Application of 3D NMR for Structure Determination of Peptide Natural Products

Article abstract of DOI:10.1021/acs.joc.5b01486

Despite the advances in NMR, structure determination is often slow and constitutes a bottleneck in natural products discovery. Removal of this bottleneck would greatly improve the throughput for antibiotic discovery as well as other therapeutic areas. Overall, faster structure methods for structure determination will serve the natural products community in a broad manner. This report describes the first application of 3D NMR for elucidation of two microbially produced peptide natural products with novel structures. The methods are cost-effective and greatly improve the confidence in a proposed structure.

Full text of DOI:10.1021/acs.joc.5b01486

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Article
Application of 3D NMR for Structure
Determination of Peptide Natural Products
Fan Zhang, Navid Adnani, Emmanuel Vazquez-Rivera, Doug
R. Braun, Marco Tonelli, David R. Andes, and Tim S Bugni
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.5b01486 • Publication Date (Web): 14 Aug 2015
Downloaded from http://pubs.acs.org on August 20, 2015
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Application of 3D NMR for Structure Determination of
Peptide Natural Products
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Fan Zhang,† Navid Adnani,† Emmanuel VazquezꢀRivera,‡ Doug R. Braun,† Marco Tonelli,§ David R.
Andes,∥ and Tim S. Bugni*,†
†Pharmaceutical Sciences Division, University of Wisconsin–Madison, Madison, Wisconsin 53705,
United States
‡Molecular & Environmental Toxicology Center, University of Wisconsin–Madison, Madison,
Wisconsin 53705, United States
§National Magnetic Resonance Facility at Madison, University of Wisconsin–Madison, Madison,
Wisconsin 53706, United States
∥Department of Medicine, University of Wisconsin–Madison, Madison, Wisconsin 53705, United States
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
* To whom correspondence should be addressed. Tel.: 1ꢀ608ꢀ263ꢀ2519. Eꢀmail: tim.bugni@wisc.edu.
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ABSTRACT: Despite the advances in NMR, structure determination is often slow and constitutes a
bottleneck in natural products discovery. Removal of this bottleneck would greatly improve the
throughput for antibiotic discovery as well as other therapeutic areas. Overall, faster structure methods
for structure determination will serve the natural products community in a broad manner. This report
describes the first application of 3D NMR for elucidation of two microbially produced peptide natural
products with novel structures. The methods are cost effective and greatly improve the confidence in a
proposed structure.
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INTRODUCTION
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Drug resistant infectious diseases, in particular Gramꢀnegative infections, continue to threaten global
health as well as contemporary medical practices.1 For example, as pathogens become increasingly
resistant, semiꢀroutine procedures such as organ transplants and joint replacements will become
increasingly dangerous. Combined with the emergence of panꢀresistant pathogens, new antibiotics are
sorely needed. Although natural products have provided the antibiotic basis upon which many medical
practices were built, discovering novel antibiotics has become more challenging. Nonetheless, advances
in sequencing and genomics has not only indicated that there is more potential than previously thought,
but also have provided tools for nearly routine analyses of the genetic potential of bacteria to produce
small molecules, many of which would have antibiotic potential.2,3 In parallel, analytical strategies that
take advantage of increasing sensitivity of mass spectrometers as well as the speed and resolving power
of UPLC systems have enabled rapid identification of novel antibiotics.3–12 Combining these new tools
with the bacterial diversity found in underexplored ecological niches such as insect symbionts,13 thermal
vents,14 caves,15 coal mines,16 and marine ecosystems17 has demonstrated great promise for discovering
the next generation of antibiotic therapies.
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To address the challenges of resistant infectious disease, the potential of these genetic and
analytical methods must be fully realized. Our lab has focused on LCMSꢀbased metabolomics as a
discovery platform to discover novel natural products from marine invertebrateꢀassociated bacteria.
This has led to a high discovery rate of novel natural products.8–11 As these methods pave the way for
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rapid discovery, structure determination becomes a limiting factor for providing valuable lead
structures. Therefore, we have developed methods such as isotopic labeling followed by 13Cꢀ13C
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gCOSY11,18,19 for rapid assembly of carbon backbones in polyketides and 13Cꢀ15N NMR11 to assist with
thiazolines and other heterocyclic systems. While our previous studies demonstrated a high level of 13C
enrichment could be achieved for molecules of acetate origin, questions remained surrounding
isotopically labeling peptides. For example, typical growth media used were rich in natural abundance
precursors such as amino acids from sources like yeast extract and peptone. On the other hand, if
uniform labeling could be achieved for both 13C and 15N, structure determination of peptideꢀbased
natural products could be achieved using biomolecular NMR strategies such as 3D NMR techniques.
