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Nov 7, 2017 - isotopic profile of [M + H]+ in the mass spectrum of 1 revealed the presence of three ... Hz) between H-5 and H-6 and meta-coupling (2.0 Hz) between H-6 and H-8 defined ..... heterocyclic core of the caulamidines is biosynthesized, so ... 3 E. Patridge, P. Gareiss, M. S. Kinch, D. Hoyer, Drug Discov. Today ...

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This article can be cited before page numbers have been issued, to do this please use: D. Milanowski, N. Oku, L. Cartner, H. Bokesch, R. T. Williamson, J. Saurí, Y. Liu, K. Blinov, Y. Ding, X. Li, D. Ferreira, L. A. Walker, S. Khan, M. Davies-Coleman, J. A. Kelley, J. McMahon, G. E. Martin and K. Gustafson, Chem. Sci., 2017, DOI: 10.1039/C7SC01996C.

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Volume 7 Number 1 January 2016 Pages 1–812

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Unequivocal determination of caulamidines A and B: application and validation of new tools in the structure elucidation tool box† Received 00th January 20xx, Accepted 00th January 20xx DOI: 10.1039/x0xx00000x www.rsc.org/

Dennis J. Milanowski,a‡ Naoya Oku,a§ Laura K. Cartner,ab Heidi R. Bokesch,ab R. Thomas Williamson,c Josep Saurí,c Yizhou Liu,c Kirill A. Blinov,d Yuanqing Ding,e Xing-Cong Li,e Daneel Ferreira,e Larry A. Walker,e Shabana Khan,e Michael T. Davies-Coleman,f∇ James A. Kelley,g James B. McMahon,a Gary E. Martin,*c and Kirk R. Gustafson*a Ambiguities and errors in the structural assignment of organic molecules hinder both drug discovery and total synthesis efforts. Newly described NMR experimental approaches can provide valuable structural details and a complementary means of structure verification. The caulamidines are trihalogenated alkaloids from a marine bryozoan with an unprecedented structural scaffold. Their unique carbon and nitrogen framework was deduced by conventional NMR methods supplemented by new experiments that define 2-bond heteronuclear connectivities, reveal very long-range connectivity data, or visualize the 35,37Cl isotopic effect on chlorinated carbons. Computer-assisted structural elucidation (CASE) analysis of the spectroscopic data for caulamidine A provided only one viable structural alternative. Anisotropic NMR parameters, specifically residual dipolar coupling and residual chemical shift anisotropy data, were measured for caulamidine A and compared to DFT-calculated values for the proposed structure, the CASE-derived alternative structure, and two energetically feasible stereoisomers. Anisotropy-based NMR experiments provide a global, orthogonal means to verify complex structures free from investigator bias. The anisotropic NMR data were fully consistent with the assigned structure and configuration of caulamidine A. Caulamidine B has the same heterocyclic scaffold as A but a different composition and pattern of halogen substitution. Caulamidines A and B inhibited both wild-type and drug-resistant strains of the malaria parasite Plasmodium falciparum at low micromolar concentrations, yet were nontoxic to human cells.

Introduction Intricate structures of secondary metabolites evolved to interact with important cellular biopolymers, consequently they represent prevalidated templates for exploring biologically relevant chemical space. Many approved drugs are based on natural product structures and these compounds continue to provide effective 1-4 scaffolds for drug development. A recent resurgence in natural products discovery and development has been driven by factors 5-7 that include better bioactivity screening outcomes, advances in DNA sequencing and bioinformatics that facilitate biosynthetic 8,9 engineering and prediction of the resulting chemical structures, and innovative applications of mass spectrometry and molecular networking that afford new avenues for natural product discovery.10-14 Regardless of the approach utilized, successful natural product studies require the ability to define the precise

molecular structures of the isolated compounds. This is especially critical for potential drug development applications or when deduced natural product structures are the target of organic synthesis efforts. Since NMR is the most powerful and informationrich spectroscopic technique for assigning the structure of noncrystalline compounds, some recently described NMR experimental methods that facilitate the elucidation of ever more complex organic structures provide new opportunities to advance natural product discovery, development, and synthesis. A suite of standard 2D NMR experiments have been routinely used to characterize organic structures of natural and synthetic origin for more than two decades. These include HSQC and HMBC heteronuclear correlation pulse sequences to establish interatomic connectivities, COSY and TOCSY to define proton spin systems, and NOESY and ROESY to probe spatial proximity relationships. However, these well-established NMR experimental approaches can sometimes be insufficient to definitively establish the intricate

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skeletal frameworks and functional group assemblages encountered in many natural products. Incomplete spectroscopic characterization or interpretational mistakes can lead to ambiguous or incorrect structural assignments, as highlighted in some recent reviews.15,16 Several recently reported NMR techniques that can extend the range of heteronuclear correlations, or establish the number of bonds defined by these correlations, provide additional 17-24 means to elaborate complex chemical structures. These new experimental capabilities can be especially useful for characterizing highly proton-deficient compounds. Other NMR methodologies that are applicable to chlorinated metabolites provide direct visualization of 1-bond chlorine isotope effects on carbons to define sites of Cl-substitution.25-27 In addition, anisotropy-based NMR experiments provide alternative means to evaluate the global correctness of molecular frameworks and configurations that are 17,28 These complementary to traditional NMR data interpretation. data provide a robust means to verify structural assignments in an objective manner that is free of potential investigator bias. In the current study, all of these contemporary structure elucidation strategies were applied in a concerted fashion to unambiguously establish the structures of two novel heterocyclic marine alkaloids, caulamidines A (1) and B (2) (Fig.1).

Fig. 1 Structures of caulamidines A (1) and B (2).

Results and discussion Previous studies of an extract from the bryozoan Caulibugula intermis that were conducted in the early 2000’s provided the caulibugulones, a series of isoquinoline quinones and iminoquinones, as the principal cytotoxic agents.29 During final C18 HPLC purification of the caulibugulones two structurally distinct minor constituents, named caulamidines A (1) and B (2), were also obtained. Spectroscopic characterization of these compounds using the instrumentation and experimental techniques available at that time permitted the proposal of a tentative structure for caulamidine A (1), but convincing proof of the structure was lacking. Caulamidine B (2) was only isolated as a trace constituent, and while it was clearly related to 1, we were unable to assign its structure. Decomposition of 1 while converting between free base and salt forms of the alkaloid stalled our structural studies until a recent recollection of the bryozoan provided an additional supply of compounds 1 and 2. A characteristic 27:27:9:1 A+2 pattern for the + isotopic profile of [M + H] in the mass spectrum of 1 revealed the presence of three chlorine atoms, and HRFABMS established a molecular formula of C23H21Cl3N4, which required 14 indices of

hydrogen deficiency. Extensive NMR analyses (Table 1) allowed assignment of a substituted hexahydro-2,6-naphthyridine ring system in 1 (rings A and D) with two N-methyl groups (δH/C 1 13 3.00/37.2 and 3.24/35.8). H- C HMBC correlations from the N-1 methyl group to a carbon signal at δ 174.0 indicated an adjacent C-2 exocyclic imine functionality, while a correlation to a signal at δ 47.4 (C-25) established the presence of a neighboring methylene group (Fig. 2 A and B). The C-25 methylene protons (δΗ 3.18, 3.38) showed COSY correlations to a second methylene group (δΗ 1.73, 2.25) and these C-24 protons exhibited HMBC cross-peaks to the two quaternary ring-junction carbons, C-10 (δC 58.9) and C-23 (δC 39.8). In a similar manner, the N-13 methyl group showed HMBC correlations to the flanking exocyclic imine (δC 159.1) and 13 methylene (δC 52.6) carbon signals. The disparity in the C NMR shifts of the two exocyclic imine resonances suggested these functionalities were associated with different molecular scaffolds. The C-12 methylene protons (δH 3.66, 3.87) were coupled to the C11 methine proton, which showed HMBC correlations to C-2, C-10, and C-23. The deshielded chemical shift of H-11 (δH 5.02) and C-11 (δC 54.8) suggested a chlorine substituent at C-11. The N-1, C-2, N-3, C-10 constellation constitutes an amidine functionality in 1, as does N-13, C-14, N-15, and C-23. Carbon and proton resonances for two 1,2,4-trisubstituted benzene moieties in 1 were readily assigned. Ortho-coupling (8.4 Hz) between H-5 and H-6 and meta-coupling (2.0 Hz) between H-6 and H-8 defined the proton distribution in the C-ring, while nitrogen-substitution at C-4 was based on its deshielded chemical shift (δC 156.0). The link between C-9 and the C-10 bridgehead was based on an HMBC correlation between H-8 and C-10, which established the presence of a fused pyrrole moiety (B). Tentative assignment of a chlorine substituent at C-7 was consistent with its chemical shift of δC 126.3. The second benzene moiety (F) had a similar distribution of protons as defined by their coupling patterns, while the presence of a nitrogen at C-16 (δC 143.9), and a chlorine substituent at C-19 (δC 125.8) was proposed based on their chemical shift values. Attachment of a methylene group attached at C-21 was evident from HMBC correlations from H-20 (δH 6.96) to C22 (δC 29.6), and conversely from the H-22 protons (δH 2.28 and 2.48) to C-16 (δC 143.9) and C-21 (δC 125.4). The isolated H-22 methylene protons had numerous further HMBC correlations into rings A and D that established its connection to the C-23 bridgehead. Thus, both N-15 and C-22 were incorporated into a fused 6-membered ring (E) situated between rings D and F. Establishment of the 5- and 6-membered rings containing the C-2 and C-14 imino carbons, respectively, was consistent with the 13 disparity observed for the DFT-calculated C shifts for the dihydroindole-derived (C-2, calculated δC 173.8) and tetrahydroquinoline-derived (C-14, calculated δC 156.8) systems that are fused to the 2,6-naphthyridine core of 1. The deshielded chemical shifts of B-ring carbons compared with corresponding Ering signals were attributed to the ring strain associated with the configuration of the fused 5-membered B-ring. An extensive set of 1 15 H- N HMBC correlations (Fig. 2 C) that were readily observed with our current NMR spectrometer (600 MHz, 3 mm cryogenic probe) but lacking in our original set of NMR data (500 MHz, 5 mm room temperature probe), provided strong evidence for placement of the nitrogen atoms within the structural framework of 1. We then applied the recently developed LR-HSQMBC18-20 and 21 HSQMBC-TOCSY pulse sequences, which can extend the range of heteronuclear correlations to observe 4J and even some 5J and 6J correlations. These experiments complement the traditional HMBC

