Electronic Supporting Information      

Molecular and genetic basis for early stage structural diversifications in

hapalindole-type alkaloid biogenesis

Qin Zhua and Xinyu Liua*

aDepartment of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260

*To whom correspondence should be addressed: xinyuliu@pitt.edu

Electronic Supplementary Material (ESI) for Chemical Communications.This journal is © The Royal Society of Chemistry 2017

ESI Materials and Methods General methods. All polymerase chain reactions (PCRs) were carried out on a C1000 thermal

cycler (Bio-Rad). DNA sequencing was performed by Elim BioPharm Inc. Preparative-scale reverse-

phase HPLC was performed using a Dionex instrument equipped with Luna C18 columns (21 x 250

mm and 4.6 x 250 mm) (Phenomenex). Analytical reverse-phase HPLC was performed using a

Dionex UHPLC with a photo-diode array UV/Vis detector (Thermo Fisher Scientific) and a 4.6 x 250

mm Luna C18 column (Phenomenex). HRMS analysis was conducted using a Q Exactive Benchtop

Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) equipped with a Dionex RSLC

(Thermo Fisher Scientific). NMR spectrum was recorded on a Bruker Avance III 700 MHz

spectrometer equipped with a 1H/13C/15N triple-resonance inverse probe (1.7mm 'microprobe’).

Materials. Synthetic oligonucleotides for gene amplification by PCR were purchased from Life

Technologies or Integrated DNA Technology. Kappa HiFi DNA polymerase was obtained from Kappa

Biosystems. Restriction endonucleases, T4 DNA ligase and Antarctic phosphatase were purchased

from New England Biolabs. LB broth and agar used for culturing E. coli were obtained from Teknova.

All other reagents including inorganic salts and cofactors were purchased from Sigma-Aldrich or

Fisher Scientific unless otherwise stated.

Strains and plasmids. E. coli TOP10 cell (Life Technologies) was used for routine cloning and

plasmid propagation. E. coli C43(DE3) cell (Lucigen) was used for protein expression. pQTEV cloning

plasmid was obtained from Addgene.

GenBank# for WelU1, WelU2, WelU3, AmbU4, WelC1 and WelC3 proteins described in this

work: WelU1: AHI58823 WelU2: AHI58818 WelU3: AIH14742 AmbU4: AHB62757

WelC1: AHI58845 WelC3: AHI58829

Protein expression. Genes coding WelU1, WelU2, WelU1(27-228), WelU2(27-226), WelC1 and

WelC3 proteins were amplified from H. welwitschii UTEX B1830 genomic DNA using oligo pairs 1/2,

3/4, 5/2, 6/4, 7/8 and 9/10 (Table S1) respectively. They were ligated to the BamHI/NotI sites of

cloning vector pQTEV. Positive clones were identified by restrictive digestion and sequence verified.

WelU3-coding gene was synthesized by Bio Basic Inc (Ontario, Canada) and ligated to pQTEV

analogously. Each pQTEV construct was transformed to E. coli C43(DE3) cells (Lucigen).

Subsequent heterologous expressions and purifications were carried out in an identical fashion as

described for AmbP1 and WelP1 proteins in reference 1 using IMAC. The purified proteins were

analyzed by SDS-PAGE to ensure homogeneity (Figure S10), concentrated with 10 kDa cutoff

concentrator tubes, assayed, and the remainder was flash-frozen using liquid nitrogen and stored at -

80 C for later assays. Approximate yields for each protein are: WelU1 (1.0 mg/L), WelU2 (insoluble),

WelC1 (1.2 mg/L), WelC2 (1.1 mg/L), WelU1(27-228) (5.0 mg/L), WelU2(27-226) (3.8 mg/L), WelU3

(1.5 mg/L).