These approaches would also provide a gateway to automated backbone assignment of structures
containing typical amino acids. Therefore, we evaluated isotopic labeling in a medium that contained
typical quantities of nonꢀenriched components. To demonstrate proof of concept for both isotopic
labeling and 3D NMR strategies, we tested the methods using two novel antibiotic peptides, one of
which was prioritized as part of an antibiotic drug discovery program. In particular, peptide 2 showed
broad spectrum activity including against Gramꢀnegative pathogens. Although peptide 2 did not show
high potency, the difference between the MIC (22.7 ꢁM) and MBC (45.5 ꢁM) was small indicating
potential for Gramꢀnegative infections. The overall goal of this study was to provide a basis for
reducing structure determination times from months to hours or days.
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RESULTS AND DISCUSSION
Uniform labeling peptides with 13C and 15N
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Our previous studies showed that fermentation of actinobacteria upon addition of uniformly 13Cꢀ
labeled glucose and subsequent purification yielded 13Cꢀlabeled natural products.19 However, peptides
could be more challenging since in most cases Uꢀ13C glucose was utilized for 13C enrichment in natural
products which have been biosynthesized via a polyketide pathway.11,20 For the 15N incorporation,
15NH4Cl was evaluated as the nitrogen source. As mentioned, a potential issue was 15N incorporation
with peptides in rich media. To evaluate the 13C and 15N incorporation, a marine invertebrateꢀderived
Streptomyces sp. (strain WMMB 705) that produced two novel peptides that we named eudistamides A
(1) and B (2) was grown in multiple media and extracts were analyzed by LCꢀMS data (Supporting
Information). Although the medium containing 15NH4Cl as the only nitrogen source (M3, Supporting
Information) yielded the highest 15N abundance of the peptides, taking into consideration the various
factors such as the price of the medium, the yield, and the 13C and 15N abundance, the labeled medium
containing peptone and yeast extract (20 g soluble starch, 10 g [Uꢀ13C] glucose, 2.5 g peptone, 2.5 g
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yeast extract, 5 g 15NH4Cl, 5 g CaCO3 per liter of artificial seawater) was used for producing 13Cꢀ and
15Nꢀlabeled peptides.
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Strategies and experiments for the resonance assignment of uniformly 13C, 15N-labeled peptides
Backbone Assignment Strategy
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A typical triple resonance 3D NMR spectrum observes correlations of three resonances, a
proton, a carbon, and a nitrogen. Many 3D NMR experiments have been designed for assigning the
backbone of 15N/13C isotopically labeled protein, such as HNCO,21 NH(CA)CO,22 HNCA,21
CBCANH,23 CBCA(CO)NH,24 HN(CO)CA,25 HCACO,21 HCA(CO)N,21 H(CCO)NH,26 C(CO)NH,26
and HBHA(CBCACO)NH.27 Ideally, standard triple resonance backbone assignment of a peptide has
relied on a combination of the CBCANH, CBCA(CO)NH, and NHCO spectra (Figure 1; a–c). The
general idea was that the CBCANH correlated each NH group with the Cα and Cβ chemical shifts of its
own residue (strongly) and of the preceding residue (weakly), whereas the CBCA(CO)NH only
correlates the NH group to the preceding Cα and Cβ chemical shifts. The NHCO experiment provides
the connectives between each NH group with the carbonyl carbon of the preceding residue, thus
establishing the backbone assignment. In our experience, the quality of the CBCANH and
CBCA(CO)NH spectra were sometimes not sufficient in terms of S/N. The Cα or Cβ resonances were,
for example, not visible above the noise level. In this case, the combination of HNCA and HN(CO)CA
experiments (Figure 1; e–f) which were more sensitive provided the same information as the CBCANH
and CBCA(CO)NH spectra, except without the Cβ resonances. The HN(CA)CO experiment (Figure 1;
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d) was powerful for linking each NH group with the carbonyl carbon of the same residue, particularly
helpful to the assignment of depsipeptides. A depsipeptide is a peptide in which one or more of its ꢀ
C(O)NHR groups are replaced by the corresponding ester, ꢀC(O)OR. Taking eudistamides A (1) and B
(2) for example, no correlation for Cꢀ1 could be seen in NHCO spectrum, whereas HN(CA)CO
experiment could correlate NHꢀ7 with the carbonyl carbon of the ester (Cꢀ1). Therefore, our backbone
assignment strategy used three 3D NMR experiments HNCO, CBCANH, and CBCA(CO)NH to
establish the connectivity of the backbone as the first step. The other 3D NMR experiments NHCA,
HN(CO)CA, NH(CA)CO were utilized to make further confirmation regarding to some complicated
cases, such as overlapping carbonyl carbon signals and any crosspeak that was very weak or missing in
either CBCANH or CBCA(CO)NH spectrum. We were thereby able to make unambiguous backbone
assignment of the eudistamides A and B and provided a general strategy for nonꢀPro containing peptide
natural products. However, due to the absence of an amide proton in proline, these 3D NMR
experiments cannot be used to sequentially connect proline residue to the preceding residue. The
CDCA(NCO)CAHA (Figure 1; h) was designed for the sequential assignment of proline residues28 and
the other option is to rely on the observation of NOEs to neighboring residues.