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experiment, which typically detects 2J and 3J, and only rarely 4J 1 13 correlations. The additional long-range H- C correlations detected Table 1. NMR spectroscopic data for caulamidine A (1) in CD3CN. in these experiments, including some 2J/3J couplings not seen in the HMBC data set, fully supported the proposed heterocyclic structure of 1 (Fig. 2 D). a b position δC δN δH (mult, J in Hz) HMBC 1-N 78.9 2 174.0 3-N 241.7 4 156.0 5 117.8 7.17 (d, 8.5) 3, 4, 6, 7, 9 6 129.4 7.31 (dd, 8.4, 2.0) 4, 5, 7, 8 7 126.3 8 123.8 6.95 (bs) 4, 6, 7, 10 9 133.3 10 58.9 11 54.8 5.02 (dd, 10.8, 4.7) 2, 9, 10, 12, 23 12a b 13-N 14 15-N 16 17 18 19 20 21 22a b 23 24a b 25a b 26 27

52.6

-

159.1 143.9 124.2 127.2 125.8 127.3 125.4 29.6

87.5 216.6 -

39.8 24.7

-

47.4

-

37.2 35.8

-

3.87 (dd, 13.3, 6.6) 3.66 (dd, 13.3, 10.5) 6.94 (d, 8.2) 7.12 (dd, 8.2, 2.4) 6.96 (s) 2.48 (d, 15.9) 2.28 (d, 15.9) 2.25 (m) 1.73 (dd, 15.0, 6.2) 3.38 (ddd, 12.5, 7.5, 1.6) 3.18 (dt, 11.7, 5.9) 3.00, 3H (s) 3.24, 3H (s)

11, 13, 14, 15, 27 11, 13, 14, 15 15, 16, 19, 21 16, 19 16, 18, 19, 21, 22 10, 14, 16, 21, 23, 24 10, 13, 14, 16, 21, 23, 24 10, 14, 22, 23, 25 1, 10, 22, 23, 25 2, 3, 24 24, 26 1, 2, 3, 25 12, 13, 14, 15

a15 N assignments were based on 1H−15N HMBC correlations. The δN values were not calibrated to an external standard but were referenced to neat NH3 (δ 0.00) using the standard Bruker parameters. b1H-13C (optimized for 8.3 Hz) and 1H-15N (optimized for 8 Hz) HMBC correlations are listed.

Another very useful experiment was 1,1-HD-ADEQUATE, which provides proton-detected visualization of one-bond 13C-13C 22-24,30 homonuclear couplings. The correlations observed in a standard HMBC experiment are due to both 2-bond and 3-bond heteronuclear couplings, and the inability to distinguish between these alternatives can lead to ambiguous or biased interpretation of the data. The 1,1-HD-ADEQUATE experiment complements HMBC data by affording proton-detected 1JCC correlations, which are 2 22,31 functionally equivalent to JCH HMBC correlations. These data can thus define direct carbon-carbon connectivities, which was particularly useful for establishing the location of quaternary carbons directly adjacent to protonated ones in compound 1 (Fig. 2 E). The location of chlorine substituents in caulamidine A (1) was initially assigned solely from carbon/proton NMR chemical shift considerations. Application of a new band-selective CLIP-HSQMBC

35,37

experiment, that can visualize the Cl isotope effect on both protonated and non-protonated 13C nuclei, provided unequivocal 25 37 support for these assignments. Carbons substituted with Cl have a slightly different chemical shift compared to those substituted 35 with Cl (δυ ~ 3-5 ppb). This chemical shift differential manifests in 13 2D correlation cross peaks that are split and 1D C slices that have a distinct shoulder (Fig. 3). The bs-CLIP-HSQMBC data for 1 clearly 35,37 revealed the Cl isotope effect for C-7, C-11, and C-19, which definitively established chlorine-substitution at these positions. Once the 2D structure of 1 was firmly established, the relative configurations of the C-10, C-11, and C-23 stereogenic centers were defined by diagnostic NOE interactions (Fig. 2 F). Key NOE enhancements included those between H-8/H-22a, H-11/H-24a, and H-22b/H-24b. These NOEs were confirmed from NMR experiments with the TFA salt of 1, which provided greater dispersion of the proton signals (Supplemental Information). The

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Journal Name Fig. 3. The 35,37Cl isotope effect observed in the bs-CLIP-HSQMBC experiment 1D 13C slices for C-7 and C-19 of caulamidine A (1). The isotope shifts of 5.1 and 4.6 ppb for C-7 and C-19 correspond to shifts of 0.77 and 0.69 Hz, respectively. While isotope effects of this magnitude have been observed in the past in 1D 13C NMR spectra, it is far easier to obtain the resolution to observe these effects using the bs-CLIP-HSQMBC experiment.

Fig. 4. Experimental (exptl, blue) and computed ECD spectra of (10S,11S,23S)caulamidine A at the B3LYP/6-31G** (gas, green) and B3LYP/6-311++G** (lbs, black) levels in the gas phase and at the B3LYP-SCRF(COSMO)/6-311++G**//B3LYP/6311++G** (sol, red) level in MeOH.

Fig. 2. (A) and (B) Selected 1H-13C HMBC correlations for caulamidine A (1). (C) 1H15 N HMBC correlations. (D) Additional correlations in LR-HSQMBC with respect to 1 H-13C HMBC (orange arrows) and additional correlations in HSQMBC-TOCSY with respect to LR-HSQMBC and HMBC (green arrows) (E) Key 1,1-HD-ADEQUATE correlations revealed quaternary carbons adjacent to protonated centers. (F) NOESY and ROESY correlations used to assign the relative configuration.

data for 1 using the ACD Laboratories Structure Elucidator CASE program19,32-34 to identify and rank potential alternative structures. This revealed only one other plausible structure, compound 3 (Fig. 5), based on the NMR connectivity data and predicted vs. calculated 13 C chemical shift values. A suite of computational studies was then

performed to compare the experimental and calculated values of chemical shifts, coupling constants, and free energy levels of the proposed and alternate structures 1 and 3, respectively. By all these criteria, the assigned structure of caulamidine A (1) was confirmed. The critical value of the LR-HSQMBC and 1,1-HD-ADEQUATE experiments was underscored by results from the CASE analyses. When only data from conventional NMR methods including HSQC and HMBC were used, the CASE program ran for 250 hours without ever generating a single structure. When LR-HSQMBC and 1,1-HDADEQUATE data were added to the input file, the program ran for less than one second and structure 1 was the top candidate. Ultimate verification of the structural proposal for caulamidine A was accomplished using both residual chemical shift anisotropy (RCSA) measurements35-37 and residual dipolar couplings (RDC).38-40 These NMR phenomena, which result from partial alignment of molecules in an anisotropic medium, carry rich structural information. The measured RDCs result from changes in heteronuclear (1H-13C) couplings and RCSAs arise from changes in 13 the C chemical shielding tensor. Anisotropic NMR data can be employed to define the relative orientations of bonds and shielding or dipolar coupling tensors, regardless of the distance between them. They provide a powerful, independent means to assess the global correctness of a proposed structure and configuration, whether the structure is proposed by an investigator or derived 17,28 from CASE program output. Applications of RDCs to biomolecules were first reported more than 20 years ago,41,42 while the first reported utilization of RDCs in small molecule structural analysis were those of Shapiro and co-workers.43,44 Subsequently, application of RDCs in small molecule structure elucidation/confirmation has been the subject of several chapters4547 40 and a recent perspective paper. To date, there has not been a review of the applications of RDCs in natural product structural determination, but the recent chapter of Gil and Navarro-Vázquez

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absolute configuration of caulamidine A (1) was then established by comparing its experimental ECD spectrum (Fig. 4) with the DFTcalculated simulations of exciton coupling between the aromatic chromophores (see Supplemental Information for molecular modelling and computational details). This comparison permitted assignment of the absolute configuration of 1 as (10S, 11S, 23S). In addition to traditional interpretation and assignment of the spectroscopic data for caulamidine A, we also employed computerassisted structure elucidation (CASE) analysis of the NMR and HRMS

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Fig. 5. Comparison of the experimental vs DFT-calculated RDC (red) and RCSA (blue) values for caulamidine A (1), the CASE-generated alternative structure 3, and configurational isomers 4 and 5. The Q-value is a quantitative similarity measurement for the DFT-calculated RDC and RCSA values for the structure compared to the experimentally measured data. RDC values define the orientation of the C-H bond vectors for protonated carbons, whereas RCSAs describe the chemical shift tensors for all carbons in the molecule’s skeleton.17 For proton-deficient molecules, RCSA data can provide a better assessment of global structural correctness than sparsely available RDCs

for obtaining reliable RCSA measurements using a polymeric gel (Supplemental Information) and specially constructed NMR tubes.35 It is in this context that RCSA and RDC values were experimentally determined for caulamidine A and then compared to the DFTcalculated values for structure 1 and the alternative structure 3 (Fig. 5) generated using the Structure Elucidator CASE program. The Qfactor, which is a quantitative assessment of the quality of the fit between experimental and calculated values, was almost three times worse for 3 than for 1. The difference between calculated and experimental 13C chemical shifts was also significantly higher for 13 13 structure 3 [dN( C) = 4.323] compared to 1 [dN( C) = 2.685]. These analyses, where the correct structure should have the lowest Q and 13 dN( C) values, ruled out structure 3 and conclusively affirmed the structure assigned for caulamidine A (1). We also examined the fit of the experimental RDC/RCSA data to the two other energetically feasible stereoisomers of 1. Here again, the best agreement was

obtained to 1 itself, with the two other viable isomers, 4 and 5, giving substantially higher Q-values (Fig. 5). The structural assignment of caulamidine A (1) was thus facilitated by an array of new heteronuclear NMR experiments, along with CASE analysis and advanced DFT computational techniques. The structure was then validated by analysis of the RDC and RCSA anisotropy parameters, which provide an orthogonal and unequivocal means of confirming the overall correctness of a 17 molecular structure. The only other application of both RDCs and RCSAs in the structural elucidation of a new natural product was the 37 recent study of homodimericin A. Caulamidine B (2) was isolated as a glassy solid and its molecular formula, established by HRESIMS as C23H21ClBr2N4, was similar to 1 except for halogen content. By using the same conventional NMR experiments and the more recent heteronuclear experiments that were employed with caulamidine A, the carbon

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these effects in small molecules has been hampered by the difficulty of separating the isotropic from the anisotropic component of the chemical shift. Only recently has a practical approach been described

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does survey applications of RDCs for natural product structure 47 confirmation. The fundamentals of residual chemical shift anisotropy (RCSA) have been recognized for many years, but the ability to measure

