ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2009, Vol. 35, No. 4, pp. 497–503. © Pleiades Publishing, Ltd., 2009.Original Russian Text © T.F. Berdnikova, A.S. Shashkov, G.S. Katrukha, O.A. Lapchinskaya, N.V. Yurkevich, A.A. Grachev, N.E. Nifant’ev, 2009, published in BioorganicheskayaKhimiya, 2009, Vol. 35, No. 4, pp. 550–556.

497

INTRODUCTION

Ray fungus

Amycolatopsis

orientalis

subsp.

eremo-mycini

was previously [1–3] shown to produce mainlythe highly active antibiotic eremomycin, which refersto the dalbaheptide family and has a structure [4] shownbelow.

2

During the study of the component composi-

tion of the antibiotic complex in the culture liquid ofthis producing strain, a minor component, eremomycinB, also possessing antimicrobial activity toward a num-ber of test organisms, was found. We herein report theisolation and establishment of the structure of the afore-mentioned new antibiotic, a minor component of theeremomycin complex.

CH3

O

NNH

H3C

NHH3C

H

H

HO

O

N

O

Cl O

O

NH

HN

OHHO OH

NH

O

HO H H

H

COOHH

O

HH

CONH2O

O

O OH

OHOH

O

O

HN

CH3

M

H3CHO

O OHCH3

NH2

H3C

P5

P4

P1 P2

X1X2

P6

X4

P7

X3

X5

E

D

X8

X7X6

P8

e

g

f

P3

α1 α2 α3 α4α5

α6

α7

2

C B A6

3 2 6

4

3 2

61

2

5

3

5

1

1

2

5

6

5

2

4

2

Eremomycin, M = HEremomycin Ç, M = CH2COOH

12

H H

H

The Structure of Antibiotic Eremomycin B

T. F. Berdnikova

a

, A. S. Shashkov

b,

1

, G. S. Katrukha

a

, O. A. Lapchinskaya

a

, N. V. Yurkevich

a

, A. A. Grachev

b

, and N. E. Nifant’ev

b

a

Gauze Research Institute of New Antibiotics, Russian Academy of Medical Sciences, ul. Bol’shaya Pirogovskaya 11, Moscow, 119021 Russia

b

Zelinskii Institute of Organic Chemistry, Russian Academy of Sciences, Leninskii pr. 47, Moscow, 119991 Russia

Received January 16, 2009; in final form, January 26, 2009

Abstract

—A new biologically active component, antibiotic eremomycin B, was isolated from the culture liq-uid of

Amycolatopsis orientalis

subsp.

eremomycini

, the producing strain for antibiotic eremomycin. Its struc-ture was established by NMR spectroscopy and mass spectrometry. Eremomycin B was shown to differ fromeremomycin by the presence of an

N

-carboxymethyl substituent in the disaccharide eremosamine fragment.

Key words: antibiotics, eremomycin, eremomycin B, eremosamine, NMR, structure

DOI:

10.1134/S1068162009040128

1

Corresponding author; phone/fax: +7(499)-135-8784; e-mail: shash@ioc.ac.ru.

2

Abbreviation: Dnsp, 3,5-dinitrosulfopenyl.

498

RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

Vol. 35

No. 4

2009

BERDNIKOVA et al.

RESULTS AND DISCUSSION

During the study of the composition and chemicalnature of the minor components generated in the pro-cess of the biosynthesis of eremomycin by the

A

.

orientalis

subsp.

eremomycini

producing strain, wehave found a new component, eremomycin B, besidesthe reported minor components dechloroeremomycin,deamidoeremomycin, deseremosaminyl eremomycin,and deamido deseremosaminyl eremomycin [5].

