Article pubs.acs.org/jnp
Synthesis of Natural Cyclopentapeptides Isolated from Dianthus chinensis Shengping Zhang,† Zaid Amso,† Luis M. De Leon Rodriguez,‡ Harveen Kaur,† and Margaret A. Brimble*,†,‡ †
School of Chemical Sciences, The University of Auckland, 23 Symonds Street, Auckland, 1142, New Zealand Maurice Wilkins Centre for Molecular Biodiscovery, 23 Symonds Street, The University of Auckland, Auckland, 1142, New Zealand
‡
S Supporting Information *
ABSTRACT: The first syntheses of the naturally occurring cyclic peptides dianthin I (1), pseudostellarin A (2), and heterophyllin J (3) are described. The linear protected peptide precursors were prepared efficiently via Fmoc-solid-phase synthesis and subsequently cyclized in solution under dilute conditions. The structures of the synthetic cyclopentapeptides were confirmed by NMR spectroscopy and mass spectrometry and were in agreement with the literature data reported for the natural products.
P
conformation.13 This conformation is especially favored when the peptide sequence contains residues such as glycine, proline, and N-methylated amino acids that can induce torsion angles similar to the ones observed for β-turn structures, thereby bringing the N- and C-termini into close proximity.14 In addition, side reactions, such as epimerization and dimerization, can also occur during the cyclization. These side reactions have a negative impact on the final yield and therefore should be minimized. Compounds 1−3 contain proline and glycine residues within their structures; hence it was envisaged that cyclization of their linear precursors in solution phase would not be problematic by adopting conventional cyclization protocols. The achiral glycine was selected as the C-terminal residue for the cyclization site in order to avoid racemization and minimize steric hindrance around the carbonyl activated group.12 Peptide dimerization is minimized by conducting the macrolactamization reaction at a very dilute peptide concentration (less than 1 mM).13,15 Importantly, dianthin I (1) contains one serine residue within its sequence, while both pseudostellarin A (2) and heterophyllin J (3) have one tyrosine residue instead. Given the different pKa values of the hydroxy functionality of serine and tyrosine (∼16 and 10.4, respectively), one would expect that the hydroxy group of tyrosine could be readily deprotonated in the presence of a base, such as N,N-diisopropylethylamine (DIPEA), and subsequently undergo undesirable reactions with coupling reagents, such as O-(benzotriazol-1-yl)-N,N,N′,N′-
lants of the Caryophyllaceae family are a rich source of cyclic peptides that exhibit exceptional and effective biological activities, including estrogen-like, cytotoxic, and cell proliferative activities.1,2 Dianthus chinensis L., a member of the Caryophyllaceae family, is a small herb distributed in the northern part of mainland China, as well as other countries including Korea and Mongolia. This plant, known as “Qu mai” in Mandarin, is an important traditional Chinese medicine used for the treatment of an array of health problems, in particular urinary tract problems and various types of cancer.3 Recently, three cyclic peptides, dianthin I (1), pseudostellarin A (2), and heterophyllin J (3) (Figure 1), were isolated from the aerial parts of D. chinensis.4 Their primary structures and absolute configuration were determined and shown to consist of four amino acids adopting the L-configuration and an achiral glycine residue. Cyclopentapeptides consisting solely of naturally occurring amino acids are rarely isolated from Nature and, due to the small to medium ring size involved, are also challenging and seldomly synthesized.5,6 Herein, we report the first total synthesis of these three naturally occurring cyclopentapeptides along with their structural confirmation.
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RESULTS AND DISCUSSION Peptide macrolactamization is an essential strategy when undertaking the preparation of cyclic peptides.7,8 However, despite abundant reports of methods for the cyclization of linear peptides,9−12 the effectiveness of an individual process is still highly dependent on the peptide sequence. In fact, the success of peptide cyclization is predominantly dictated by the propensity of linear peptides to attain a suitable “ring-closure” © XXXX American Chemical Society and American Society of Pharmacognosy
Received: February 21, 2016
A
DOI: 10.1021/acs.jnatprod.6b00152 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 1. Structures of dianthin I (1), pseudostellarin A (2), and heterophyllin J (3).
