Total structure of the peptide antibiotic components of thiopeptin by 1H and 13C NMR spectroscopy

Tetrahedron Letters No. 39, pp 3649 - 36~0 @ Pergaman Press Ltd. 1978. Printed inGreat Britain.

TOTAL STRUCTURE

OF THE PEPTIDE

OF THIOPEPTIN

Otto D. Hensens* Merck Thiopeptin,

Laboratories,

antibiotics

is a valuable

as a lactic-acidosis

complex was characterized

by Miyairi

components

although

raphy5

to thiostrepton,

describeda. batches their

components

Subsequent

indicated

by the subscripts

Table 1:

thiopeptin

allowed

We wish

of the components

Thiopeptin

as mixtures. formulae

system.

into two distinct

thiopeptin

similar

ester (Amide)

Bis-Des-Deala

(Amide)

*from microanalytical Correlations thiopeptins the molecule

Pure components

which

differences

arbitrarily

in

designated

Ala, Afi, Aja, A4a and B, from Ba by multiple

prepara-

from the "a" components,

but

in our fermentation.

components*

“b” Series

Ba - C71H64NI6016S6

Bb - C71%2N1801~+6

Ala- C72H66NI6016S6

Ati- C72Ha4Nl6OlaS6

A~Q- C68H62NI6016S6

A4b- C68H8oNI6016S6

A3a- C66H79NI7OISS6

A3b- C65H77N17015S6

and lH and 13C NMR data.

between

were made

crystallog-

to that previously

Consistent series

Bb was separated

A2 was not produced

for thiopeptin

by X-ray

by lH and 13C NMR techniques.

A3b and A4b were not separated

Acid

Des-Deala

B and four

by 'H NMR at 300 MHz and HPLC of various

in Table 1.

whereas

determined

"a" Series

Methyl

and

The antibiotic

to report here on the structure

chromatography

of cases a two compound

a and b as summarized

ThiOpeptinS

primarily

by silicagel

them to be grouped

from some batches

Molecular

of which was largely

components

were separated

analysis

tive tic on silicagel.

were analyzed

in swine and chickens'

in sheep and cattle'.

et al3 and shown to consist of a major component

the structure

in the majority

lH NMR spectra

were isolated

preventive

by 13C NMR spectroscopy6.

of the various

Thiopeptin

to a group of highly modified

growth promotant

No structures for any of the components have been reported 4 experiments , primarily on thiopeptin B, have demonstrated a close re-

and its side-chain

determination

New Jersey 07065

Al, AZ, A3 and A4.

acid hydrolysis

lationship

Rahway,

by Streptomyces tateyamensis and belonging

peptide

was shown to be effective

minor

COMPONENTS

and Georg Albers-Schunberg

Sharp & Dohme Research

produced

sulfur-containing

ANTIBIOTIC

BY 'H AND 13C NMR SPECTROSCOPY

the majority

to within

are circled

of resonances

in the 'H NMR spectra

0.04 ppm and differences in Figure

1 and which

3649

were noted

of thiostrepton

at four different

shall now be discussed.

and the

sites of

lb.

3650

39

Deala PI- Deala 01 Dsala 121

/

Deala 13)

Thz(4) Thiosirepton:

Thiapeptin

X -0:

R-

Ba L Rb: X’S:

RI = CH3CH2-

R-

R1 = CH3

ThzW- ip-Alacn-oeala(ll-Alalll \ Val orIle / 0

Thr(l) ' !hz,3, \ Thr(Z)But \

AU

and Alb

X - 5; R -

R1 0

&andA&:X-S:R-

CH3

0

HZ

AjaandAB:X*S;R*H

RI- CH3 RI= CHj

Fig. 1. Structure

d Thicqeptinr

I CYs-Thstn-Thzl2I Thrtn 0 CYS Thz Pip

*

Thiostreptine residue Qulnaldic acid precursor Thiazdine residue Thiazde ring Piperldine ring

Nature of Amino Acids Amino acid analysis of the acid hydrolysate of the thiopeptin components gave Thr, Val and Ala in the molar ratio of 1:1:2 indicating that the Ile residue in thiostrepton is replaced by Val.

That

this residue is attached to the quinaldic acid precursor portion was confirmed by high-resolution mass spectral data of the TMS derivatives of all components. The highest peak at m/e 521.2687 corresponds to fragment a (talc. 521.2686) (Fig. 2) which can readily aromatize by losing TMSiOH (m/e 431.2187, talc. 431.2185). The 'H NMR spectrum7 of thiostrepton in CDC13 reveals eight methyl doublets, one triplet and one singlet in the region O-2

ppm whereas the thiopeptins, instead of the triplet, have

an extra doublet at 60.98 (Val

CH3,

J

=

7

Hz).

