Highly N-methylated linear peptides produced by an atypical sponge-derived Acremonium sp.

Article abstract of DOI:10.1021/np0503653

RHM1 (1) and RHM2 (2) are highly N-methylated linear octapeptides produced by an atypical strain of Acremonium sp., cultured from a marine sponge collected in Papua New Guinea. The known peptaibiotic efrapeptin G (3) was also isolated from this fungus. The planar structures of 1 and 2 were assigned based on 1D- and 2D-NMR experiments and fragmentation patterns from ESIMS. The absolute configuration of 1 was determined via Marfey's method; the absolute configuration of 2 is proposed to be identical. Efrapeptin G (3) displayed potent cytotoxicity against murine cancer cell lines, while RHM1 (1) and RHM2 (2) showed weak cytotoxicity against murine cancer cell lines; efrapeptin G (3) and RHM1 (1) also demonstrated antibacterial activity.

Full text of DOI:10.1021/np0503653

J. Nat. Prod. 2006, 69, 83-92
83
Highly N-Methylated Linear Peptides Produced by an Atypical Sponge-Derived Acremonium sp.
Claudia M. Boot,† Karen Tenney,† Frederick A. Valeriote,‡ and Phillip Crews*,†
Department of Chemistry and Biochemistry and Department of Ocean Sciences, UniVersity of California, Santa Cruz, California 95064, and
Henry Ford Health System, Department of Internal Medicine, DiVision of Hematology and Oncology, Detroit, Michigan 48202
ReceiVed September 26, 2005
RHM1 (1) and RHM2 (2) are highly N-methylated linear octapeptides produced by an atypical strain of Acremonium
sp., cultured from a marine sponge collected in Papua New Guinea. The known peptaibiotic efrapeptin G (3) was also
isolated from this fungus. The planar structures of 1 and 2 were assigned based on 1D- and 2D-NMR experiments and
fragmentation patterns from ESIMS. The absolute configuration of 1 was determined via Marfey’s method; the absolute
configuration of 2 is proposed to be identical. Efrapeptin G (3) displayed potent cytotoxicity against murine cancer cell
lines, while RHM1 (1) and RHM2 (2) showed weak cytotoxicity against murine cancer cell lines; efrapeptin G (3) and
RHM1 (1) also demonstrated antibacterial activity.
It is not currently possible to predict the chemical signatures of
filamentous fungi isolated from marine sources.1 Low molecular
weight (<500 amu) bioactive polyketides often dominate the
secondary metabolites isolated from terrestrial fungal cultures. Two
important therapeutically active compounds produced by well-
studied fungal genera Penicillium and Aspergillus illustrate this
chemistry. Mycophenolic acid (C17H20O6), discovered over 70 years
ago from Penicillium sp.,2 is an immunosuppressive agent (1995:
mycophenolate mofetil, first marketed in the U.S.) that has also
shown promising antiviral properties.3 The members of the well-
defined structural class headed by mevinolin (C24H36O5), originally
isolated from Aspergillus, are potent HMG-CoA inhibitors of
cholesterol biosynthesis (1987: first FDA approval of lovastatin).4
Exploration of structural diversity of marine-derived fungal
natural products has been in progress for more than a decade. Early
discoveries of the chloriolins5 and the fumiquinazolines6 validated
the hypothesis that marine-derived strains of fungi could serve as
useful sources of unprecedented molecular frameworks. Compounds
such as asperazine7 from the sponge-derived, chemically rich
Aspergillus niger, or the aigilomycins8 from a mangrove isolate of
Aigialus parVus, provided further validation that new chemotypes
could be discovered from genera previously isolated from terrestrial
sources. Similarly, the outstanding cytotoxic properties of the
gymnastatins from Gymnascella9 as well as the trichoverroids and
their related macrolides, especially isororidin A from Myrothecium
Verrucaria,10 are excellent examples of structures with therapeutic
potential obtained from sponge-derived fungi.
This work describes the constituents of a marine sponge-derived
Acremonium strain grown in saltwater culture. At the outset we
recognized numerous previous studies on terrestrial members of
this genus, with the antibiotic cephalosporin C11 as the most notable
metabolite produced. Only five previous studies had examined the
chemistry of marine-derived strains of Acremonium, resulting in
different biosynthetic products isolated from distinct sources. This
interesting pattern included reports of (a) polyketides12 and hyd-
roquinones13 from algal-derived cultures, (b) ketide-terpenes from
a strain isolated from submerged wood,14 (c) two families of
alkaloids from a tunicate-derived fungus,15 and (d) diterpene
glycosides from a holothurin source.16 In contrast to these outcomes,
our initial NMR spectra of the crude extracts from the sponge-
derived Acremonium culture displayed signals characteristic of
polypeptides. These findings are described below with the structure
elucidation of two new octapeptides, RHM 1 (1) and RHM2 (2),
and efrapeptin G (3),17 a mitochondrial ATPase inhibitor previously
isolated only from the genus Tolypocladium.
Results and Discussion
A crude extract from a small-scale (100 mL) culture of an
Acremonium sp. (UCSC coll. no. 021172 cKZ) cultured from a
Teichaxinella sp. marine sponge (coll. no. 02172; collection
locale: Milne Bay, Papua New Guinea) displayed potent cytotox-
icity in a primary screen using leukemia and solid tumor murine
and human cancer cell lines (Table 1).18 This prompted the growth
of a larger-scale culture (20 L) to facilitate the purification of
metabolites from dichloromethane-soluble broth (EFD) and mycelial
extracts (TFD) (Table 1, Figure S1). Bioassay against a murine
lymphocytic cell line (L1210) and Staphylococcus epidermidis and
NMR data were used to guide the fractionation, resulting in two
new related linear octapeptides, RHM1 (1) and RHM2 (2), and the
known peptaibiotic efrapeptin G (3).
A dereplication approach provided the identity of 3, which
displayed an ESIMS peak at m/z 1649.4. Natural product data bases
were searched for compounds with masses between 1647 and 1651,
which resulted in 11 hits. Refining the search to metabolites of
fungal origin revealed efrapeptin G as the single remaining match.
A comparison of analytical data for 3 and efrapeptin G indicated
similar profiles; the FTMS data, m/z 1648.0914, required the [M]+
molecular formula of C83H143N18O16 (∆ 1.0 mmu of calcd) and
confirmed that it was a salt. The presence of 15 amino acid units
and a carboxy terminal imminium ion moiety was also evident from
diagnostic resonances in the NMR spectrum as discussed below
and consistent with prominent ESIMS fragment ions (m/z 703.5
and 945.6) observed via fragmentation at the amide bond between
the 10th and 11th amino acids of efrapeptin G.
The next challenge was to unequivocally define the sequence of
the 15 amino acids present in 3. The arguments advanced to
establish the structure of efrapeptin G17a were based on amino acid
analysis, a comparison of four FABMS fragment ions which
highlighted the difference between efrapeptins F and G, and the
results of directed biosynthetic experiments.19 To proceed, we
undertook a detailed analysis of the literature spectra of efrapeptin
G and data we collected for 3 (Figures S2a, S2b, S3, and S4). The
FAB mass spectrum for efrapeptin G was included in the
supplementary literature,17a and these data are overlaid in Figure 1
with the FABMS we obtained on 3. Three types of fragment ions20
identified in efrapeptin G were used to set the proposed amino acid
sequence, and these consisted of (a) type B, N-terminal acylium
ions, (b) type Y′, C-terminal amino fragments, and (c) type Z,
C-terminal alkyl fragments. There were only four fragment ions
discussed in the structure elucidation of efrapeptin G (boxed values,
* To whom correspondence should be addressed. Tel: 831-459-2603.
