Synthesis of 7-15N-oroidin and evaluation of utility for biosynthetic studies of pyrrole-imidazole alkaloids by microscale 1H-15N HSQC and FTMS

Article abstract of DOI:10.1021/np900638e

Numerous marine-derived pyrrole-imidazole alkaloids (PIAs), ostensibly derived from the simple precursor oroidin, 1a, have been reported and have garnered intense synthetic interest due to their complex structures and in some cases biological activity; however very little is known regarding their biosynthesis. We describe a concise synthesis of 7-¹⁵N-oroidin (Id) from urocanic acid and a direct method for measurement, of ¹⁵N incorporation by pulse labeling and analysis by 1D ¹H-¹⁵N HSQC NMR and FTMS. Using a mock pulse labeling experiment, we estimate the limit of detection (LOD) for incorporation of newly biosynthesized PIA by 1D ¹H-¹⁵N HSQC to be 0.96 μg equivalent of ¹⁵N-oroidin (2.4 nmole) in a background of 1500 μg of unlabeled oroidin. (about 1 part per 1600). 7-¹⁵N-Oroidin will find utility in biosynthetic feeding experiments with live sponges to provide direct information to clarify the pathways leading to more complex pyrrole-imidazole alkaloids.

Full text of DOI:10.1021/np900638e

428
J. Nat. Prod. 2010, 73, 428–434
Synthesis of 7-15N-Oroidin and Evaluation of Utility for Biosynthetic Studies of
†
1
Pyrrole-Imidazole Alkaloids by Microscale H-15N HSQC and FTMS
Yong-Gang Wang,‡ Brandon I. Morinaka,§ Jeremy Chris P. Reyes,‡ Jeremy J. Wolff,⊥ Daniel Romo,*,‡ and
Tadeusz F. Molinski*,§,|
Department of Chemistry, Texas A&M UniVersity, P.O. Box 30012, College Station, Texas 77842-3012, Bruker Daltonics, 40 Manning Road,
Billerica, Massachusetts 01821, and Department of Chemistry and Biochemistry, and Skaggs School of Pharmacy and Pharmaceutical
Sciences, UniVersity of California, San Diego, 9500 Gilman DriVe, La Jolla, California 92093-0358
ReceiVed October 8, 2009
Numerous marine-derived pyrrole-imidazole alkaloids (PIAs), ostensibly derived from the simple precursor oroidin,
1a, have been reported and have garnered intense synthetic interest due to their complex structures and in some cases
biological activity; however very little is known regarding their biosynthesis. We describe a concise synthesis of 7-15N-
oroidin (1d) from urocanic acid and a direct method for measurement of 15N incorporation by pulse labeling and analysis
1
by 1D H-15N HSQC NMR and FTMS. Using a mock pulse labeling experiment, we estimate the limit of detection
1
(LOD) for incorporation of newly biosynthesized PIA by 1D H-15N HSQC to be 0.96 µg equivalent of 15N-oroidin
(2.4 nmole) in a background of 1500 µg of unlabeled oroidin (about 1 part per 1600). 7-15N-Oroidin will find utility in
biosynthetic feeding experiments with live sponges to provide direct information to clarify the pathways leading to
more complex pyrrole-imidazole alkaloids.
The polycyclic pyrrole-imidazole alkaloid (PIA) family has
received much attention recently from the synthetic organic
community1 because of their challenging molecular structure, dense
functionality, and potential biological activity. All are believed to
derive from a simple common sponge metabolite, most likely
oroidin (1a) or its analogues hymenidin (1b) and clathrodin (1c),
the structures of which differ only in bromine content.2 Representa-
tive of the members of this family are the dimeric PIAs sceptrin
(2),3 ageliferin (3),4 massadine (4),5 palau’amine (5),6 and axinel-
lamines A and B (6a,b)7 and higher-order tetrameric PIAs.8 In
addition, compact monomeric polycyclic PIAs, such as agelastatins
A,9 B,10 C, and D11 (7a-d) and dibromophakellin12 appear to be
derived from oxidative intramolecular cyclizations of oroidin (1a)
or hymenidin (1b). PIAs are of contemporary interest due to their
potent biological activity and intriguing biosyntheses. For example,
(-)-agelastatin A (7a), an antitumor PIA,10 is 1.5-16 times more
active than cisplatin against a range of tumor cell lines and potently
inhibits osteopontin, an adhesion glycoprotein transcriptionally
regulated by the Wnt ꢀ-catenin pathway and implicated in tumor
progression, dissemination, and metastases.13
The widely accepted hypothesis for biosynthesis of dimeric and
tetrameric PIAs involves dimerization of oroidin (1a) followed by
consecutive oxidative transformations leading to more densely
functionalized polycyclic alkaloids.8 In 1982, Bu¨chi reported a
“biomimetic” synthesis of the simple PIA racemic dibromophakellin
by oxidative cyclization of 9,10-dihydrooroidin.14 The structure of
sceptrin (2) is highly suggestive of a formal [2+2] cycloaddition
of two molecules of hymenidin (1b) to 2; however, despite many
attempts, Faulkner, Clardy, and co-workers were unable to achieve
photodimerization of 1b to 2,3 suggesting that an alternative
nonconcerted mechanism may be responsible for the biosynthesis.
