Supplementary Information
Cycloexpansamines A and B: Spiroindolinone alkaloids from a
marine isolate of Penicillium sp. (SF-5292)
Running Head: Spiroindolinone alkaloids from a marine isolate of Penicillium sp.
Chiwook Lee,1,2 Jae Hak Sohn,2 Jae-Hyuk Jang,3 Jong Seog Ahn,3 Hyuncheol Oh,1 Jonas
4
Baltrusaitis , In Hyun Hwang5, and James B. Gloer5
1
College of Pharmacy, Wonkwang University, Iksan, Korea; 2College of Medical and Life
Sciences, Silla University, Busan, Korea; 3Chemical Biology Research Center, Korea Research
Institute of Bioscience and Biotechnology (KRIBB), Cheongwon, Korea; 4PhotoCatalytic
Synthesis Group, University of Twente, Enschede, The Netherlands, and 5Department of
Chemistry, University of Iowa, Iowa City, IA, USA
Correspondence. Professor Hyuncheol Oh, College of Pharmacy, Wonkwang University, Iksan
570-749, Korea. E-mail: hoh@wonkwang.ac.kr
List of Supplementary Information
Experimental Section
Physical Properties of Compounds 1 and 2
Figure S1. Experimental ECD spectrum (top) and TDDFT-calculated ECD spectrum
(bottom) with energy-minimized model of 1.
Figure S2. 1H NMR spectrum of cycloexpansamine A (1, 400 MHz, pyridine-d5)
Figure S3.
13
C NMR spectrum of cycloexpansamine A (1, 100 MHz, pyridine-d5)
Figure S4. HMQC spectrum of cycloexpansamine A (1, 400 MHz, pyridine-d5)
Figure S5. HMBC spectrum of cycloexpansamine A (1, 400 MHz, pyridine-d5)
Figure S6. COSY spectrum of cycloexpansamine A (1, 400 MHz, pyridine-d5)
Figure S7. NOESY spectrum of cycloexpansamine A (1, 600 MHz, pyridine-d5)
Figure S8. 1H NMR spectrum of cycloexpansamine B (2, 600 MHz, pyridine-d5)
Figure S9.
13
C NMR spectrum of cycloexpansamine B (2, 100 MHz, pyridine-d5)
Figure S10. HSQC spectrum of cycloexpansamine B (2, 600 MHz, pyridine-d5)
Figure S11. HMBC spectrum of cycloexpansamine B (2, 600 MHz, pyridine-d5)
Figure S12. COSY spectrum of cycloexpansamine B (2, 600 MHz, pyridine-d5)
Figure S13. NOESY spectrum of cycloexpansamine B (2, 600 MHz, pyridine-d5)
Figure S14. Expanded NOESY spectrum of cycloexpansamine B (2, 600 MHz,
pyridine-d5)
Figure S15. IR spectrum of cycloexpansamine A (1)
Figure S16. IR spectrum of cycloexpansamine B (2)
2
Experimental Section
General experimental procedures. Optical rotations were recorded on a Perkin Elmer 341
digital polarimeter. UV data for compound 1 were obtained in MeOH using a Varian Cary III
UV/vis spectrophotometer and ECD data were recorded in MeOH with an Olis Cary-17
spectrophotometer (1-cm cell). UV data for compound 2 were obtained in MeOH using a
Beckman Coulter UV/vis spectrophotometer. IR spectra were obtained using a Varian 640-IR
spectrometer. NMR spectra (1D and 2D) were recorded in pyridine-d5 or CDCl3 using a JEOL
JNM ECP-400 spectrometer (400 MHz for 1H and 100 MHz for
spectrometer (600 MHz for 1H and 150 MHz for
relative to tetramethylsilane (H/C = 0).
All
13
13
C) or JEOL JNM ECA-600
C), and chemical shifts were referenced
13
C NMR multiplicities were determined by
analysis of DEPT and/or HMQC data, and are consistent with the position assignments.
HMQC/HSQC and HMBC experiments were optimized for 1JCH = 140 Hz and nJCH = 8 Hz,
respectively. ESIMS data were obtained using a Q-TOF micro LC-MS/MS instrument (Waters,
USA) at Korea University, Seoul, Korea.
Solvents for extractions and flash column
chromatography were reagent grade and used without further purification. Solvents used for
HPLC were analytical grade.
Flash column chromatography was carried out using YMC
octadecyl-functionalized silica gel (C18). HPLC separations were performed on a Shiseido
Capcell Pak C18 column (10  250 mm; 5 m particle size; 2 ml/min) or Agilent prep-C18
column (21.2  150 mm; 5 m particle size; 5 ml/min). Compounds were detected by UV
absorption at 210 nm.
Energy minimization and ECD calculations.
Initial structures were obtained from a
Spartan’10 conformation search using MMFF,1 then further fully optimized using the 6-31G(d)
basis set2,3 combined with the M06-2X functional4 followed by frequency calculations using the
3
same level of theory. Absence of the imaginary frequencies confirmed that all conformers were
minima on the potential energy surface.