Scheme 1. Synthesis of (A) Dianthin I (1), (B) Pseudostellarin A (2), and Heterophyllin J (3)a
a Reagents and conditions: (i) Fmoc-Gly-HMPP (2 equiv), DIC (2 equiv), CH2Cl2−DMF (1:1), rt, 5 h; (ii) 20% piperidine in DMF, rt, 2 × 10 min; (iii) Fmoc-amino acid (4 equiv), HBTU (3.9 equiv), DIPEA (8 equiv), DMF, 75 °C, 25 W, 5 min; (iv) 20% piperidine in DMF, 75 °C, 35 W, 1 min, repeat once more, 50 W, 3 min; (v) repeat (iii) and (iv) until sequence completion; (vi) TFA−iPr3SiH−H2O (95:2.5:2.5), rt, 2 h (>95% purity); (vii) HBTU (3 equiv), 6-Cl-HOBt (3 equiv), DIPEA (5 equiv), CH2Cl2−DMF (4:1), rt, 22 h; (viii) HMPB linker (2 equiv), DIC (2 equiv), CH2Cl2−DMF (2:1), rt, 5 h; (ix) Fmoc-glycine (3 equiv), DIC (3 equiv), DMAP (0.02 equiv), DMF, rt, 1 h; (x) 1% TFA in CH2Cl2, rt, 4 ×3 min; (xi) 50% TFA in CH2Cl2, rt, 20 min.
in high purity (Scheme 1A). On the other hand, the super-acidlabile 4-(4-hydroxymethyl-3-methoxyphenoxy)butyric acid (HMPB) linker was used for the synthesis of linear precursors 5 and 6 of pseudostellarin A (2) and heterophyllin J (3), respectively, in order to preserve the tBu protecting group on the hydroxy group of tyrosine during peptide cleavage. In this case, the HMPB linker was first attached to the resin using a DIC-mediated reaction, which was then followed by the coupling of Fmoc-glycine using DIC−DMAP. After sequence elongation the protected linear peptide was cleaved from the linker using a 1% TFA solution in CH2Cl2, and the resulting peptides 5 and 6 were used directly for the macrolactamization reaction without further purification (Scheme 1B). The key cyclization to afford the three cyclopentapeptides 1−3 from 4−6, respectively, was performed successfully by addition of a mixture of the appropriate linear peptide, HBTU, and 6-chloro-1-hydroxybenzotriazole (6-Cl-HOBt) in CH2Cl2−
tetramethyluronium hexafluorophosphate (HBTU). We therefore decided to prepare dianthin I (1) from its unprotected linear precursor 4, while pseudostellarin A (2) and heterophyllin J (3) were prepared from their tyrosine hydroxy groupprotected linear precursors 5 and 6, respectively. The three linear precursors of cyclopentapeptides 1−3 were synthesized on aminomethyl polystyrene resin using FmocSPPS (Scheme 1). For unprotected dianthin I precursor 4, a commercially available hydroxymethylphenoxypropionic acid (HMPP) linker bound to Fmoc-glycine initially was attached to the resin using N,N-diisopropylcarbodiimide (DIC), followed by removal of the Fmoc group with 20% piperidine in DMF. The coupling of the remaining amino acids was carried out using HBTU and DIPEA in DMF. Following the final Fmoc deprotection, treatment of the resin with 95% trifluoroacetic acid (TFA) released the resin-bound peptide with simultaneous removal of the protecting group on serine, giving compound 4 B
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DMF (4:1) at a rate of 10 mL h−1 to a stirring solution of DIPEA in CH2Cl2 to give a final peptide concentration of 0.7− 0.8 mM.2 The reaction was generally complete within 20 h after addition of the reagents. HPLC analysis indicated that the cyclic peptides were the major products with no detectable linear starting material present. The reaction mixture was then concentrated and treated with 50% TFA in CH2Cl2 if side-chain protecting group removal was required. Finally, the title cyclopentapeptides 1−3 were purified by semipreparative RPHPLC in 71%, 76%, and 68% yield, respectively. The cyclopeptides were characterized by HPLC, MS, 1H and 13C NMR, IR, optical rotation, and CD spectroscopy (see Supporting Information). The spectroscopic data of synthetic compounds 1−3 were in agreement with those reported for the corresponding natural products,3 thus confirming the elucidated structures (Tables 1−3). Furthermore, the NMR data indicated that proline adopted a trans conformation in all three cyclopentapeptides, thus indicating a relatively high-energy threshold for interexchange between the cis and trans conformers.2 