Recent speculation on the order of biosynthesis of 2 and 3 revolves
around ionic mechanisms of ring expansion, supported by the
demonstrated conversion of 2 to 3 under thermal conditions,1f,15,16
or a ring-contraction pathway from an ageliferin-type dimer
generated by a Diels-Alder-type cycloaddition of two molecules
of hymenidin (1b).2 For example, one speculative hypothesis
invokes a cycloaddition in the biosynthesis of palau’amine6b and
axinellamine, followed by a stepwise electrophilic chlorination and
ring contraction. Remarkably, none of the above hypotheses have
been tested experimentally in live sponges.
We describe here a synthesis of 15N-labeled oroidin (1d) and
optimization of conditions for detection of 15N-incorporation by
microscale FTMS and 1H-15N HSQC using a “mock” pulse-
labeling experiment with 1d and natural oroidin (1a). We estimate
the limits of detection of these methods and determine their
suitability for use in field incorporation for biosynthetic studies of
complex PIAs with living sponges. This information can be used
to answer questions of the biosynthesis of PIAs by sponges in the
genera Agelas, Stylissa, and Ptilocaulis.
Results and Discussion
Several syntheses of oroidin have been described in the literature,
and the synthetic approaches utilized can be classified into two
categories.17 The first involves synthesis of the required 2-ami-
noimidazole ring by condensation of a guanidine derivative18 or
cyanamide19 with an R-haloketone. An alternative strategy involves
amination of an imidazole ring via azido or diazonium transfer
reagents.20 We elected the latter strategy for brevity and the utility
of the commercially available starting material urocanic acid, as
demonstrated by Lovely.1o,21 This strategy enabled a late-stage
introduction of the 15N-label, which is ideal for cost considerations
(Scheme 1). Disconnection of the amide bond leads to the known
4,5-dibromo-2-trichloroacetylpyrrole 8,21 readily synthesized from
commercially available 2-trichloroacetylpyrrole, and the 15N-labeled
allylic amine 9, which could be obtained by a lithiation/azidation
sequence at the 2-position of the N-protected urocanic acid ester
10. The 15N label would be introduced toward the end of the overall
sequence by Gabriel synthesis employing the potassium salt of
commercially available 15N-phthalimide and an activated derivative
of the alcohol, which, in turn, could be obtained by reduction of
the carbomethoxy group of ester 10.
† Dedicated to the late Dr. John W. Daly of NIDDK, NIH, Bethesda,
Maryland, and to the late Dr. Richard E. Moore of the University of Hawaii
at Manoa for their pioneering work on bioactive natural products.
* To whom correspondence should be addressed. Tel: +1 (858) 534-
7115. Fax: +1 (858) 822-0386. E-mail: tmolinski@ucsd.edu. Tel: +1 (979)
845-9571. Fax: +1 (979) 862-4880. E-mail: romo@tamu.edu.
‡ Texas A&M University.
§ Department of Chemistry and Biochemistry, UC San Diego.
⊥ Bruker Daltonics.
| Skaggs School of Pharmacy and Pharmaceutical Sciences, UC San
Diego.
Fisher esterification of urocanic acid with methanol (Scheme 2)
and subsequent protection gave the known N-trityl imidazole
10.1021/np900638e  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/22/2010

Synthesis of 7-15N-Oroidin
Journal of Natural Products, 2010, Vol. 73, No. 3 429
Scheme 1. Retrosynthetic Analysis of 7-15N-Oroidin (1d) from
Urocanic Acid
Scheme 2. Synthesis of 15N-Labeled Oroidina
Figure 1. Oroidin (1a) and other representatives of the pyrrole-
imidazole alkaloid (PIA) family from marine sponges.
the 15N-labeled pthalimide 15. Cleavage of phthalimide 15 with
NH2NH2 furnished the 15N-allylic amine 9, which was acylated with
4,5-dibromo-2-trichloroacetylpyrrole (8) to provide 15N-pyrrole-
carboxamide 16 in good overall yield. Removal of the trityl group
from 16 with anhydrous methanolic HCl, generated in situ from
acetyl chloride and MeOH, afforded the corresponding 2-azido
imidazole,20c which was hydrogenated in the presence of Lindlar’s
catalyst to provide 7-15N-oroidin (1d, >98% atom 15N).