No symmetry constraints were used during the
optimization. Solvent methanol and the PCM solvation model5 were used in Gaussian 09 Rev.
B0.1.6
Time-dependent density functional (TDDFT) calculations were performed to calculate ECD
spectra at the optimized geometry using the same basis set and functional. A total of 20 excited
states were calculated, and only singlet excited states were considered. For TDDFT calculations,
B3LYP/aug-cc-pVDZ combined with COSMO solvation were used as implemented in NWChem
6.3.7 This combination of density functional and basis set has been shown to provide good ECD
simulations.8 To visualize the ECD spectra, SpecDis version 1.62 software by Dr. Torsten Bruhn
was used.9
1
Shao, Y. et al. Advances in methods and algorithms in a modern quantum chemistry program package.
Phys. Chem. Chem. Phys. 8, 3172-3191 (2006).
2
Ditchfield, R., Hehre, W. J. & Pople, J. A. Self‐Consistent Molecular‐Orbital Methods. IX.
An
Extended Gaussian‐Type Basis for Molecular‐Orbital Studies of Organic Molecules. J. Chem. Phys. 54,
724-728 (1971).
3
Hehre, W. J., Ditchfield, R. & Pople, J. A. Self-Consistent Molecular Orbital Methods. XII.
Further
Extensions of Gaussian—Type Basis Sets for Use in Molecular Orbital Studies of Organic Molecules
J. Chem. Phys. 56, 2257-2261 (1972).
4
Zhao, Y. & Truhlar, D. The M06 suite of density functionals for main group thermochemistry,
thermochemical kinetics, noncovalent interactions, excited states, and transition elements: two new
functionals and systematic testing of four M06-class
functionals and 12 other functionals
Theor.
Chem. Acc. 120, 215-241 (2008).
5
Mennucci, B.; Tomasi, J. Continuum solvation models: A new approach to the problem of solute’s
charge distribution and cavity boundaries. J. Chem. Phys. 1997, 106, 5151-5158.
4
6
Frisch, M. J. et al. Gaussian 09, revision B.01. Gaussian, Inc., Wallingdorf, CT, 2009.
7
Valiev, M. et al. NWChem: A comprehensive and scalable open-source solution for
large
scale
molecular simulations. Comput. Phys. Commun. 181, 1477-1489 (2010).
8
Autschbach, J. Time-dependent density functional theory for calculating origin- independent
rotation and rotatory strength tensors. ChemPhysChem 12, 32249
optical
3235 (2011).
Bruhn, T.; A.Schaumloffel; Y.Hemberger; Bringmann, G. SpecDis, Version 1.62, University of
Wuerzburg, Germany, 2013.
PTP1B assay procedures.
PTP1B (human, recombinant) was purchased from BIOMOL
Research Laboratories, Inc. The enzyme activity was measured in a reaction mixture containing
2 mM p-nitrophenyl phosphate (pNPP) in 50 mM citrate, pH 6.0, 0.1 M NaCl, 1 mM EDTA and
1mM dithiothreitol (DTT). The reaction mixture was placed in a 30 oC incubator for 30 min, and
the reaction was terminated by the addition of 10 N NaOH. The amount of produced pnitrophenol was estimated by measuring the increase in absorbance at 405 nm. The nonenzymatic hydrolysis of 2 mM pNPP was corrected by measuring the increase in absorbance at
405 nm obtained in the absence of PTP1B enzyme. A known PTP1B inhibitor, ursolic acid (IC50
= 3.08 M) was used as a positive control in the assay.10,11
10
Na, M. et al. Inhibition of protein tyrosine phosphatase 1B by ursane-type triterpenes isolated
from
Symplocos paniculata. Planta Med. 72, 261-263 (2006).
11
Zhang, W. et al. Ursolic acid and its derivative inhibit protein tyrosine phosphatase 1B, enhancing
insulin receptor phosphorylation and stimulating glucose uptake. Biochem
1505-1512 (2006).
5
Biophy
Acta
1760,
Physical Properties of Compounds 1 and 2
Cycloexpansamine A (1): yellow solid; []25D + 33 (c 1.39, MeOH); UV (MeOH) λmax (log ε)
263 (3.78), 348 (3.26), and 365 (3.37) nm; CD (MeOH) λmax (Δε) 227 (+23), 242 (−11), 267
(+21), 305 (−0.16), 322 (+0.74), and 360 (−2.8) nm; IR (CHCl3) max 3234, 2954, 2916, 1716,
1660, 1650, 1608, 1459, 1372 cm-1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 450.2380
[M + H]+ (calcd for C26H32N3O4, 450.2393).
Cycloexpansamine B (2): yellow solid; []25D + 20 (c 0.15, MeOH); UV (MeOH) max (log )
263 (4.12), 362 (3.83); IR (CHCl3) max 3284, 2955, 2921, 1715, 1650, 1603, 1546, 1459, 1367,
755 cm-1; 1H and
13
C NMR data, see Table 2; HRESIMS m/z 454.2349 [M + H]+ (calcd for
C25H32N3O5, 454.2342).