Fax: 831-459-2935. E-mail: phil@chemistry.ucsc.edu.
† University of California, Santa Cruz.
‡ Ford Health System.
10.1021/np0503653 CCC: $33.50
© 2006 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/07/2006

84 Journal of Natural Products, 2006, Vol. 69, No. 1
Boot et al.
Chart 1
Table 1. Bioactive Properties of Extracts and Pure Compounds
as 1408,Y′13), and each of these agreed with our experimental data.
We observed an additional 20 fragment ions, which were used to
verify the sequence of amino acids (Figure 1). The eight underlined
values in Figure 1 were present in the FABMS spectrum of
efrapeptin G but were not discussed in the literature. There are an
additional eight FABMS peaks observed for both efrapeptin G and
3, which are related to side-chain fragmentation including m/z 250.1,
292.2, 377.2, 618.4, 729.3, 772.3, 814.3, and 857.6. This analysis
confirmed that the two samples were identical and bolstered
confidence that the original structure of efrapeptin G was correct.
from Strain 021172c
relative
MICb
(µg/mL)
sample
potencya
EtOAc extract (100 mL culture)
CH2Cl2 extract of broth (20 L culture)
efrapeptin G (3)
92
580
3300
16
nt
125
80
RHM1 (1)
50
CH2Cl2 extract of mycelia (20 L culture)
RHM2 (2)
RHM1 (1)
34
250
>400
25
1.0
4.0
nt
vancomycin
0.63
The lack of published NMR data for efrapeptin G (3) stimulated
us to assemble a complete data set as shown in Table 2. All NMR
resonances for the 83 carbons, the 130 protons attached to carbon,
and the 13 protons attached to nitrogen could be located and
identified with the aid of a DEPT spectrum. Some assignments
could be readily made (Table 2) on the basis of literature values
for a hydrolysis product of efrapeptin G,17a which was composed
of the imminium capping group and the last two amino acids (Iva
and Leu). The individual resonances of the remaining sets of closely
a Potency against murine L1210 (lymphocytic leukemia) is calculated
by multiplying the reciprocal of the dilution required for potent
inhibition (zone of 500 units) × 100/mg/mL; larger values correspond
to greater potency; potency is relative to the value for 3. b MIC is the
minimum inhibitory concentration against Staphylococcus epidermidis;
nt ) not tested.
Figure 1), described as m/z 1185 (observed as 1184, Z11), 1284
(barely visible in spectrum, Z12), 1299 (Y′12), and 1410 (observed

Highly N-Methylated Linear Peptides
Journal of Natural Products, 2006, Vol. 69, No. 1 85
Figure 1. FABMS fragmentation (m/z) for efrapeptin G (3). Codes: underlined values this work and ref 17a; boxed values discussed in
ref 17a; values ( 1 amu (+*/-**).32
related functional groups were only provisionally made and were
assisted by published NMR data for the nonstandard amino acids
Pip, Iva, and â-Ala21 as well as calculated NMR data (ACD v 8.0).
Finally, the NMR analysis added the penultimate evidence to verify
the structure of 3, and the configuration shown is identical to that
of efrapeptin G.
nonmethylated Leu. One of the other nonstandard amino acids that
composes 1 (N-MeVal or N-MeIle) may have a retention time
similar to Leu, resulting in a misidentified peak in the AA analysis.
The substructural features responsible for the partial formula
C10N9O11, based on the 10 carbonyl carbons observed by 13C NMR
and heteroatoms established by MS, were revisited to identify the
terminal carboxylic acid. This partial formula consists of C9N9O9
from (a) the single primary amide, (b) the three secondary amides,
and (c) the five tertiary amides, resulting in a residual atom count
of CO2. This observation along with the lack of resonances in the
δ 60-80 region could only be rationalized by attaching a carboxylic
acid group to C-48. Finally, all 10 degrees of unsaturation were
accounted for by these aforementioned structural elements.
The three substructures A-C (Figure 3) were assembled after
obtaining 2D-NMR data including gHMBC, gHSQC, gHMQC,
gCOSY, and TOCSY. A natural abundance 15N gHMBC NMR
experiment was also conducted in order to clarify overlapping
proton shifts due to the large number of aliphatic amino acids. While
the data did not provide new correlations for the construction of
substructures, the shifts included in Table 3 verified the presence
of the nine amide groups discussed above.
The justification for the substructures proposed in Figure 3 for
1 hinged primarily on the 1H-1H COSY and gHMBC data, while
the biggest challenge involved the unambiguous placement of the
seven isopropyl, isobutyl, and sec-butyl moieties. The terminus of
substructure A was established through correlations from the acetyl
methyl group (δ 1.80) to the acetyl carbonyl, C-2 (δ 169.0), and
from the secondary NH of Gln1 to C-2 and the amide carbonyl,
C-7 (δ 171.4). The additional backbone portion was deduced on
the basis of the correlations from the Ile2 NH proton (δ 8.14) to
C-7 and its amide carbonyl, C-13 (δ 172.6). The MeLeu3 R proton
(δ 5.07) also correlated to C-13 and the MeLeu3 amide carbonyl,
C-20 (δ 169.9), and the final key correlation was from the Ile4 R
proton, H-21 (δ 4.46), to C-20. The side-chain assignments of the
R and â protons were provided through COSY (Table 3), while
TOCSY data confirmed that the spin systems were properly
assigned for the δ and γ carbons. Other highlights of the logic to
assign side-chain protons included the multiplicity and shifts at
CH2-4 to locate the propianomide group as well as the dt for H-3,
and the COSY correlation to H-4b (δ 1.66 ddt) indicated it was
flanked by one geminal and three vicinal protons. The 13C shift at
C-4 (δ 28.5) was also diagnostic of this environment, as it matched
the calculated shift (δ 27.5) for this substructure. Similarly, the
The structures of 1 and 2 were assembled in an iterative process.
This began with identifying their constitutive amino acid groups
via a combination of degradation and NMR studies. Next, the
subunits were assembled on the basis of gHMBC correlations
considered in parallel with the fragmentation patterns observed by
ESIMS. The characterization of RHM1 (1), isolated as a crystalline
white solid, proceeded with establishing the molecular formula
C53H97N9O11 based on FTMS data [m/z 1036.7386 (MH)+, ∆ 0.5
mmu of calcd]. The ESIMS data included fragment ions at m/z
891.6, 764.5, 637.4, 626.5, and 411.3, as shown in Figure 2.
Shifts from the 13C NMR, and carbon types derived from a DEPT
experiment, revealed the peptide backbone features of 1 (Table 3).
Ten carbonyls (between δ 174 and 169), eight R carbons (between
δ 59 and 52), five N-methyl carbons (between δ 31 and 29), and
NMR resonances characteristic of side-chain methyl carbons for
five isoleucines (Iles), one valine (Val), and one leucine (Leu) were
observed. Features of the 1H NMR (DMSO-d6, Figure S4) supported
conclusions drawn from the 13C data including three NH protons
(δ 8.31, 8.14, and 7.93), eight R protons (δ 5.20, 5.17, 5.10, 5.07,
4.78, 4.52, 4.46, and 4.31), and five N-methyl groups (δ 3.08, 3.02,
two at 2.95, and 2.94). A sharp methyl singlet located at δ 1.80
(overlapping with five other aliphatic protons) suggested an N-acetyl
terminus.