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Figure 1. Connectives observed in seven different types of 3D NMR experiment. (a) The CBCANH
experiment correlates the chemical shift of amide resonance with the Cα and Cβ of both interꢀ and intraꢀ
residue. (b) The CBCA(CO)NH experiment only correlates the amide resonance with Cα and Cβ of the
preceding residue. (c) The NHCO experiment provides the connectives between the amide of a residue
(NHi) with the carbonyl carbon of the preceding residue (COiꢀ1). (d) The NH(CA)CO experiment shows
the amide resonance (NHi) is correlated with the carbonyl carbon of the same residue (COi), as well as
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that of the preceding residue (COiꢀ1). (e) The NHCA experiment correlates the amide resonance (NHi)
with the Cα of the same residue and preceding residue. (f) The NH(CO)CA experiment only correlates
the resonances of the amide with the Cα of the preceding residue. (g) The HCCHꢀTOCSY correlates a
1H/13C pair to all other protons in the same aliphatic sidechain. (h) The thick arrow represents pathways
utilizing the 1J (Cα/δ, N) coupling, whereas the thin arrow represents the less efficient pathway via 2J
(Cα, N). Protons directly involved in the transfer pathways are shown in bold font.
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Sidechain Assignment Strategy
Depending on the structure, sidechains can be assigned using TOCSY and HSQC. However, the
most useful NMR experiment for sidechain assignment is an HCCHꢀTOCSY spectrum (Figure 1; g).29
The general principle behind using the HCCHꢀTOCSY spectrum is as follows: Using the known Cα and
Cβ chemical shifts from the backbone assignment, the side chain chemical shifts would be assigned by
viewing 2D slices at each carbon shift, typically at the Cα and Cβ chemical shifts. Each “slice” contains
resonances for the protons attached to carbons in that 13Cꢀ13C spin system. The HCCHꢀTOCSY
experiment combined with 13C chemical shift ranges of the common amino acids provides a very
powerful means for the assignment of the aliphatic sidechain. Some of the proteinogenic amino acids,
such as Alanine, Serine, Threonine and Glycine were easily identified since Alanine, Serine, and
Threonine’s Cβ chemical shifts were unique compared to those of the other amino acids and Glycine has
no Cβ. Valine, Isoleucine and Proline could also be easily identified because they have lower than
normal Cα chemical shifts. For the aromatic sidechain assignment, such as Tyrosine, Phenylalanine,
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Tryptophan, and Histidine, additional 2D NMR data are needed to complete the whole sidechain
assignment.
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Structure elucidation of Eudistamides A (1) and B (2)
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Eudistamide A (1) was obtained as a white powder with a molecular formula of C61H90N14O13,
determined by HRESIMS. Interpretation of HCCHꢀTOCSY data (Table S1; Supporting Information) of
the 13Cꢀ and 15Nꢀdoubly labeled 1 established the amino acid residues Leucine (Leu; 2×), Arginine
(Arg), Proline (Pro), Glycine (Gly), Glutamine (Gln), Alanine (Ala), Valine (Val), and Phenylalanine
(Phe), which were further confirmed by natural abundance experiments, 1Hꢀ1H COSY, HSQC and
HMBC data (Table S3; Supporting Information). The Pro residue was assigned as cis based on the 13C
NMR chemical shifts of the βꢀ and γꢀcarbon atoms (ꢂδβγ). ꢂδβγ for transꢀPro is regularly less than 5
ppm while ꢂδβγ for cisꢀPro is regularly between 5 and 10 ppm.30 ꢂδβγ for the proline residue in 1 was
5.8 ppm, supporting the assignment as cis. The 3ꢀ(2ꢀMethylphenyl)acrylic acid (Meꢀacyl) moiety was
established by HMBC, whereas geometry of the double bond (C66 and C67) was assigned as E on the
basis of large coupling constant (15.5 Hz) through the double bond.
The connectivity of the partial structures Thr−Phe−Leu2−Val−Ala−Gln−Gly and Pro−Arg−Leu1
was established by using 3D NMR data which were discussed for the backbone assignment (CBCANH,
CBCACONH, NHCO, HNCA, NH(CO)CA, NH(CA)CO; Table S1; Supporting Information) of the
13Cꢀ and 15Nꢀdoubly labeled 1. The HMBC correlation from Hꢀ62 to Cꢀ1 and the ROESY correlation
between H3ꢀ63 and Hꢀ2 connected Leu1 to Thr via an ester on the basis of the chemical shift of Cꢀ62 (δc
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68.5). Moreover, for the uncommon amino acid residue, the HMBC correlations from Hꢀ66 and Hꢀ67 to
Cꢀ65 supported the linkage of 3ꢀ(2ꢀMethylphenyl)acrylic acid (Meꢀacyl) moiety to Thr. The ROESY
correlations between Hꢀ24a and H2ꢀ22 secure the ring closure by connecting the Gly unit to Pro, which
satisfied the 24 degrees of unsaturation deduced from the molecular formula.
