dioxane-1 N KOH, rt, 1 h; (c) TMS-diazomethane, MeOH,
rt, 1 h; (d) Pd(OH)2, 4.4% HCOOH in MeOH, H2, rt, 4 h; (e) 1.25
M HCl in MeOH, 110 °C, 30 min; (f) TFAA, dichloromethane, 110
°C, 5 min.
consistent in yield, and detection of the desired products
by LC-MS was not successful. Development of an
alternative approach to circumvent these problems was
clearly warranted. Therefore, the authentic standards of
5 were converted into 3-methoxyaspartic acid (OMeAsp)
by oxidation with RuCl3-NaIO4 (CCl4-MeCN-0.1 M
sodium phosphate buffer ) 2:2:3, pH 7.6),17 and then
isolated as their N-Cbz derivatives by treating the dried
reaction mixtures with CbzCl followed by solvent-
partitioning (Scheme 4). LC-MS analysis of the N-Cbz-
OMeAsp products resulted in very broad, low intensity
peaks, so the amino acid derivatives were methylated
with TMS-diazomethane to give 15. While the peak
shape and peak intensity of the dimethyl ester 15 were
improved, it was not possible to resolve the four diaste-
reotopic standards by HPLC, even when they were
chromatographed on chiral supports (Regis Sumipax OA-
3100 L-Val and Regis Pirkle D-phenylglycine). Successful
separation of the four diastereomers was ultimately
achieved by replacing the Cbz group with a trifluoroacetyl
group (Scheme 4)18 and then analyzing the products by
GC-MS on a Chirasil-L-Val capillary column. Although
a molecular ion for N-TFA-OMeAsp dimethyl ester (16)
was not observed under the GC-MS conditions, diag-
nostic fragment ions, for example, m/z 196 [M- ‚COOMe-
MeOH]+ and m/z 103 [C(OMe)COOMe]+ (Figure 1), were
(15) Boger, D. L.; Zhou, J.; Borzilleri, R. M.; Nukui, S.; Castle, S. L.
J. Org. Chem. 1997, 62, 2054-2069.
(16) Scho¨llkopf, U.; Groth, U.; Deng, C. Angew. Chem., Int. Ed. Engl.
1981, 20, 798-799.
(17) Oxidation without using a buffered solution did not give
OMeAsp.
J. Org. Chem, Vol. 70, No. 17, 2005 6845

Oku et al.
FIGURE 1. Fragmentation and the typical EI-MS spectrum of N-TFA-OMeAsp dimethyl ester (16).
used to identify each diastereomer which eluted in the
order (2R,3R), (2S,3S), (2R,3S), and (2S,3R) with satis-
factory peak separation (tR: 15.5, 15.8, 16.4, and 16.7
min, respectively).
and 2 is somewhat surprising considering the structural
similarities and comparable inhibitory activity against
HIV for these two peptides. However, this finding is
consistent with the observation that the âOMeTyr Câ
proton in 1 is a broadened singlet at δ5.03 while the same
proton in 2 appears as a 9.3 Hz doublet at δ4.24.
Assignment of the complete absolute stereochemistry of
neamphamide A (1) and the âOMeTyr residue in pap-
uamide B (2) should now simplify the efforts required to
synthesize these peptides. In addition, the method de-
veloped for analyzing the âOMeTyr residues should be
applicable to other naturally occurring peptides that
contain this amino acid derivative.
With appropriate standards in hand, stereochemical
assignments of the âOMeTyr residues in 1 and 2 were
initiated. The peptides were oxidized with RuCl3-NaIO4,
hydrolyzed in 6 N HCl (110 °C, 6 h), and the hydrolysates
were successively derivatized with HCl-MeOH and
TFAA to provide GC-MS samples. The extracted ion
traces for the m/z 196 and m/z 103 fragment ions from
N-TFA-OMeAsp dimethyl ester (16) both clearly showed
the elution of (2S,3S)-16 (tR 15.8 min) from the oxidative
degradation product of 1 and (2R,3S)-16 (tR 16.4 min)
from that of 2 (Supporting Information). The complete
oxidation, hydrolysis, derivatization, and analysis se-
quence was repeated with both parent peptides, and the
results were consistent each time. This allowed assign-
ment of a (R)-âOMe-L-Tyr residue in 1 and a (R)-âOMe-
D-Tyr residue in 2.19
In conclusion, we have defined the absolute configu-
ration of the hydroxy acid moiety and the two unusual
amino acid residues in neamphamide A (1) as (2R,3R,4R)-
Htmha, (2R,3R,4S)-Agdha, and (R)-âOMe-L-Tyr, respec-
tively. This is the first time the complete stereostructure
has been assigned for a member of this family of anti-
HIV sponge depsipeptides. In addition, a new analytical
procedure was established to determine the absolute
configuration of âOMeTyr residues in peptide natural
products. Application of this technique to papuamide B
(2) revealed the presence of (R)-âOMe-D-Tyr in 2, which
is epimeric at CR to the corresponding residue in 1. The
stereochemical divergence of the âOMeTyr residues in 1
Experimental Section
GC-MS Analysis. Chiral GC-MS analyses were executed
with a gas chromatograph-mass selective detector system
equipped with a Chirasil-L-Val capillary column (25 m × 0.25
mm i.d., 0.16 µm film thickness), using helium as a carrier
gas at a linear velocity of 29.2 cm/s. Temperatures were set to
250 °C at the injection port and 220 °C at the transfer line to
the detector. Samples were injected splitless and chromato-
graphed under the oven temperature program of 60 °C for 3
min, 5 °C/min heat ramp to 200 °C, and the same temperature
held for 10 min. Electron-impact ionization at 70 eV was
employed for mass spectral detection, and elution of N-TFA-
OMeAsp dimethyl ester (16) was traced by diagnostic fragment
ions at m/z 196 and m/z 103.