Amino acid (AA) analysis was not straightforward; it indicated
1
the presence of glutamic acid (Glu), Ile, and Leu, while the H
NMR indicated a -CH-CH-(CH3)2 backbone of a Val group.
Consideration of the AA analysis and 1H and 13C NMR data side-
by-side revealed the initial assessment of Leu and Glu to be
incorrect. A glutamine (Gln) group was identified from signature
diastereotopic NH2 protons (δ 7.26 and 6.74) upfield of the
backbone NH protons and individually coupled to C-5 and C-6
(gHMBC data) and to each other (1H-1H COSY). The observation
of glutamic acid by AA analysis can be attributed to hydrolysis of
glutamine during the workup step. The presence of N-MeLeu is
illustrated by both a gHMBC correlation from an N-Me group, H3-
14 (δ 2.94) to C-15, and the multiplicity of H-15, a dd (J ) 4.5
and 12.0 Hz), rather than a ddd, which would be expected for a

86 Journal of Natural Products, 2006, Vol. 69, No. 1
Boot et al.
Table 2. Provisional Assignments of 13C/1H NMR (125/500 MHz, MeOH-d4) Resonances for Efrapeptin G (3)
1H δ; mult.
lit. 1H δa mult.
(J Hz) no.
type
13C δ
lit. 13C δa
(J Hz) no.
Ac
Me
CdO
Pip
R
CH3
C
17.5
171.7
2.18; s 3H
CH
57.5, 56.7, 56.1
5.14; bd (6.0) 1H, 5.05; bt (4.0) 1H,
4.86; m 1H
â
γ
δ
ꢀ
CH2
CH2
CH2
CH2
27.5, 26.7, 26.6
21.7, 21.6, 21.4
26.2, 26.0, 25.8
45.5, 44,9, 42.2
1.93-1.58; m 6H
2.26; m 3H, 1.93-1.58; m 3H
1.56-1.41; m 6H
4.34; m 2H, 4.22; m 1H
3.75-3.69; m 2H, 3.41-3.36; m 1H
CdO
Aib
C
175.6, 174.0, 173.5
NH
8.28; s 1H, 8.18; s 1H, 8.09; s 1H
7.91; s 1H, 7.89; s 1H
Rc
C
58.7, 58.4, 58.3, 58.3, 58.1
27.8, 27.3, 25.6, 25.2, 24.1
27.1, 26.9, 25.3, 24.4, 23.9
178.0, 177.6, 177.4, 176.9, 175.6
â1
CH3
CH3
C
1.56-1.41; m 15H
1.56-1.41; m 15H
â2
CdO
Iva
NH
R
7.89; s 1H, 7.19; s 1H
C6
CH3
CH2
1.9, 61.3
23.6, 23.3
34.2, 28.3
61.8
23.6
27.9
â1
â2
1.56-1.41; m 6H
1.93-1.58; m 4H
1.37; s 3H
1.9; m 1H, 2.15;
m 1H
γ
CH3
C
8.7, 8.6
178.4, 177.0
8.3
176.8
0.95-0.84; m 6H
0.85; t (7.32) 3H
CdO
Leu
NH
R
7.93; d (7.0) 1H, 7.10; d (9.5) 1H
4.16; ddd (3.0, 6.5, 10.5) 1H
3.93; ddd (4.5, 6.0, 10.5) 1H
1.93-1.58; m 2H,
CH
54.6, 54.1
41.1, 37.4
53.2
41.7
3.92; m 1H
1.61-1.77; m 2H
â
CH2
1.56-1.41; m 2H
γ
CH2
CH3
CH3
C
6.4, 26.2
25.5
23.1
22.1
169.9
1.93-1.58; m 2H
1.61-1.77; m 1H
1.00;b d (6.14) 3H
1.02;b d (6.14) 3H
δ1
21.7, 21.6
20.8, 21.1
176.4, 176.1
0.95-0.84; m 6H
δ2
0.95-0.84; m 6H
CdO
â-Ala
NH
R
7.77; m 1H
3.85; m 1H,
2.92; ddd (6.0, 10.0, 17.0) 1H
CH2
34.2
â
CdO
CH2
C
37.0
173.7
2.62; m 1H, 2.44; m 1H
Gly
NH
7.87; m 1H
R
CdO
CH2
45.5
175.0
3.75-3.69; d (17.5) 2H
Ala
NH
7.74; d (7.5) 1H
R
CH
CH3
53.5
17.5
173.5
4.01; dq (3.5, 7.5) 1H
1.56-1.41; m 3H
â
CdO
Imminium Cap
2′
CH2
46.0
45.9
3.41-3.36; m 1H, 3.75-3.69; m 1H
3.43; m 1H, 3.64;c
m 1H
3′
4′
6′
CH2
CH2
CH2
20.2
43.6
55.7
19.9
43.6
55.6
2.06; m 2H
2.06; m 2H
3.41;c m 2H
3.81; m 1H
3.73; m 1H
2.15; m 2H
3.03; m 1H, 3.18;
m 1H
3.41-3.36; m 2H
3.81; ddd (6.5, 9.5 10.0) 1H
3.75-3.65; m 1H
2.13; m 2H
7′
8′
CH2
CH2
19.2
32.0
19.2
32.2
3.03; m 1H, 3.18; m 1H
8a′
1′′
1
C
166.9
166.1
NH
CH
CH2
8.36; d (4.5) 1H
4.34; m 1H
3.64; m 1H, 3.41-3.36; m 1H
46.2
58.0
46.9
57.9
4.37; m 1H
3.58; m 1H, 3.39;
m 1H
2
3
CH2
41.1
41.1
1.93-1.58; m 1H,
1.20; ddd (3.5, 10.5, 14.0) 1H
1.93-1.58; m 1H
0.95-0.84; m 3H
0.95-0.84; m 3H
1.69; m 1H, 1.22;
m 1H
1.66; m 1H
0.94; d (6.34) 3H
0.94; d (6.34) 3H
4
5
6
CH
CH3
CH3
25.6
21.8
23.7
25.8
21.8
23.8
a Literature values for 11.17a b,c Values are interchangeable.

Highly N-Methylated Linear Peptides
Journal of Natural Products, 2006, Vol. 69, No. 1 87
Figure 2. ESIMS fragmentation pattern:1 m/z for RHM1 (1),2 m/z for RHM2 (2).
side-chain protons attached to C-8 and C-15 could be deciphered
on the basis of the multiplicities of the â protons (H-9, multiplet
via coupling to a CH3; H-16a ddd) and the reasonable agreement
in the observed shift at C-16 (δ 36.4) versus the calculated shift (δ
41).
The configuration of each amino acid was determined by
Marfey’s method, and the derivatives were analyzed with LCMS.
The hydrolysis step converted the glutamine to glutamic acid, as
was the case with the amino acid analysis, so R and S standards
for glutamic acid were also examined. Retention times and masses
of standards matched the R isomer of glutamic acid and the S
stereoisomer for the N-Me-Leu and N-Me-Val and the 2S, 3S
stereoisomer for both Ile and N-Me-Ile. Thus the stereostructure
of 1 was determined to be Ac-(R)-Gln-(2S,3S)-Ile-(S)-N-Me-Leu-
(2S,3S)-Ile-(S)-N-Me-Val-(2S,3S)-N-Me-Ile-(2S,3S)-N-Me-Ile-(2S,3S)-
N-Me-Ile-OH.