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The advanced Marfey’s method31 was applied to assign the absolute configurations of the amino
acid residues from acid hydrolysis of 1. The 1ꢀfluoroꢀ2,4ꢀdinitrophenylꢀ5ꢀleucineꢀamide (FDLA)
derivatives of the hydrolysate of 1 and authentic
analysis. The absolute configuration of all amino acids were established by comparison of their HPLC
retention time and molecular weights with those of corresponding authentic ꢀ and ꢀstandards. Upon
Dꢀ and Lꢀamino acids were subjected to LCꢀMS
D
L
analysis, the amino acid residues Arg, Pro, Ala, Val, alloꢀThr and Phe were determined to have the Lꢀ
configuration, whereas the Gln and Leu were deduced to have the
Supporting Information).
Dꢀconfiguration (Table S5;
Eudistamide B (2) was isolated a white amorphous powder with a molecular formula of
C72H99N11O18 as determined by HRESIMS. A detailed analysis of HCCHꢀTOCSY (Table S2;
Supporting Information) of the 13Cꢀ and 15Nꢀdoubly labeled 2 indicated an amino acid content as
follows: Thr (×2), Tyr (×2), Ser, Lys, 3ꢀOHꢀLeu (×2), Leu, and Phe. CBCANH, CBCA(CO)NH
NHCO, HNCA, NH(CO)CA, and NH(CA)CO spectra (Table S2; Supporting Information) revealed the
amino acids sequence as 3ꢀOH−Leu1−Thr1−Tyr1−Ser−3ꢀOHꢀLeu2−Lys−Tyr2−Leu−Phe−Thr2, which
was further confirmed by ESIMS/MS fragmentation analysis (Figure S22; Supporting Information).
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The linkage of 3ꢀ(2ꢀMethylphenyl)acrylic acid (Meꢀacyl) moiety to Thr2 was supported by the HMBC
correlation from Hꢀ76 to Cꢀ74. The ROESY correlation between H3ꢀ72 and Hꢀ2 and consideration of
unsaturation deduced from the molecular formula connected 3ꢀOHꢀLeu1 to Thr2 via an ester on the basis
of the chemical shift of Cꢀ71 (δc 68.5).
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The absolute configurations of the amino acids were determined using acid hydrolysis followed
by the advanced Marfey’s method. The chromatographic comparison between Marfey’s derivatives of
the hydrolysate of 2 and appropriate amino acid standards assigned the Lꢀconfigurations for Ser and Leu
and
Dꢀconfigurations for Lys, Phe, alloꢀThr, and Tyr (Table S4; Supporting Information). The (2R, 3S)
and (2R, 3R)ꢀ3ꢀhydroxyleucine were synthesized following Bonnard’s procedure32 and derivatized with
Lꢀ and L/DꢀFDLA respectively. Since enantiomers would exhibit identical retention behavior under nonꢀ
chiral HPLC conditions, the (2R, 3S)ꢀDꢀFDLA derivative showed the same retention time as (2S, 3R)ꢀLꢀ
FDLA, whereas the (2R, 3R)ꢀDꢀFDLA derivative showed the same retention time as (2S, 3S)ꢀLꢀFDLA.
By comparing the above the four retention times of the 3ꢀOHꢀLeu standards with the
LꢀFDLA
derivatives of the hydrolysate of 2, both the 3ꢀOHꢀLeu in 2 were assigned as 2S, 3R (Table S6;
Supporting Information). The 2S, 3R configuration of the two 3ꢀhydroxyleucines in 2 were also
confirmed by Jꢀbased analysis and NOESY data. The large coupling constants observed from DQFꢀ
COSY in MeOD between Hꢀ2 and Hꢀ3 (11.5 Hz), and between Hꢀ28 and Hꢀ29 (11.0 Hz) established
their antiꢀrelationship. The ROESY correlation between Hꢀ4 and NHꢀ7, as well as the ROESY
correlation between Hꢀ30 and NHꢀ33 established the rotamer (A4) in Figure 2.
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Figure 2. J-based configuration analysis of C-2 and C-3 of compound 2.
Compounds 1 and 2 were tested for antibacterial activity against Pseudomonas aeruginosa,
Methicillinꢀresistant Staphylococcus aureus (MRSA), Escherichia coli and Bacillus subtilis. Compound
2 showed antibacterial activity against MRSA, E. coli and B. subtilis, with the MIC values of 22.7, 22.7
and 2.8 µM, respectively, while compound 1 did not show any detectable antibacterial activities at 52.2
µM. Compound 2 was also tested using a murine thigh model infected with either E. coli ATCC 25922
or S. aureus ATCC 25923 (Figure 3). Over the 16ꢀfold intraperitoneal dose range, no toxicity
associated with compound administration was observed (Figure 3; a and c). Conversely, intravenous
dose levels above 8 mg/kg resulted in animal death (Figure 3; b and d). Modest, but broad spectrum
(both Gramꢀpositive and Gramꢀnegative bacteria) efficacy was observed with two highest tolerated dose
levels given by both administration routes. Compared to untreated control animals, compound 2
reduced the organism burden in the thighs of immunocompromised mice by more than 0.5 log10
CFU/Thigh.