a Reagents and conditions: (a) AcCl, MeOH, reflux, quant.; (b) TrCl, Et3N,
DMF; (c) DIBAL-H, THF; (d) TBSCl, imidazole, 74% for 3 steps; (e) n-BuLi,
TsN3, THF, 94%; (f) TBAF, THF, 82%; (g) MsCl, Et3N, THF; (h) potassium
phthalimide-15N, DMF, 42% for 2 steps; (i) NH2NH2, EtOH, 50 °C; (j) 4,5-
dibromo-2-trichloroacetylpyrrole, Na2CO3, DMF, 76% for 2 steps; (k) AcCl,
MeOH/EtOAc, 99%; (l) H2/Pd-Lindlar, MeOH/THF, 84%.
derivative 10.22 Reduction of ester 10 (DIBAL-H) afforded the
corresponding alcohol 11 in excellent yield. Initial attempts to
execute a direct azidation of alcohol 11 at the 2-imidazole position
with the unprotected alcohol using known procedures (n-BuLi/
TsN3)20c-e gave none of the desired azido product. Instead, the
product obtained was tentatively assigned the structure of a triazene-
The 1H, 13C, and 15N NMR spectra and mass spectra were fully
consistent with the structure and location of the 15N label as
depicted. The FTMS of natural oroidin (1a, Figure 1) shows the
expected 1:2:1 pattern for the pseudomolecular ion [M + H]+
associated with the 79Br and 81Br isotopomers (m/z 387.93990,
∆mmu ) -0.41 mmu). The FTMS spectrum of 7-15N-oroidin (1d)
(Figure 2b) shows the same ion pattern, except the distribution is
increased by 1 amu (m/z 388.93719, ∆mmu ) -0.16). Less than
2% of the 14N isotopomer was detected, which is consistent with
the reported purity of the 15N-pthalimide (>98% atom 15N) employed
in the synthesis.
1
imidazole derivative based on H NMR and mass spectrometric
analysis.23 When the TBS-protected imidazole 12 was subjected
to the previously employed conditions for azido transfer, azide 13
was obtained in excellent yield. Deprotection of silyl ether 13 with
TBAF afforded alcohol 14, which was then subjected to mesylation
(MsCl, Et3N). Without purification, the mesylate was treated with
the potassium salt of 15N-phthalimide (>98% atom 15N) to provide
Two general methods are available for measuring incorporation
of the stable isotope 15N into natural products by pulse labeling:

430 Journal of Natural Products, 2010, Vol. 73, No. 3
Wang et al.
Figure 2. MALDI FTMS (7 T) expansions of the pseudomolecular
ion [M + H]+ of oroidin for (a) natural 1a (C11H12Br214N5O) and
(b) 7-15N-oroidin (1d), C11H12Br214N415NO, >98% atom 15N. Mass
accuracy ) 1 ppm.
mass spectrometry and NMR. In both techniques, the level of
isotopic enrichment from de novo biosynthesis of derived PIAs must
exceed the background 15N present of extant PIAs within the sponge
and, for MS in particular, 15N background contributions from other
nitrogen atoms in the molecule. As a stable isotope label, 15N is
preferable to 13C because the natural abundance of the heavier to
lighter isotope for N (∼0.3%) is only about one-fourth of C
(∼1.1%). 15N incorporation will, therefore, be easier to detect.
Ideally for 15N incorporation experiments, mass spectrometry should
differentiate the contributions to the M + 1 peak from 15N and
13C,24 a task best suited to Fourier transform MS (FTMS) with
exceptionally high resolution (7 T field, R ) 240 000), which should
be capable of resolving the nominal M + 1 peak of the pseudo-
molecular ion into individual isotopomer contributions from species
containing one 13C or one 15N. Baseline separation of the isoto-
pomers was readily achieved by MALDI FTMS (Figure 3), allowing
quantitation of the 15N isotopomer peak. For oroidin (1a), the critical
difference in the mass of 12C-15N containing ions versus the heavier
13C-14N isotopomer amounts to only 0.00632 amu, a baseline
separation not readily achievable by high-resolution mass spec-
trometers with conventional analyzers (e.g., double sector, TOF).
Quantitation of the 15N isotopomer can be made from integration
of ion current intensities of the [M + 1] components, 15N-12C and
14N-13C (Figure 3). Assuming the 13C abundance in natural and
synthetic 15N-labeled oroidin are identical, the 13C peak intensity
may be used as the denominator in the ratio h (eq 1), where hN and
hC are the respective measured peak heights of the 15N-12C and
14N-13C component ions.
Figure 3. MALDI FTMS (7 T) expansions of m/z and peak height,
h, for the M + 1 peak of the pseudomolecular ions of oroidin
(C11H11Br2N5O). Mass accuracy ) 1 ppm (R ) 240 000). (a)
Natural 1a. (b) 1a + 1d (0.04 molar equiv). (c) 1a + 1d (0.10
molar equiv). Peak assignments of [M + H]+ in (c): m/z 388.94372,
12
12
C
13CH12Br2N5O+; m/z 388.93739; C11H12Br2N415NO+. The
10
unrelated peak at m/z 388.9305 (indicated with an *) is tentatively
assigned to some loss of H from the dominant 79Br/81Br isotopomer,
C11H1079Br81BrN5O+ ([M]+, ∆mmu ) -0.5).