A similar approach was employed to characterize RHM2 (2). It
was isolated as an amorphous white solid with the molecular
formula C52H95N9O11 obtained through FTMS for 2 [m/z 1022.7442
(M + H)+, ∆ 0.2 mmu]. Four fragment ions similar to those seen
in 1 were also observed for 2 at m/z 877.6, 750.5, 623.4, and 397.3
(Figure 2). The molecular formula difference between compounds
1 and 2 amounted to 14 amu, signifying one less CH2. Consistent
with this minor difference, the 13C NMR and DEPT spectra for
compound 2 were similar to compound 1 with 10 carbonyl carbons,
eight R carbons, and five N-Mes. However, the 13C shifts for the
side-chain methyl carbons were diagnostic of four Ile, two Val,
and one Leu residue (Table 4), indicating one of the Ile groups of
1 was replaced as Val in 2. The 1H NMR spectra for 2 also
contained evidence for a substantial population of rotational isomers
at the Gln1 NH, the Val2 NH, the Ile5 NH, and protons 21, 33, and
40 (Figure S19).
Exhaustive analysis of 2D-NMR for 2 including gHSQC,
gHMQC, gHMBC, gCOSY, and TOCSY aided the construction
of five substructures, D-H (Figure 4). Substructure D possessed
the same terminal group as 1. This consisted of an N-Ac group
attached to the Gln, established through gHMBC correlations from
the acetyl methyl group (δ 1.81) to the acetyl carbonyl C-2 (δ 168.8)
and from the secondary NH of Gln1 to C-2 and the amide carbonyl,
C-7 (δ 171.2). Extending this unit into an Ac-Gln1-Val2-MeIle3 (D)
array was based on the additional 2D-NMR correlations (Table 4).
The first set (Figure 4) was from the Gln1 R proton, H-3 (δ 4.31)
to C-2 and C-7 (δ 171.2) and from the Val2 NH (δ 8.13) to C-7,
along with an additional correlation between the Val2 NH and the
Val2 R carbon (δ 52.3). The connection between the second and
third amino acids to complete substructure M was made through
gHMBC correlations from H-8 to C-12 (δ 172.1), from N-MeLeu3
N-Me (δ 3.02) to C-12 and C-14 (δ 57.1), and from H-14 (δ 5.05)
to C-19 (δ 172.5). The side-chain R and â proton assignments of
2 were also established through COSY (Table 4), while TOCSY
data confirmed that the spin systems had been properly assigned
for the δ and γ protons.
A dipeptide fragment (substructure B) consisting of two N-Me
amino acids was identified in the interior backbone. Initially, the
necessary 2D correlations were not observed to immediately unravel
its exact placement, but this issue was resolved from the MS data.
The N-MeVal was defined on the basis of gHMBC correlations
from the H3-27 (δ 3.02) to R carbon C-28 (δ 57.2) and from H-28
(δ 5.10) to the amide carbonyl C-32 (δ 172.3) and to C-29 (δ 26.7),
with the latter correlation securing the position of the sole isopropyl
group. The N-MeIle was characterized by observing the clean
doublet for H-34 (δ 5.20, Figure 3) coupling only to H-35 (δ 2.03).
The pattern for these two protons indicated that a sec-butyl must
be attached to C-34. The gHMBC correlation from H3-33 (δ 2.94)
to C-32 defined the peptide bond between these two amino acids.
The final substructure (C) also contained a dipeptide fragment
made up of the two remaining N-MeIle groups. The N-Me groups
were located on the basis of gHMBC correlations from H3-40 (δ
3.08) to R carbon C-41 (δ 58.8) and C-39 (δ 172.1) and from H3-
47 (δ 2.94) to C-48 (δ 55.2). Both N-MeIle7 and N-MeIle8 R protons
H-41 and H-48 (δ 4.78 and δ 5.17) were clean doublets requiring,
as noted above, the attachment of sec-butyl groups to C-41 and
C-48. Finally, both N-MeIle7 and N-MeIle8 R (δ 4.78 and 5.17)
protons displayed gHMBC correlations to the amide carbonyl of
N-MeIle7, C-46 (δ 169.9).
At this point there were two possible ways to embed the
carboxylic acid group to complete the structure of 1. Alternative I
consisted of joining substructures A-B-C with C-53 as the
carboxylate position, whereas possibility II had an A-C-B
sequence with C-39 as the carboxylate location. The MS data proved
essential for making an unambiguous choice between the two
possible connectivities. Four intense ESIMS fragment ions were
observed along with the [M + H]+ ion for 1. These acylium ions
were generated through cleavage (type B) at the amide bond (Figure
2). The smallest fragment ion, m/z 411.3, was consistent with the
NMR-derived sequence of the first three amino acids of substructure
A: Ac-Gln1-Ile2-N-MeLeu3. According to the 2D-NMR data, the
fourth amino acid must be Ile4. The next ion in the series was at
m/z 637.4, reflecting the addition of 226.1 amu, equivalent to the
combined masses of N-MeVal and Ile, and required their connection
in this sequence (Figure 2), validating proposed structure I. The
subsequent mass fragment ions, m/z 764.5 and 891.6, required the
successive addition of two N-MeIle groups (Figure 2), consistent
with the sequence established by NMR.

88 Journal of Natural Products, 2006, Vol. 69, No. 1
Boot et al.
Table 3. 13C/1H NMR Data (500 MHz/125 MHz, DMSO-d6) for RHM 1 (1)
position
1H δ; mult. (J Hz)
13C δ; type
gHMBC
gCOSY
15N δ
OAc-Gln1
1
2
1.80; s
22.5; CH3
169.0; C
C-2
NH
3
4
5
6
7.93; d (8.5)
C-2, C-7
C-7
H-3
Gln NH, H-4b
H-3
124.3
4.31; dt (5.0, 8.5)
1.80; m, 1.66; ddt (13, 8.5, 5.0)
2.03; m
52.1; CH
28.5; CH2
31.6; CH2
173.7; C
NH2
7
7.26; s, 6.74; s
C-5, C-6
115.4
124.3
171.4; C
Ile2
NH
8
8.14; d (8.5)
4.52; t (9.0)
1.80; m
1.44; m, 1.06; m
0.77; m
C-7, C-13
52.5; CH
H-10
Ile NH, H-9
H-8
9a
36.0; CH
24.0; CH2
10.6; CH3
14.9; CH3
172.6; C
31.1; CH3
53.8; CH
36.4; CH2
24.5; CH
21.2; CH3
20.8; CH3
169.9; C
10b
11c
12d
13
0.77; m
MeLeu3
14e
15
2.94; s
5.07; dd (4.5, 12.0)
1.78; m, 1.55; ddd (4.5, 11.0, 14.5)
1.80; m
0.77; m
0.77; m
C-15
C-13, C-16, C-20
115.4
115.4
H-16a
H-15
16
17
18f
19f
20
Ile4
NH
21
22
8.31; d (8.0)
4.46; dd (8.5, 9.5)
1.80; m
1.44; m, 1.06 m
0.77; m
C-20
C-20, C-22
H-21
Ile NH, H-22
H-21
52.7; CH
35.5; CH
24.0; CH2
10.5; CH3
14.9; CH3
170.3; C
30.7; CH3
57.2; CH
26.7; CH
18.8; CH3
17.9; CH3
172.3; C
30.0; CH3
55.5; CH
32.6; CH
23.6; CH2
10.4; CH3
14.8; CH3
172.1; C
29.9; CH3
58.8; CH
32.2; CH
23.4; CH2
10.4; CH3
14.6; CH3
169.9; C
23b
24c
25d
26g
27e
28
0.77; m
MeVal5
MeIle6
3.02; s
C-28
C-29, C-32
116.2
117.8
5.10; d (11.0)
2.19; dq (6.5, 11.0)
0.77; m
H-29
H-28
29
30h
31h
32
0.77; m
33e
34
2.94; s
5.20; d (11.0)
2.03; m
1.39; m, 1.30; m
0.77; m
0.77; m
C-32
H-35
H-34
35a
36b
37c
38d
39
MeIle7
40e
41
3.08; s
4.78; d (11.0)
1.80; m
1.39; m, 1.13; m
0.77; m
0.77; m
C-39, C-41
C-39, C-42, C-46
116.0
117.4
H-42
H-41
42
43b
44c
45d
46
MeIle8OH
47e
48
2.94; s
5.17; d (11.0)
2.03; m
1.39; m, 1.30; m
0.77; m
0.77; m
29.9; CH3
55.2; CH
32.6; CH
23.2; CH2
10.2; CH3
14.6; CH3
170.1; C
C-46, C-48
C-46
H-49
H-48
49a
50b
51c
52d
53g
OH
n.o.