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Figure 3. In vivo time kill experiment with compound 2 using a neutropenic mouse thigh model.
Each symbol represents the mean and standard deviation from four thighs of two mice infected with
either E. coli or S. aureus. The error bars represent the standard deviation. Five single doses (4, 8, 16,
32, and 64 mg/kg) of compound 2 were administered to mice. (a) E. coli infected murine thighs treated
with five single doses of 2 by intraperitoneal route. (b) E. coli infected murine thighs treated with five
single doses of 2 by intravenous route. (c) S. aureus infected murine thighs treated with five single
doses of 2 by intraperitoneal route. (d) S. aureus infected murine thighs treated with five single doses of
2 by intravenous route. b and d regimens do not include the 16, 32, and 64 mg/kg since the mice died
following administration.
Although the cyclopetides and cyclodepsipeptides have been encountered frequently as bacterial
secondary metabolites, eudistamides A (1) and B (2) differ markedly from the known peptides by virtue
of the presence of a unique 3ꢀ(2ꢀMethylphenyl)acrylic acid moiety, which has not been previous
reported in a peptide. Only some analogs of 3ꢀ(2ꢀMethylphenyl)acrylic acid residue were rarely
descried in peptides, such as WS9326s,33 pepticinnamins,34 and mohangamides35 incorporating a 2ꢀ
pentenyl cinnamic acid moiety, skyllamycins A and B36,37 incorporating a 2ꢀpropenyl]ꢀcinnamic acid
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moiety, and corprisamides A and B bearing 2ꢀheptatrienyl cinnamic acid moiety.38 Moreover, another
remarkable feature of compound 2 is the high content of the βꢀHydroxyꢀαꢀamino acids, including
genetically coded proteinogenic amino acids Ser and Thr, and nonstandard amino residue 3ꢀOHꢀLeu.
Members of the βꢀHydroxyꢀαꢀamino acid class occur as constituents of many important antibiotics, such
as vancomycin39 and lysobacin.40 Furthermore, compounds 1 and 2 could be produced in sufficient
quantities by scale up microbial fermentation, which would greatly facilitate followꢀup mechanistic and
preclinical studies.
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CONCLUSION
The introduction of 3D NMR has dramatically improved the speed and reliability of the protein
assignment process.22,42 However, we expanded the scope of 3D NMR experiments from protein
applications to a strategy that is universally applicable for labeled peptides. We have demonstrated a
rapid and efficient structure elucidation protocol for complex peptides containing typical amino acids.
The protocol we propose consists of a typical set of 3D NMR experiments for backbone and sidechain
assignment of uniformly labeled peptides with 13C and 15N. While 3D NMR experiments may not be
optimal for natural product peptides, this proof of concept provides a basis for using triple resonance
strategies for rapidly establishing the backbone of peptide natural products. Development of specific
NMR experiments would be advantageous. The triple resonance experiments used in this study were
highly complementary to more traditional methods such as HMBC and ROESY. This report describes
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the first application of 3D NMR to two microbially produced peptide natural products with novel
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structures. The methods are cost effective and greatly improve the confidence in a proposed structure.
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EXPERIMENTAL SECTION
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General Experimental Procedures
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Optical rotations were measured on a Polarimeter. UV spectra were recorded on a UVꢀVis
Spectrophotometer. IR spectra were measured with a FT–IR Spectrophotometer. 1D and 2D NMR
spectra were obtained in DMSO with a NMR spectrometer equipped with a 13C/15N{1H} cryoprobe. 3D
NMR spectra were obtained in DMSO with a NMR spectrometer with a (1H{13C,15N}) cold probe.
HRMS data were acquired with a QTOF mass spectrometer (Ionization method: Spray Voltage (+)
4000; Spray Voltage (–) 3700; Capillary temperature 320; full MS scan 200–2000). MSMS data were
acquired with a hybrid quadrupoleꢀOrbitrap mass spectrometer. RP HPLC was performed using a
HPLC system and a Phenomenex Luna C18 column (250 × 10 mm, 5 µm), as well as a preparative
HPLC and Phenomenex Gemini C18 column (250 × 30 mm, 5 µm). The Advanced Marfey’s method
utilized a HPLC coupled with a mass spectrometer.
Biological Material. Ascidian specimens were collected in September 2011 from the Florida Keys
(24°33.416’, 81°21.611’). Identification was confirmed by Shirley ParkerꢀNance. A voucher specimen
for Eudistoma olivaceum (Van Name, 1902)42 is housed at the University of WisconsinꢀMadison. For
cultivation, a sample of ascidian (1 cm3) was rinsed with sterile seawater, macerated using a sterile
pestle in a microꢀcentrifuge tube, and dilutions were made in sterile seawater, with vortexing between
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steps to separate bacteria from heavier tissues. Dilutions were separately plated on three media: ISP2
supplemented with artificial seawater,43 R2A,44 and M4.45 Each medium was supplemented with 50
µg/mL cycloheximide and 25 µg/mL nalidixic acid. Plates were incubated at 28 °C for at least 28 days,
and strain WMMB 705 was purified from an ISP2 isolation plate.