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and nitrogen molecular framework of 2 was revealed to be the same as 1 (Supplemental Information). However, examination of 1 1 H- H couplings and some key HMBC correlations indicated that halogen substitutions in the aromatic C- and F-rings of caulamidine B were different from caulamidine A. The H-5 singlet (δ 7.37) showed a 3-bond HMBC correlation to N-3 while the ortho-coupled H-8 doublet (δ 6.89, 8.4 Hz) correlated with the bridgehead C-10. This supported halogen substitution at C-6 in 2 instead of C-7 as in 1. In a similar manner, HMBC correlations from the H-17 singlet (δ 7.13) to N-15 and from the H-20 doublet (δ 6.86, 8.4 Hz) to C-22 required halogen substitution at C-18 in 2. The regiochemistry of chlorine- vs bromine-substitution in caulamidine B (2) was defined using a high-resolution, band-selective HSQC experiment recently described by the Molinski laboratory to assign halogen substitution 26,27 patterns in a series of polyhalogenated natural products. This is 35,37 Cl isotope effect, but it is a sensitive technique for detecting the only applicable for chlorinated carbons that are also protonated. The bs-HSQC experiment provides characteristic split 2D cross 13 peaks and shoulders on the 1D C slices for Cl-substituted carbons, similar to the bs-CLIP-HSQMBC experiment employed with 1. Using 35,37 this technique, the Cl isotope effect was clearly observed for C11 (Supplemental Information), which then required Br-substitution at C-6 and C-18 in 2. The relative configuration was assigned from diagnostic NOE interactions measured in 2 that were similar to those observed in 1, while the absolute configuration was established as (10S, 11S, and 23S) by comparing the experimental and DFT-calculated ECD spectra (Supplemental Information). We recently found that the eudistidines, a different class of 48 heterocyclic marine alkaloids, exhibited antimalarial activity, thus, we also evaluated the caulamidines for antimalarial effects against chloroquine-sensitive (D6) and chloroquine-resistant strains of the 49 Plasmodium falciparum parasite. Caulamidines A (1) and B (2) showed similar inhibitory effects against both strains of P. falciparum with IC50 values that ranged from 8.3-12.9 µM (Supplemental Information). Caulamidine A (1), was also tested for cytotoxic activity in the single dose (40 µM) NCI-60 cell screen. At this concentration it showed only modest growth inhibition against a very small subset of human cell lines, revealing a significant concentration differential between its antimalarial activity and cytotoxic effects.

Conclusions Caulamidines A (1) and B (2) share a novel heterocyclic scaffold with no close precedents in the chemical literature. A likely precursor is tryptamine or tryptophan, but it is not apparent how the unique heterocyclic core of the caulamidines is biosynthesized, so biosynthesis arguments to help define and rationalize the structures were not viable. However, unambiguous assignment of the caulamidine structures was possible using a suite of powerful new NMR techniques, complemented with CASE analysis and DFTcomputational studies. New NMR pulse sequences were utilized to extend long-range heteronuclear connectivities via 4J and even 5J 1 couplings, while the 1,1-HD-ADEQUATE experiment revealed JCC couplings that are functionally equivalent to 2-bond HMBC 31 correlations. The additional heteronuclear correlation data provided by these experiments supplemented more traditional NMR methods and permitted verification of the structural framework of the caulamidines. The value of these experiments were evident from the dramatic reduction in time for successful CASE analysis that resulted when they were included in the

caulamidine NMR data sets being analyzed. High-resolution 2D 35,37 Cl isotope NMR experiments, which allow visualization of the effect, clearly defined sites of chlorination for both protonated (bsHSQC) and quaternary (bs-CLIP-HSQMBC) carbons. The assigned structure and configuration of caulamidine A (1) was then confirmed by anisotropic NMR experiments to assess the 3D relative orientation of C-H bonds (RDC) and carbon chemical shielding tensors (RCSA), which are not dependent on the distances between bonds and atoms. These data provided a complementary means to assess the global correctness of a molecular structure and thus verify or refute the constitution and configuration of an 17 Prior application of these new NMR assigned structure. methodologies have largely focused on proof-of-principle studies using known compounds or the application of a single technique to resolve unanswered structural questions. While a number of these NMR techniques were used in the structural assignment of 37 homodimericin A, the breadth of NMR experiments employed to unequivocally define the caulamidine structures is heretofore unprecedented. As illustrated by our caulamidine studies, concerted application of contemporary NMR and computational techniques can provide valuable data to help correctly define the complex organic structures often found in natural products. They provide additional means to deduce and evaluate 2D structural assignments as well as to confirm stereochemical features. In concerted applications, these recent advancements provide powerful new tools that can help resolve challenging structural problems, while reducing misassignments and the resulting propagation of incorrect structures. The continuing development and application of new NMR methods that expand the boundaries for data acquisition and structural characterization will further advance natural products discovery, development, and total synthesis efforts.

Acknowledgements We gratefully acknowledge D. Newman (NCI) for organizing and documenting the collections, and the Natural Products Support Group at NCI-Frederick for extraction. This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. This project was also funded in part with Federal funds from the National Cancer Institute, National Institutes of Health, under contract HHSN261200800001E and by the USDA Agricultural Research Service Specific Cooperative Agreement No. 58-6408-1-603. MTD-C acknowledges support from the Fulbright Foundation as a Fulbright Senior Research Fellow in the Molecular Targets Laboratory of the NCI-Frederick. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.

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Unequivocal determination of caulamidines A and B: application and validation of new tools in the structure elucidation tool box

a

Molecular Targets Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702-1201, United States b Basic Science Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702-1201, United States c NMR Structure Elucidation, Process Research and Development, Merck & Co. Inc., Rahway, New Jersey 07065, United States d Molecule Apps, LLC, Corvallis, Oregon 97330, United States e National Center for Natural Products Research and Department of BioMolecular Sciences, School of Pharmacy, University of Mississippi, Oxford, Mississippi 38655, United States f Department of Chemistry, Rhodes University, Grahamstown, South Africa g Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702-1201, United States Supplemental Information: S3-5 S6 S7 S8 S9 S10 S11-12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30

General experimental, animal material, isolation Mass guided LC-MS purification of caulamidines A (1) and B (2) Table SI 1. NMR data for caulamidine A (1) LR-HSQMBC & HSQMBC-TOCSY correlations for caulamidine A (1) Table SI 2. NMR data for caulamidine B (2) LR-HSQMBC, HSQMBC-TOCSY, and NOE correlations for caulamidine B (2) ECD and computational analysis of caulamidines A (1) and B (2) Figure SI 1. Optimized geometries of (10S,11S,23S)-caulamidines A (1) and B (2) Figure SI 2. Experimental and DFT calculated ECD spectra of caulamidine A (1) Figure SI 3: Key molecular orbitals in the calculated ECD of caulamidine A (1) Figure SI 4. Experimental and DFT calculated ECD spectra of caulamidine B (2) Table SI 3. Calculated ECD parameters for caulamidine A (1) Table SI 4. Calculated interatomic distances for caulamidines A (1) and B (2) Table SI 5. Comparison of DFT-calculated and experimental 13C NMR chemical shift values for caulamidine A (1) and caulamidine B (2) Table SI 6. Experimental RDC data of caulamidine A (1) Table SI 7. Experimental RCSA data of caulamidine A (1) 35 Cl/37Cl isotope effect observed in bc-HSQC for C-11 of caulamidine B (2) Antimalarial screening assay References 1 Caulamidine A (1) H NMR spectrum (CD3CN) 13 Caulamidine A (1) C NMR spectrum (CD3CN) Caulamidine A (1) COSY spectrum (CD3CN) Caulamidine A (1) HSQC spectrum (CD3CN) Caulamidine A (1) 1H-13C HMBC spectrum (CD3CN) Caulamidine A (1) NOESY spectrum (CD3CN) S1

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Dennis J. Milanowski,a,‡ Naoya Oku,a,§ Laura K. Cartner,a,b Heidi R. Bokesch,a,b R. Thomas Williamson,c Josep Saurí,c Yizhou Liu,c Kirill A. Blinov,d Yuanqing Ding,e Xing-Cong Li,e Daneel Ferreira,e Larry A. Walker,e Shabana Khan,e Michael T. Davies-Coleman,f,∇ James A. Kelley,g James B. McMahon,a Gary E. Martin,*,c Kirk R. Gustafson*,a

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S35 S36 S37 S38 S39 S40 S41 S42 S43 S44 S45 S46 S47 S48-49 S50-56 S57 S58 S59 S60 S61 S62 S63 S64 S65 S66 S67

Caulamidine A (1) ROESY spectrum (CD3CN) Caulamidine A (1) 1H-15N HMBC spectrum (CD3CN) Caulamidine A (1) 1H-13C LR-HSQMBC spectrum (CD3CN) optimized for 2Hz Caulamidine A (1) 1H-13C HSQMBC-TOCSY spectrum (CD3CN) optimized for 8Hz + 60 ms mixing time Caulamidine A (1) 1H-13C HSQMBC-TOCSY spectrum (CD3CN) optimized for 4Hz + 40 ms mixing time 1 Caulamidine B (2) H NMR spectrum (CD3CN) 13 Caulamidine B (2) C NMR spectrum (CD3CN) Caulamidine B (2) COSY spectrum (CD3CN) Caulamidine B (2) HSQC spectrum (CD3CN) Caulamidine B (2) 1H-13C HMBC spectrum (CD3CN) Caulamidine B (2) NOESY spectrum (CD3CN) Caulamidine B (2) ROESY spectrum (CD3CN) Caulamidine B (2) 1H-15N HMBC spectrum (CD3CN) Caulamidine B (2) 1H-13C LR-HSQMBC Spectrum (CD3CN) optimized for 2Hz Caulamidine B (2) 1H-13C LR-HSQMBC Spectrum (CD3CN) optimized for 8Hz + 60 ms mixing time Caulamidine A (1) HD-J-HSQC spectra for RDC measurement Caulamidine A (1) {1H}-13C spectra for RCSA measurement Coordinates of caulamidine A (1) from DFT geometry optimization GIAO chemical shielding tensors of caulamidine A (1) Table SI 8. NMR Data for Caulamidine A (1) TFA Salt in CD3CN 1 Caulamidine A (1) TFA salt H NMR spectrum (CD3CN) 13 Caulamidine A (1) TFA salt C NMR spectrum (CD3CN) Caulamidine A (1 TFA salt) COSY spectrum (CD3CN) Caulamidine A (1) TFA salt HSQC spectrum (CD3CN) Caulamidine A (1) TFA salt 1H-13C HMBC spectrum (CD3CN) Caulamidine A (1) TFA salt ROESY spectrum (CD3CN) Caulamidine A (1) TFA salt 1D NOE spectrum-1 (CD3CN) Caulamidine A (1) TFA salt 1D NOE spectrum-2 (CD3CN) Caulamidine A (1) TFA salt 1D NOE spectrum-3 (CD3CN) Caulamidine A (1) TFA salt 1D NOE spectrum-4 (CD3CN)