The CD spectra of compounds 1−3 were also recorded in order to gain further structural information. Typically, cyclic pentapeptides are known to be composed of γ- and β-turns in solution.16 These features are observed in the CD spectra of compounds 1−3. The CD spectrum of compound 1 showed two minima at 197 and 215 nm (Figure S5, Supporting Information), assigned to a random coil and a β-turn I configuration, respectively. The CD spectrum of compound 2 showed a broad negative signal with a minimum around 210 nm (Figure S10, Supporting Information), which suggested a β-turn I, and the CD spectrum of compound 3 contains a broad negative signal at around 223 nm (Figure S15, Supporting Information), which is commonly assigned to a γ-turn.16,17 While cyclization of the linear protected precursors of 2 and 3 proceeded smoothly, it is often desirable to use unprotected linear peptides in order to eliminate the extra step required for protecting group removal. We therefore investigated the cyclization reaction using a linear peptide containing an unprotected tyrosine residue. Accordingly, the unprotected linear precursor 7 of pseudostellarin A (2) (Scheme 2) was synthesized following the protocol described for the preparation of 4 (Scheme 1). Cyclization of 7 using the conventional HBTU, 6-Cl-HOBt, and DIPEA coupling mixture afforded one major product, for which the molecular mass determined by high-resolution mass spectrometry (HRMS) (Figure S16, Supporting Information) indicated formation of the tetramethyluronium pseudostellarin A derivative 8 (Scheme 2). Given that the cyclization reaction was carried out with an excess of coupling reagent (HBTU, 3 equiv), the deprotonated phenoxide side chain of tyrosine is able to react with HBTU, thus forming the tetramethyluronium derivative. Subsequent treatment of this tetramethyluronium derivative with aqueous 0.1 M NaOH solution afforded pseudostellarin A (2) within 2 h (Figure 2). Thus, although pseudostellarin A (2) can also be prepared from its linear unprotected peptide precursor 7, one extra step was still required for the conversion of 8 to 2, which is equal to the number of steps required to prepare 2 from its linear protected precursor 5. In summary, three naturally occurring cyclic pentapeptides, namely, dianthin I (1), pseudostellarin A (2), and heterophyllin J (3), were prepared successfully in good yield. Spectroscopic
Table 1. 1H and 13C NMR Spectroscopic Data (400 MHz, C5D5N) of Synthetic and Naturally Occurring Dianthin I (1) synthetic dianthin I (1) δH (J in Hz)
δC
δH (J in Hz)
δC
3.67 (1H, m) 4.83 (1H, dd, 16.2, 9.5)
43.9
3.69 (1H, d, 16.1) 4.84 (1H, dd, 16.1, 9.5) 8.17 (1H, d, 9.5)
43.9
residue Gly1 α
NH CO Phe2 α
8.16 (1H, d, 7.2)
5.39 (1H, m)
55.8
β
3.44 (1H, m)
40.3
171.2
3.90 (1H, m) γ δ ε ζ NH CO Pro3 α β γ δ
CO Ser4 α β NH CO Phe5 α β γ δ ε ζ NH CO
natural product
7.59 7.44 7.27 8.67
(2H, (2H, (1H, (1H,
m) m) m) d, 8.7)
138.8 130.7 129.7 128.3
171.1 5.40 (1H, dd, 13.6, 8.8) 3.45 (1H, dd, 13.6, 7.5) 3.94 (1H, dd, 13.6, 7.5) 7.57 7.42 7.25 8.68
(2H, (2H, (1H, (1H,
overlap) t, 7.5) overlap) d, 8.8)
171.6 4.46 1.76 2.06 1.49 1.76 3.44 3.67
(1H, (1H, (1H, (1H, (1H, (1H, (1H,
d, 7.0) m) m) m) m) m) m)
61.9 32.6 22.2 49.1
58.2 62.0
4.46 (1H, 1.75 (1H, 2.05 (1H, 1.50 (1H, 1.75 (1H, 3.41 (1H, 3.61 (1H, 7.6)
d, 7.6) overlap) m) m) overlap) overlap) dd, 10.8,
7.44 (2H, m) 7.27 (2H, m) 7.27 (1H, m) 10.78 (1H, d, 6.9)
58.4 35.7
140.5 130.5 129.1 127.0 171.8
138.7 130.5 129.2 127.2
62.0 32.5 22.1 49.0
175.9 5.09 (1H, dd, 13.0, 6.5) 4.25 (2H, t, 6.5) 8.66 (1H, d, 6.5)
174.2 4.34 (1H, m) 3.73 (1H, m) 3.90 (1H, m)
40.3
171.6
176.0 5.08 (1H, q, 6.52, 6.52, 6.51) 4.26 (2H, d, 6.5) 8.63 (1H, d, 6.9)
56.1
58.0 61.9 174.1
4.30 (1H, overlap) 3.76 (1H, d, 11.2) 3.89 (1H, dd, 11.2, 3.7) 7.45 (2H, d, 7.5) 7.30 (2H, t, 6.5) 7.25 (1H, overlap) 10.86 (1H, d, 6.9)
58.4 35.6
140.4 130.4 129.0 126.9 171.7
analysis of these synthetic compounds was consistent with the reported data for the natural products.