The disadvantage of analysis of 15N-content in oroidin and other
PIAs by MS is the presence of at least five N atoms, each
contributing to the intensity of the 15N-12C component of the M
+ 1 peak. Consequently, it may be expected that the actual LOD
by FTMS will be higher.
h ) hN/hC
(1)
Consequently, h can be used to reflect small changes in the 15N
content.25 From the ion current peak heights measured in the FTMS
spectrum of natural oroidin (1a), h was found to be 0.1487 (
0.0175. A prepared “mock” labeled sample (Mix1), consisting of
a 1:0.005 mixture of oroidin (1a) and 7-15N-oroidin (1d), showed
an enhanced h of 0.1881 ( 0.0135, corresponding to a ∼4% 15N
enrichment. A second sample (Mix2, 1:0.100 1a:1d) gave h ) 1.024
( 0.0369, corresponding to ∼88% 15N enrichment. We estimate
that the limit of detection (LOD, (3σ) for 15N incorporation
corresponds to an increase in intensity, ∆h ∼5.2%. Given that
natural abundance of 15N is approximately 0.3% atom, this would
Direct detection of 15N incorporation by 15N NMR spectroscopy
is impractical due to low natural abundance and severely compro-
mised sensitivity of this I ) 1/2 nucleus, which possesses a low-
magnitude, negative magnetogyric ratio (γ ) -4.31726570 MHz
26
1
T-1). Conveniently, H-15N HSQC circumvents most of these
problems by indirect detection of bonded 1H-15N couplets through
magnetization transfer from 15N and observation of the more
1
sensitive H nucleus. Because four of the five N atoms in oroidin
(1a) are bonded to H, 2D 1H-15N HSQC can provide bond-specific
information about fractional enrichment at an individual N atom
in oroidin and, by extension, more complex PIAs by measurement
1
amount to observation of as little as 10.4 parts per thousand of 15
N
of the H-15N couplet intensity. The intensity of cross-peaks in
incorporation per N atom of oroidin (1a) by de novo synthesis.
Detection of this small amount will, of course, be made more
difficult by the presence of unlabeled oroidin (1a) within the sponge.
the 2D 1H-15N HSQC spectrum of oroidin will vary for each N-H
couplet depending on efficiency of magnetization transfer that
1
largely depends upon the magnitude of the JNH scalar coupling

Synthesis of 7-15N-Oroidin
Journal of Natural Products, 2010, Vol. 73, No. 3 431
1
Figure 4. 2D H-15N HSQC (600 MHz, 1:1 DMSO-d6/benzene-d6) of natural oroidin (1a, depicted tautomer is arbitrary). N locants are
labeled following the numbering scheme of Assman and Ko¨ck (ref 32). ns ) 16, T2, T1 ) 2K × 256; F2, F1 ) 2K × 1K; d1 ) 1.5 s.
constant. Nevertheless, the intensity of each cross-peak will scale
linearly as a function of 15N abundance.
When put to practice, it is easier and more meaningful to measure
15N enrichment from the volume integration of cross-peaks of 2D
HSQC spectra orsbest of allsfrom the 1D version of the HSQC
experiment, where evolution of the F1 dimension (15N chemical
shift) is eliminated and acquisition time is devoted to improving
S/N in the detected dimension F2 (1H chemical shift). We define
here ∆I as the difference of cross-peak intensity, I, of the 15N-
labeled N-H couplet of interest (in this case, the NH(CdO) signal
of 1d) in a sample of oroidin (1a), spiked with a known amount of
7-15N-oroidin (1d), compared to the intensity, I0, of oroidin recorded
under the same conditions (eq 2). The values of I are normalized
to the intensity of the pyrrole N-H couplet of oroidin, Ipy, in each
experiment.
∆I ) (I - I0)/I0
(2)
We elected to use the pyrrole N-H couplet of oroidin (1a) (δH
) 13.14, s; δN 169.1) as the control because of its ready assignment
1
from H-15N HMBC, lack of tautomerism, and likely origination
in a different pathway unaffected by biosynthetic incorporation of
15N into the amino-imidazole fragment (see discussion).
The exquisite mass sensitivity of the triple-tuned {13C,15N}1H
1.7 mm 600 MHz microcryoprobe was used to full advantage in
this work for characterization of the 15N content of oroidin samples.