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General Experimental Methods. CD spectra were obtained in CH3CN or MeOH with a Jasco J-720 spectropolarimeter using a microvolume disk cell (0.1 mm thickness). The free base of caulamidines A (1) and B (2) were prepared by washing with 1% aqueous triethylamine followed by H2O. NMR spectra were acquired in CD3CN using a Bruker AVANCE III NMR spectrometer equipped with either a 3 mm 1

13

spectra were also acquired with a Varian Inova spectrometer equipped with a 5 mm room temperature 1

13

probe and operating at 500 MHz for H and 125 MHz for C. Spectra were referenced to the residual nondeuterated solvent signals at δH 1.93 and at δC 1.30. 1H-13C HMBC data sets were acquired using JCH values of 3.5 Hz, 8.3 Hz, and 11 Hz. 1H-15N HMBC data sets were acquired using a JNH value of 8.0 Hz. LR-HSQMBC were optimized for 2 Hz coupling and 1,1-HD-ADEQUATE for 40 Hz. Anisotropic NMR data were acquired for 1mg of caulamidine A in a pHEMA (poly-(2-hydroxyethyl methacrylate)) gel cross-linked with EGDMA (ethylene glycol dimethylacrylate) with a HEMA monomer concentration of 60% (v/v) and a cross-linking ratio of 0.07% (v/v).1 Weak and strong alignment conditions were achieved with an NMR stretching tube with inner diameters of 4.2mm and 3.2mm for the wide and thin sections, respectively.2 RDCs were measured with the HD-J-HSQC (homonuclear decoupled J-resolved HSQC) experiment,3 with a recycling delay of 1.5s, an F1 acquisition time of 256ms on a spectral window of 600Hz, an F2 acquisition time of 120ms, and a transient number of 96 for both weak and strong alignment conditions. Carbon RCSA were measured with the {1H}-13C experiment with a recycling delay of 1.5s, an acquisition time of 0.55s, and transient numbers of 25600 and 76800 for weak and strong alignment conditions, respectively. All anisotropic NMR measurements were conducted at 25oC on a Bruker 500MHz spectrometer equipped with a ProdigyTM probe. (+)HRESIMS data were acquired on an Agilent Technology 6530 Accurate-mass Q-TOF LC/MS. Positive-ion, fast-atom bombardment mass spectra (HR-FABMS) were obtained on a double-focusing mass spectrometer using a sample matrix of nitrobenzyl alcohol. Preparative reversed-phase HPLC was run on an Agilent 1260 Infinity HPLC using a Phenomenex Luna-C18(2) (5µ, 100Å,150 x10 mm) column with 0.1% formic acid or a Dynamax C18 (60 Å, 1 x 25 cm) column with 0.1% TFA. Animal Material. Samples of the marine bryozoan Caulibugula intermis were collected and identified by P. L. Colin (Coral Reef Research Foundation) in the South Pacific near Palau. Animal material was frozen shortly after collection and maintained frozen prior to extraction. Voucher specimens for the original collection (0CDN1079, C011545) and later recollections (0YYA1176-T, C034489 and 0YYA0799-J, C034487) are maintained at the Smithsonian Institution, Washington, D.C.

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TCI or 1.7 mm TXI cryogenic probe, and operating at 600 MHz for H and 150 MHz for C. NMR

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In situ photograph of Calibugula intermis Isolation. The frozen bryozoan from the original collection (227.7 g) was ground and extracted with H2O to yield 25.9 g of aqueous extract after lyophilization. The animal material was then extracted with CH2Cl2-MeOH (1:1) followed by MeOH (100%) to give 5.14 g of combined organic extract after removal of the solvent. The crude organic extract was fractionated by solvent-solvent partitioning as described previously.4 The methyl tert-butyl ether (MTBE) soluble material (1.19 g) was repeatedly chromatographed on Sephadex LH-20 (2 × 125 cm) eluting with hexane-CH2Cl2-MeOH (2:5:1), monitoring at 254 nm. Final purification was achieved by C18 HPLC (Dynamax 60 Å, 1 x 25 cm) eluted with a linear H2O/CH3CN gradient (0.1% TFA vol/vol) from 0 to 100% CH3CN over 30 min to give a total of 3.7 mg of caulamidine A (1). The Caulibugula intermis recollections (981 g) were extracted in a similar manner to provide a total of 8.7 g of organic solvent extract. Solvent partitioning and mass-guided HPLC purification using a Phenomenex Luna-C18(2) (5µ, 100Å,150 x10 mm) column and a linear gradient from 95% H2O/5% CH3CN to 100% CH3CN over 20 minutes (all solvents contained 0.1% formic acid) provided 14.8 mg caulamidine A (1) and 4.7 mg caulamidine B (2).

Caulamidine A (1): glassy solid; [α]D -5.6 (c 0.1, CH3CN); UV (CH3CN) λmax 320 (sh, ε 4,100) 282 (ε 15,700), 220 (ε 19,500) nm; CD (CH3CN, 8.19 × 10-4 M) λext (∆ε) 314 (1.37), 302 (0.0), 265 (-5.22), 237 (0.0), 229 (1.84), 223 (0.0), 206 (-4.17) nm; 1H NMR and 13C data, see Table SI 1; HRFABMS [M + H]+ m/z 459.0924, calcd for C23H2235Cl3N4, 459.0910 (∆ 1.4 mDa).

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Caulamidine B (2): glassy solid; [α]D -2.7 (c 0.1, CH3OH); UV (CH3OH) λmax 293 (ε 6,740) 234 (ε 11,850) nm; CD (CH3OH, 1.82 × 10-4 M) λext (∆ε) 296 (0.36), 293 (0.0), 267 (-6.45), 247 (0.0), 239 (13.64), 217 (0.0), 209 (-2.64) nm; 1H NMR and 13C data, see Table SI 2; HRESIMS [M + H]+ m/z

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546.9891, calcd for C23H2235Cl79Br2N4, 546.9900 (∆ -0.9 mDa).

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Caulamidine B

Caulamidine A

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Mass guided LC-MS purification of caulamidines A (m/z 458.6-459.6 ) and B (m/z 550.6-551.6).

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Table SI 1. NMR data for caulamidine A (1) in CD3CN. Position' 1:N' 2' 3:N' 4' 5' 6' 7' 8' 9' 10' 11' 12a' '''''b' 13:N' 14' 15:N' 16' 17' 18' 19' 20' 21' 22a' '''''b' 23' 24a' '''''b' 25a' '''''b' 26' 27'

13

15

! '( C/ N)' 78.9' 174.0' 241.7' 156.0' 117.8' 129.4' 126.3' 123.8' 133.3' 58.9' 54.8' 52.6' '' 87.5' 159.1' 216.6' 143.9' 124.2' 127.2' 125.8' 127.3' 125.4' 29.6' '' 39.8' 24.7' '' 47.4' '' 37.2' 35.8'

1

∀ ! '(mult,'Hz)'

'' '' '' '' 7.17'(d,'8.5)' 7.31'(dd,'8.4,'2.0)' '' 6.95'(bs)' '' '' 5.02'(dd,'10.8,'4.7)' 3.87'(dd,'13.3,'6.6)' 3.66'(dd,'13.3,'10.5)' '' '' '' '' 6.94'(d,'8.2)' 7.12'(dd,'8.2,'2.4)' '' 6.96'(bs)' '' 2.48'(d,'15.9)' 2.28'(d,'15.9)' '' 2.25'(m)' 1.73'(dd,'15.0,'6.2')' 3.38'(ddd,'12.5,'7.5,'1.6)' 3.18'(dt,'11.7,'5.9)' 3.00'3H'(s)' 3.24'3H'(s)'

HMBC' '' '' '' '' N3,'C4,'C6,'C7,'C9' C4,'C5,'C7,'C8' '' C4,'C6,'C7,'C10' '' '' C2,'C9,'C10,'C12,'C23' N13,'N15,'C11,'C14,'C27' N13,'N15,'C11,'C14' '' '' '' '' N15,'C16,'C19,'C21' C16,'C19' '' C16,'C18,'C19,'C21,'C22' '' C10,'C14,'C16,'C21,'C23,'C24' N13,'C10,'C14,'C16,'C21,'C23,'C24' '' C10,'C14,'C22,'C23,'C25' N1,'C10,'C22,'C23,'C25' N3,'C2,'C24' C24,'C26' N1,'N3,'C2,'C25' N13,'N15,'C12,'C14'

' '

LR:HSQMBC*' '' '' '' '' C8'(4J),'C10'(4J)' C9'(4J),'C10'(5J)' '' C5'(4J),'C9'(2J)' '' '' C14'(4J)' '' C16'(5J),'C27'(3J)' '' '' '' '' C20'(4J)' C21'(4J)' '' C17'(4J)' '' '' '' '' '' C2'(4J),'C9'(4J)' C23'(3J)' C23'(3J)' C23'(5J)' C16'(5J),'C23'(4J)'

'HSQMBC:TOCSY**' 1,1:HD:ADEQUATE' '' '' '' '' '' '' '' '' '' C4,'C6' '' C5,'C7' '' '' '' C7,'C9' '' '' '' '' N13'(3J),'N15'(5J)' C10,'C12' C9'(4J)' C11' C9'(4J)' C11' '' '' '' '' '' '' '' '' '' C16,'C18' N15'(4J),'C22'(5J)' C17' '' '' C23'(4J)' C19,'C21' '' '' '' '' '' C21,'C23' '' '' '' C23' '' C23,'C25' C10'(4J),'C22'(4J)' C24' C10'(4J),'C22'(4J)' C24' '' '' '' ''

'

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Additional correlations for caulamidine A (1) obtained from LR-HSQMBC with respect to HMBC are highlighted in red. Additional correlations obtained from HSQMBC-TOCSY with respect to both HMBC and LR-HSQMBC are highlighted in green.