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EXPERIMENTAL SECTION
General Experimental Procedures. All reagents were purchased from commercial sources and used without further purification. Solvents for routine peptide synthesis and RP-HPLC were purchased as synthesis grade and HPLC grade, respectively. Aminomethyl polystyrene resin was purchased from Rapp Polymere (Tuebingen, Germany). Fmoc-Gly-O-CH2-Phi-OCH2-CH2-COOH (Fmoc-GlyHMPP) was purchased from Polypeptide Laboratories Group (Limhamn, Sweden). HBTU and 6-Cl-HOBt were supplied by GL Biochem (Shanghai, People’s Republic of China). DIC, DIPEA, triisopropylsilane (iPr3SiH), and piperidine were purchased from C
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Table 2. 1H and 13C NMR Spectroscopic Data (400 MHz, C5D5N) of Synthetic and Naturally Occurring Pseudostellarin A (2) synthetic pseudostellarin A (2) residue Gly1 α
NH CO Pro2 α β γ δ CO Tyr3 α β γ δ ε
δH (J in Hz) 4.08 (1H, dd, 14.6, 6.7) 4.30 (1H, dd, 14.0, 4.2) 9.16 (1H, d, 8.4)
β γ δ NH CO Ala5 α β NH CO
δH (J in Hz) 4.06 (1H, br s)
residue
42.6
Gly1 α
dd, 7.9,
62.7
4.52 (1H, br s)
62.7
m) m) m) m) m) br s)
30.3
1.26 1.90 1.60 1.93 3.35 4.20
30.3
5.31 (1H, dd, 16.0, 9.0) 3.45 (2H, m) 7.42 (2H, d, 8.4)
25.3 48.1
57.3 38.3 128.9 131.3
(1H, (1H, (1H, (1H, (1H, (1H,
m) overlap) overlap) overlap) m) br s)
γ
25.3
δ
48.2
5.32 (1H, dd, 17.5, 9.8) 3.48 (2H, m) 7.43 (2H, d, 8.0)
158.0
57.4 37.8 129.1 131.5
NH CO Tyr4 α β
158.1 8.41 (1H, br s)
172.6
173.2
dd, 15.6,
56.2
4.65 (1H, br s)
56.2
m) m) m) d, 6.5) d, 6.5) d, 8.0)
40.9
1.88 2.26 1.70 0.83 0.85 9.47
40.4
25.6 22.4 23.3
(1H, (1H, (1H, (3H, (3H, (1H,
overlap) m) overlap) d, 6.5) d, 6.5) overlap)
173.0 58.4 18.2 174.7
γ δ ε ζ NH CO Ala5 α β NH CO
25.6 22.3 23.5 173.6
4.95 (1H, d, 7.0) 1.59 (3H, d, 7.0) 9.21 (1H, br s)
δH (J in Hz) 4.06 (1H, dd, 14.7, 5.8) 4.39 (1H, dd, 14.5, 4.5) 8.66 (1H, t, 4.8)
51.1 18.1 175.5
natural product
δC 42.7
δH (J in Hz) 3.99 (1H, dd, 12.3, 3.9) 4.43 (1H, dd, 12.3, 3.9) 8.84 (1H, br s)
169.4 4.60 2.05 2.16 1.75 2.05 3.39
(1H, (1H, (1H, (1H, (1H, (1H,
m) m) m) m) m) m)
63.0 30.4 25.5 47.7
4.00 (1H, m) CO Val3 α β γ
172.7
7.14 (2H, d, 8.0)
8.21 (1H, d, 9.1)
5.01 (1H, m) 1.62 (3H, d, 7.1) 8.97 (1H, d, 4.4)
NH CO Pro2 α β
170.0
172.5
4.65 (1H, 8.3) 1.93 (1H, 2.23 (1H, 1.93 (1H, 0.86 (3H, 0.90 (3H, 9.30 (1H,