This

doublet

is coupled to a hextet (outer satellites not observed) at 61.92 (Val B-CE, J = 6 Hz) which in turn is coupled to a doublet at HO

62.88 (Val a-Cg, J = 6 Hz). Moreover, the 13C NMR spectrum of thiostrepton7has 15 resonances in the O-35 *OTMS a

1 x CH carbons. One CH2 resonance is missing which occurs at

Thiostrepton

(R-CH2CH3)

“‘/e 535

Thiopeptins

(R - CH3)

m/e 521

Fig.

ppm region whereas

all thiopeptins have only 14, comprising 10 x CH3, 3 x CH2 and

2

23.0 ppm for thiostrepton. The presence of Val is thus firmly established.

3651

No. 39

Nature of Piperidine Ring 'b" Series: Comparison protons

in thiostrepton

piperidine

of the chemical

and thiopeptin

ring structure.

neighbouring

ring currents

shifts and coupling

Alb indicate

The chemical

shifts

of the thiazole

constants

that both compounds

of the -CH2CH2-

rings and analyze

protons

for the piperidine have the same AIare anomalous

due to the

as a first order four spin system

Jgem characterized by the following coupling constants: = 12.5 Hz; Jiem3b = 19 Hz; JGc4b = vie = 6 Hz; J;ic4b = 12.5 Hz; JGC40 = J3b 68 = l.?;l$ J30 68 = 3.5 Hz. On the basis'of its J30,4a homoallylic couplil)lgto the protons it C3, H-i was assigned to'the unresolved broad singlet at 65.26 in Ay, and 65.23 in thiostrepton. at C3 via an amineand removes

the homoallylic

"a" Series: second

enamine

coupling

The piperidine

fragment.

giving

of d-TFA exchanges two doublets

to H-66 which now appears

ring protons

order spin system which becomes

a -CH2CH2CH-

Addition

equilibrium

(Jgem = 12.5 Hz)

as a very sharp singlet.

of the "a" series are characterized

almost

All five resonances

both the allylic protons

for H-4o and H-48

first order on protonation

with

move to lower field on addition

by a complex d-TFA suggesting

of the acid including

the singlet

for H-6 (64.47 in Ala) which undergoes the same shift as the resonance for H-2 (64.44, 8 dd, J = 3.5, 10 Hz, axial) suggesting an amine flanked by two CH groups. It is proposed therefore that the saturated from comparison

piperidine

ring system

of the 13C NMR spectra

(161.0 ppm in thiostrepton)

C6 at 60.7 ppm, both of which

the "a" series.

undergo

comes

to the imine carbon C2 whereas

(C2) in the 45-80 ppm region, the same upfield

Confirmation

has one extra carbon at 160.9 ppm

protonation

similar

to that assigned

shift with

to

d-TFA.

of Modified !l%reunineResidue, Thr(2l

The -CHNH- protons thiostrepton, whereas

in thiopeptin

thiopeptins, It remains

of Thr(2)

in the thiopeptins

e.g. the a-CH and NH doublets

was not immediately

the 13C NMR evidence

a peak at 189.9 ppm appears

changing

peptide

content

compared

readily

found appreciably

on gated decoupling

was carefully

in 20% CD30H/CDC13 but is observed

downfield

analogue

for these shifts

In both series

in the

to thiostrepton

(Table 1) and hence

for the low field shifts as follows.

manner

Thiocarbonyl

by the relationship

a thioamide

observed

group was entertained which 3 . Further

for the -CHNH- protons

groups appear downfield

6c=S = 1.45 Aczo - 46.5 ppm

amide carbonyl

in thiostrepton

(162.8 ppm).

Moreover,

of the thiazole-4-carboxamide

with

of their oxygen analogues 10 . On calculation, this that assigned

(161.1 ppm) and that observed

the thiocarbonyl

provides

further

resonance

to the

in thiazole-4-carbox-

at 189.7 ppm in the thio-

strong support.