Initial in vitro assay with WelC1, WelC3, WelU1 using substrate 1: To examine whether WelC1,

WelC3, WelU1 has the ability to convert 1 to 2. Assays in the scale of 100 µL were set up at pH=6.5

(MES buffer) and 7.5 (HEPES buffer) with 0.5 mM of 1 in the presence of either WelC1, WelC3, or

WelU1 (10 µM, final concentration). The enzymatic reactions were allowed to proceed at 30 ºC for 6

hour before being quenched with EtOAc extraction. EtOAc layer was dried under a stream of nitrogen

gas, redissovled in MeOH and subjected to HPLC analysis.

In vitro assays with WelU1, WelU2, WelU3, WelU(27-228) and AmbU4: For a typical standalone

assay, substrate 4 was synthesized using WelP1 and immediately used for the subsequent reactions.

A100 µL reaction was set up that contains 1 mM 3, 1.5 mM GPP, 10 uM WelP1 and 20 mM MgCl2 in

50 mM Tris buffer (pH 9.0). The reaction was incubated at 30 °C for 1h and quenched by a 1 mL

EtOAc extraction. The EtOAc layer was dried under a stream of nitrogen. To this crude preparation of

4 was added the buffer at the desired pH (MES buffer for pH=5.0, 6.0 and Tris buffer for pH=7.0, 8.0

and 9.0) with 10% DMSO to 100 µL and the U enzyme (5 µM final concentration). The reaction was

incubated at 37 °C for 5-120 min and quenched with 1 mL EtOAc extraction. The EtOAc fraction was

dried and dissolved in 100 uL MeOH for HPLC analysis. For a coupled assay with WelP1, a 100-µL

reaction was set up at either pH=9.0 (Tris buffer, 50 mM) or pH=6.0 (MES buffer, 50 mM) that

contains 1 mM 3, 1.5 mM GPP, 20 mM MgCl2, 50 µM WelP1 and 5 uM WelU1. The reaction was

incubated at 30 °C for 30 mins before quenched by 1 mL EtOAc extraction. The EtOAc crude was

dried and dissolved in MeOH for HPLC analysis. For preparative scale reaction for isolating WelU1

and WelU3 enzymatic product, assays were scaled to 10 mL scale proportionally and the crude

materials were purified by semi-preparative HPLC.

Table S1. Oligos used for clonings

Oligo # Oligo Name Oligo Sequence

1 WelU1-5' GATCGGATCCATGAAACGAAATTTTATCATTG

2 WelU1-3' GATCGCGGCCGCTCAAGTTTCAGCTGGTTCG

3 WelU2-5' GATCGGATCCATGAAGCGAAATTTGATGATTG

4 WelU2-3' GATCGCGGCCGCTTATGTTCCGGTGGGTTCTG

5 WelU1_5'-trucated GATTGGATCCGCAAGTGCTGTTTCGATTC

6 WelU2_5'-trucated GATTGGATCCGCAGGTGCTACTTTAATTACGATC

7 WelC1-5' GATCGGATCCATGATTAGAATTATTGCTATTG

8 WelC1-3' GATCGCGGCCGCTTAATTACTACAGATGTTCAAAG

9 WelC3-5' GATCGGATCCATGACTGCTGAAAACCGACTG

10 WelC3-3' GATCGCGGCCGCTCAAAATGTTGGCAACCAATTTAC

Figure S1. a) In vitro characterization of WelU1/C1/C3 using 1 as a substrate. b) Metal dependency of WelU1 at pH=9. c) Thermostability of WelU1 at pH=9. Chromatographs shown were derived from HPLC analysis, selectively monitored at 280 nm.

a)

22.0 24.0 26.0 28.0 30.0 (min)

1 Std1 +

WelU1/C1/C3

b)

22.00 24.00 26.00 (min)

42

WelU1, pH=930 ºC, 30 min

EDTA (10 mM)

MgCl2 (10 mM)

ZnCl2 (10 mM)

CaCl2 (10 mM)

4 Std

c)

22.00 24.00 26.00 (min)

42WelU1, pH=9, 30 min

4 Std

30 ºC

37 ºC

42 ºC

60 ºC

Figure S2. 1H NMR spectral overlay of 12-epi-fischerinole U 2 generated enzymatically from 4 with WelU1 with that reported in reference 2.