δC
9.44 (1H, overlap) 169.7
4.54 (1H, 4.5) 1.71 (1H, 1.93 (1H, 1.71 (1H, 1.93 (1H, 3.34 (1H, 4.20 (1H,
synthetic heterophyllin J (3)
4.30 (1H, d, 13.4)
7.14 (2H, d, 8.4) ζ NH CO Leu4 α
natural product
δC 42.6
Table 3. 1H and 13C NMR Spectroscopic Data (400 MHz, C5D5N) of Synthetic and Naturally Occurring Heterophyllin J (3)
(1H, (1H, (3H, (3H, (1H,
m) m) d, 6.7) d, 6.7) d, 9.1)
61.6 31.7 19.4 20.2
4.59 (1H, 2.04 (1H, 2.12 (1H, 1.70 (1H, 1.98 (1H, 3.34 (1H, 5.9) 3.93 (1H,
overlap) m) m) m) m) dd, 13.6,
7.35 (2H, d, 8.5) 7.13 (2H, d, 8.5)
59.2 37.6
128.8 131.3 116.7 158.1
9.37 (1H, d, 8.0)
25.6 47.7
173.1 4.59 2.40 0.95 1.03 8.03
(1H, (1H, (3H, (3H, (1H,
overlap) m) d, 5.6) d, 5.6) d, 7.0)
61.6 31.4 19.6 20.4 173.0
4.84 (1H, overlap) 3.47 (1H, dd, 11.3, 7.5) 3.59 (1H, dd, 11.3, 7.5) 7.34 (2H, d, 7.0) 7.14 (2H, d, 7.0)
59.5 37.2
129.0 131.4 116.8 158.2
9.49 (1H, overlap) 172.2
4.87 (1H, m) 1.62 (3H, d, 7.1) 9.43 (1H, d, 8.3)
63.1 30.4
m)
172.5 4.87 (1H, m) 3.47 (1H, dd, 13.6, 7.5) 3.56 (1H, dd, 13.6, 7.5)
42.9
169.6
172.9 4.60 2.38 0.95 1.04 8.01
δC
50.5 17.5 174.1
172.6 4.84 (1H, overlap) 1.61 (3H, d, 5.9) 9.51 (1H, overlap)
50.7 17.4 174.6
Bruker micrOTOFQ mass spectrometer. All the analytical RP-HPLC experiments were carried out using an analytical column (XTerra MS C18, 125 Å, 4.6 mm × 150 mm, 5 μm) on a Dionex Ultimate 3000 System with a 35 min linear gradient of 5−75% solvent B (where solvent A was 0.1% TFA in water and solvent B was 0.1% TFA acetonitrile) at a flow rate of 1 mL min−1, and UV signals were detected at wavelengths 210, 225, 254, and 280 nm. Semipreparative RP-HPLC was performed on a Waters 600 system using a semipreparative column (XTerra MS C18 prep column, 125 Å, 19 mm × 300 mm, 10 μm) at a flow rate of 5 mL min−1 with a suitable gradient of 5−75% solvent B adjusted according to the elution profiles from analytical RP-HPLC chromatography. LC-MS were obtained on either an Agilent Technologies 1120 Compact LC equipped with a Hewlett-Packard 1100 MSD mass spectrometer or an Agilent Technologies 1260 Infinity LC equipped with an Agilent Technologies 6120 Quadrupole mass spectrometer using an analytical column (Phenomenex Gemini C18, 110 Å, 50 mm × 200 mm, 5 μm) with a 35 min linear gradient of 5−75% solvent B at a flow rate of 0.3 mL min−1.