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Sequencing. Genomic DNA was extracted using the UltraClean Microbial DNA Isolation kit (Mo Bio
Laboratories, Inc.). 16S rDNA genes were amplified using 100–200 ng genomic DNA template with
the primers 8–27F (5' to 3' GAGTTTGATCCTGGCTCAG) and 1492R (5' to 3'
GGTTACCTTGTTACGACTT). The following PCR conditions were used: 94 °C for 5 min, followed
by 30 cycles of 94 °C for 30 s, 55 °C for 1 min, 72 °C for 1.5 min, with a final step of 72 °C for 5 min.
The PCR bands were excised from the gel and purified using the QIAquick Gel Extraction kit
(QIAGEN). One ꢁL purified product was sequenced. Sequencing reactions were performed by the UW
Biotechnology Center and reactions were sequenced with an ABI 3730xl DNA Analyzer. WMMB 705
were identified as Streptomyces sp. by 16S sequencing, and demonstrated 99% sequence similarity to
Streptomyces sp. FXJ6.141 (accession number GU 339421).
Fermentation, Extraction, and Isolation. Two 10 mL seed cultures (25 × 150 mm tubes) in medium
ASWꢀA (20 g soluble starch, 10 g glucose, 5 g peptone, 5 g yeast extract, 5 g CaCO3 per liter of
artificial seawater) were inoculated with strain WMMB 705 and shaken (200 RPM, 28 °C) for four
days. Twoꢀliter flasks (2 × 500 mL) containing ASWꢀA medium with Diaion HP20 (7% by weight)
were inoculated with 8 mL from the culture tube and shaken at 200 rpm at 28 °C for seven days.
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Filtered HP20 was washed with water and extracted with acetone. The acetone extract was
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chromatographed on Diaion HP20ss, eluting sequentially with methanol:water (10:90, and 95:5). The
95% methanol eluate was evaporated to dryness to afford a yellow oil (460 mg), and then it was
subjected to RP HPLC (30%/70% to 60%/40% ACN/H2O with H2O containing 0.1% acetic acid over 30
min, 25 ml/min, followed by 90/10% to 100% of the same solvents for 3 min, 25 ml/min, and a hold at
100%/0% of the same solvents) using a Phenomenex Gemini C18 column (250 × 30 mm, 5 µm),
yielding a fraction containing 1 (50 mg, tR 11.5 min), and compound 2 (21 mg, tR 12.5 min). The
fraction containing 1 was further subjected to RP HPLC (55ꢀ65% MeOHꢀH2O with H2O containing
0.1% acetic acid over 25 min, 4.0 mg/ml) using a Phenomenex Luna C18 column (250 × 10 mm, 5 µm),
yielding 1 (22 mg, tR 19 min). For 13C and 15N incorporation, the same procedure was used (1 × 250
mL) with labeled medium ASWꢀA (20 g soluble starch, 10 g U13Cꢀglucose, 2.5 g peptone, 2.5 g yeast
extract, 5 g 15NH4Cl, 5 g CaCO3 per liter of artificial seawater).
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Antibacterial Assay. Eudistamides A (1) and B (2) were tested for antibacterial activity against MRSA
(ATCC #33591), E. coli (ATCC #25922), B. subtilis and P. aeruginosa (ATCC #27853), and MICs
were determined using a dilution antimicrobial susceptibility test for aerobic bacteria.46 Eudistamides A
(1) and B (2) were dissolved in DMSO, serially diluted to 10 concentrations (0.125–64 ꢁg/mL), and
tested in a 96ꢀwell plate. Vancomycin was used as a control and exhibited an MIC of 0.69 ꢁmol against
MRSA and 0.69 ꢁmol against B. Subtilis. Gentamicin was used as a control and exhibited an MIC of
8.4 ꢁmol against E. coli and 8.4 ꢁmol against P. aeruginosa. Peptides 1 and 2 were tested in triplicate,
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and vancomycin and gentamicin were also tested in triplicate. Six untreated media controls were
included on each plate. The plates were incubated at 35 °C for 18 h. The MIC was determined as the
lowest concentration that inhibited visible growth of bacteria.
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Eudistamide A (1): white solid, [α]25D; −51 (c 0.1, MeOH); UV (MeOH) λ (log ε) 209 (3.55), 224
(3.17), 280 (3.23); IR (ATR) υmax 3300, 2953, 1648, 1535, 1519, 1450, 1320, 1242, 1072, 760, 677 cmꢀ
1; 1H and 13C NMR (see Table S3; Supporting Information); HRMS [M+H]+ m/z 1227.6882 (calcd for
C61H91N14O13, 1227.6890).