a -hInterchangeable carbons; n.o.: not observed; low-field diastereotopic protons are labeled “a”, high-field diastereotopic protons are labeled
“b”.
Three additional partial structures of N-Me amino acid subunits
Making the final unique choice among the several possible final
structures for 2 was based primarily on ion fragmentation observed
in ESIMS (Figure 2). The connectivities deduced for D by NMR
matched the tripeptide required by the mass of the smallest fragment
ion, m/z 397.3. Similar to the case for 1, the next highest fragment
ion, m/z 632.4, required the addition of an N-MeVal and Ile to
rationalize the mass increment of 226.1 amu. To adhere to the mass
requirements, substructure E must be followed by G. Substructures
D and F could not be consecutive due to the correlations of H-14
and NH of Ile5 to distinct carbonyls (C-19 and C-25, respectively).
This allows the construction of the sequence D-E-F with only G
and H undefined. The correlation from H3-46 (δ 2.94) to C-45
meant substructure G could not be the terminal amino acid in the
and one dipeptide subunit were identified in a straightforward
manner on the basis of the data of Table 4. The methylated amino
acids consisted of E as N-MeVal, and G and H as N-MeIles were
each identified in a straightforward fashion. The remaining dipeptide
substructure F, composed of Ile5-N-MeIle6, was established through
gHMBC correlations from N-H (δ 8.29) to C-26 (δ 52.6) and C-25
(δ 169.7) and from Ile5 R proton H-26 (δ 4.52) to C-31 (δ 172.0),
along with correlations from H3-32 (δ 3.08) to C-31 and C-33 (δ
58.7). At this point there were several ways to connect the five
substructures of 2 (D-H). When no correlations were present
(carbons 35, 42, and 49), protons were assigned on the basis of the
shifts for parallel amino acids from compound 1.

Highly N-Methylated Linear Peptides
Journal of Natural Products, 2006, Vol. 69, No. 1 89
facilitate further studies. The antimicrobial assay against S. epi-
dermidis directed fractionation to RHM1 (1), which possesses
antibiotic properties (MIC 25-50 µg/mL, Table 1). Efrapeptin G
(3) was slightly less active against S. epidermidis (MIC 80 µg/mL,
Table 1), while RHM2 (2) did not exhibit antimicrobial activity
(MIC > 400 µg/mL, Table 1). Both 1 and 2 exhibited mild
cytotoxicity against murine L1210 cells in a disk diffusion soft agar
colony-forming assay18 (Table 1). Alternatively, 3 exhibited potent
cytotoxicity against murine L1210 cells and against HCT-116 with
an IC50 of 3.5 × 10-3 µg/mL. Developmental therapeutics work
has been initiated on this compound including clonogenic studies,
and these results will be reported elsewhere.
Previous studies suggest that the C-terminal blocking group may
play a role in the biological activity of the efrapeptins,17 possibly
accounting for the differential bioactivity against murine cell lines
between efrapeptin G (3) and the RHMs (1 and 2). Efrapeptin G
(3) is largely composed of unusual amino acids such as isovaline,
aminoisobutyric acid, â-alanine, and pipecolic acid, which may also
contribute to the differences in bioactivity. Efrapeptins are most
likely produced by a nonribosomal peptide synthetase (NRPS), due
to the presence of a high proportion of unusual amino acids and
modified C- and N-termini. RHMs may also be the product of an
NRPS, as indicated by the large number of methylated amino acids
and the presence of (R)-glutamine.
Previously reported N-methylated peptides from marine-derived
fungi include the cyclin-dependent kinase inhibitors dictyonamides22
from an unidentified fungus, cytotoxic scytalidmides23 from Scyta-
lidium sp., and the aspergillamides24 produced by an Aspergillus.
Compounds 1 and 2 are most similar to the dictyonamides, which
are linear and contain a large proportion of N-methylated and
aliphatic amino acids. However, 1 and 2 are markedly different
from other peptides of this group by the presence of the (R)-
glutamine. The production of chlorofusin by the fungi genera
Fusarium,25 the trikoningins by Trichoderma,26 and integramides
by Dendrodochium27 have all preceded the RHMs as natural
products from fungi containing at least one (R)-amino acid.
However, none of these strains were marine-derived, nor are the
structures methylated at the amide nitrogens, making 1 and 2 the
first N-methylated peptides with an (R)-amino acid from a marine-
derived fungus.
Figure 3. Substructures A-C for RHM1 (1) illustrating key
gHMBC and gCOSY correlations.
molecule; therefore the sequence of the substructures for 2 was
D-E-F-G-H with C-52 assigned as the carboxylic acid group.
The configuration of each amino acid in 2 is postulated to be the
same as 1 on the basis of the congruent origin of biosynthetic
materials for 1 and 2, resulting in the sequence Ac-(R)-Glu-(S)-
Val-(S)-N-MeLeu-(S)-N-Me-Val-(2S,3S)-Ile-(2S,3S)-N-Me-Ile-(2S,3S)-
N-Me-Ile-(2S,3S)-N-Me-Ile-OH.
The remaining issue deserving comment for 2 was the 1H NMR
differences between 1 and 2 in the region of NH and R proton
shifts. Unexpectedly, a minimum set of four amide bond rotamers
could be observed for 2, while two (one major, one minor) rotational
isomers could be observed for 1. The differing circumstances can
be seen by comparing the intense and small doublets in the ratio
85:15 observed for H-41 (δ 4.78) in 1 versus the cluster for 2 of at
least four distinct H-33 doublets (δ 4.70-4.80) in the ratio of 38:
33:20:9 (A:B:C:D), as shown in Figure S19. Other distinct rotational
isomers visible for 2 include three for the Ile5 NH (26:35:39) and
Gln1 NH (32:47:21), two at the Val2 NH (55:45) and R proton H-40
(58:42), and four at H-21 (31:24:23:20). The presence of rotational
isomers in 1 and 2 can also be observed in the 13C NMR (Figure
S20). For example, each of the R and γ carbons of N-MeVal5 (C-
28, C-30, C-31) of 1 demonstrated the minor rotamer, which was
Experimental Section
General Experimental Procedures. The NMR spectra for 1 were
recorded at 500 MHz (1H) and 125 MHz (13C). NMR spectra for 2
were recorded at 600 MHz (1H) and 150 MHz (13C). The natural
abundance 15N gHMBC experiment used the same pulse sequence as
standard gHMBC experiments. High-resolution mass measurements
were obtained from a FT-ICR hybrid mass spectrometer, which
combines ion trap and Fourier transform cyclotron resonance technology
into a single instrument. Additional mass spectra were acquired with a
benchtop Mariner ESITOF mass spectrometer. Preparative HPLC was
performed using columns of 6 µm ODS; semipreparative HPLC was
performed using columns of 5 µm ODS.