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Table SI 2. NMR data for caulamidine B (2) in CD3CN. Position' 1:N' 2' 3:N' 4' 5' 6' 7' 8' 9' 10' 11' 12' '' 13:N' 14' 15:N' 16' 17' 18' 19' 20' 21' 22' '' 23' 24' '' 25' '' 26' 27'

13

15

! '( C/ N)' 80.3' 174.4' 240.3' 159.1' 119.7' 122.5' 123.8' 125.1' 130.4' 58.4' 54.7' 52.7' '' 89.0' 159.7' 216.3' 146.9' 125.2' 120.2' 124.3' 129.2' 122.5' 29.3' '' 39.6' 24.6' '' 47.4' '' 37.2' 35.7'

1H'(mult,'Hz)' '' '' '' '' 7.37'(s)' '' 7.12'(d,'10.6)' 6.89'(d,'8.4)' '' '' 5.01'(dd,'11.1,'6.5)' 3.87'(dd,'13.5,'6.5)' 3.66'(t,'11.3)' '' '' '' '' 7.13'(bs)' '' 6.99'(dd,'8.0,'2.1)' 6.86'(d,'8.4)' '' 2.38'(d,'15.9)' 2.28'(d,'15.9)' '' 2.23'(dd,'15.1,'5.9)' 1.74'(dd,'15.1,'5.9)' 3.41'(dd,'13.0,'6.8)' 3.18'(dt,'12.1,'6.0)' 3.01'3H'(s)' 3.23'3H'(s)'

HMBC' '' '' '' '' N3,'C4,'C6,'C9' '' C5,'C6,'C9' C4,'C6,'C10' '' '' C2,'C9,'C10,'C12,'C23' N13,'N15,'C10,'C11,'C14,'C27' N13,'N15,'C11,'C14' '' '' '' '' N15,'C16,'C19,'C21' '' C17,'C18,'C20,'C21' C16,'C18,'C22' '' C10,'C14,'C16,'C20,'C21,'C23,'C24' N13,'C10,'C14,'C16,'C20,'C21,'C23,'C24' '' C22,'C23,'C25' N1,'C10,'C22,'C23,'C25' N3,'C2,'C23,'C24' N1,'C24,'C26' N1,'N3,'C2,'C25' N13,'N15,'C12,'C14'

S9

LR:HSQMBC' '' '' '' '' C8'(4J),'C10'(4J)' '' C4'(4J),'C8'(2J),'C10'(4J)' C5'(4J),'C11'(4J),'C23'(4J)' '' '' '' '' C10'(3J)' '' '' '' '' C20'(4J)' '' '' '' '' '' '' '' '' '' C26'(3J)' '' '' ''

HSQMBC:TOCSY' '' '' '' '' '' '' '' '' '' '' '' C9'(4J)' C9'(4J)' '' '' '' '' '' '' C16'(4J),'C22'(4J)' '' '' '' '' '' '' '' '' '' '' ''

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Additional 1H-13C correlations for caulamidine B (2) obtained from LR-HSQMBC with respect to HMBC are highlighted in red. Additional correlations obtained from HSQMBC-TOCSY with respect to both HMBC and LR-HSQMBC are highlighted in green. NOESY and ROESY correlations are in blue.

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ECD and computational analysis of caulamidines A (1) and B (2) Based on extensive NMR analysis, the absolute configurations (AC) of caulamidines A (1) and B (2) were determined to be either 10S, 11S, 23S or the 10R, 11R, 23R enantiomer. Electronic circular dichroism (ECD) data were computed to facilitate the AC assignments.5-8 The 10S, 11S, 23S configuration OPLS_2005 force field in MacroModel,9 yielding seven conformers with only one within an energy cutoff of 19 kJ/mol. This lowest energy conformer was used for the geometry optimization followed by harmonic vibrational frequency computation at the B3LYP/6-31G** and B3LYP/6-311++G** levels in the gas phase (Figure SI 1), and subsequently by calculation of excitation energies and rotatory strengths at the B3LYP/6-31G** and B3LYP/6-311++G** levels in the gas phase, and at the B3LYPSCRF(COSMO)/6-311++G**//B3LYP/6-311++G** level in MeOH (Figure SI 2). All computations at the quantum mechanics levels were performed using the Gaussian 09 software packages.10 The simulated ECD spectra at the above levels overall match the experimental ECD curve. Molecular

orbital

analysis

was

carried

out

at

the

B3LYP-SCRF(COSMO)/6-

311++G**//B3LYP/6-311++G** level in MeOH (Figure SI 3). Interestingly, orbitals O115 and O118 involve a

13 10

π bonding, and orbitals O120 and O122 a 1013π ∗ bonding, both delocalizing 13 electrons at 10

atoms including N-1 – C-9 and Cl at C-7. Similarly, orbitals O115 and O117 also involve a and orbitals O121 and O123 a

13 10

13 10

π bonding,

π ∗ bonding, both involving 13 electrons at 10 atoms including N-13 – C-

21 and Cl at C-19. The experimentally observed low amplitude positive Cotton effect (CE) at 323 nm is attributed to the electronic transition (ET) at 309 nm from orbital O118 to its unoccupied LUMO orbital O120 (Table SI 3, Figure SI 3). The broad negative CEs in the 313 - 250 nm region are generated by the ETs at 316, 283, 281, and 279 nm. The negative CE at 323 nm is predominantly attributed to the ET at 316 nm from HOMO (O119) to LUMO (O120), and that at 269 nm is mainly contributed by ET at 279 nm from orbital O118 to O121. The high amplitude positive CE at 233 nm is contributed by the ETs at 239 (O118O124 and O125), 238 (O116O120 and O121), and 237 (O117O120) nm. Noticeably, only the ET at 239 nm partially relates to the C-Cl antibonding orbital O125, indicative of the inability to differentiate the (11R)- and (11S)- configurations by ECD spectroscopy. However, the NOESY correlation between H-11 and H-24β supports an (11S)- configuration. This assignment is confirmed by the fact that the H-11-H-24β distances were optimized as 2.06 and 3.95 Å for the (11S)- and (11R)configurations, respectively, at the B3LYP/6-311++G** levels in the gas phase (Table SI 4). Additionally, the calculated total nuclear spin-spin coupling constant J values also support the (11S)S11

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was employed for the conformational random search with an energy window of 130 kJ/mol by using the

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configuration. The J values for H-11/H-12α and H-11/H-12β with the (11S)- configuration were calculated as 11.7 and 6.4 Hz, consistent with the experimentally observed values of 10.8 and 6.4 Hz, respectively, whereas those for the (11R)- configuration were computed as 4.4 and 2.0 Hz, respectively, at the mPW1PW91-SCRF(PCM)/6-311++G**//B3LYP/6-311++G** level in acetonitrile.11 Therefore the

at 214 nm is contributed by the ETs at 228, 226, 221, 217, 205, 204, and 201 nm (Table SI 3 and Figure SI 3). ECD computation was also carried out to assign the AC of caulamidine B (Figure SI 1), using the same protocols. As analyzed above, the diagnostic CEs in the ECD spectrum of caulamidine A are generally contributed by the ETs from

13 10

π to

13 10

π ∗ , in which some of the 13 electrons are rarely

delocalized onto the chlorine atoms. Thus, it may be assumed that the presence of the bromine atoms in caulamidine B wouldn’t significantly change the shape of the ECD curve. Since the experimental ECD curve of caulamidine B is highly similar to that of caulamidine A, the AC of caulamidine B was mandatorily assigned as (10S,11S,23S)-. This was confirmed by the excellent agreement of the calculated ECD spectrum of (10S,11S,23S)- caulamidine B with its experimental ECD spectrum (Figure SI 4).

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AC of caulamidine A can be unambiguously assigned as (10S,11S,23S). The experimentally observed CE

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Figure SI 1. Optimized geometries of (10S,11S,23S)- caulamidines A (1) and B (2) at the B3LYP/6311G++ level in the gas phase.

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Figure SI 2. Experimental (exptl) and computed ECD spectra of (10S,11S,23S)- caulamidine A at the B3LYP/6-31G** (gas) and B3LYP/6-311++G** (lbs) levels in the gas phase and at the B3LYPSCRF(COSMO)/6-311++G**//B3LYP/6-311++G** (sol) level in MeOH.

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Figure SI 3. Molecular orbitals involved in key transitions in the calculated ECD spectrum of (10S,11S,23S)- caulamidine A at the B3LYP-SCRF(COSMO) /6-311++G**//B3LYP/6-311++G** level in MeOH.

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Figure SI 4. Experimental (exptl) and computed ECD spectrum of (10S,11S,23S)- caulamidine B at the B3LYP-SCRF(COSMO)/6-311++G**//B3LYP/6-311++G** (sol) level in MeOH.

S16

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Excited State

E/ev

λ/nm

f

Rlen

State#

Related Orbitals

1

119→120

3.92

316.0

0.12

-77.5

2

118→120

4.01

309.3

0.13

99.7

4

119→122, 119→123

4.38

283.4

0.24

-40.2

5

119→122, 119→123

4.42

280.7

0.01

-17.9

6

118→122

4.44

279.1

0.15

81.2

7

118→121

4.45

278.6

0.17

-78.7

13

118→124, 118→125

5.18

239.4

0.02

40.3

14

116→120, 116→121

5.21

237.8

0.02

52.8

16

117→120

5.24

236.7

0.17

34.8

20

119→128

5.43

228.4

0.01

-31.7

22

115→120, 118→126

5.48

226.1

0.05

-54.4

29

113→120, 118→127

5.62

220.6

0.03

-60.3

32

117→122

5.72

216.9

0.05

-75.0

47

114→122, 119→137

6.06

204.7

0.07

-66.7

49

118→136/5

6.08

203.9

0.03

-29.6

52

115→123, 119→137

6.16

201.4

0.08

-31.4

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Table SI 3. Calculated Transition States, Related Excitation Energies (E), Wave Lengths (λ), Oscillator Strengths (f) and Rotatory Strengths in Length Form (Rlen) of (10S,11S,23S)- caulamidine A (1) at the B3LYP-SCRF(COSMO)/6-311++G**//B3LYP/6-311++G** Level in MeOH.

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Distance

A

B

H-5 to H-6

2.50

-

H-5 to Me-26

4.81

4.79

H-7 to H-8

-

2.47

H-8 to H-12α

2.31

2.31

H-8 to H-22α

2.93

2.94

H-8 to Me-27

3.46

3.47

H-11 to H-12β

2.41

2.41

H-11 to H-12α

3.05

3.05

H-11 to H-24β

2.06

2.05

H-11 to H-24α

3.63

3.62

H-12βto Me-27

2.23

2.23

H-12α to Me-27

2.90

2.91

H-17 to Me-27

3.77

3.76

H-17 to H-18

2.49

-

H-19 to H-20

-

2.48

H-20 to H-22β

2.51

2.51

H-20 to H-22α

3.24

3.24

H-22β to H-24α

2.98

2.98

H-22β to H-25α

2.36

2.37

H-24αto H-25α

2.38

2.38

H-24αto H-25β

2.54

2.54

H-24βto H-25β

2.38

2.38

H-24βto H-25α

3.05

3.05

H-25βto Me-26

2.40

2.40

H-25αto Me-26

2.60

2.61

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Table SI 4. Important Interatomic Distances in the Geometries of (10S,11S,23S)-caulamidines A (1) and B (2) Optimized at the B3LYP/6-311++G** Level in the Gas Phase (Å).

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13

Table SI 5. Comparison of the DFT-calculated and experimentally measured C NMR chemical shift values for caulamidine A (1) and caulamidine B (2) in CD3CN.