We have found that eremomycin B is not absorbedon KB-

2 [

Na

+

]

cation-exchange resin during the isola-tion of eremomycin from the culture liquid by the pro-cedure in [6], but could be isolated from the eluate afterKB-

2 [

Na

+

]

as an individual substance using two subse-quent chromatographic separations, first, on sulfoca-tionite CDV-

3 [

H

+

]

and, then, on CM-cellulose. The iso-lation and purification of eremomycin B was monitoredby paper chromatography and paper electrophoresiswith the subsequent visualization with Pauli’s reagentand bioautography using the test microorganism

Bacil-lus

subtilis

ATCC 6633.The investigation of the physicochemical and bio-

logical characteristics of the new antibiotic showed(Table 1) that eremomycin B, as well as eremomycin, iseasily soluble in water and aqueous solutions of acidsand bases and insoluble in most organic solvents; itsUV spectrum in 0.01 M HCl has an absorption maxi-mum (

λ

max

) at 280 nm, characteristic for dalbaheptideantibiotics. Eremomycin B is a less basic compoundthan eremomycin, as it migrates slower to the cathodeupon paper electrophoresis in acidic pH. Treating ere-momycin B with potassium 4-chloro-3,5-dinitrobenze-nesulfonate under the conditions in [7] and the subse-quent electrophoretic analysis of the reaction mixturedemonstrated the formation of mono-

N

-

and di-

N

,

N

'-3,5

-dinitrosulfophenyl derivatives of the antibiotic,which is evidence of the presence of two reactive amino

groups in eremomycin B, instead of three as in eremo-mycin [4]. Eremomycin B possesses slightly less anti-bacterial activity as compared to eremomycin (Table 1).

According to the mass spectrometry data, themolecular weight of eremomycin B (1616) is 59 auhigher than that of eremomycin (1557). The analyticaldata obtained (Table 1) allowed us to assume the pres-ence of the substituent at one of the three amino groupsof eremomycin B. The structure of this substituent thatdistinguishes eremomycin B from eremomycin wasestablished by

1

H and

13

C NMR spectroscopy using thedata of

1

ç/

1

ç

COSY, TOCSY, and ROESY homonu-clear and

1

ç/

13

ë

gHSQC and gHMBC heteronucleartwo-dimensional techniques.

The analysis of the one-dimensional and two-dimensional spectra of eremomycin B and the compar-ison of the data obtained with the results of the similaranalysis carried out earlier for eremomycin [4, 8](Table 2) showed that the two antibiotics have a similaraglycone and differ in the structure of their disaccha-ride moiety. The doubling of some peaks referring tocertain carbon atoms and protons in aromatic rings Aand B and the disaccharide fragment g-f observed in thespectrum of eremomycin B, as opposed to those in thespectrum of eremomycin, is obviously due to the differ-ence in the temperature of registration of the spectra foreremomycin (

70°ë

) [8] and eremomycin B (

55°ë

),rather than a structural difference (Table 2). The lowertemperature retards the rotation of the disaccharidefragment attached to the sterically shielded oxygenatom at C-4 of ring B around the glycoside bonds. Thisresults in the appearance of the signals of two preferen-tial conformers.

The presence of the signals of the carboxymethylgroup

–ëç

2

ë(é)éç

with chemical shifts

δ

ë

equal to172.33 (M1, carbonyl) and 44.26 (M2, the methyleneunit) and

δ

ç

= 3.57 (the two-proton singlet of the meth-

Table 1.

Physicochemical and biological characteristics of eremomycin and eremomycin B

Parameter Eremomycin Eremomycin B

Molecular weight,

m

/

z

(MALDI-MS) 1558 [

M

+ H]

+

; 1580 [

M

+ Na]

+

; 1596 [

M

+ K]

+

1617 [

M

+ H]

+

; 1639 [

M

+ Na]

+

; 1655 [

M

+ K]

+

λ

max

in UV spectrum of the solution in 0.01

M

HCl 280 280

R

f

paper chromatography* S

1

(

see the Experimental

)

0.62 0.41

Mobility (cm) upon paper electrophoresis in E

1

electrolyte (see the Exper-imental)

7.2 5.2

Solubility

in water, 0.01 M HCl, and NaOH Good Good

in acetone, alcohols, and EtOAc Insoluble Insoluble

The number of free amino groups (the method of partial substitution [7]) 3 2

Biological activity towards

B. sabtilis

ATCC 6633, MSC**,

µ

g/ml 0.2 0.4

Notes: * Visualization with Pauli’ reagent and by bioautography using the test organism

B. subtilis

ATCC 6633.** MSC, minimum suppressing concentration.

RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

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No. 4

2009

THE STRUCTURE OF ANTIBIOTIC EREMOMYCIN B 499

Table 2.

Chemical shifts (

δ

, ppm) of the signals in the

1

H and

13

C-NMR spectra of eremomycin [8] and eremomycin B andthe registered correlation peaks in the ROESY and HMBC spectra of eremomycin B that corroborate its structure (the atomindices are given in the structural formulae of eremomycin and eremomycin B)

No.*Eremomycin Eremomycin B

Atom indexCorrelation peaks in the spectra

δ

C

δ

H

δ

C

δ

H

ROESY HMBC

1 176.40 178.23 X8

α

7

2 174.46 175.37 X3

α

3, P7

3 172.11 172.99 X5

4 171.80 172.61 X6

α

5,

α

6

– 172.33 M1 M2

5 171.29 172.05 X4

α

3, P7

6 171.23 172.26 X1

α

1,

α

2, P4

7 169.39 170.13 X2

α

2,

α

3

8 167.88 168.56 X7

α

6,

α

7

9 157.21 157.93 D3 D2, D4

10 157.21 157.9; 157.4 B3

11 156.41 156.6; 155.8 A4

12 155.68 156.36 D5 D4

13 155.06 155.78 E4 E2, E5, E6

14 153.34 154.9; 153.9 B5

15 150.76 151.38 C4 C2, C6

16 138.20 138.86 C1

α

2

17 137.92 139.47 D1 α7

18 136.31 7.16 137.05 7.16 E2 α5, E6 α5, α6

19 134.92 135.69 B1

20 134.22 134.8; 134.2 B4

21 133.88 134.0 A1

22 130.70 7.39 131.53 7.40 C6 C2, P6 e2ax, e2eq, P6

23 129.42 7.57 130.0; 130.4 7.55; 7.66 A2

24 128.79 6.96 129.49 6.97 A6 P8 e1, e2eq

25 128.35 129.08 C5 C2, C6

26 127.60 7.13 128.30 7.14 E6 α5, E2

27 127.28 7.71 128.01 7.72 C2 C6, P6 P5

28 126.56 127.07 E1 α5, E5

29 125.56 7.49 126.18 7.40; 7.59 C3

30 123.55 7.06 124.36 7.03; 7.18 A3

31 122.17 123.01 E3 E5

32 122.09 5.32 123.2; 122.8 5.47; 5.09 A5

33 119.01 7.04 119.68 7.05 E5

34 118.27 119.08 D6 D2, D4, E2, α7

35 109.09 6.59 109.71 6.61 D2 D2, α7

36 107.71 5.51 108.7; 107.0 5.59; 5.43 B2

37 104.80 5.63 105.4; 105.3 5.66; 5.58 B6

500

RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 4 2009

BERDNIKOVA et al.

Table 2. (Contd.)