This low-volume, high-mass sensitivity cryoprobe allowed mea-
surements of samples of only ∼1.5 mg in 35 µL (acquisition times,
∼4 h). Figure 4 shows the 2D 1H-15N HSQC experiment for
oroidin (1a) with respective N assignments (also, see the Experi-
mental Section for accurate δ’s). A single dominant cross-peak was
observed in the 2D 1H-15N HSQC of 7-15N-oroidin (1d) (not
shown), confirming the location of the 15N label at the amide group
1
Figure 5. 1D H-15N HSQC experiments (1H δ, 1.7 mm micro-
cryoprobe, 600 MHz) of 1a with N-H assignments. Labels indicate
N atom assignments made from a separate 1H-15N HMBC
experiment (not shown). (a) 1500 µg of 1a, no added [15N]-1d. (b)
1500 µg 1a + 10 µg 1d. (c) 1500 µg 1a + 5.0 µg 1d. (d) 1500 µg
3
N-H (δH ) 8.75, t, JHH ) 6.0 Hz, H7; δN ) 107.8, Ν7), as
1a + 2.5 µg. Relaxation delay, d1 ) 1.50 s, optimized for 1JN-H
)
expected.
1
Figure 5a-c shows 1D H-15N HSQC experiments of oroidin
90 Hz; 1H π/2 pulse ) 12 µS, 15N π/2 pulse ) 34 µS; NA ) 6K;
dummy scans ) 16; NI ) 1; T2 ) F2 ) 8K points (no zero fill).
See Supporting Information for calculations of limit of detection,
mean and SD.
(1a) spiked with different amounts of 7-15N-oroidin (1d), and the
signals of each N-H couplet, normalized to the N1-pyrrole couplet,
are reported in Table 1 (note that the integrals are not exactly

432 Journal of Natural Products, 2010, Vol. 73, No. 3
Wang et al.
1
Table 1. Integrals from H-15N 1D HSQC of Natural Oroidin
(1a, 1.5 mg/ 30 µL) and 1a Spiked with 1da
aliquot µg 1d
N-12
N-1
N-14
N-7
N-16
0.0
2.5
5.0
10.0
1.26
1.03
1.65
0.63
1.00
1.00
1.00
1.00
1.05
0.48
1.18
0.00
2.17
2.57
3.83
5.74
2.14
2.23
2.55
3.02
a Integrals of H-N couplets corresponding to N-12, N-14, and N-16
vary due to tautomerism.
Figure 7. Possible biosynthesis of oroidin (1a) and conversion to
stevensine (17).
caribica28) produced 14C-labeled stevensine (17),29 the simplest
monomeric PIA related to oroidin (1a), upon exposure to U-14C-
labeled histidine or C5-14C-ornithine, but not U-14C-arginine (Figure
7).27 Paradoxically, U-14C-histidine was incorporated into 17, but
this would require an additional C1 unit from an unidentified source
to complete the imidazole ring and a nonintuitive substitution of
NH2 at C-2 of the imidazole ring. No simple mechanism for this
scenario presents itself. Alternatively, citrulline from the N-
carbamoylation of ornithine may be the precursor to the side chain
of oroidin; however ornithine is also a precursor for the biosynthesis
of the pyrrole group. This hypothesis would not be resolved by
labeled ornithine incorporation experiments. Ornithine, if it is the
precursor to oroidin (1a), must undergo decarboxylation, amide
coupling with a suitable pyrrole carboxylate group, N-carbamoy-
lation to citrulline, oxidative N-insertion into the side chain, and
aromatization of the heterocyclic product to give the nascent
2-aminoimidazole ring (Figure 7).
These uncertainties in the origin of oroidin (1a) are, in theory,
addressable by suitable 15N-labeled amino acid incorporation
experiments. Unfortunately, low levels of radioactivity and specific
incorporation levels were observed with amino acids in the Kerr
experiment ([U-14C]-His, 1460 dpm (0.026%) and [U-14C]-ornithine,
1300 dpm (0.024%), respectively27), presumably a consequence
of the low numbers of cultured cells used (5 × 107) and higher
metabolic mobilization and dilution of 14C-labeled amino acids into
other committed pathways of primary metabolism. Consequently,
detection of 15N incorporation into PIAs using 15N-labeled amino
acids may be precluded by the limits of detection. For the above
reasons, late-stage precursors (e.g., 7-15N-oroidin (1d), sceptrin (2),
and ageliferin (3)) are attractive for mapping the origin of
downstream polycyclic PIAs since these molecules should be
committed intermediates. Measurement of their rates of incorpora-
tion into PIAs in living sponges of the genera Agelas, Stylissa, and
Ptilocaulis may also provide critical data on the rates of marine
alkaloid secondary metabolism, for which there is no current
information.