Eudistamide B (2): white solid, [α]25D; +37 (c 0.1, MeOH); UV (MeOH) λ (log ε) 207 (3.32), 223
(3.17), 279 (2.82); IR (ATR) υmax 3290, 2932, 1738, 1647, 1532, 1517, 1455, 1314, 1244, 1072, 761,
669 cmꢀ1; 1H and 13C NMR (see Table S4; Supporting Information); HRMS [M+H]+ m/z 1406.7253
(calcd for C72H100N11O18, 1406.7248).
Acid Hydrolysis of Eudistamides A (1) and B (2). Separate solutions of compounds 1 and 2 (0.5 mg
each) in 6 N HCl (1 mL) were hydrolyzed at 110 °C for 4 h and dried under vacuum.
Determination of Proteinogenic Amino Acid Configurations.
previously reported.47 The hydrolysate was mixed with 1 N NaHCO3 (40 ꢁL), and 35 ꢁL of
(10 mg/mL in acetone). Each solution was stirred at 45 °C for 1 h, cooled to room temperature,
quenched with 1 N HCl (40 ꢁL) and dried under vacuum. Similarily, the stander ꢀand ꢀamino acid
Lꢀ and L/DꢀFDLA were synthesized as
LꢀFDLA
L
D
were derivatized separately. The derivatives of the hydrolysate of compound 1 and the standard amino
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acids were subjected to LCꢀMS analysis with a Phenomenex Kinetex C18 reversedꢀphase column (2.6
ꢁm, 100 x 4.6 mm) at a flow rate of 0.5 mL/min and with a linear gradient of H2O (containing 0.1%
formic acid) and MeOH (90:10 to 0:100 over 15 min, and a hold at 100% MeOH for 5 min). The
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absolute configurations of the amino acids were determined by comparing the retention times of the
L
ꢀ
and
acids in 1 were assigned based on a comparison of retention time of amino acid standards derivatized
with ꢀFDLA. In a similar manner, the derivatives of the hydrolysate of compound 2 and the standard
amino acids were subjected to LCꢀMS analysis with a Phenomenex Luna C18 reversedꢀphase column
Dꢀamino acids derivatives, which were identified by MS. The absolute configurations of the amino
L
(5.0 ꢁm, 250 x 4.6 mm) at a flow rate of 1.0 mL/min and with a linear gradient of H2O (containing 0.1%
formic acid) and ACN (90:10 to 0:100 over 30 min, and a hold at 100% ACN for 5 min). The retention
times and ESIMS data for LꢀFDLA derivatives of the hydrolysates and the standard amino acids are
summarized in Table S5 and S6 (Supporting information).
Synthesis of (2R,3S)-3-Hydroxyleucine and (2R,3R)-3-Hydroxyleucine and advanced Marfey’s
analysis of 3-hydroxyleucines. (2R,3S)ꢀ3ꢀHydroxyleucine and (2R,3R)ꢀ3ꢀHydroxyleucine were
synthesized using the procedures by Bonnard et al.32 We obtained 20.0 mg of major precursor (S)ꢀ1ꢀ
((2R,5S)ꢀ5ꢀisopropylꢀ3,6ꢀdimethoxyꢀ2,5ꢀdihydropyrazinꢀ2ꢀyl)ꢀ2ꢀmethylpropanꢀ1ꢀol and 2.5 mg of
minor precursor (R)ꢀ1ꢀ((2R,5S)ꢀ5ꢀisopropylꢀ3,6ꢀdimethoxyꢀ2,5ꢀdihydropyrazinꢀ2ꢀyl)ꢀ2ꢀmethylpropanꢀ1ꢀ
ol. The hydroxyleucine precursors were hydrolyzed following the general peptide acid hydrolysis
process. The major precursor gave a mixture of (2R,3S)ꢀ3ꢀhydroxyleucine and Dꢀvaline, and the minor
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precursor gave a mixture of (2R,3R)ꢀ3ꢀhydroxyleucine and
D
ꢀvaline. These two hydrolysate mixtures
were derivatized with
LꢀFDLA and
L/
D
ꢀFDLA, respectively, resulting in four products: (2R, 3S)ꢀ3ꢀ
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hydroxyleucineꢀLꢀFDLA, (2R, 3S)ꢀ3ꢀhydroxyleucineꢀDꢀFDLA, (2R, 3R)ꢀ3ꢀhydroxyleucineꢀLꢀFDLA and
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(2R, 3R)ꢀ3ꢀhydroxyleucineꢀDꢀFDLA. Among these four products, (2R, 3S)ꢀ3ꢀhydroxyleucineꢀDꢀFDLA
will show the same retention time as (2S, 3R)ꢀ3ꢀhydroxyleucineꢀLꢀFDLA, and (2R, 3R)ꢀ3ꢀ
hydroxyleucineꢀDꢀFDLA will show the same retention time as (2S, 3S)ꢀ3ꢀhydroxyleucineꢀLꢀFDLA,
under the nonꢀchiral HPLC condition we used in advanced Marfey’s analysis. The resulting four
derivatization mixtures were subjected to LCꢀMS analysis with a Phenomenex Luna C18 reversedꢀphase
column (5.0 ꢁm, 250 x 4.6 mm) at a flow rate of 1.0 mL/min and with a linear gradient of H2O
(containing 0.1% formic acid) and ACN (25:75 to 65:35 over 50 min). By comparing these four
retention times with the retention time of hydrolysateꢀLꢀFDLA derivative of compound 2, the two 3ꢀ