Sigma-Aldrich (St. Louis, MO, USA). TFA, acetonitrile (HPLC grade), and DMF (AR grade) were provided by Scharlau (Barcelona, Spain). CH2Cl2 (AR grade) was purchased from ECP Limited (Auckland, New Zealand). HMPB linker was supplied by Novabiochem (Merck, Germany). The following Fmoc-amino acids were purchased from GL Biochem: Fmoc-Pro-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Leu-OH, and Fmoc-protected amino acids with the following side chain protection: Fmoc-Ser(tBu)OH and Fmoc-Tyr(tBu)-OH (tBu = tert-butyl). Optical rotations were measured at the sodium D line (589 nm) at 20 °C using a PerkinElmer 341 instrument. Infrared spectra were recorded on a PerkinElmer Spectrum 100 infrared spectrometer, and 64 scans were collected for each spectrum at a resolution of 4 cm−1. Nuclear magnetic resonance experiments were performed on a Bruker AVANCE 400 spectrometer (1H 400 MHz; 13C 100 MHz) in deuterated pyridine. High-resolution mass spectra were obtained on a D
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Scheme 2. Formation of the Tetramethyluronium Pseudostellarin A Derivative 8a
a
Reagents and conditions: (i) HBTU (3 equiv), 6-Cl-HOBt (3 equiv), DIPEA (5 equiv), CH2Cl2−DMF (4:1), rt, 22 h. TFA−iPr3SiH−H2O (95:2.5:2.5) for 2 h at rt. The resin was then filtered and washed with neat TFA (2 × 3 mL). Cold Et2O was added to the combined TFA filtrates, and the precipitated product was isolated by centrifugation (4000 rpm, 6 min). The precipitate was dissolved in 50% aqueous acetonitrile and lyophilized. The fully protected linear peptides 5 and 6 were synthesized on aminomethyl polystyrene resin on a scale of 0.2 mmol using an HMPB linker. At first a mixture of HMPB linker (2 equiv) and DIC (2 equiv) in CH2Cl2−DMF (2:1) was added to the preswollen resin and agitated for 5 h. The resulting HMPB-attached resin was then treated with a solution of Fmoc-Gly (3 equiv), DIC (3 equiv), and DMAP (0.02 equiv) in DMF for 1 h followed by removal of the Fmoc group using 20% piperidine in DMF (5 mL, 2 × 10 min). Peptide elongation was completed using the same method as mentioned above. The fully protected linear peptides 5 and 6 were released from the resin by treatment of 1% TFA in CH2Cl2 (4 × 3 min), followed by removing the resin by filtration. The solvent was then evaporated under reduced pressure, and the residue was dissolved in 70% aqueous acetonitrile and lyophilized. Unprotected linear dianthin I (4): tR 13.5 min (22.1% B); EIMS m/z 554.2 [M + H]+; EIMS/MS m/z (%) 554.2 (100) (calcd for C28H36N5O7, 554.2). Protected linear pseudostellarin A (5): tR 15.5 min (36.1% B); EIMS m/z 576.2 [M + H]+; EIMS/MS m/z (%) 576.2 (100) (calcd for C29H46N5O7, 576.3). Protected linear heterophyllin J (6): tR 14.6 min (34.2% B); EIMS m/z 562.2 [M + H]+; EIMS/MS m/z (%) 562.2 (100) (calcd for C28H44N5O7, 562.3). Unprotected linear pseudostellarin A (7): tR 10.0 min (24.9% B); EIMS m/z 520.2 [M + H]+; EIMS/MS m/z (%) 520.2 (100) (calcd for C25H38N5O7, 520.3). Dianthin I (1). To a stirred solution of DIPEA (200 μL, 1.15 mmol) in CH2Cl2 (230 mL) was added a mixture of 4 (126 mg, 0.23 mmol), HBTU (262 mg, 0.69 mmol), and 6-Cl-HOBt (117 mg, 0.69 mmol) in CH2Cl2−DMF (4:1, 50 mL) via a syringe pump at rt for 2 h, and the resulting reaction solution was stirred for another 20 h. The reaction solution was then concentrated under reduced pressure, diluted with 0.1% (v/v) TFA−water, and subjected to purification using semipreparative RP-HPLC to give 1 as a colorless solid (88 mg, 71%); tR 16.8 min (38.5% B); [α]24D 64.6 (c 0.10, MeOH); IR 3280, 2937, 1630, 1529, 1444, 1207, 1174, 746, 700 cm−1; 1H NMR and 13C NMR data, see Table 1; HREIMS m/z 558.2431 [M + Na]+ (calcd for C28H33N5NaO6, 558.2323). Pseudostellarin A (2). To a stirred solution of DIPEA (174 μL, 1.0 mmol) in CH2Cl2 (200 mL) was added a mixture of 5 (115 mg, 0.2 mmol), HBTU (228 mg, 0.6 mmol), and 6-Cl-HOBt (102 mg, 0.6 mmol) in CH2Cl2−DMF (4:1, 50 mL) via a syringe pump at rt within 2 h, and the resulting reaction solution was allowed to stir for another 20 h. After concentration under reduced pressure, the reaction was treated with 50% TFA in CH2Cl2 (5 mL) for 20 min followed by TFA removal under nitrogen flow. Further purification of the remaining