Figure 6. Linear regression of 1H-15N HSQC cross-peak integral
ratio, (I - I0)/I0, for the NH(CdO) signal in “mock” pulse-labeling
experiments with natural abundance 1a (1500 µg, I0) spiked with
measured aliquots of 1d (µg, x-scale). Observed values, I, for the
1
NH-CdO H-15N couplet are normalized to the intensity Ipy of
the pyrrole N-H cross-peak. For the NH(CdO) peak in the natural
abundance sample (no added 1d), I ) I0 and I0/Ipy ) 1.00. Error
bars are (SD.
proportional to the stoichiometric ratios of attached H’s). As
predicted, the value of ∆I increases linearly with the amount of
added 7-15N-oroidin (1d), and the slope of this “standard addition”
linear regression (Figure 6) gives the proportionality constant c )
0.34 for ∆I per µg of added 1d. It should be noted that this value
will be highly dependent upon the pulse sequence parameters and
conditions used for NMR acquisition.
Constant c will be useful for estimation of absolute %N
incorporations in PIAs labeled during in-field incorporation experi-
ments of secondary metabolism within living sponges. Although it
is advisible to measure the relative HSQC intensities of the pyrrole
N-H couplet and NH(CdO) couplets of the specific PIA in
question, it may be reasonably expected that the magnetic properties
of these couplets in more complex PIAs are comparable to those
of oroidin (1a). We estimate the limit of detection for 15N
enrichment by HSQC in 1a to be approximately 1.5 parts per
thousand (based on (3 SD). This is more sensitiVe than the LOD
of 15N-enrichment by FTMS, the reason being that HSQC detects
the couplet 15N-1H at each N atom and eliminates background
“dilution” by other 15N’s; however the precision is inferior to FTMS
replicate measurements.
The use of precursors labeled with the spin I ) 1/2 stable isotope
15N is particularly attractive, as it provides bond-specific information
regarding the flow of intermediates in the biosynthesis of complex
PIAs. Judicious design and placement of the label in 15N-labeled
oroidin is important. Because the primary NH2 group of the
aminoimidazole group is potentially metabolically labile through
deamination reactions, we chose to label the amide bond in 15N-
oroidin. Quantitation of 15N incorporation into proteins has been
made from integration of cross-peak volumes of 2D HSQC
Biosynthetic Considerations. Two phases of PIA biosynthesis
can be considered: the assembly of oroidin (1a) and downstream
conversion of the latter to more complex PIAs. Despite much
speculation on the biosynthesis of PIAs, only one experimental
study in living marine sponges has appeared to date. The precursor
for the imidazole side chain is probably an amino acid, and both
histidine and ornithine have been proposed.27 Kerr and co-workers
found that cultured cells of Teichaxinella morchella (Stylissa

Synthesis of 7-15N-Oroidin
Journal of Natural Products, 2010, Vol. 73, No. 3 433
experiments;30 however, in the present work with natural products
we show that it is far simpler to integrate the corresponding 1D
HSQC spectra.
synthetic compounds were made at the Laboratory for Biological Mass
Spectrometry (Texas A&M University), Scripps Research Institute
(TOF-MS), or University of California, San Diego (EIMS) mass
spectrometry facilities. MALDI FTMS were recorded on a 7 T Bruker
Apex QFTMS. LCMS was carried out on a ThermoFisher Accela
UHPLC coupled to an MSQ single quadrupole mass spectrometer
operating in positive ion mode, unless otherwise stated. Semipreparative
HPLC was carried out on a Varian SD200 system equipped with a
dual-pump and UV-1 UV detector under specified conditions. All
solvents for purification were of HPLC grade. See Supporting Informa-
tion for other procedures.
Finally, although the ratio of sceptrin to ageliferin found in one
sample of Agelas conifera is ∼10:1,31 the ratios of monobrominated
and dibrominated analogues of sceptrin and ageliferin vary slightly
from approximately 1:1 to 3:4; however, these data do not clearly
indicate an ordering for a bromination-dimerization sequence in
the biosynthesis of dimeric PIAs. Propagation of 7-15N-oroidin (1d)
along the biosynthetic pathway to complex PIAs will carry the 15
N
Synthesis of 7-15N-Oroidin (1d). To a solution of amide 16 (380
mg, 0.58 mmol) in 20 mL of EtOAc was added MeOH (1.17 mL, 28.9
mmol) followed by dropwise addition of AcCl (2.06 mL, 28.9 mmol)
(to generate HCl) at 0 °C. The solution was stirred for 1.5 h at 25 °C,
and during that time a precipitate formed. The reaction mixture was
filtered to separate the solid product of azidoimidazole hydrochloride
salt (240 mg, 99%), which was of sufficient purity for the next reaction:
IR (thin film) 3115, 233, 2150, 1611, 1555, 1439 cm-1; 1H NMR (500
MHz, CD3OD) δ 4.11 (d, J ) 5 Hz, 2H), 6.35 (dt, J ) 16, 3.5 Hz,
label through symmetrical and nonsymmetrical intermediates, and
detection of residual 15N in strategically chosen N-H bonds in the
molecule should reveal timing, ordering, and molecularity of the
C-C bond-forming events and the extent of coupling of de novo
enzymatic reactions. HPLC analysis of PIA-containing extracts from
A. conifera (Agelasidae), collected in the Florida Keys by Ko¨ck
and co-workers,31 shows the presence of sceptrin (2) and ageliferin
(3); however, some specimens of Agelas sceptrum collected by us
contained sceptrin (2) but were devoid of the expected precursors
1a-c. If 1a-c are precursors to other PIAs, as is highly probable,
this implies that the rates of conversion of 1a-c to higher-order
PIAs are highly dependent upon the sponge species, their biosyn-
thetic capacities, and possibly the habitats they occupy. 7-15N-
Oroidin (1d) should find great value in systematic studies of the
time-dependent biosynthesis of PIA natural products by pulse
labeling in live sponges to answer some of these questions.