Nature of Side Chain Thiopeptins which

of

of all other resonances.

as a split peak when measured

gives hczo = 163.0 ppm for 15~~8 = 189.9 ppm in very good agreement

amide in DMSO-d6

The reason

analyzed.

of those in (J = 10 Hz)

mixture and is therefore assigned to a carbon adjacent to a slowly ex7 NH group . Microanalytical data on the thiopeptins support a higher sulfur

support was obtained

corresponding

downfield

solvent

account

in a predictable

were

in the latter appear at 65.87 and 68.32

Ala they occur at 66.85 and 69.79 respectively.

clear until

a singlet

fully deuterated

would

Aa

in the 140-190 ppm region attributed

Al, has one extra carbon at 56.8 ppm

Nature

characterizes

of Ala and Alb.

Ba/b and Ala/~

can be readily

have

distinguished

two Deala residues

in their side chain as in thiostrepton

from the other Deala residue

in the bicyclic

portion.

No.

3652

Thiopeptin singlet residues tained

B, forms a sodium salt and was assigned

at 63.93 and are therefore are missing

from facile

completely

are close structural

the first reported

components

Ba/b and Ala/~

examples

sequence,

structurally.

However,

of naturally

can be envisaged

acid artefacts

authentic

occurring

as being

simply

sample

of thiostrepton,

of thiopeptin

Dr. Jerrold

Liesch

Fujisawa

for performing

for mass spectral

thioamides.

for both Deala

to the products

ob-

Ba/b

of thiopeptins

in being,

as far as we

Biosynthetically,

related by an oxidation

the other components

of this work will be published

Mr. Carl Homnick

have a three proton

The two series

they are unique

A4a/4b

(and Ala/s)

the

or reduction

and A3af3b being

des-Deala

respectively.

elsewhere.

AeknowZedgements: We would like to express our appreciation

mixture,

acid; Ala/u

and are identical

the thiopeptins

of thiostrepton.

or esterification

A full report

a carboxylic

The lH and 13C NMR resonances

of Ba/b and Ala/s.

defines

analogues

are aware,

and bis-des-Deala

esters.

in A3a/b and for only one in A4&

acid hydrolysis

This therefore

and hydrolysis

methyl

39

Pharmaceutical

to the Squibb Institute

Co., Japan for samples

the amino acid analyses,

for an

of thiopeptin

and to Mr. Jack Smith and

data.

References and Notes: 1.

K. Mine, N. Miyairi,

N. Takano,

Antimicrob. Agents and Chemother.

S. Mori and N. Watanabe,

3 496 (1972). 2.

L.A. Muir and A. Barreto,

3.

N. Miyairi,

T. Miyoshi,

U.S. Patent 4.061.732 (1977).

H. Aoki, M. Kohsaka,

H. Ikushima,

K. Kunugita,

H. Imanaka, Antimicrob. Agents and Chemother. 1 192 (1972); i&m. 4.

I. Muramatsu,

Y. Motoki,

E. Hikawa,

A. Hagitani

and N. Miyairi,

B. Anderson,

6.

K. Tori, T. Tokura,

7.

'H NMR and 13C NMR spectra were obtained

D.C. Hodgkin

and M.A. Viswamitra,

range 3.5-5.0 mgf0.4 ml.

ification

of carbons

l'et.Letters 185 (1976).

(SW) of 4000 Hz and concentrations

13c NMR spectra were obtained with AT 0.4-0.6

Slow exchange

adjacent

of peptide

using a

lH NMR spectra were measured

angle 50' on samplej of ca. 60 mgj0.4 ml

in 5 mm tubes at 45'C.

(London) 225 233 (1970).

and G. Lucacs,

at 300 MHz and 75 MHz respectively

in the FT mode.

(AT) of 1 and 2 set with sweep width

pulse flipping

Nature

K. Okabe, M. Ebata, H. Otsuka

SC-300 MHz NMR spectrometer

tion times

J. Antibiot. 23 113 (1970).

ibid. 25 537 (1972); I. Muramatsu,

and H. Suzuki, ibid. 30 383 (1977).

M. Aoyama

5.

Varian

H. Sakai and

to the NH groups because

and 20% CD30D/CDC13

in the latter solvent

of *H-isotope

in the

set, SW 15000 HZ and

in 20% CD30H/CDC13

NH protons

using acquisi-

allows

induced upfield

ident-

shifts of

ca. 0.1 ppm. The absolute

8.

configuration

and will be discussed C. Roussel,

9. 2847

at this new asymmetric

center was tentatively

assigned

A. Liden, M. Chanon,

J. Metzger

and J. Sandstrom,

J. Amer. Chem. SOC. 98

(1976).

10. H-O. Kalinowski

as R

in the full paper.

and H. Kessler,

AngeW. &em. Intern. Edit. (EngZ.) 13 90 (1974).

(Received in USA 12 June 1978; received in UK for publication 27 July 1978)