6
30
20
10
0
190
240
290
340
390
440
190
240
290
340
390
440
-10
-20
20
10
0
-10
-20
Figure S1. Experimental ECD spectrum (top) and TDDFT-calculated ECD spectrum
(bottom) with energy-minimized model of 1.
7
Figure S2. 1H NMR spectrum of cycloexpansamine A (1, 400 MHz, pyridine-d5)
Figure S3.
13
C NMR spectrum of cycloexpansamine A (1, 100 MHz, pyridine-d5)
9
JF34(3)-4-3-PYR-C-4.jdf
JF34(3)-4-3-HMQC-4.jdf
Y : parts per Million : 13C
200.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0
20.0
(Millions)
1.0 3.0
5.0
JF34(3)-4-3-PYR-H-8.jdf
9.0
8.0
X : parts per Million : 1H
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0
1.0
(Billions)
Figure S4. HMQC spectrum of cycloexpansamine A (1, 400 MHz, pyridine-d5)
10
2.0
3.0
JF34(3)-4-3-PYR-C-4.jdf
Y : parts per Million : 13C
210.0200.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0
(Millions)
0
JF34(3)-4-3-PYR-H-8.jdf
JF34(3)-4-3-PYR-BC-3.jdf
9.0
8.0
X : parts per Million : 1H
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0
(Billions)
Figure S5. HMBC spectrum of cycloexpansamine A (1, 400 MHz, pyridine-d5)
11
1.0
(Millions)
0
3.0 6.0
JF34(3)-4-3-PYR-H-8.jdf
JF34(3)-4-3-PYR-H-8.jdf
Y : parts per Million : 1H
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
JF34(3)-4-3-COSY-PYR-3.jdf
9.0
8.0
X : parts per Million : 1H
7.0
6.0
5.0
4.0
3.0
2.0
1.0
Figure S6. COSY spectrum of cycloexpansamine A (1, 400 MHz, pyridine-d5)
12
0 2.0 4.0 6.0 8.0
(Millions)
(Millions)
0
3.0 6.0
JF34(3)-4-3-PYR-H-8.jdf
JF34(3)-4-3-PYR-H-8.jdf
Y : parts per Million : 1H
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
JF34(3)-4-3-NOESY-3.jdf
9.0
8.0
X : parts per Million : 1H
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0 0
3.0 6.0
(Millions)
Figure S7. NOESY spectrum of cycloexpansamine A (1, 600 MHz, pyridine-d5)
13
9.0
Figure S8. 1H NMR spectrum of cycloexpansamine B (2, 600 MHz, pyridine-d5)
14
Figure S9.
13
C NMR spectrum of cycloexpansamine B (2, 100 MHz, pyridine-d5)
15
JF34(3)-4-5-2-C-7.jdf
JF34(3)-4-5-2-HSQC-4.jdf
0.6
Y : parts per Million : 13C
170.0160.0 150.0 140.0 130.0 120.0 110.0 100.0
90.0
80.0
70.0
60.0
50.0
40.0
30.0
abundance
20.0 0
0.2
0.4
JF34(3)-4-5-2-H-4.jdf
8.0
X : parts per Million : 1H
7.0
6.0
5.0
4.0
3.0
2.0
1.0
-10.0 10.0
(thousandths)
Figure S10. HSQC spectrum of cycloexpansamine B (2, 600 MHz, pyridine-d5)
16
30.0
JF34(3)-4-5-2-C-6.jdf
JF34(3)-4-5-2-HMBC-4.jdf
Y : parts per Million : 13C
200.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0
abundance
20.0 0 0.2 0.4 0.6 0.8
JF34(3)-4-5-2-H-4.jdf
7.0
X : parts per Million : 1H
6.0
5.0
4.0
3.0
2.0
1.0
-10.0 0 10.0 20.0 30.040.0
(thousandths)
Figure S11. HMBC spectrum of cycloexpansamine B (2, 600 MHz, pyridine-d5)
17
1.0
abundance
0
JF34(3)-4-5-2-H-4.jdf
JF34(3)-4-5-2-H-4.jdf
Y : parts per Million : 1H
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
JF34(3)-4-5-2-COSY-4.jdf
9.0
8.0
X : parts per Million : 1H
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0
0.2 0.4
abundance
Figure S12. COSY spectrum of cycloexpansamine B (2, 600 MHz, pyridine-d5)
18
0.6
0.8
1.0
abundance
0
JF34(3)-4-5-2-H-4.jdf
JF34(3)-4-5-2-H-4.jdf
Y : parts per Million : 1H
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
JF34(3)-4-5-2-NOESY-4.jdf
8.0
X : parts per Million : 1H
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0
abundance
Figure S13. NOESY spectrum of cycloexpansamine B (2, 600 MHz, pyridine-d5)
19
1.0
Figure S14. Expanded NOESY spectrum of cycloexpansamine B (2, 600 MHz, pyridine-d5)
20
Figure S15. IR spectrum of cycloexpansamine A (1)
21
Figure S16. IR spectrum of cycloexpansamine B (2)
22