1H), 6.43 (d, J ) 16 Hz, 1H), 6.89 (d, J ) 2 Hz, 1H), 7.31 (s, 1H); 13
C
NMR (125 MHz, CD3OD) δ 42.3 (d, JC-N ) 114 Hz), 100.8, 107.3,
115.2, 116.3, 117.5 (d, JC-N ) 22.5 Hz), 129.4 (d, JC-N ) 106 Hz),
131.8, 132.3, 142.6, 162.4 (d, JC-N ) 174 Hz); HRESIMS m/z 416.9171
[M + H]+ (calcd for C11H12Br2N615NO, 416.9163).
To a solution of the azidoimidazole hydrochloride salt (210 mg, 0.50
mmol) in 30 mL of MeOH/EtOAc (1:1) was added Lindlar’s catalyst
(100 mg, 5% Pd on CaCO3, poisoned with Pb). The resulting mixture
was stirred under atmospheric hydrogen at 25 °C for 6 h and then
filtered with MeOH through a pad of Celite. The filtrate was
concentrated under reduced pressure to leave a residue, which was
purified by flash column chromatography on silica gel (gradient elution:
10:1:0 f 1:1:0.01 CH2Cl2/MeOH/NH4OH) to furnish 7-15N-oroidin (1d)
(165 mg, 84%): IR (thin film) 3304, 2363, 2339, 1620, 1507, 1415
Conclusion
A concise, gram-scale synthesis of 15N-labeled oroidin from
commercially available and inexpensive urocanic acid has been
achieved. 15N-Oroidin, or its debromo analogues, may be utilized
in biosynthetic feeding experiments to elucidate likely intermediates
in the biosynthesis of complex members of the pyrrole-imidazole
marine alkaloid family, including palau’amine and axinellamine.
FTMS detection of 15N offers unparalleled sensitivity (picomole),
and 1H-15N HSQC provides bond-specific information. The utility
1
cm-1; H NMR (500 MHz, CD3OD) δ 4.00 (d, J ) 6 Hz, 2H), 5.87
3
3
(dt, JNH ) 16 Hz, JHH ) 6 Hz, 1H), 6.30 (d, J ) 16 Hz, 1 H), 6.47
(s, 1H), 6.83 (d, J ) 1 Hz, 1H); 13C NMR (125 MHz, CD3OD) δ 43.1
(d, 1JC-N ) 106 Hz), 100.7, 107.0, 115.1, 118.5, 122.2, 123.6 (d, 2JC-N
1
1
) 15 Hz), 129.7 (d, JC-N ) 99 Hz), 132.2, 152.8, 162.4 (d, JC-N
)
175 Hz); HRESIMS m/z 390.9289 [M
+
H]+ (calcd for
C11H12Br2N415NO, 390.9258).
1
of H-15N HSQC experiments for detection of site-specific 15N
See Supporting Information for additional procedures.
incorporation in a mock pulse-labeling experiment has been
demonstrated. Several advantages of 15N pulse labeling of biosyn-
thetic pathways are apparent, especially for in-field experiments
carried out with living marine invertebrates. The 15N NMR chemical
shift was exploited through indirect 1H-15N HSQC to afford
enhancement of otherwise weak 15N signals by polarization transfer
through N-H couplets. 15N incorporation was further amplified by
Isolation of Natural Oroidin (1a) from Stylissa caribica. A sample
of Stylissa caribica (wet wt. 24.5 g) was collected in the Bahamas
(June 2008) and extracted with 1:1 CH2Cl2/MeOH (rt, overnight, 2 ×
500 mL). The solvent was removed under reduced pressure and the
residue partitioned between hexanes (3 × 250 mL) and 9:1 MeOH/
H2O (250 mL). The aqueous MeOH layer was concentrated and further
partitioned between n-BuOH (3 × 250 mL) and H2O (250 mL).
Removal of the solvent from the upper layer gave an n-BuOH extract
(787 mg), a portion of which (191 mg) was adsorbed onto a solid-
phase extraction cartridge (C18) and eluted with 4:1 CH3CN/H2O,
followed by THF. The first-eluting fraction (116 mg) was separated
by gradient HPLC (C18, 10:90 to 60:40 CH3CN/H2O + 0.1% TFA) to
give oroidin 1a (8.8 mg, 1.0 wt % weight).