No.*Eremomycin Eremomycin B

Atom indexCorrelation peaks in the spectra

δC δH δC δH ROESY HMBC

38 103.87 6.61 104.32 6.63 D4 D4

39 102.30 5.55 104.3; 103.7 5.35; 5.81 g1

40 98.03 5.37 99.0; 98.4 5.42; 5.43 f1 f2eq f2eq, f2ax

41 93.27 5.08 93.92 5.10 e1 e2eq e2eq, e2ax, P8, A6

42 80.00 4.03 81.2; 80.1 4.29; 3.83 g2

43 77.14 4.03 78.1; 77.9 3.76; 3.49 g3

44 76.44 3.79 76.9; 76.7 4.40; 4.19 g5

45 75.62 3.53 76.39 3.53 e4 Me-e3, e6, e2eq e2ax, e6

46 75.30 5.47 76.11 5.49 P8 e5, α6

47 75.20 3.48 75.2 3.67; 3.54 f4 f2eq f2ax, f6

48 71.78 5.59 72.52 5.62 P6 C2, C6, α2 C6

49 70.33 3.63 70.79; 70.45 3.50; 3.76 g4

50 67.03 3.85 67.73 3.89 e5 e4, e6 Me-e3, e6, P8

51 66.62 4.58 67.41 4.78; 4.53 f5

52 62.31 4.27 63.92 4.30 α6 α5, E6

53 61.76 4.24 62.35 4.24 α1 P3, P4, P5 P1,2; P4, P5

54 61.76 3.64 62.36 3.65; 3.96 g6

55 59.83 5.45 60.37 5.50 α2

56 59.46 4.75 60.72 4.72 α7 D2

57 57.45 58.03 e3 e4, e2eq, Me-e3

58 57.11 62.72 f3 f2eq, M2, Me-f3

59 55.34 4.56 56.17 4.60 α5 E2, E6 E2, E6, α6

60 55.21 6.53 55.86 6.60; 6.51 α4

61 53.10 4.94 53.81 4.96 α3 P7 P7, e2eq, Me-e3

– – 44.26 3.57 M2 f2eq, f2ax, Me-f3

62 39.76 1.88 40.20 1.89 P4 α1, P1, P2, P3 α1, P3, P1,2

63 39.40 2.14; 2.36 38.16 2.14; 2.36 f2ax f2eq

f3 f1, f4f1, Me-f3

64 39.08 2.39; 2.54 39.75 2.40; 2.56 e2ax e2eq

e1, e4, C6e1, A6, C6, α3

65 36.95 2.67 37.58 2.68 P7 α3

66 32.66 2.89 33.31 2.91 P5 α1 α1, C2

67 24.53 1.73 25.16 1.75 P3 α1, P1, P2, P4

68 22.78 0.96 23.58 0.96 P1 P2, P3, P4 P3

69 22.03 0.95 22.60 0.97 P2 P1, P3, P4 α1, P4

70 18.85 1.68 19.50 1.71 Me-e3 e4 α3, e5

71 17.51 1.40 17.30 1.41 Me-f3 f5, M2

72 17.91 1.42 18.75 1.45 e6 e4, e5

73 17.59 1.28 17.9 1.31 f6

Note: *The number of the signal of the carbon atom in the 13C-NMR spectrum of eremomycin [8].

RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 4 2009

THE STRUCTURE OF ANTIBIOTIC EREMOMYCIN B 501

ylene unit) in the spectra of eremomycin B points to thedifference in the structures of the antibiotics under con-sideration. The localization of the substituent men-tioned in the disaccharide fragment of the molecule(see the formula and Fig. 1) followed from the analysisof the correlation peaks in the ROESY and HMBCspectra. Thus, the ROESY spectrum (Fig. 2) containedcorrelation peaks between the protons of the carboxym-ethyl methylene unit and protons of the methyl group inthe fMe-f3 residue (Fig. 1), and also the axial and equa-torial protons of f2 (the deoxy unit). These correlation

peaks can be observed only in the case of the localiza-tion of the carbonyl fragment at the amino group of thef residue of eremosamine (see the formula and Fig. 1).This is also corroborated by the correlation peak in thecoordinates of the chemical shifts of methylene protons(M2) of the carboxymethyl group and the carbon atomf3 in the HMBC spectrum (Fig. 3). The presence of thementioned N substituent in the eremosamine residue isalso corroborated by the downfield shift of the signal f3in the 13C NMR spectra when passing from eremomy-cin to eremomycin B (Table 2, signal no. 58).