Biological Materials. Strain 021172cKZ was cultured via previously
reported methods5 from a Teichaxinella sp. marine sponge collected
with scuba in 2002, during an expedition to Papua New Guinea (sponge
collection no. 02172, voucher maintained at UCSC). Strain 021172cKZ
was identified as Leucosphaerina indica (3% different) via molecular
methods10 and as an atypical Acremonium sp. through morphology-
based identification. When grown on potato dextrose agar media, the
strain demonstrated large, creamy, white moist areas that were
considered sporodochia. The white color was unusual, as most
Acremonium spp. are buff to some shade of salmon. Microscopically,
the conidiophores were branched, which is also unusual for Acremo-
nium, and the conidia were pointed at both ends and contained two
guttules (oil droplets).28 The strain is hereto referred to as a notably
atypical Acremonium sp. due to the large genetic difference presented
by the molecular method and its unusual morphological characteristics.
The fungus is maintained as a cryopreserved glycerol stock at UCSC.
1
also observed in the H NMR. Similarly, at least three rotamers
are visible in the 13C NMR of 2, which are most apparent at the
N-MeVal4 and Val2 γ carbons (C-10, C-11 and C-23, C-24 in Figure
S20), the N-MeLeu3 R carbon (C-14), and the N-Me of N-MeIle6
(C-32).
1
Data from a variable-temperature H NMR (VT NMR) experi-
ment was sought to further define the presence of the rotamers
discussed above for 1 and 2. The latter was chosen for this analysis
because it contained several well-resolved proton signals. As
expected, the apparent doubled doublet for H-40 (Figure S19)
coalesced to a single doublet (J ) 10.5 Hz, Figure S19) at 160 °C.
The coalescence temperature of protons H-21, H-14, and H-33 was
not reached due to limitations of the solvent (DMSO-d6). Another
interesting feature of the VT NMR experiment was the eventual
collapse and upfield shift of the NH protons (Ile5, Val2, and Gln1,
Figure S19) due to a faster exchange rate at the increased
temperature. The results of the VT NMR experiment suggest the
four stable isomers we observed can be attributed to the arrangement
of R groups in multiple sets of trans-trans, trans-cis, cis-trans,
or cis-cis conformations.
The final step was to explore the bioactivity of the three
compounds, each of which was isolated in sufficient amounts to

90 Journal of Natural Products, 2006, Vol. 69, No. 1
Boot et al.
Table 4. 13C/1H NMR Data (600 MHz/150 MHz, DMSO-d6) for RHM2 (2)
position
1H δ; mult. (J Hz)
13C δ; type
gHMBC
gCOSY
OAc-Gln1
1
2
1.81; m
22.3; CH3
168.8; C
C-2
NHa
3
7.94; d (8.5)
C-2, C-3
4.31; dt (4.0, 9.0)
1.81; m, 1.66; ddt (13.0, 9.0, 5.0)
2.03; m
51.9; CH
28.4; CH2
31.5; CH2
173.5; C
C-2, C-4, C-5, C-7
C-3, C-5, C-6
C-6
4
5
6
NH2
7
7.20; m, 6.80; m
C-6
171.2; C
Val2
NHa
8
8.13; d (8.5)
4.47; t (8.7)
1.43; m
0.77; m
0.77; m
C-7, C-8
C-9, C-10, C-12
H-8
52.3; CH
35.4; CH
17.7; CH3
18.6; CH3
172.1; C
31.0; CH3
57.1; CH
36.3; CH2
24.3; CH
23.0; CH3
20.7; CH3
172.5; C
29.9; CH3
55.3; CH
26.6; CH
17.8; CH3
18.6; CH3
169.7; C
Val NH, H-9
H-8
9
10b
11b
12
MeLeu3
13c
14
3.02; s
5.05; dd (4.5, 12.0)
1.78; m, 1.55; ddd (4.0, 10.5, 14.5)
1.81; m
0.77; m
0.77; m
C-12, C-14
C-15, C-16, C-19
H-15a, H-16
H-14
H-14
15
16
17d
18d
19
MeVal4
20c
21a
22
2.94; s
5.09; d (11.0)
2.20; m
0.77; m
0.77; m
C-21
C-22, C-23, C-24
H-22
H-21
23b
24b
25e
NHa
26
Ile5
8.29; d (8.5)
4.52; t (9.3)
1.81; m
1.43; m, 1.14; m
0.77; m
C-25, C-26
C-25, C-27, C-30, C-31
52.6; CH
35.8; CH
23.9; CH2
10.5; CH3
14.8; CH3
172.0; C
27
28f
29g
30h
31
H-28a to C-29, C-30
0.77; m
MeIle6
32
3.08; s
4.79; d (11.0)
1.80; m
1.43; m, 1.14; m
0.77; m
0.77; m
30.5; CH3
58.7; CH
32.1; CH
23.9; CH2
10.6; CH3
14.8; CH3
169.6; C
29.8; CH3
53.8; CH
32.5; CH
23.4; CH2
10.6; CH3
14.6; CH3
170.1; C
29.8; CH3
55.1; CH
32.5; CH
23.3; CH2
10.1; CH3
14.4; CH3
170.0; C
C-31, C-33
C-32, C-34
33a
34
H-34
H-33
35f
36g
37h
38e
39c
40a
41
MeIle7
2.94; s
5.12; d (11.0)
2.03; m
1.23; m, 1.07; m
0.77; m
0.77; m
C-38
C-41, C-45
H-41
H-40
42f
43g
44h
45
MeIle8OH
46c
47
2.94; s
5.16; d (11.0)
2.03; m
1.23; m, 1.07; m
0.77; m
0.77; m
C-45
C-48, C-52
H-48
H-47
48
49f
50g
51h
52
OHi
10.78; bs
a Multiple proton signals appear due to rotation about amide bonds. b -hInterchangeable carbons. i In dioxane-d8; low-field diastereotopic protons
are labeled “a”, high-field diastereotopic protons are labeled “b”.
Culture Conditions. The large-scale culture (20 L) was grown in
Czapek-Dox media made with filtered Monterey Bay seawater-based
media adjusted to pH 7.3 with shaking (150 rpm) for 21 days at room
temperature (28 °C).
