fluorescent and photoactivatable derivatives of marine · pdf file"no photocrosslinking...

Fluorescent and photoactivatable

derivatives of marine natural products

Thomas Lindel

TU Braunschweig, Institute of Organic Chemistry

Workshop

Design of chromophore systems with specific properties and applications

in medicine as biomarkers and antitumoral agents

Timișoara, April 18-19, 2013

-conotoxin MVIIA in solution

(R. A. Atkinson et al., Biochemistry 2000, 3908)

- blocks Ca2+-channels of type N

FDA approval 2004

indication: opioid-resistent pain

maximal dose 20 g/d

Conus magus

-Conotoxin MVIIA (= ziconotide, "Prialt")

H-C-K-G-K-G-A-K-C-S-R-L-M-Y-D-C-C-T-G-S-C-R-S-G-K-C-NH2

B. M. Oliveira et al., Biochemistry 1984, 5087.

approved

ecteinascidin 743: N,O-acetal moiety

necessary for activity

N

NN

N

O

NH

H

O

H

O

O

P

O O

O

alkylation of the exocyclic amino

group of guanine (minor groove)

Ecteinascidin 743 (= trabectidin, Yondelis)

preferred base triplets

Review: D'Incalci and Galmarini, Mol. Cancer Ther. 2010, 9, 2157

approved

Eribulin (E7389, Halaven)

antitumor

eastern section of halichondrin B,

made by Chemical Synthesis (62 steps,

based on Kishi's work, JACS 1992, 3162)

Eribulin mesylate, a novel

microtubule destabilizing agent,

has recently gained approval in

the US for patients who have

received at least two

chemotherapeutic regimens for

the treatment of metastatic breast

cancer (MBC), with prior therapy

including an anthracycline and a

taxane.

Mechanism:

suppresses microtubule polymerization,

but has no effect on microtubule

depolymerization

Review: Glück et al., Cancer Treat. Rev. 2012, 38, 143

Eribulin (E7389, Halaven) approved

Estimation on marine natural products:

25000 characterized

2500 with known biological activity

250 available in sufficient quantity

25 with atomically resolved bioactivity

2.5 in the clinic

total synthesis of

marine natural

products

technology for

chemical target

search

biological

activity

eunicellane diterpenoid

from Eunicella labiata

Salvá et al.,

J. Nat. Prod. 1997, 485

www.flickr.com/photos/agvnono/44461449/sizes/o/in/photostream/

www.chem.unep.ch/pops/termites/termite_ch2.htm

cubitene

from the defense secretion of the East

African termite Cubitermes umbratus

Prestwich et al., JACS 1978, 2560

Corals and termites

Progress: 9 steps shorter, > 5-fold yield

Kristina Simon et al., Angew. Chem. Int. Ed. 2012, 51, 10889

Kodama et al.,

Chem. Lett. 1996, 809

24 steps from D-mannitol

< 1% overall yield

+

our route

15 steps from S-carvone

5.2 % overall yield

There are more cubitanoids

calyculones

from the Caribbean sea whip

Eunicea calyculata

(Fenical et al., JOC 1984, 1417;

JOC 1991, 1227; Rodríguez et al.,

Pure Appl. Chem. 2012, 1847)

Review: Wefer, Simon, Lindel, Phytochem. Rev. 2012, online

cytostatic

Cytostatic (‒)-dibromophakellstatin

Isolation:

31 mg from 170 kg of Phakellia mauritiana (0.000018 % of the wet weight)

"The sponge was extracted twice with methanol-dichloromethane (1:1)

(200 L per extraction)."

Pettit et al., J. Nat. Prod. 1997, 180.