1H NMR (700 MHz, CDCl3) of 2 isolated from WelU1 in vitro enzymatic assay

1H NMR (500 MHz, CD2Cl2) of 2 isolated from H. welwitschii IC-52-3

Adapted with permission from Stratmann, K. et al. J Am Chem Soc 116, 9935–9942 (1994). Copyright (1994) American Chemical Society

1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0 ppm

Figure S3. In vitro assay shows compound 5 is not a substrate for WelU1

5.0 15.0 25.0 35.0 (min)

Rel

ativ

e ab

sorb

ance

(280

nm

)

5 std

5 + WelU1 pH=6, 1 h

Figure S4. Bioinformatical analysis of WelU1 using (a) Plotter (http://wlab.ethz.ch/protter/start/) and (b) SignalP (http://www.cbs.dtu.dk/services/SignalP/) reveals the potential structural role of N-terminal peptide sequence.

http://wlab.ethz.ch/protter/#

# Measure Position Value Cutoff signal peptide? max. C 27 0.462 max. Y 27 0.541 max. S 18 0.749 mean S 1-26 0.575 D 1-26 0.554 0.510 YESName=Sequence SP='YES' Cleavage site between pos. 26 and 27: ANA-AS D=0.554 D-cutoff=0.510 Networks=SignalP-TM

a)

b)

Figure S5. Bioinformatical analysis of WelU2 using (a) Plotter (http://wlab.ethz.ch/protter/start/) and (b) SignalP (http://www.cbs.dtu.dk/services/SignalP/) reveals the potential structural role of N-terminal peptide sequence.

# Measure Position Value Cutoff signal peptide? max. C 27 0.532 max. Y 27 0.564 max. S 23 0.731 mean S 1-26 0.523 D 1-26 0.549 0.510 YESName=Sequence SP='YES' Cleavage site between pos. 26 and 27: ANA-AG D=0.549 D-cutoff=0.510 Networks=SignalP-TM

a)

b)

Figure S6. Summary of relative and absolute quantities of 1 and its biogenetic derivatives 1a versus 2 and its biogenetic derivatives 2a, 3-6 from (b) H. welwitschii UTEX B1830 and (c) IC-52-3. The compounds with number 3-6 are not identical to those described in the main text with the same number. The numbering system (3-6) here is solely designed for this figure. The relative quantity shown in (b) was derived from the HPLC analysis of H. welwitschii UTEX B1830 crude metabolites and the absolute quantity shown in (c) was derived from what was reported in reference 2.

(R=none, R'=Me)

15.0 20.0 25.0 30.0 35.0 40.0 45.0-2.5

10.0

24.0 mAU

min

WVL:275 nm

NH

NC(R)

Cl

HH

NH

NC(R)H

H

NH

(R)CN

HH

NH

(R)CN Cl

H

NH

Me

CN

Cl

HMe

MeO

NR'

Me(R)CN Me

Me

O

O

H

Cl

H

NR'

Me(R)CN Me

Me

O

O

H

Cl

H

NH

(R)CN Cl

HH

12-epi-fischerindole U (2)

12-epi-fischerindole G (2a)

12-epi-hapalindole C (1)

12-epi-hapalindole E (1a) welwitindolinone B (5) welwitindolinone C (6)welwitindolinone A (4)12-epi-fischerindole I (3)

2 (R=none)

2a(R=none)

4

6 (R=S R'=H)

6

1(R=none)

3(R=none)

6 (R=S R'=Me)

Compounds %4 (R=none) 1.1

6 (R=S, R'=H) 7.16 (R=none, R'=Me) 3.3

2 (R=none) 20.72a (R=none) 26.61 (R=none) 2.63 (R=none) 22.0

6 (R=S, R'=Me) 16.6

Compound Name Quantity isolated1 (R=none) 12-epi-Hapalindole C isonitrile 11 mg

1 (R=S) 12-epi-Hapalindole C isothiocyanate 1 mg1a (R=none) 12-epi-Hapalindole E isonitrile 93 mg