To identify the antibiotic obtained, we carried outthe search for an analogue using a computer database ofnatural biologically active compounds (BNPD) devel-oped by J. Bérdy (Hungary) [9] and also the worldwidearrays of scientific and patent information available viaSTN (the Scientific and Technical Network) service.The search was performed for molecular weight (1616)and absorption maximum in the UV–VIS area (λmax =280 nm) for the solution of eremomycin B in 0.01 MHCl. We failed to find data on the antibiotic that matchthe characteristics underlying the search. This is evi-dence that eremomycin B is a previously unknown rep-resentative of dalbaheptide antibiotics of the vancomy-cin group.

OO OH

OHOH

O

O

HN

CH3

CH2

H3CHO

H

HOOC

H

Fig. 1. Interatom contacts registered in ( ) ROESY and( ) gHMBC spectra corroborating the localization ofthe carboxymethyl substituent at the amino group of the fresidue of eremosamine. See also Figs. 2 and 3.

f6

4.01.41.61.82.02.22.42.6

3.9

3.8

3.7

3.6

3.5

3.4

f4

f4

f2eqe2eq e2ax

f2ax

P4

P3

e4

e5

g6

M2

Me–f3Me–e3 e6

ppm

ppm

Fig. 2. The fragment of the ROESY spectrum for eremomycin B (D2O, 55°C), corroborating the localization of the carboxymethylsubstituent at the amino group of the f residue of eremosamine. The signal marking at the spectrum axes is given in Table 2, in theformula, and in Fig. 1.

5

5

1 33

2

g

f

1

502

RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 4 2009

BERDNIKOVA et al.

EXPERIMENTAL

Potassium 4-chloro-3,5-dinitrobenzenesulfonatewas synthesized as described in [7]; cation-exchangeresins SDV-3 and KB-2 were from Reakhim (RussianFederation); CM cellulose was from Serva (Germany);and antibiotic eremomycin was obtained on a pilotplant of the Gauze Research Institute of New Antibiot-ics, Russian Academy of Medical Sciences [6]. 1H- and13C NMR spectra were registered on an AVANCE 600(Bruker, Germany) spectrometer in D2O at 55°C.Chemical shifts are given relative to 3-trimethylsilyl-2,2,3,3-tetrasdeuteropropionic acid (TSP, δH = 0.0, δC =1.4).The standard Bruker software was used for thetwo-dimension experiments. A time shift of 300 ms wasused in the ROESY experiment. The gHMBC methodwas optimized for a constant of 8 Hz.

The UV–VIS spectra of the isolated antibiotic wererecorded on a UV-1601 PC (Shimadzu, Japan) spectro-photometer; mass spectra were taken on an Ultraflex IIMALDI ToF/ToF (Bruker Daltonics, Germany) instru-ment equipped with a 355-nm (Nb) UV laser in themode of generation of positively charged ions using areflectron; the accuracy of the masses measured was0.001%. The solution of the sample (1 µl) and the solu-tion of 2,5-dihydroxybensoic acid in 20% aqueous ace-tonitrile containing 0.5% TFA (0.3 µl) were mixed onthe target; the resulting mixture was air dried.

Paper chromatography was carried out on FN-14(Filtrak, Germany) paper using 4 : 1 : 1 n-BuOH–AcOH–H2O as the developing system. Electrophoresiswas performed at 550 V for 3 h on FN-14 (Filtrak, Ger-

many) paper in a V-shaped Durrum’s apparatus [10] inelectrolytes Ö1 (pH 1.7; 30% aqueous ëç3ëééç) andÖ2 (pH 1.1; 30 : 30 : 40 85% aq. HCOOH–CH3COOH–H2O) [7]. The electrophoretic mobility of the antibioticwas determined from the value of the migration of thesubstance from the start line to the cathode (in cm).Antibiotics were visualized by the method of bioautog-raphy suggested by A. Haese and U. Keller [11] usingB. subtilis as a test organism, and also the specificreagents in peptide chemistry [12] ninhydrin (for thedetection of primary amino groups), Pauli’s reagent(for the detection of the residues of phenol-containingamino acids), and ortho-chlorotolidine (for the detec-tion of peptide bonds CONH–).