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13

Position

DFT-calculated C shift (ppm)

Observed C shift (ppm)

DFT-calculated C shift (ppm)

Observed 13C shift (ppm)

2 4 5 6 7 8 9 10 11 12 14 16 17 18 19 20 21 22 23 24 25 26-Me 27-Me

173.8 156.9 120.4 130.2 127.9 123.6 133.2 57.9 54.9 54.0 156.8 143.9 125.6 128.1 128.2 126.9 123.9 30.5 41.3 26.9 49.0 38.9 37.7

174.0 156.0 117.8 129.4 126.3 123.8 133.3 58.9 54.8 52.6 159.1 143.9 124.2 127.2 125.8 127.3 125.4 29.6 39.8 24.7 47.4 37.2 35.8

174.5 159.3 123.4 122.7 124.7 123.5 130.3 57.1 54.7 53.9 157.5 146.4 127.6 119.6 125.9 127.7 121.4 30.1 41.5 25.8 49.0 38.8 36.8

174.4 159.1 119.7 122.5 123.8 125.1 130.4 58.4 54.7 52.7 159.7 146.9 125.2 120.2 124.3 129.2 122.5 29.3 39.6 24.6 47.4 37.2 35.7

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2

1 13

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Bond C23-H23 C24-H24 C6-H6 C4-H4 C1-H1 C21-H21 C26-H26 C28-H28a/b C14-H14a/b C9-H9a/b* C15-H15a/b C29-H29a/b/c† C17-H17a/b/c * †

RDC (Hz) 6.9 8.1 -1.9 9.4 9.1 6.9 -4 overlap with gel signal overlap with gel signal 0.2 0.5 -0.7 -0.2

Methylene RDCs are reported as the averages of the two individual CH RDCs. Methyl group RDCs are utilized in analogy to previously described analysis,12,13, except that a C-H to C-N conversion factor of 6.3-1 was used specifically for the N-methyl.

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Table SI 6. Experimental RDC data of caulamidine A (1)

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Atom

RCSA*† (Hz)

C12 C8 C19 C2 C20 C23 C4 C6 C22 C3 C5 C1 C21 C24 C10 C26 C28 C14 C11 C17 C29 C9 C15

-0.2 -1.5 -0.3 -2.6 -1.5 -2.1 -3.4 -2.5 3.5 0.1 -2.3 -1.8 -1.4 -2 overlap with gel signal 0.1 -0.5 1.7 overlap with gel signal 0.1 0.4 0.8 0.5

*

Resonances are first referenced relative to TMS (tetramethylsilane) at 0 ppm. In order to compensate for a potential referencing error due to TMS evaporation during the relatively lengthy NMR measurements, the strong alignment spectrum was further shifted upfield by 0.5 Hz relative to the weak alignment spectrum, on the basis of a slightly improved Q-factor. † Values in Hz are based on a spectrometer frequency of 500 MHz.

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Table SI 7. Experimental RCSA data of caulamidine A (1)

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The Cl/ Cl isotope effect detected by bs-HSQC for C-11 of caulamidine B (2). 37

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Antimalarial Screening Assay

The antimalarial activity was determined against chloroquine sensitive (D6) and chloroquine resistant (W2) strains of Plasmodium falciparum by measuring plasmodial lactate dehydrogenase (LDH) activity

or W2 strain of P. falciparum (200 µL, with 2% parasitemia and 2% hematocrit in RPMI 1640 medium supplemented with 10% human serum and 60 µg/mL Amikacin) was added to the wells of a 96- well plate containing 10 µL of serially diluted test samples. The plate was incubated at 37 ºC, for 72 h in an environment of 90% N2, 5% O2, and 5% CO2. Plasmodial LDH activity was determined by mixing 20 µL of the incubation mixture with 100 µL of the Malstat reagent and incubating at room temperature for 30 min. Twenty microliters of a 1:1 mixture of NBT/PES (Sigma, St. Louis, MO) was added and the plate was further incubated in the dark for 1 h. The reaction was then stopped by adding 100 µL of a 5% acetic acid solution and the absorbance was read at 650 nm. Artemisinin and chloroquine were included as the drug controls. The in vitro cytotoxicity of samples to mammalian cells was also tested to determine the selectivity index of the antimalarial activity. Vero cells (monkey kidney fibroblasts) were seeded into a 96-well plate at a density of 25,000 cells/well and grown for 24 h. Test samples at different concentrations were added and cells were further incubated for 48 h. Cell viability was determined by the Neutral Red method.15 Doxorubicin was included as the drug control. IC50 values were obtained from the dose response curves.

Cytotox IC50

P. falciparum strain Sample Caulamidine A

D6 ( IC50 µM) 11.3

W2 ( IC50 µM) 8.3

Vero cells

Caulamidine B

12

12.9

NC

Chloroquine Artemisinin

0.02 0.03

0.37 0.02

NC= no cytotoxicity at 50 µM

S23

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according to the procedure of Makler and Hinrichs.14 A suspension of red blood cells infected with the D6

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(1) Gil-Silva, L.F.; Santamaría-Fernández, R.; Navarro-Vázquez, A.; Gil, R.R. Chemistry Eur. J, 2016, 472-476. (2) Nath, N.; Schmidt, M.; Gil, R.R; Williamson, R.T.; Martin, G.E.; Navarro-Vázquez, A.; Griesinger, C.; Liu, Y. J. Am. Chem. Soc. 2016, 138, 9548-9556. (3) Castañar, L.; García,M.; Hellemann, E.; Nolis, P.; Gil, R.R.; Parella, T. J. Org. Chem. 2016, 81, 11126-11131. (4) Bokesch, H. R.; Blunt, J. W.; Westergaard, C. K.; Cardellina II, J. H.; Johnson, T. R.; Michael, J. A.; McKee, T. C.; Hollingshead, M. G.; Boyd, M. R. J. Nat. Prod. 1999, 62, 633-635. (5) Ding, Y.; Li, X.-C.; Ferreira, D. J. Org. Chem. 2007, 72, 9010-9017. (6) Ding, Y.; Li, X.-C.; Ferreira, D. J. Nat. Prod. 2009, 72, 327-335. (7) Ding, Y.; Li, X.-C.; Ferreira, D. J. Nat. Prod. 2010, 73, 435-440. (8) Li, C.-S.; Ding, Y.; Yang, B.-J.; Miklossy, G.; Yin, H.-Q.; Walker, L. A.; Turkson, J.; Cao, S. Org. Lett. 2015, 17, 3556-3559. (9) MacroModel; version 9.9 ed.; Schrödinger LLC, New York, NY: 2011. (10) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J., J. A.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J.; Gaussian 09 Revision A.1; Gaussian Inc., Wallingford CT, 2009. (11) Li, C.-S.; Ren, G.; Yang, B.-J.; Miklossy, G.; Turkson, J.; Fei, P.; Ding, Y.; Walker, L. A.; Cao, S. Org. Lett. 2016, 18, 2335-2338. (12) Ottiger, M.; Bax, A. J. Am. Chem. Soc. 1999, 121, 4690-4695. (13) Sánchez-Pedregal, V.M.; Santamaría-Fernández, R.; Navarro-Vázquez, A. Org Lett., 2009, 11, 14711474. (14) Makler, M. T.; Hinrichs, D. J. Am. J. Trop. Med. Hyg. 1993, 48, 205-210. (15) Borenfreund, E.; Babich, H.; Martin-Alguacil, N. In vitro Cell. Dev. Biol. 1990, 26, 1030-1034

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Supporting Information References

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Caulamidine A (1) 1H NMR Spectrum (600 MHz, CD3CN)

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Caulamidine A (1) 13C NMR Spectrum (150 MHz, CD3CN)

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Caulamidine A (1) COSY Spectrum (CD3CN)

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Caulamidine A (1) HSQC Spectrum (CD3CN)

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Caulamidine A (1) 1H-13C HMBC Spectrum (CD3CN) Optimized for 8.3 Hz

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Caulamidine A (1) NOESY Spectrum (CD3CN)

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Caulamidine A (1) ROESY Spectrum (600 MHz, CD3CN)

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Caulamidine A (1) 15N-1H HMBC Spectrum (CD3CN) Optimized for 8.0 Hz

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Caulamidine A (1) 1H-13C LR-HSQMBC Spectrum (CD3CN) Optimized for 2Hz.

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Caulamidine A (1) 1H-13C HSQMBC-TOCSY Spectrum (CD3CN) Optimized for 8Hz + 60 ms Mixing Time

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Caulamidine A (1) 1H-15N HSQMBC-TOCSY Spectrum (CD3CN) Optimized for 4Hz + 40 ms Mixing Time

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Caulamidine B (2) 1H NMR Spectrum (600 MHz, CD3CN)

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Caulamidine B (2) 13C NMR Spectrum (150 MHz, CD3CN)

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Caulamidine B (2) COSY Spectrum (CD3CN)

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Caulamidine B (2) HSQC Spectrum (CD3CN)

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Caulamidine B (2) 1H-13C HMBC Spectrum (CD3CN) Optimized for 8.3 Hz

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Caulamidine B (2) NOESY Spectrum (CD3CN)

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Caulamidine B (2) ROESY Spectrum (CD3CN)

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Caulamidine B (2) 15N-1H HMBC Spectrum (CD3CN) Optimized for 8.0 Hz

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Caulamidine B (2) 1H-13C LR-HSQMBC Spectrum (CD3CN) Optimized for 2Hz

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Caulamidine B (2) 1H-13C LR-HSQMBC Spectrum (CD3CN) Optimized for 8Hz + 60 ms Mixing Time

Chemical Science

Caulamidine A (1) HD-J-HSQC Spectra for RDC Measurement Showing a Representative Region. Spectra from weakly and strongly aligned samples are shown in red and blue respectively.

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Caulamidine A (1) {1H}-13C Spectra for RCSA Measurement Showing a Representative Region. Spectra from weakly and strongly aligned samples are shown in red and blue, respectively.

