Isolation, Structure Determination, and Biological Properties of Cyclothiazomycins B1 and B2 Takanori Murakami, Takatsugu Kimura Toshikatsu Okuno, Hyouta Himeno, Masaru Hashimoto (Hirosaki University)

DHHA1

CH3 O O NH2 H N N N N H NH O H HN O NH H C CONH2 3 O O N O H N H CH3 N N S N S Cyclothiazmycin B1: H O DHHA1 = (Z)-isomer S N H3C H COOH Cyclothiazmycin B2: HN N O S DHHA1 = (E)-isomer CH2 S N O N N N H S S Isolation of Cyclothiazomycin B1 and B2 Hyphal Swelling by A307 Streptmyces sp. A307 strain culture broth (4.8 L) centrifuge supernatant residue MeOH-CHCl3 (1:1) supernatant mycelial cake supernatant (150 mg) residue MeOH Sep-Pak ODS (CH3CN:H2O) extracts residue active fraction (eluted with 80:20) 1) remove MeOH 2) AcOEt ODS HPLC

aqueous suspension extracts Cyclothiazomycin B2 Cyclothiazomycin B1 centrifuge (3.0 mg) (20 mg)

1 H-NMR spectra (-d5 + D2O) Cyclothiazomycin B2

Cyclothiazomycin B1 Properties of Cyclothiazomycin B1 (C61H69N21S7O13) ESIMS m/z = 1528 [M+H]+, 1550 [M+Na]+ (aq.CH3CN-NaCl) ESIMS m/z = 1545 [M’+D]+

(CH3CN-D2O) (suggesting 16 exchangeable protons) HRESIMS m/z = 1528.3448 (many candidates)

COSY and HSQC 53 non-exchangeable protons

13C-NMR and HSQC 61 carbons (60 by 1D-NMR) Hydrolysates

S UV (PDA) 271, 300, 315 nm S

HOOC N N CH3 IR (KBr) 3400, 1675, 1516 cm-1 saramycetic acid O Gly1, Arg1, Pro1, Asx1, HOOC analysis Cys, (not quantitative) H N S 2 N Suggested 21 nitrogens (due to nitrogen rule) CH3 N hetero atom 7 sulfurs COOH FGHSQC (pyridine-d5-D2O)

13 Chemical Shift Alteration in C-NMR by D2O

pyridine-d5/D2O (3:1)

pyridine-d5/H2O (3:1)

overlay 70 60 50 40 ppm 1H, 13C Chemical Shifts ROESY and HMBC

NH 3.50 NH H 3.50 158.42 H 42.05 H2N H 1.90 H2N N 2.07 Cys N 8.69 Cys Arg H 26.69 H mAla H mAla H H 5.33 2.35 29.00 COOH Arg COOH 2.60 2.57 28.78 H H 55.03 H H C H N H3C S 33.13 57.27 N 3 S 9.13 O 174.96 H O H 69.14 H 175.02 H N 5.05 H3.95H N 172.67 O H H O 9.15 4.10 O O 3.95H H H N NH 4.04 HN NH Gly O 10.57 H 80.48 H 5.66 O H H 169.12 (S) Gly 46.29 H 36.79 H O 4.17 DHHA2 H N Tzn2 O DHHA2 H N Tzn2 4.27 132.22 6.42 S S 170.86 H DHHA1 H 2.02 130.12 167.75 H 10.67 H H C H3C13.69 H N 3 HN CH N 10.48 CH3 2.14 NH 3 DHHA1 13.96 127.11 N Tzl2 N Tzl2 131.99 Asn 5.40 171.29 O Asn O 6.68 H S(Tzl3) H S (Tzl3) 173.70 H H 168.60 Tzn1 3.73 H O CONH N 78.92 Tzn1 O CONH2 N 3.93 2 148.29 48.00 8.38 H 5.23 39.12 177.66 36.76 H H H H N 3.75 N N N 120.09 H H H H H 52.91 3.62 H S 3.21 S H H H 22.78 5.80 104.98 Tzl1 S 62.58 3.59H 6.85 S H Tzl1 1.50 H 166.65 H 1.78 O 5.80 O H N 134.23 HN 34.14 10.96H 170.42 H DHA Pro H DHA 21.92 1.93 Pro O 8.23 O H H CH H O N CH3 H O NH 2.85 N 3 132.81 N 9.73 2.45 H 50.49 139.63 172.63 H H 9.72 118.65 80.18 5.90 H 162.71 H Ala N H 8.11 N H H H N N 151.97 N 169.50 N H Ala 6.08 37.25 H 4.16 HMBC Pyr S H 4.28 S H Pyr Tzn3 S Tzn3 S ROE (strong) Tzl2 Tzl2 H ROE (weak) (Tzl3) (Tzl3) MALDI-TOF/TOF Analysis of Cyclothiazomycin B1