1
the enhanced mass sensitivity of the triple-tuned H{13C,15N} 600
MHz 1.7 mm microcryoprobe. Biosynthetic studies with alkaloids
bearing N-H groups that are not complicated by exchange or
tautomerism afford the best cases for application of the technique.
In principle, even alkaloids lacking N-H couplets (tertiary N
compounds) are amenable to study by detection of long-range
1H-15N couplets from HMBC experiments. The use of 15N stable
isotope avoids more complex logistics associated with radioactive
tracers (e.g., 14C, 3H) that often preclude laboratory operations
aboard seagoing research vessels. The results of field incorporation
experiments with 7-15N-oroidin (1d) will be reported in due course.
MALDI FTMS of Oroidin (1a) and 7-15N-Oroidin (1d). Matrix
solution (1 µL of saturated R-cyano-4-hydroxycinnamic acid (CHCA)
solution in 50:50:0.2 MeOH/H2O/TFA) was added to 1 µL of the
sample solution (1 pmol/µL) and the sample (approximately 1 pmol)
deposited onto a stainless steel target. For each sample, 200 shots were
acquired for each mass spectrum (acquisition time, ∼1 min each), and
24 spectra were co-added. Data were processed using Bruker software,
and error analysis was carried out using standard deviation of replicate
MS measurements (N g 5).
Experimental Section
General Experimental Procedures. IR spectra were recorded on
thin films using a Jasco 4100 FTIR by attenuated total reflectance (ATR)
on samples mounted on a 3 mm ZnSe plate. 1H and 13C NMR spectra
were recorded on a Bruker DRX 600 MHz equipped with 1.7 mm
{13C,15N}1H 600 MHz CPTCI microcryoprobe, JEOL 500 MHz
spectrometer equipped with a 5 mm {13C }1H probe, or Varian 400
and 500 MHz spectrometers equipped with a 5 mm {13C}1H room-
temperature probe and 5 mm {1H}13C cryoprobe, respectively. NMR
1D 1H-15N HSQC Experiments. Samples for mixing experiments
were prepared by addition of measured aliquots of a standard solution
of 1d (1.25 mg/10 mL MeOH) to a sample of 1a (1500 µg). The
samples were concentrated by removal of solvent (MeOH + 0.05%
TFA) and redissolved in 1:1 DMSO-d6/benzene-d6 (35 µL) before
transferring ∼30 µL to a NMR tube (φ ) 1.7 mm) using a gastight
syringe. 1D 1H-15N HSQC NMR spectra were acquired with 8K data
1
points in T2 (no zero-fill) and optimized for JN-H ) 90 Hz. Each
1
spectra are referenced to residual solvent signals (CHCl3, H, δ 7.26
experiment was preceded by 16 dummy scans, and ∼6K scans were
collected. The corresponding 1H-15N integrals were averaged and
normalized against the N1 pyrrole couplet of 1 (IP ) 1.000). See Table
ppm; CDCl3, 13C, δ 77.16 ppm) or the internal offset for 15N assigned
by the instrument manufacturer (Bruker). HRMS measurements of

434 Journal of Natural Products, 2010, Vol. 73, No. 3
Wang et al.
1 (numbering system follows that of Assman and Ko¨ck32). H-15N
assignments: δH, δN (1:1 DMSO-d6/C6D6): 13.14, 169.1 (N1); 8.75,
107.8 (N7); 13.54, 134.7 (N12); 12.78, 137.7 (N14); 8.21, 62.2 (N16).
1
(8) Grube, A.; Kock, M. Org. Lett. 2006, 8, 4675–4678.
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Acknowledgment. We thank A. Jansma (UCSD) for assistance with
NMR, J. Pawlik (UNC Wilmington), and the captain and crew of the
RV Seward Johnson for logistical support during collecting expeditions
and in-field assays. This Investigation was supported by a Ruth L.
Kirschstein National Research Service Award NIH/NCI T32 CA009523.
We are grateful to the NIH (GM052964 D.R. and T.F.M.) and the
Welch Foundation (A-1280) for generous support.
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Note Added after ASAP Publication: This paper was published
on the Web on Jan 22, 2010, with an omission in the Acknowledgment
paragraph. The corrected version was reposted on Mar 1, 2010.
1
Supporting Information Available: Synthetic procedures and H
and 13C NMR spectra for 1d, 9-16, and other intermediates and
1
calculation of LOD for H-15N HSQC experiments. This material is
available free of charge via the Internet at http://pubs.acs.org.
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therein.
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precursors derived from petrochemical-based feedstock. In practice,
this will be inconsequential, since corrections can be made with the
appropriate normalization.
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NP900638E