hydroxyleucines in the peptide was assigned as 2S, 3R. Detailed retention times of four isomers and
hydrolysate could be found in Table S6 (Supporting Information). 1H NMR data of (S)ꢀ1ꢀ((2R,5S)ꢀ5ꢀ
isopropylꢀ3,6ꢀdimethoxyꢀ2,5ꢀdihydropyrazinꢀ2ꢀyl)ꢀ2ꢀmethylpropanꢀ1ꢀol (CDCl3, 500 MHz): δ 4.09 (1H,
m), 3.99 (1H, m), 3.73 (3H, s), 3.69 (3H, s), 3.63 (1H, br), 2.25 (1H, m), 2.01, (1H, m), 1.64 (1H, br),
1
1.04 (3H, d, J = 7.0 Hz), 1.03 (3H, d, J = 7.0 Hz), 0.99 (3H, d, J = 7.0 Hz), 0.71 (3H, d, J = 7.0 Hz). H
NMR data of (R)ꢀ1ꢀ((2R,5S)ꢀ5ꢀisopropylꢀ3,6ꢀdimethoxyꢀ2,5ꢀdihydropyrazinꢀ2ꢀyl)ꢀ2ꢀmethylpropanꢀ1ꢀol
(CDCl3, 500 MHz): δ 4.10 (1H, t, J = 3.5 Hz), 3.92 (1H, t, J = 3.5 Hz), 3.65 (3H, s), 3.64 (3H, s), 3.59
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(1H, br), 2.20 (1H, m), 1.76, (1H, m), 1.51 (1H, br), 0.98 (3H, d, J = 7.0 Hz), 0.85 (3H, d, J = 7.0 Hz),
0.84 (3H, d, J = 7.0 Hz), 0.63 (3H, d, J = 6.9 Hz).
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Organisms, media, and antibiotic of in vivo studies. Eudistamide B (2) was tested for antibacterial
activity in vivo studies. One isolate of E. coli ATCC 25922 and S. aureus ATCC 25923 were used for
these studies. Organisms were grown, subcultured, and quantified using MuellerꢀHinton broth (MHB)
and agar (Difco Laboratories, Detroit, MI).
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Animals. Six weekꢀold, specific pathogenꢀfree, female ICR/Swiss mice weighing 24 to 27 g were used
for all studies (Harlan SpragueꢀDawley, Indianapolis, IN). Animals were maintained in accordance with
the American Association for Accreditation of Laboratory Animal Care (AAALAC) criteria. All animal
studies were approved by the Animal Research Committees of the William S. Middleton Memorial VA
Hospital and the University of Wisconsin.
Murine thigh infection model. Mice were rendered neutropenic (neutrophils < 100/mm3) by injecting
cyclophosphamide (Mead Johnson Pharmaceuticals, Evansville, IN) intraperitoneally 4 days (150
mg/kg) and 1 day (100 mg/kg) before thigh infection. Previous studies have shown that this regimen
produces neutropenia in this model for 5 days. Broth cultures of freshly plated bacteria were grown to
logarithmic phase overnight to an absorbance of 0.3 at 580 nm (Spectronic 88; Bausch and Lomb,
Rochester, N.Y.). After a 1:10 dilution into fresh MHB, bacterial counts of the inoculum ranged from
106.3ꢀ6.9 CFU/ml. Thigh infections with each of the isolates were produced by injection of 0.1 ml of
inoculum into the thighs of isofloraneꢀanesthetized mice 2 h before therapy.
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In vivo time kill and PAE. Two hours after thigh infection with either with single doses of compound
2 (4, 8, 16, 32, and 64 mg/kg) by the intraperitoneal or intravenous route. Groups of two treated and
untreated mice were sacrificed at each treatment endpoint (0 hour at the start of therapy and 6 hours
after treatment). The thighs (four per treatment group) were immediately removed upon euthanasia and
processed for CFU determination. The burden of organisms in the thigh was measured by viable plate
counts of tissue homogenates. The impact of each dose on the burden of organisms over time the study
period.
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ACKNOWLEDGMENT
This work was supported by funding from the University of WisconsinꢀMadison School of Pharmacy.
This work was also funded in part by the U19 AI109673, NIH, and NIGMS Grant R01 GM107557. We
thank the Analytical Instrumentation Center (AIC) at the University of WisconsinꢀMadison School of
Pharmacy for the facilities to acquire spectroscopic data, especially MS data. This study made use of
the National Magnetic Resonance Facility at Madison (NMRFAM), which is supported by NIH grants
P41RR02301 (BRTP/NCRR) and P41GM66326 (NIGMS).
Supporting Information Available The NMR data, MS data, and Marfey’s analysis for compounds 1
and 2. This material is available free of charge via the Internet at http://pubs.acs.org
REFERENCES
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