should be carboxylic acid

the last nitrogen Stereochemistry of Amino Acids

OH NO O NO O 2 H 2 H O C N acidic N NH2 hydrolysate NH2 HN S S NH CH3 CH3 O2N O2N aq. NaHCO3 F HN COOH C O 40ºC, 2 hr HO Marfey's regent (FDAA) R

LL : DL = 5:1 (L-Cys : D-Cys = 10 : 1)

UV (330 nm)

Ion chromatography (LC-ESIMS)

Racemate! Cyclothiazomycin B1

S S O H (R) N N N N H (R) N S HOOC H S H O N (R) H CH3 H O S N N (R) S S H3C N O O H N O N HN N H (S) H2NOC NH O CH3 H O N H2N N N (S) H N H H O N O

H3C H Structure of Cyclothiazomycins B2

B2 HPLC analysis B1 in Pyridine-d5 Slowly isomerized into 0 hr cyclothiazomycin B1

80 hr 200 hr 5.0 10.0time (min)

1 cyclothiazomycin B2 H-NMR spectra of Cyclothiaomycins （in DMSO-d6)

cyclothiazomycin B1 Distribution of the chemical shift alterations NH Possibilities H H + - H N H 1. Conformational isomer? 2 N H H (B1)-(B2)  alteration should be observed over the H COOH molecule (S) H H H N H3C S O H 2. Diastereomer due to the hemithioacetal? H H H N O It hardly occurs by entropic factor. O H HN NH O H H 3．Diastereomer about the DHHA moieties? H (R) O (Z) H S N Most plausible. But DHHA2 in both are Z- DHHA1 H (Z) configuration on the basis of ROEs. H3C NH HN CH3 (Z) O DHHA2 N H S Diastereomer of DHHA1!! H (R) H O CONH2 N (Z) H H H N N H H H H S (S) H H H S O Consideration by H HN H Molecular Orbital Calculation （HF631G*) H O H N CH3 H O NH H (E)-isomer H (Z)-isomer 5.93 ppm 6.56 ppm H N H (Z) (R) N N H S H S

Chemical shift alteration localizes around DHHA1. Cyclothiazomycin B1 Cyclothiazomycin B2 NH NH H N H N 2 N 2 N H H COOH COOH H H3C H H H3C H N S N S O H O H H N O H N O O O HN NH HN NH O H O H O H S N O H S N ZZ H3C HN H Z NH CH3 E NH HN CH3 O N N S O S H O H H3C CONH2 N O CONH2 N H N H H N N N S H S H H S H S O HN O HN H H O O N CH3 O NH N CH3 O NH H H H H H N H N N N N N S S S S

Cyclothiazomycin B1 Cyclothiazomycin B2 Inhibition of DNA-Dependent RNA Synthesis

transcription RNA polymerase × DNA

mRNA Cyclothiazomycin B1 blank cyclothiazomycin B1 translation SP6 on ribosome employed T7 thiostrepton RNA polymerase RNA

Transcription of DNA for protein dihydrofolate reductase (dihydrofolate reductase)