Biological Assays. The disk diffusion soft agar colony-forming assay
is used to identify extracts and pure compounds with potent cytotoxicity
and solid tumor selectivity. Activity is expressed in zone unit
differentials between solid tumor cells (murine Colon38 and human
ColonH116 and LungH125) and leukemia (murine L1210, human
CEM) or normal cells (CFU-GM). Selectivity is defined as a zone unit
differential greater than or equal to 250.18 The antimicrobial assay is
carried out in microtiter format as previously described.29
methanol three times, each for 24 h. The XAD-16 column was washed
with water followed by methanol, and the eluent was concentrated in
vacuo. The resulting crude oil was partitioned between 90% aqueous
methanol and hexanes, followed by a 50% aqueous methanol-
dichloromethane partition. Both the soft agar disk diffusion and
antimicrobial assay of the crude extracts from 021172cKZ indicated
that the broth- and mycelial-derived dichloromethane fractions (EFD
and TFD, respectively) contained the active components. The EFD (1.44
g) was fractionated with preparatory HPLC (25 to 75% MeCN in H2O
with 0.1% formic acid over 50 min) followed by two consecutive rounds
of semipreparative HPLC (50 to 100% MeCN in H2O with 0.1% formic
acid over 30 min, and 42 to 48% MeCN in H2O with 0.1% formic
acid over 20 min) to afford 21.1 mg of 3 and 13.3 mg of 1. The TFD
(4.76 g) was fractionated with preparatory HPLC (25 to 75% MeCN
in H2O with 0.1% formic acid over 30 min), followed by semiprepara-
Extraction and Isolation. The broth and mycelia were separated
through vacuum filtration and extracted independently: the broth with
a column of XAD-16 resin, and the mycelia by soaking in 1 L of

Highly N-Methylated Linear Peptides
Journal of Natural Products, 2006, Vol. 69, No. 1 91
Acknowledgment. This work was supported by the National
Institutes of Health (RO1 CA 47135). We thank B. Gerstenberger for
assistance with synthesis of N-Me amino acids, J. Cunniff and D. Kage
at Thermo Electron for providing FTMS data, and N. Sevova at Notre
Dame for providing FABMS data. We would also like to thank C. Bui
for AA analysis, A. Amagata for assistance with fungal cultures, and
S. Pomponi for sponge identification. Mr. M. Waddington, Accugenix,
a division of Acculabs, INS, is acknowledged for molecular identifica-
tion, and Dr. D. A. Sutton, Department of Pathology, University of
Texas Health Science Center at San Antonio, is acknowledged for
taxonomic identification of the fungal strain.
Supporting Information Available: 1H and 13C NMR and FABMS
1
for compound 3, and H, 13C NMR, gHMBC, gCOSY, and TOCSY
for compounds 1 and 2 along with VT NMR figures are available free
of charge via the Internet at http://pubs.acs.org.
References and Notes
(1) For a recent review of the entire chemistry of marine-derived fungi
(273 compounds discovered to 2002), see: Bugni, T. S.; Ireland, C.
M. Nat. Prod. Rep. 2004, 21, 143-163.
Figure 4. Substructures D-H for RHM2 (2) illustrating key
gHMBC correlations.
(2) (a) Clutterbuck, P. W.; Oxford, A. E.; Raistrick, H.; Smith, G.
Biochem. J. 1932, 26, 1441-1458. (b) Campbell, I. M.; Calzadilla,
C. H.; McCorkindale, N. J. Tetrahedron Lett. 1966, 7, 5107-5111.
(3) (a) Cole, R. J.; Cox, R. H. Handbook of Toxic Fungal Metabolites;
Academic Press: New York, 1981. (b) Seebacher, G.; Mallinger,
R.; Laufer, G.; Grimm, M.; Griesmacher, A.; Weigel, G.; Wolner,
E.; Muller, M. M. AdV. Exp. Med. Biol. 1998, 431, 801-803. (c)
Covarrubias-Zu´niga, A.; Gonzalez-Lucan, A.; Dominguez, M. M.
Tetrahedron 2003, 59, 1989-1994.
tive HPLC (50 to 88% MeCN in H2O with 0.1% formic acid) to afford
345 mg of 1 and 33.5 mg of 2.
RHM1 (1): fine white crystals; [R]25 -125.0 (c 0.08, MeOH);
D
UV (MeOH) λmax (log ꢀ) 205 nm (4.33); 1H NMR (500 MHz, DMSO-
d6) and 13C NMR (125 MHz, DMSO-d6), see Table 3; FTMS m/z [M
+ H]+ 1036.7386 (calcd for C53H98N9O11 1036.7391).
RHM2 (2): fine white crystals; [R]25 -137.5 (c 0.08, MeOH);
D
(4) (a) Endo, A.; Hasumi, K.; Sakai, K.; Kanabe, T. J. Antibiot. 1985,
38, 920-925. (b) Rosen, T.; Heathcock, C. Tetrahedron 1986, 42,
4909-4951. (c) McKenney, J. M. Clin. Pharm. 1988, 7, 21-36.
(5) Cheng, X. C.; Varoglu, M.; Abrell, L.; Crews, P.; Lobkovaky, E.;
Clardy, J. J. Org. Chem. 1994, 59, 6344-6348.
UV (MeOH) λmax (log ꢀ) 207 nm (4.47); 1H NMR (600 MHz, DMSO-
d6) and 13C NMR (150 MHz, DMSO-d6), see Table 4; FTMS m/z [M
+ H]+ 1022.7242 (calcd for C52H96N9O11 1022.7244).
Efrapeptin G (3): white solid; 1H NMR (500 MHz, MeOH-d4) and
13C NMR (125 MHz, MeOH-d4), see Table 2; FTMS m/z [M]+
1648.0914 (calcd for C83H143N18O16 1648.0924).
(6) Numata, A.; Takahashi, C.; Matsushita, T.; Miyamoto, T.; Kawai,
K.; Usami, Y.; Matsumura, E.; Inoue, M.; Ohishi, H.; Shingu, T.
Tetrahedron Lett. 1992, 33, 1621-1624.
Preparation of (2S,3R)-N-Me-Ile and (2R,3S)-N-Me-Ile.30 The
procedure consisted of combining N-Boc-(2R,3S)-Ile (155.6 mg)
dissolved in THF under an N2 atmosphere with anhydrous CH3I (0.335
mL) at 0 °C followed by the addition of 60% oil suspension of NaH
(70 mg) while gently stirring under a flow of N2. The resulting mixture
was stirred for 16 h at room temperature under N2, then diluted with
saturated aqueous NH4Cl (5 mL) and extracted with diethyl ether (5
mL). The organic layer was extracted with saturated aqueous NaHCO3-
water (1:1) three times (2.5 mL). The aqueous layers were combined
and adjusted to pH 3 with 10% citric acid and extracted with EtOAc
(2.5 mL) two times. The organic layers were combined and concentrated
through rotary evaporation, yielding 80.4 mg of pale yellow oil. The
material was deprotected by dissolving 18.0 mg of the crude oil in 1
mL of CH2Cl2 and adding 1 mL of TFA while stirring under an N2
atmosphere at 0 °C for 1.5 h. The same procedure was carried out
with N-Boc-(2S,3R)-Ile (131.4 mg), resulting in 70.8 mg of a pale
(7) Varoglu, M.; Corbett, T. H.; Valeriote, F. A.; Crews, P. J. Org. Chem.
1997, 62, 7078-7079.
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1997, 38, 5675-5678. (b) Amagata, T.; Bio, M.; Ohta, T.; Minoura,
K.; Numata, A. J. Chem. Soc., Perkin Trans 1 1998, 3585-3599.
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Valeriote, F. A.; Crews, P. J. Med. Chem. 2003, 46, 4342-4350.
(b) Laurent, D.; Guella, G.; Roquebert, M. F.; Farinole, F.; Mancini,
I.; Pietra, F. Planta Med. 2000, 66, 63-66.
(11) Abraham, E. Cephalosporins and Penicillins: Chemistry and Biology;
Flynn, E. H., Ed.; Academic Press: New York, 1972.
(12) Chen, C. Y.; Imamura, N.; Nishijima, M.; Adachi, K.; Sakai, M.;
Sano, H. J. Antibiot. 1996, 49, 998-1005.