N

CCl3

O

Br

BrH

N

N

HNNH

O

O

Br

Br

5 steps

10 %

Jacquot, Zöllinger, Lindel, Angew. Chem. 2005, 2336

enantioselective: Zöllinger, Mayer, Lindel, Synlett 2007, 2756

SAR studies: Moldovan, Zöllinger, Jones, Kelter, Fiebig, Lindel, Eur. J. Org. Chem. 2012, 685

New ring C-functionalized derivatives of dibromophakellstatin

N

N

HN

NH

O

O

OHBr

Br

N

N

HN

NH

O

O

OBr

Br

ready for target search

Rareș-Petru Moldovan et al., Eur. J. Org. Chem. 2012, 681

N

N

HN

NH

O

O

OBr

Br

N

N

HN

NH

O

O

OBr

Br

Goals of photoaffinity labeling

A. Blencowe, W. Hayes, Soft Matter 2005, 1, 178-205.

L. Dubinsky, B. P. Krom, M. M. Meijler, Bioorg. Med. Chem. 2012, 20, 554-570.

Which targets interact with a given natural product?

How does a given natural product interact with a given target?

Photoactivatable and fluorescent marine natural product derivatives

Lindel group

azide analog of oroidin: target unknown

photohemiasterlin: target tubulin?

fluorescent psammaplin:

target HDAC?

Chemical target search: key task to natural product chemistry

the old players new candidates?

Hemiasterlins

natural product hemiasterlin

from the marine sponges

Hemiasterella minor and

Cymbastela sp. (Andersen et al.)

cytotoxic (IC50 2-10 nM)

non-covalent bonding of HTI-286

to -tubulin in the ,-tubulin

dimer (modeling)

a "photohemiasterlin",

made by chemical

synthesis

research tool

Wartmann, Lindel, Eur. J. Org. Chem. 2013, 1649

L-Phototryptophan

Ac2O, HOAc, microwave

(40 W), 45 °C, 2 h, sealed

tube

50%

Aspergillus amino acylase

(ABCR), cat. CoCl2-6 H2O,

phosphate buffer (pH 7),

37 °C, 16 h

+

[]D23 -24.5 (c = 0.44, MeOH).

24%, ee 98% 31%, ee 93%

4 steps from 6-bromoindole


Phototryptophan is safe enough.

50 100 150 200 250 300 350 400

-24

-20

-16

-12

-8

-4

0

4

8

He

atF

low

(m

W)

Temp (°C)

Phototryptophan

Tryptophan

exothermal

endothermal

DSC (Differential scanning calorimetry)

start: 30°C

end: 400°C

heating rate: 10K/min


Photoindoles for chemical target search

S

R

Analytical HPLC of enantiomers on a Chiracel OD column


A photo hemiasterlin

photo analogon of

hemiasterlin from the

sponge Cymbastela sp.

(eastern half according to

Andersen et al.; coupling

DCC/HOBt)

target tubulin

photoreaction:


Photoactivatable amino acids and proteins

P. G. Schultz et al., ChemBioChem 2007, 2210:

Crystal structure of 4-

(trifluoromethyldiazirinyl)-

phenylalanyl-tRNA synthetase

"No photocrosslinking product was

observed" (after photolysis).

It would be good to have something

chemically more selective.

Irradiation of diazirines

W. Kirmse, W. Kilian, S. Steenken, J. Am. Chem. Soc. 1990, 112, 6399-6400. E. J. Dix,

J. L. Goodman, J. Phys. Chem. 1994, 98, 12609-12612.

p-Methoxyphenyl(trifluoromethyl)carbene: singlet

B3LYP/6-311G (2d, 2p)

see also: M.-G. Song, R. S. Sheridan, J. Phys. Org. Chem. 2011, 24, 889-893.

singlet

Raimer, Lindel, Chem. Eur. J. 2013, online

+

350 nm, 10 mM (1:1),

dry DCM, Ar, rt, 2 h

no C-D bond !