1a (R=S) 12-epi-Hapalindole E isothiocyanate 4 mg2 (R=none) 12-epi-Fischerindole U isonitrile 4 mg

2 (R=S) 12-epi-Fischerindole U isothiocyanate 2 mg2a (R=none) 12-epi-Fischerindole G isonitrile 4 mg3 (R=none) 12-epi-Fischerindole I isonitrile 10 mg4 (R=none) welwitindolinone A isonitirle 2 mg

5 (R=S, R'=H) welwitindolinone B isothiocyanate 10 mg5 (R=S, R'=Me) N-methylwelwitindolinone B isothiocyanate 5+12 mg6 (R=S, R'=H) welwitindolinone C isothiocyanate 14 mg

6 (R=none, R'=Me) N-methylwelwitindolinone C isonitrile 47mg6 (R=S, R'=Me) N-methylwelwitindolinone C isothiocyanate 110mg

hapalindoles and welwitindolinones isolated from H. welwitschii IC-52-3 (47g dry tissue)

a)

b)

c)

Figure S7. 1H NMR spectral overlay of 12-epi-hapalindole C 1 generated enzymatically from 4 with WelU3 with that reported in reference 2.

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

0.37

0.23

0.61

1.59

0.55

0.85

1.09

0.57

0.99

0.99

1.05

0.56

0.79

0.79

1.03

0.68

0.17

0.72

1H NMR (700 MHz, CDCl3) of 1 isolated from WelU3 in vitro enzymatic assay

1H NMR (500 MHz, CDCl3) of 1 isolated from H. welwitschii IC-52-3

Adapted with permission from Stratmann, K. et al. J Am Chem Soc 116, 9935–9942 (1994). Copyright (1994) American Chemical Society

Figure S8. In Vitro characterization of a heterologously expressed and purified AmbU4 in ambiguine biogenesis. Assay was conducted in an identical fashion as described for WelU1 and WelU3 with 4 as the substrate. Product was analyzed by HPLC and monitored at 280 nm. Authenticity of the product was validated by 1H NMR spectral analysis that matched the reported data described in reference 3. .

Figure S9. Biomimetic total synthesis of hapalindole-type alkaloids using aza-Prins cyclization as a key step as illustrated in reference 4. Substrate S1, when treated with DDQ, gave stereoselective formation of adduct S5 where substituents at C10, C11 and C15 are in an all-equatorial configuration. The reaction is presumed to proceed through the benzylic cation S2, which undergoes conformational change to S3 under thermodynamic control that further leads to imminium ion S4 for an aza-Prins clyclization.

22.0 25.0 30.0

4 6

(min)

4 Std

4 + AmbU4pH=6, 30 min

rela

tive

abso

rban

ce

(280

nm

)

NCNH6

12-epi-hapalindole U

RNH S1

RNH S2

RNH

RNH

1011

12

15

S3

S5

DDQ , Δ

(R=NCS, CN, NHCHO)

RNH S4

Figure S10. SDS-PAGE of representative proteins heterologously expressed and purified in this work.

Reference Cited: 1. X. Liu, M. L. Hillwig, L. M. Koharudin and A. M. Gronenborn, Chem. Commun. (Camb.), 2016, 52, 1737-1740. 2. K. Stratmann, R. E. Moore, R. Bonjouklian, J. B. Deeter, G. M. L. Patterson, S. Shaffer, C. D. Smith and T. A. Smitka, J. Am. Chem. Soc., 1994, 116, 9935-9942. 3. S. Li, A. N. Lowell, F. Yu, A. Raveh, S. A. Newmister, N. Bair, J. M. Schaub, R. M. Williams and D. H. Sherman, J. Am. Chem. Soc., 2015, 137, 15366-15369. 4. Z. Lu, M. Yang, P. Chen, X. Xiong and A. Li, Angew. Chem. Int. Ed. Engl., 2014, 53, 13840-13844.

WelU1WelC3

WelC1

20.6

28.934.8

80.0

Ladder(kD)

49.1

WelU2(27-226)

15

2520

50

75

Ladder(kD)

37

WelU1(27-228)

a) b)