(13) Abdel-Lateff, A.; Konig, G. M.; Fisch, K. M.; Holler, U.; Jones, P.
G.; Wright, A. D. J. Nat. Prod. 2002, 65, 1605-1611.
(14) Verracarin A, isororidin A, and verrol-4-acetate; see ref 10b.
(15) The oxepinamides and fumiquinazones: Belofsky, G. N.; Anguera,
M.; Jensen, P. R.; Fenical, W. M. Chem. Eur. J. 2000, 6, 1355-
1360.
1
yellow oil. The identity of the products was confirmed with H NMR
and LCMS; the purities were found to be sufficient for use as standards
for the Marfey’s analysis.
(16) The virescenosides come from both marine fungi: (a) Afiyatullov,
S. S.; Kuznetsova, T. A., Isakov, V. V.; Pivkin, M. V.; Prokof’eva,
N. G.; Elyakov, G. B. J. Nat. Prod. 2000, 63, 848-850. (b)
Afiyatullov, S. S.; Kalinovsky, A. I.; Kuznetsova, T. A.; Isakov, V.
V.; Pivkin, M. V.; Dmitrenok, P. S.; Elyakov, G. B. J. Nat. Prod.
2002, 65, 641-644. (c) Afiyatullov, S. S.; Kalinovsky, A. I.;
Kuznetsova, T. A.; Pivkin, M. V.; Pfrkof’eva, N. G.; Dmitrenok, P.
S.; Elyakov, G. B. J. Nat. Prod. 2004, 67, 1047-1051; and terrestrial
fungi: (d) Cagnoli-Bellavita, N.; Ceccherelli, P.; Mariani, R.;
Polonsky, J.; Baskevitch, Z. Eur. J. Biochem. 1970, 15, 356-559.
(17) (a) Gupta, S.; Krasnoff, S. B.; Roberts, D. W.; Renvich, J. A. A.;
Brinen, L. S.; Clardy, J. J. Org. Chem. 1992, 57, 2306-2313, 13C
Determination of R/S Configuration Using Marfey’s Method.31
Approximately 1 mg of 1 was hydrolyzed with 1 mL of 6 N HCl at
110 °C for 20 h. The hydrolysate was evaporated to dryness and treated
with the following: 0.100 mL of 1 M NaHCO3 and 0.050 mL of a 10
mg/mL solution of 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (L-
FDAA). The mixture was heated at 80 °C for 30 min, cooled to room
temperature, and quenched with 0.050 mL of 2 N HCl. Then 0.300
mL of MeCN was added and samples were evaluated with LCMS. A
linear gradient with aqueous MeCN with 0.01 M TFA was run on an
analytical C-18 RP column over 40 min (25 to 100%), and retention
times of standards were compared with the derivatized hydrolysate of
1. Retention times (min) of standards: (S)-Gln (13.2), (R)-Gln (13.4),
(2S,3S)-N-Me-Ile (24.2), (2R,3R)-N-Me-Ile (25.4), (2S,3R)-N-Me-Ile
(24.3), (2R,3S)-N-Me-Ile (25.6), (2S,3S)-Ile (22.6), (2R,3R)-Ile (24.9),
(2S,3R)-Ile (22.5), (2R,3S)-Ile (24.8), (S)-N-Me-Val (22.3), (R)-N-Me-
Val (23.5), (S)-N-Me-Leu (23.6), (R)-N-Me-Leu (24.5), (S)-Glu (15.4),
(R)-Glu (15.9). Retention times (min) for the hydrolysate of 1: (R)-
Glu (15.9), (S)-N-Me-Val (22.4), (2S,3S)-Ile (22.6), (S)-N-Me-Leu
(23.6), (2S,3S)-N-Me-Ile (24.1).
1
and H NMR comparisons in Table 2 are made to compound 11 in
this publication. (b) Drasnoff, S. B.; Gupta, S.; St. Leger, R. J.;
Renwick, J. A. A.; Roberts, D. W. J. InVertebr. Pathol. 1991, 58,
180-188.
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Hoffmann, J.; Pan, M.; McLaughline, S. J. Exp. Ther. Oncol. 2002,
2, 228-236.
(19) Krasnoff, S. B.; Gupta, S. J. Chem. Ecol. 1991, 17, 1953-1962.
(20) For nomenclature of the ions, see: Roepstorff, P.; Fohlman, J.
Biomed. Mass Spectrom. 1984, 11 601.

92 Journal of Natural Products, 2006, Vol. 69, No. 1
Boot et al.
(21) Pip can be found in (a) the apicidins: Singh, S. B.; Zink, D. L.;
Liesch, J. M.; Mosely, R. T.; Dombrowski, A. W.; Bills G. F.; Darkin-
Rattray, J. S.; Schmatz, D. M.; Goetz, M. A. J. Org. Chem. 2002,
67, 815-825; and (b) Sch 217048: Hegde, V. R.; Puar, M. S.; Chan,
T. M.; Dai, P.; Das, P. R.; Patel, M. 1998, 63, 9584-9586. (c) â-Ala
is found in the theonellapeptolides: Roy, M. C.; Ohtani, I. I., Ichiba,
T.; Tanaka, J.; Satari, R.; Higa, T. Tetrahedron 2000, 56, 9079-
9092. Pip, Iva, and â-Ala are all present in (d) adenopeptin:
Hayakawa, Y.; Adachi, M.; Kim, J. W.; Shin-Ya, K.; Seto H.
Tetrahedron 1998, 54, 15871-15878.
(26) Auvin-Guette, C.; Rebuffat, S.; Vuidepot, I.; Massias, M.; Bodo, B.
J. Chem. Soc., Perkin Trans. 1 1993, 2, 249-255.
(27) Singh, S. B.; Herath, K.; Guan, Z. Q.; Zink, D. L.; Dombrowski, A.
W.; Polishook, J. D.; Silverman, D. C.; Lingham, R. B.; Felock, P.
J.; Hazuda, D. J. Org. Lett. 2002, 4, 249-255.
(28) Personal communication, Dr. D. A. Sutton, Department of Pathology,
University of Texas Health Science Center.
(29) Ralifo, P.; Crews, P. J. Org. Chem. 2004, 69, 9025-9029.
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Kameyama, T. J. Antibiot. 2001, 54, 22-28.
(31) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591-596.
(32) Within the B and Y′ groups certain fragment ions appear 1 amu lower
than expected (specified by * in Figure 1) due to loss of a proton via
a molecular rearrangement. One possibility is through double-bond
formation within the imminium group as previously noted in ref 17a.
Fragment ions also appear 1 amu higher than expected in select Y′
fragments (as indicated by ** in Figure 1).
(22) Komatsu, K.; Shigemori, H.; Kobayashi, J. J. Org. Chem. 2001, 66,
6189-6192.
(23) Tan, L. T.; Cheng, S. C.; Jensen, P. R.; Fenical, W. J. Org. Chem.
2003, 68, 8767-8773.
(24) Toske, S. G.; Jensen, P. R.; Kauffman, C. A.; Fenical, W. Tetrahedron
1998, 54, 13459-13466.
(25) Duncan, S. J.; Gruschow, S.; Williams, D. H.; McNicholas, C.;
Purewal, R.; Hajek, M.; Gerliz, M.; Martin, S.; Wrigley, S. K.; Morre,
M. J. Am. Chem. Soc. 2001, 123, 554-560.
NP0503653