Conversion of âOMeTyr (5) into N-TFA-OMeAsp Di-
methyl Ester (16). A solution of each deprotected amino acid
standard (100 µg) in a mixture of MeCN (40 µL) and H2O (30
µL) was stirred with NaIO4 (2.4 mg) until the salt crystals
disappeared. To this solution were added CCl4 (40 µL) and a
1 mg/mL solution of RuCl3 hydrate in 0.1 M sodium phosphate
buffer (30 µL, pH 7.6), and the biphasic mixture was vigorously
stirred at room temperature for 16 h. After removal of the
solvent, a half portion of the oxidation product was reacted
with CbzCl (20 µL) in a mixture of 1 N KOH (50 µL) and
freshly distilled 1,4-dioxane (50 µL). After the mixture was
stirred for an hour at ambient temperature, 100 µL of 1 N
KOH was added to the reaction mixture and the unreacted
reagent was extracted with EtOAc (100 µL × 3). The aqueous
layer was then acidified with 2 N HCl (150 µL) and extracted
again with EtOAc (100 µL × 5), which yielded N-Cbz-
(18) In a preliminary GC-MS experiment using D- and L-N-Cbz-
Asp dimethyl esters as the models, both compounds eluted ap-
proximately 5 min after the thermal gradient reached the maximum
temperature limit of the Chirasil column (200 °C) with poor mutual
separation. This suggested that other derivatization of the amino group
instead of Cbz was required to improve the chromatographic behavior
of the methylated OMeAsp derivatives.
(19) It should be noted that the Cahn-Ingold-Prelog priority for
Câ of 5 changes following the RuCl3-NaIO4 oxidation. For example,
(R)-âOMe-L-Tyr yields (2S,3S)-OMeAsp via this oxidative degradation.
6846 J. Org. Chem., Vol. 70, No. 17, 2005

Complete Stereochemistry of Neamphamide A
OMeAsp. This material was further esterified with TMS-
diazomethane (10 µL, 2 M solution in n-hexane) in MeOH (50
µL) by stirring the reaction mixture for 1 h to give N-Cbz-
OMeAsp dimethyl ester (15), followed by deprotection of the
N-Cbz group by hydrogenolysis on 20% Pd(OH)2 on carbon (1
mg suspension in 100 µL of 4.4% HCOOH in MeOH) under a
H2 atmosphere at room temperature for 4 h. The glass-wool
filtrate of the reaction suspension was concentrated and again
methylated with 1.25 M HCl in MeOH (100 µL) at 110 °C for
20 min, followed by TFA-amidation with dichloromethane/TFA
anhydride (1:1, 100 µL) at the same temperature for 5 min to
give N-TFA-OMeAsp dimethyl ester (16). The derivatization
product was dissolved in MeCN (15 µL), and a 1 µL aliquot
was subjected to GC-MS analysis. Retention times (min): 15.5
for (2R,3R)-, 15.8 for (2S,3S)-, 16.4 for (2R,3S)-, and 16.7 for
(2S,3R)-16.
µL aliquot of the product dissolved in MeCN (33 µL) was
subjected to GC-MS analysis. Retention times (min): 15.8 for
N-TFA-OMeAsp dimethyl ester (16) from 1 and 16.4 for 16
from 2.
Acknowledgment. We thank Prof. M. V. D’Auria
at the University of Naples “Federico II”, Naples, Italy,
for helpful discussions. M.A.L. gratefully acknowledges
the financial support of the National Institutes of Health
(AI-50888). The content of this publication does not
necessarily reflect the views or policies of the Depart-
ment of Health and Human Services, nor does mention
of trade names, commercial products, or organizations
imply endorsement by the U.S. government.
Analysis of the âOMeTyr Residues in Neamphamide
A (1) and Papuamide B (2). A 200-µg portion of each peptide
was oxidized with RuCl3-NaIO4 as described in the previous
section. The dried reaction mixture was dissolved in 1% MeOH
in H2O, applied to a short column of reversed-phase polymer
(PolysorbTM MP-1, 100 mg), and desalted by washing with
the same alcoholic water mixture. The peptide was recovered
from the column by elution with MeOH and then hydrolyzed
in degassed 6 N HCl (200 µL) at 110 °C for 6 h. Half of the
hydrolysate was derivatized with HCl-MeOH followed by
dichloromethane/TFA anhydride as described above, and a 1
Supporting Information Available: Experimental pro-
cedures; 1H NMR spectra of 3 at 0 and 24 h in CD3OD; positive
ESI-MS spectra of 3 obtained before and after 60 h-NMR
experiments; 1H NMR, gHSQC, and 1D gNOESY spectra of 7
in CD3OD; 1H NMR, gHSQC, and 1D gNOESY spectra of 8 in
1
D2O; H NMR and gHSQC spectra of 4 from 1 and synthetic
(2R,3R,4R)-4; GC-MS analysis of 16 derived from 1 and 2.
This material is available free of charge via the Internet at
http://pubs.acs.org.
JO0508853
J. Org. Chem, Vol. 70, No. 17, 2005 6847