Figure 2. HPLC spectrum of (A) synthetic pseudostellarin A (2), (B) tetramethyluronium byproduct (8), and (C) the reaction mixture of 8 treated with aqueous 0.1 M NaOH solution. The HPLC profiles were stacked for clarity. A syringe pump (New Era Pump System Inc., model NE-1000) was used for dropwise addition. All CD spectra were recorded using a Pi Star-180 (Applied Photophysics, Surrey, UK) spectrometer at 20 °C in 1 mm quartz cuvettes (Hellma Analytics, Mullheim, Germany) with a cell of 0.1 cm path length in the range from 190 to 300 nm at 0.5 nm intervals with a 5 s response time. Each CD spectrum measurement represents the average of three scans obtained with a 2 nm optical bandwidth. A baseline spectrum was collected with the solvent alone (100% methanol) and then subtracted from the raw peptide spectra. The measurements were performed at peptide concentrations of 80 μM in 100% methanol. Data are expressed as mean residue ellipticities [θ] in (deg cm2 dmol−1) and calculated as θ = S/(10c × L × n), where S is the raw CD signal in millidegrees, c is the peptide concentration (M), L is the cuvette path length (cm), and n is the number of peptide bonds. Synthesis of Linear Precursors. The linear precursors of dianthin I, heterophyllin J, and pseudostellarin A, namely, peptides 4−7, were synthesized using a microwave-assisted Fmoc/tBu strategy. The deprotected linear precursors 4 and 7 were prepared on a 0.2 mmol scale using a HMPP linker. To the preswollen (in CH2Cl2) aminomethyl polystyrene resin was added a mixture of Fmoc-GlyHMPP (2 equiv) and DIC (2 equiv) in CH2Cl2−DMF (1:1), and the resulting mixture was agitated for 5 h. A Kaiser test was performed afterward to confirm completion of the coupling. After the removal of the first Fmoc group with 20% piperidine in DMF (5 mL, 2 × 10 min), the remaining amino acids in the sequence were coupled using a mixture of Fmoc-protected amino acid (4 equiv), HBTU (3.9 equiv), and DIPEA (8 equiv) in DMF (4 mL) in a CEM Discover microwave reactor. The couplings were performed for 5 min at a microwave power of 25 W with a maximum temperature of 75 °C. Fmoc deprotection was also performed under the same temperature by using 5 mL of 20% piperidine in DMF at 35 W for 1 min followed by a second deprotection at 50 W for 3 min. After the assembly step, the final peptides 4 and 7 were cleaved from the resin with a solution of E
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Journal of Natural Products
Article
residue using semipreparative RP-HPLC afforded 2 as a colorless solid (76 mg, 76%): tR 15.5 min (36.0% B); [α]24D −66.7 (c 0.075, MeOH); IR 3289, 2958, 2870, 1638, 1515, 1447, 1207, 1159, 827, 700 cm−1; 1H NMR and 13C NMR data, see Table 2; HREIMS m/z 524.2466 [M + Na]+ (calcd for C28H33N5NaO6, 524.2587). Heterophyllin J (3). Compound 3 was prepared from 6 (123 mg, 0.22 mmol) using the same method described for the synthesis of 2. Purification by semipreparative RP-HPLC gave 3 as a colorless solid (73 mg, 68%): tR 14.1 min (33.1% B); [α]24D −100.8 (c 0.10, MeOH); IR 3314, 2965, 1652, 1637, 1515, 1449, 1203, 1166, 828 cm−1; 1H NMR and 13C NMR data, see Table 3; HREIMS m/z 510.2322 [M + Na]+ (calcd for C28H33N5NaO6, 510.2431).
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00152. The HREIMS of compound 8 as well as the LC-MS profiles and IR, CD, 1H NMR, and 13C NMR spectra of compounds 1−3 (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Tel: +64 9 9238259. Fax: +64 9 3737422. E-mail: m.brimble@ auckland.ac.nz. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the China Scholarship Council for financial support (S.Z.). REFERENCES
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DOI: 10.1021/acs.jnatprod.6b00152 J. Nat. Prod. XXXX, XXX, XXX−XXX