14%

relative yields

(19F NMR)

6% 12% 37%

+ + +

+ 24% azine

p-OMe-PTD: no insertion into O-H bonds


Irradiation of p-OMe-PTD

p-Methoxyphenyl(trifluoromethyl)carbene: tyrosine labeling


Psammaplin A

Photo: www.eol.org

psammaplin A from Pseudoceratina (= Psammaplysilla) sp. (Crews et al., 1987)

antibacterial

(e.g. MRSA, MIC = 4.85 µg/mL)

antitumor

(e.g. human lung cancer A-549,

ED50 0.57 µg/mL)

histone deacetylase (HDAC) inhibition

(IC50 = 4.2 nM)

Review on tyrosine-derived -hydroxyiminoamides: Hentschel, Lindel, Synthesis 2010, 181-204

Live cell imaging of a fluorescent psammaplin A analog in L929 cells

Hentschel, Sasse, Lindel, Org. Biomol. Chem. 2012, 10, 7120

Fluorescence probably in the Golgi apparatus, only

minor fluorescence inside the nucleus, where

HDAC1 is located

Early cleavage of the disulfide bonds after

penetrating the cells

Cytotoxic N-α-(hydroxyimino)acyl cysteamine unit,

would perhaps penetrate the nuclear envelope and

inhibit HDAC1 in the nucleus

Synthesis of photopsammaplin

Fabia Hentschel, Björn Raimer 2012: cooperation with Oncotest, Freiburg

Bisdeoxypsammaplin A and its photo version


histo type cell line IC50 [µM] IC50 [µM]

bladder BXF T-24 0.908 0.54

colon CXF DiFi 0.906 2.10

stomach GXF 251 2.38 3.23

head neck HNXF CAL-27 0.816 1.05

liver LIXF 575 3.45 3.40

lung LXFA 289 1.54 1.64

breast MAXF 401 0.732 0.619

skin MEXF 1341 0.507 0.684

ovary OCXF 899 1.74 1.24

pancreas PAXF 1657 2.38 1.71

prostate PRXF DU-145 0.653 0.59

kidney RXF 486 1.54 1.08

cervix UXF 1138 3.85 2.32

R = Br

COMPARE analysis:

same mechanism

(HDAC inhibition)

thiol docking in the active site of the human

HDAC8-trichostatin A crystal structure (de

Lera group) de Lera et al., Bioorg. Med. Chem. 2011, 3637

35 nM

prodrug

11 nM

trichostatin A: 30 nM

HDAC inhibition (HeLa cell lysate, IC50)


2-Azidoimidazoles

Irradiation of 2-azidoimidazoles

N

NN

N

N

N

NN

N

N

h (>300 nm), rt,

2 h, MeOH

-N2

h (>300 nm), rt,

2 h, MeOH

-N2

Alex Sudakow et al., EJOC 2012, 681

2-Azidobenzimidazoles


Use of 19F NMR for discovery

ppm (t1) -115.0-110.0-105.0

-10

6.8

-11

4.2

188 MHz, CDCl3


Discovered.

ppm (t1)-115.00-114.50-114.00-113.50-113.00-112.50-112.00-111.50

91.0

0

5.2

9

3.0

4

2.4

7

AcOH19F NMR spectrum after irradiation of

2-azido-1-(p-fluorobenzyl)benzimidazole

in AcOH


Regioselective synthesis of 6-oxygenated 2-aminobenzimidazoles

R = CH3


Scope

strong nucleophile


How many equivalents of acid are needed?

Alex Sudakow, 2012

Bioorthogonal, photochemical peptide labeling!

Alex Sudakow, 2012

Successful monolabeling of insulin

Human insulin (C257H383N65O77S6) was

irradiated at 300 nm (Rayonet) in the

presence of azide 1 (1 eq.) in tBuOH/H2O

(1:1, + traces of DCM) at pH 4 for 2 h and

analyzed by ESI-MS (Orbitrap) after being

desalted by RP-18 chromatography.

To analyze the regioselectivity among the

carboxy groups tryptic digestion will be

necessary. LTQOT02709 #1-14 RT: 0.00-0.35 AV: 14 NL: 1.07E8T: FTMS + p NSI Full ms [130.00-2000.00]