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Structure Coordinate of Caulamidine A from DFT Geometry Optimization. C C C C C C N C C C C C N C C N C Cl C C C C C C Cl C N C C Cl H H H H H

4.021 2.729 2.528 3.604 4.880 5.099 1.679 0.556 1.110 -1.199 0.313 -1.377 -0.817 0.567 0.849 -2.105 -1.108 6.236 -2.429 -1.864 -2.005 -2.747 -3.330 -3.170 -2.940 -1.967 -0.409 -1.811 -0.158 -3.745 -1.604 -2.173 -0.837 -0.552 0.895

-1.026 -1.527 -0.675 -1.109 -0.270 0.227 -0.210 1.110 -0.556 0.667 -0.967 -0.648 -0.761 -2.029 -0.161 -1.783 0.020 0.638 0.796 -0.128 0.675 -0.515 1.864 0.964 3.101 0.798 3.135 0.291 2.113 -0.814 1.506 1.978 4.099 1.823 -0.471 1.789 0.151 1.763 -0.372 0.580 -1.713 0.255 -2.522 1.127 -2.012 2.286 -0.662 2.615 -4.231 0.742 1.212 -1.410 -0.151 -2.764 0.170 -2.509 -0.890 -3.999 1.483 -1.142 2.181 -1.747 4.074 2.055 5.086 1.441 3.908 2.752 -0.803 -4.259

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H H H H H H H H H H H H H H H H

-0.780 -0.394 0.617 1.074 0.761 1.261 0.436 1.933 -2.243 -2.387 4.162 3.453 6.097 -1.550 -3.899 -3.605

-0.469 -1.957 -0.921 0.651 4.141 2.982 2.469 2.059 0.565 -0.728 -1.342 0.098 -1.234 -2.151 -2.671 -0.254

-4.794 -3.892 0.908 1.533 -0.094 1.133 -1.761 -0.955 -3.433 -2.245 -2.556 2.140 -0.976 -0.627 2.933 3.520

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GIAO Chemical Shielding Tensors of caulamidine A. 1 C Isotropic = 73.3703 Anisotropy = 141.2073 XX= 61.7645 YX= 20.1256 ZX= 8.3978 XY= 22.6134 YY= 150.8281 ZY= -45.4062 XZ= 4.3523 YZ= -46.0083 ZZ= 7.5184 Eigenvalues: -7.9826 60.5851 167.5086 2 C Isotropic = 56.1240 Anisotropy = 151.0068 XX= 2.2535 YX= 21.9017 ZX= -31.7615 XY= 5.5815 YY= 147.1450 ZY= -30.3660 XZ= -38.4038 YZ= -29.5607 ZZ= 18.9735 Eigenvalues: -25.8332 37.4100 156.7952 3 C Isotropic = 73.9681 Anisotropy = 155.5631 XX= 11.4624 YX= 28.4309 ZX= 14.6050 XY= 30.6791 YY= 164.5843 ZY= -31.0167 XZ= 7.3042 YZ= -36.9867 ZZ= 45.8575 Eigenvalues: -1.0643 45.2916 177.6768 4 C Isotropic = 72.1485 Anisotropy = 124.9639 XX= 71.4064 YX= 14.7081 ZX= 6.0047 XY= 8.8009 YY= 141.5472 ZY= -44.4196 XZ= 12.3490 YZ= -44.2133 ZZ= 3.4921 Eigenvalues: -11.2938 72.2816 155.4578 5 C Isotropic = 62.7872 Anisotropy = 96.2986 XX= 23.5580 YX= 4.1230 ZX= -47.1358 XY= 5.5475 YY= 122.6791 ZY= -11.9696 XZ= -47.2617 YZ= -15.7686 ZZ= 42.1245 Eigenvalues: -15.4510 76.8263 126.9863 6 C Isotropic = 71.0978 Anisotropy = 141.3478 XX= 1.3397 YX= 33.9428 ZX= 19.4546 XY= 34.0807 YY= 150.8592 ZY= -30.3426 XZ= 16.5239 YZ= -32.1980 ZZ= 61.0945 Eigenvalues: -14.2618 62.2255 165.3297 7 N Isotropic = 30.5974 Anisotropy = 329.5536 XX= -90.3982 YX= 72.9496 ZX= -107.6496 XY= 101.8368 YY= 176.1129 ZY= -78.8872 XZ= -107.6878 YZ= -74.5075 ZZ= 6.0775

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Eigenvalues: -163.6262 5.1186 250.2998 8 C Isotropic = 42.1054 Anisotropy = 122.4684 XX= 36.7657 YX= 10.4281 ZX= -55.4613 XY= 38.1858 YY= 90.2246 ZY= -33.8850 XZ= -19.9424 YZ= -52.5531 ZZ= -0.6742 Eigenvalues: -29.8249 32.3900 123.7510 9 C Isotropic = 163.9295 Anisotropy = 30.7497 XX= 183.1890 YX= -9.7276 ZX= -2.1628 XY= -2.3796 YY= 149.9786 ZY= -7.5451 XZ= 4.2144 YZ= -4.5715 ZZ= 158.6209 Eigenvalues: 146.1904 161.1687 184.4293 10 C Isotropic = 133.6350 Anisotropy = 8.4992 XX= 132.1560 YX= 0.4894 ZX= 2.3603 XY= 2.4525 YY= 138.5751 ZY= -3.1071 XZ= -0.3945 YZ= -1.1990 ZZ= 130.1738 Eigenvalues: 129.0778 132.5260 139.3011 11 C Isotropic = 149.8353 Anisotropy = 10.5135 XX= 150.8640 YX= 6.5660 ZX= -1.5064 XY= 0.6875 YY= 154.3546 ZY= 5.0037 XZ= -4.6389 YZ= 2.2930 ZZ= 144.2872 Eigenvalues: 141.3115 151.3500 156.8443 12 C Isotropic = 25.6525 Anisotropy = 119.8213 XX= 61.0411 YX= -45.6530 ZX= 34.0583 XY= -22.0725 YY= -24.2707 ZY= -58.7191 XZ= 29.5146 YZ= -26.0970 ZZ= 40.1869 Eigenvalues: -48.1664 19.5904 105.5333 13 N Isotropic = 178.6561 Anisotropy = 57.0590 XX= 179.2225 YX= 16.6399 ZX= 34.0686 XY= -3.7729 YY= 206.5919 ZY= 8.9946 XZ= 54.6386 YZ= -1.7402 ZZ= 150.1540 Eigenvalues: 118.0060 201.2669 216.6954 14 C Isotropic = 146.4623 Anisotropy = 52.3242 XX= 172.5768 YX= 3.0525 ZX= -21.9418 XY= 1.9261 YY= 131.6762 ZY= 9.6545 XZ= -18.1439 YZ= 10.5424 ZZ= 135.1341 Eigenvalues: 118.4499 139.5919 181.3452 15 C Isotropic = 168.0703 Anisotropy = 18.5335

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XX= 154.2974 YX= 10.3105 ZX= -8.0709 XY= -4.0269 YY= 177.7419 ZY= 2.0173 XZ= 1.3805 YZ= 7.2840 ZZ= 172.1715 Eigenvalues: 153.0483 170.7366 180.4259 16 N Isotropic = 1.8926 Anisotropy = 324.9541 XX= 109.3593 YX= -142.5246 ZX= 88.8207 XY= -130.9505 YY= -145.5634 ZY= -10.5896 XZ= 118.5731 YZ= 24.8457 ZZ= 41.8819 Eigenvalues: -214.8154 1.9646 218.5287 17 C Isotropic = 157.1465 Anisotropy = 53.7879 XX= 142.9931 YX= -8.8105 ZX= -6.3505 XY= -14.4810 YY= 166.4434 ZY= 25.7413 XZ= -9.1196 YZ= 23.9804 ZZ= 162.0031 Eigenvalues: 137.1422 141.2923 193.0051 18 Cl Isotropic = 745.6722 Anisotropy = 445.7861 XX= 857.7816 YX= 20.1521 ZX= 222.5571 XY= 17.9074 YY= 606.9492 ZY= 11.0637 XZ= 222.9181 YZ= 9.2948 ZZ= 772.2857 Eigenvalues: 587.2543 606.8993 1042.8629 19 C Isotropic = 42.6006 Anisotropy = 133.3178 XX= 104.8531 YX= -19.7264 ZX= 32.8428 XY= -32.9676 YY= -27.9125 ZY= -24.0419 XZ= 38.3681 YZ= -38.1621 ZZ= 50.8613 Eigenvalues: -40.0820 36.4047 131.4792 20 C Isotropic = 64.8497 Anisotropy = 140.7011 XX= 109.2979 YX= -33.8811 ZX= 70.7649 XY= -33.1744 YY= 27.3681 ZY= 4.6508 XZ= 60.6524 YZ= 8.7023 ZZ= 57.8830 Eigenvalues: -6.5843 42.4829 158.6504 21 C Isotropic = 74.9504 Anisotropy = 134.3850 XX= 124.2024 YX= 0.8888 ZX= 63.4456 XY= -5.8062 YY= 55.9753 ZY= -31.4389 XZ= 68.0744 YZ= -34.6500 ZZ= 44.6735 Eigenvalues: -5.2541 65.5649 164.5404 22 C Isotropic = 63.0850 Anisotropy = 95.0787 XX= 110.5856 YX= -21.6542 ZX= 17.3036 XY= -19.1233 YY= -7.8558 ZY= -23.7352

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XZ= 18.1375 YZ= -24.2073 ZZ= 86.5251 Eigenvalues: -15.6206 78.4047 126.4708 23 C Isotropic = 69.0081 Anisotropy = 145.8769 XX= 111.3271 YX= -44.5049 ZX= 70.7496 XY= -41.9390 YY= 36.3844 ZY= 12.9909 XZ= 69.3082 YZ= 14.8894 ZZ= 59.3127 Eigenvalues: -16.3294 57.0944 166.2593 24 C Isotropic = 78.7263 Anisotropy = 147.2814 XX= 131.5354 YX= -12.2772 ZX= 69.1256 XY= -12.2632 YY= 56.5800 ZY= -33.2231 XZ= 70.0949 YZ= -30.7667 ZZ= 48.0635 Eigenvalues: -0.3500 59.6151 176.9139 25 Cl Isotropic = 744.3695 Anisotropy = 449.2518 XX= 606.3369 YX= 44.4931 ZX= 7.9014 XY= 46.1914 YY= 1017.6208 ZY= 97.2326 XZ= 16.9450 YZ= 93.6977 ZZ= 609.1508 Eigenvalues: 587.5620 601.6758 1043.8707 26 C Isotropic = 131.9597 Anisotropy = 37.9082 XX= 155.8470 YX= -3.7316 ZX= 0.6133 XY= 0.2230 YY= 103.6539 ZY= 1.3887 XZ= -10.9790 YZ= 2.7745 ZZ= 136.3782 Eigenvalues: 103.4827 135.1646 157.2318 27 N Isotropic = 168.4667 Anisotropy = 63.9112 XX= 146.8593 YX= 31.1782 ZX= 41.2037 XY= 27.8584 YY= 165.3508 ZY= -15.8643 XZ= 24.7464 YZ= -3.3920 ZZ= 193.1901 Eigenvalues: 112.0381 182.2880 211.0742 28 C Isotropic = 142.1216 Anisotropy = 55.2026 XX= 165.5857 YX= -16.7552 ZX= -11.1831 XY= -17.0915 YY= 126.2872 ZY= 10.2945 XZ= -17.2476 YZ= 14.0455 ZZ= 134.4918 Eigenvalues: 116.6099 130.8314 178.9233 29 C Isotropic = 158.5209 Anisotropy = 51.2351 XX= 139.0610 YX= -3.0297 ZX= -7.1055 XY= -10.0630 YY= 153.8083 ZY= 16.0405 XZ= -10.2752 YZ= 17.9747 ZZ= 182.6934 Eigenvalues: 136.4199 146.4652 192.6777