800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

1162.33899z=5

968.78413z=6

1452.67086z=4

1008.63069z=6

1210.35485z=5

864.68485z=7

ESI-MS spectra of

the reaction mixture

after workup

Alex Sudakow, 2012

fluorescent psammaplins

(+)-cubitene

Summary

novel carboxylic acid-selective peptide labeling

L-phototryptophan

Set-up

Lindel group, TU Braunschweig

Binding mode analysis Target identification

Photolabeling technology

Chemistry of diazirine-

based photolabeling

Synthesis of photoactivatable marine

natural products with known targets

Synthesis of fluorescent and/or

photoactivatable marine natural

products with unknown targets

New photo bio building

blocks for (bio)synthesis

Chemoselective photolabeling

of peptides and proteins

Photoaffinity-aided binding

analysis of marine natural

products on known targets

Photoaffinity-aided identifcation

of unknown targets

DFG, BASF, Merck, Honeywell,

FCI, FIOC, Evangelisches Studienwerk

Oncotest, HZI

Acknowledgements

Mona Al Batal

Santosh Kumar Adla

Werner Telle

Fabia Hentschel

Benjamin Troegel

Thomas Wartmann

Dr. Bohdan Snovydovych

Björn Raimer

Nils Marsch

Alex Sudakow

Johannes Wefer

Tim-Carlo Allmann

Maren Ballhaus

Lindel Group

Arabinose instead of ribose: sponge-inspired antimetabolites

HO N

O

OH

HO

NH

O

O

2'

HO N

O

OH

HO

NH

O

O

HO N

O

OH

HO

N

O

NH2

HO

N

NN

N

NH2

O

OH

HO

spongouridin

(1951 from Tectitethya crypta)

spongothymidin (1951)

cytarabin:

against myeloid leucemia

(1969)

vidarabin:

against viral infections

(1976)

approved

Taltobulin (= HTI-286)

cytotoxic as hemiasterlin (IC50 3 nM),

higher tubulin affinity

non-covalent bonding of HTI-286 to

-tubulin in the ,-tubulin dimer

(modeling)

clinical phase II/III

HN

NH

NOH

O

O

O

HTI-286 (= taltobulin)

in phase II

Selectivity of photoaffinity labeling

Kanoh et al., Tetrahedron 2008, 5692:

Dominance (>75%) of insertion (?) into

the O-H-bond in the case of aliphatic

alcohols in solution.

LC/MS study

upper: liquid phase (rt)

lower: solid ph. (-196 °C) CF3N

N

HN

O

O

O

NH2

Psammaplin A: cytotoxicity against mouse fibroblast cell line L929

-10

0

10

20

30

40

50

60

70

80

90

100

110

120

1 10 100 1000 10000 100000

Re

lati

ve

ac

tivit

y in

MT

T a

ssa

y [%

]

Conc. [ng/ml]

L929: 5d

Li-0061

Li-0062

MeOH

1

2

incubation

X=OH: IC50 0.42 µg/mL (0.63 µM)

X=H: IC50 0.31 µg/mL (0.49 µM)

Hentschel, Sasse, Lindel, Org. Biomol. Chem. 2012, 10, 7120

Pathways


6-Hydroxylation

2 steps from

benzimidazole, 83%

h (>300 nm), rt, 2 h

DCM/TFA (8:1)

52%

1H,15N HMBC


Ring opening: DFT calculation

singlet nitrene

-704,48

-704,47

-704,46

-704,45

-704,44

-704,43

-704,42

-704,41

0 10 20 30 40 50

To

tal E

ne

gry

[H

art

ree

]

optimization step number

Between the singlet nitrene

and the ring opened

intermediate is no energy

barrier.

Alex Sudakow, 2011

Dibromageliferin gegen bakterielle Biofilme

Biofilme sind an 80% aller bakteriellen Infektionen beteiligt.

Naturstoff

Chemische

Synthese

Lit.: Rogers, Melander, Angew. Chem. 2008, 5307; Mol. Biosyst. 2008, 614.

Wirkstoff

NH

N

HN

NH

NH

HN

NH

N

NH2

O

NH2

O

Br Br

Br

Br

Leitstruktur Dibromoageliferin

NH

N

H2N

NH2

NH2

Biofilm-hemmend (IC50 100 M)

gegen Pseudomonas aeruginosa

www.bren.ucsb.edu

fluorescent and photoactivatable derivatives of marine · pdf file"no photocrosslinking...

Documents