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30 Cl Isotropic = 825.2891 Anisotropy = 398.7389 XX= 1067.8549 YX= -88.1460 ZX= -36.1264 XY= -66.6517 YY= 691.7209 ZY= 24.1388 XZ= -61.5745 YZ= 43.7720 ZZ= 716.2915 Eigenvalues: 663.8824 720.8699 1091.1150 31 H Isotropic = 26.9536 Anisotropy = 5.4980 XX= 29.8788 YX= 1.5543 ZX= 0.9342 XY= 1.6556 YY= 26.9053 ZY= -1.4178 XZ= -0.3150 YZ= -2.3721 ZZ= 24.0767 Eigenvalues: 22.9752 27.2667 30.6189 32 H Isotropic = 28.0650 Anisotropy = 10.4456 XX= 30.1133 YX= -2.0861 ZX= -3.6483 XY= -3.1952 YY= 28.3072 ZY= 4.2407 XZ= -4.1561 YZ= 2.9942 ZZ= 25.7745 Eigenvalues: 22.6684 26.4978 35.0287 33 H Isotropic = 29.5112 Anisotropy = 10.8451 XX= 25.0117 YX= -0.6675 ZX= -1.1310 XY= -0.0672 YY= 36.4224 ZY= 1.0397 XZ= -0.8780 YZ= 2.3251 ZZ= 27.0994 Eigenvalues: 24.5992 27.1930 36.7413 34 H Isotropic = 29.0133 Anisotropy = 10.5733 XX= 25.3539 YX= 0.2474 ZX= 1.7516 XY= -0.1575 YY= 27.5358 ZY= 4.0078 XZ= 2.4191 YZ= 3.1407 ZZ= 34.1504 Eigenvalues: 24.5705 26.4073 36.0622 35 H Isotropic = 27.2668 Anisotropy = 9.9417 XX= 30.0622 YX= -0.6155 ZX= -2.5279 XY= -1.5669 YY= 21.4167 ZY= 2.0695 XZ= -3.9125 YZ= 2.5936 ZZ= 30.3215 Eigenvalues: 20.8327 27.0731 33.8946 36 H Isotropic = 29.6406 Anisotropy = 10.3484 XX= 26.1726 YX= -1.4245 ZX= 0.7071 XY= -1.5115 YY= 26.4451 ZY= 0.7749 XZ= 2.3895 YZ= 0.0732 ZZ= 36.3041 Eigenvalues: 24.6564 27.7259 36.5395 37 H Isotropic = 28.8680 Anisotropy = 10.8856 XX= 24.2393 YX= 0.6753 ZX= -0.6464

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XY= 1.2875 YY= 33.8310 ZY= 3.7728 XZ= -0.7149 YZ= 4.5161 ZZ= 28.5335 Eigenvalues: 23.7602 26.7186 36.1250 38 H Isotropic = 29.0276 Anisotropy = 10.6224 XX= 35.3426 YX= 1.4134 ZX= -2.1505 XY= 2.6187 YY= 28.0268 ZY= -4.2969 XZ= 0.6384 YZ= -3.1095 ZZ= 23.7134 Eigenvalues: 21.5754 29.3982 36.1092 39 H Isotropic = 29.7828 Anisotropy = 5.9403 XX= 31.8159 YX= -1.7767 ZX= 0.6611 XY= -3.0972 YY= 26.0882 ZY= 2.2371 XZ= 3.5257 YZ= 2.3867 ZZ= 31.4443 Eigenvalues: 24.0377 31.5678 33.7430 40 H Isotropic = 28.8647 Anisotropy = 9.0542 XX= 27.0462 YX= 4.0491 ZX= -2.9172 XY= 1.2815 YY= 33.8819 ZY= 0.1657 XZ= -2.2185 YZ= -0.3843 ZZ= 25.6660 Eigenvalues: 23.4511 28.2422 34.9009 41 H Isotropic = 28.7092 Anisotropy = 7.4608 XX= 32.0266 YX= 0.6688 ZX= 3.3941 XY= -1.2609 YY= 27.2452 ZY= 4.0218 XZ= 2.6607 YZ= 2.8103 ZZ= 26.8558 Eigenvalues: 22.9575 29.4871 33.6831 42 H Isotropic = 29.7416 Anisotropy = 6.1177 XX= 26.1179 YX= 2.4225 ZX= 0.0154 XY= -0.1327 YY= 30.5919 ZY= -2.0349 XZ= 2.1269 YZ= -2.0438 ZZ= 32.5151 Eigenvalues: 25.4854 29.9195 33.8201 43 H Isotropic = 29.9960 Anisotropy = 7.2667 XX= 33.9750 YX= 0.3413 ZX= -2.7971 XY= -2.6364 YY= 31.1269 ZY= 0.3502 XZ= -1.8709 YZ= -0.8745 ZZ= 24.8860 Eigenvalues: 24.2809 30.8665 34.8404 44 H Isotropic = 28.5233 Anisotropy = 7.6006 XX= 28.3106 YX= -3.4353 ZX= 3.0521 XY= -2.8245 YY= 24.3881 ZY= -0.0126 XZ= 0.6269 YZ= 0.6758 ZZ= 32.8711

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Eigenvalues: 22.5173 29.4622 33.5904 45 H Isotropic = 28.0484 Anisotropy = 4.0128 XX= 28.0204 YX= 2.9271 ZX= 0.4015 XY= 2.3852 YY= 28.1132 ZY= 0.7291 XZ= -2.2452 YZ= 1.0154 ZZ= 28.0116 Eigenvalues: 24.8938 28.5278 30.7236 46 H Isotropic = 24.5374 Anisotropy = 6.5512 XX= 28.5921 YX= -1.7171 ZX= 0.3344 XY= -1.3646 YY= 21.0710 ZY= 1.2504 XZ= 0.6082 YZ= 1.3616 ZZ= 23.9492 Eigenvalues: 20.2567 24.4507 28.9049 47 H Isotropic = 24.9006 Anisotropy = 11.2089 XX= 31.6052 YX= -1.8748 ZX= 1.7725 XY= -2.0262 YY= 20.1046 ZY= 0.6602 XZ= 2.5401 YZ= 0.4247 ZZ= 22.9922 Eigenvalues: 19.5315 22.7972 32.3733 48 H Isotropic = 24.4840 Anisotropy = 5.9518 XX= 25.0908 YX= -0.6856 ZX= 1.2924 XY= -0.8746 YY= 20.7083 ZY= 2.1289 XZ= 0.7234 YZ= 2.2150 ZZ= 27.6530 Eigenvalues: 19.8796 25.1206 28.4519 49 H Isotropic = 24.7006 Anisotropy = 12.1858 XX= 21.6119 YX= 3.1805 ZX= -2.7905 XY= 5.1495 YY= 29.8404 ZY= -2.7189 XZ= -2.8492 YZ= -2.5623 ZZ= 22.6494 Eigenvalues: 18.9487 22.3286 32.8245 50 H Isotropic = 24.3207 Anisotropy = 6.0724 XX= 21.5565 YX= 0.4059 ZX= -2.9132 XY= 0.4218 YY= 25.8285 ZY= 2.2790 XZ= -3.0551 YZ= 1.9485 ZZ= 25.5772 Eigenvalues: 19.6509 24.9423 28.3690 51 H Isotropic = 24.2814 Anisotropy = 4.8745 XX= 22.2474 YX= 1.7701 ZX= -2.7518 XY= 1.9463 YY= 25.9261 ZY= -0.3684 XZ= -2.6241 YZ= -0.1247 ZZ= 24.6706 Eigenvalues: 20.1435 25.1695 27.5310

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Table SI 8. NMR Spectroscopic Data for Caulamidine A (1) TFA Salt in CD3CN δH (J in Hz)

NOEb

HMBC

117.1, CH

7.39, d (8.4)

H6

C4, 7, 9

6

132.0, CH

7.47, dd (8.4, 1.8)

H5

C4, 7, 8

7

129.7, C

8

125.5, C

7.23, d (1.8)

H12, 22, 27

C4, 6, 7, 10

9

128.3, C

10

57.8, C

11

53.0, CH

5.06, dd (10.8, 6.4)

H12, 24, 27

C2, 9, 10, 12, 23

12 b

54.6, CH2

3.94, dd (14.3, 11.0)

H8, 11, 27

C11, 14

4.15, dd (14.3, 6.6)

H8, 27

C10, 11, 14, 27

Pos.

δC, typea

2

169.4, C

4

148.8, C

5

a 14

160.4, C

16

134.0, C

17

121.0, CH

7.55, d (8.6)

H18

C16, 19, 21

18

129.3, CH

7.33, dd (8.4, 2.2)

H17

C16, 19, 20

19

131.4, C

20

129.5, CH

7.15, br d (2.2)

H22, 25

C16, 18, 19, 22

21

124.5, C

22 b

30.0, CH2

2.61, d (16.1)

H20, 24, 25

C10, 14, 16, 20, 21, 23, 24

2.78, d (15.7)

H8, 20

C10, 14, 16, 20, 21, 23, 24

a 23

41.2, C

24 a

25.2, CH2

b 25 a

48.1, CH2

b

2.38, dt (15.1, 8.1)

H11, 25

C14, 22, 23, 25

2.01, ddd (14.7, 7.0, 2.2)

H22, 25

C10, 22, 23, 25

3.67, br dd (13.8, 7.3)

H24, 26

C2, 23, 24

3.42, m

H22, 24, 26

C2, 23, 24

H25

C2, 25

H8, 11, 12

C12, 14

26

39.9, CH3

3.27 (s)

27

40.6, CH3

3.53 (s)

a

multiplicity from multiplicity edited HSQC data. omitted

b

NOESY and ROESY interactions, geminal NOE's

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Caulamidine A (1) TFA Salt 1H NMR Spectrum (500 MHz, CD3CN)

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Caulamidine A (1) TFA Salt 13C NMR Spectrum (125 MHz, CD3CN)

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Caulamidine A (1) TFA Salt COSY Spectrum (CD3CN)

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Caulamidine A (1) TFA Salt HSQC Spectrum (CD3CN)

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Caulamidine A (1) TFA Salt 1H-13C HMBC Spectrum (CD3CN) Optimized for 8.3 Hz

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Caulamidine A (1) TFA Salt ROESY Spectrum (CD3CN)

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Selective (H-8) 1D NOESY spectrum of caulamidine A (1) TFA salt in CD3CN

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Selective (H-22a) 1D NOESY spectrum of caulamidine in CD3CN.

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Selective (H24a) 1D NOESY spectrum of caulamidine in CD3CN.

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Selective (H11) 1D NOESY spectrum of caulamidine in CD3CN.

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214x159mm (300 x 300 DPI)

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Page 79 of 80 Chemical Science DOI: 10.1039/C7SC01996C

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DOI: 10.1039/C7SC01996C