Isolation and purification of eremomycin B. Themother liquor (4.8 l) obtained after the absorption oferemomycin from the native solution as described in [6]and containing approximately 160 mg of eremomycinB was twofold diluted with water and acidified with1 M aqueous HCl to pH 2.5. Then, eremomycin B wasabsorbed on cationite SDV-3 [H+]. The antibiotic waswashed from the sorbent with aqueous 0.5 M NH4OH,the eluate was concentrated to 12 ml, and the antibioticwas precipitated by the addition of acetone. After filtra-tion, the precipitate contained 105 mg of raw eremomy-cin B. The product obtained was additionally purifiedby chromatography on a glass column (2 × 25 cm) withCM cellulose [H+] and elution with the linear gradientfrom an ammonium acetate buffer (pH 5.6; 0.05 MCH3COONH4–EtOH, 85 : 15) to a mixture of 0.1 MCH3COONH4–EtOH, 85 : 15 (0 50%). Isolation of

e4

4.04.55.05.5

178

176

174

172

170

168

α2/X2

ppm

ppm

α2/X1

α3/X4

α3/X2

α7/X7 α2/X2

α3/X3α6/X6

α5/X6 α1/X1

α7/X8

M2/M1 3.53.6

65

60

ppm

ppm

e4/e3

e4/e5

M2/f3

(‡)

M2

(b)

Fig. 3. (a and b) are the fragments of the 1H-13C gHMBC spectrum of eremomycin B (D2O, 55°C), corroborating the localizationof the carboxymethyl substituent at the amino group of the f residue of eremosamine. The corresponding parts of the 1H- and 13CNMR spectra are shown along the horizontal and vertical axes. The signs in the marks for cross peaks before the slash refer to pro-tons, whereas after the slash, to carbon atoms.

RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 4 2009

THE STRUCTURE OF ANTIBIOTIC EREMOMYCIN B 503

the antibiotic was monitored by paper chromatographyand paper electrophoresis (for conditions, see below).The eluate containing eremomycin B was desalted oncationite SDV-3 [H+], acidified to pH 6.5–6.8 by theaddition of 1.0 M H2SO4, and concentrated in a vacuumto a volume of 10–12 ml. The target product was pre-cipitated by the addition of acetone to give 55–60 mg ofchromatographically and electrophoretically homoge-nous eremomycin B (as a sulfate).

The reaction of eremomycin B with potassium3,5-dinitro-4-chlorobenzenesulfonate. Triethylamine(0.02 ml, 0.14 mmol) and a solution of potassium 3,5-dinit-ro-4-chlorobenzenesulfonate (5.6 mg, 0.017 mmol) in 60%acetone (0.22 ml) was added to a solution of eremomy-cin B (9.7 mg, 0.017 mmol) in water (0.15 ml). Themixture was stirred for several minutes and then kept at40°ë for 1 h during periodical stirring. The resultingyellow solution was applied on an FN-14 paper sheetfor electrophoresis in the flow of warm (40–50°ë) air.Electrophoresis was carried out at 550 V for 3 h in aV-shaped Durrum’s apparatus [10] in electrolyte Ö2.Similarly, the experiments on the partial substitution oferemomycin amino groups were performed. The mix-ture of the products of eremomycin B conversion con-tained two yellow Dnsp derivatives, one of them,mono-Dnsp-eremomycin B (with a total moleculecharge equal to zero), was at the start line, whereasanother product, di-Dnsp-eremomycin B (with a totalmolecule charge equal to –2), migrated to the anode. Inthe case of eremomycin, the electrophoregram con-tained the spots of three yellow derivatives; mono-Dnsp-eremomycin (with a total molecule charge equalto +1) migrated to cathode, and the di- and tri-Dnspderivatives of eremomycin with a charge of –1 and –3,respectively, thus migrated to anode. Thus, eremomy-cin B contains two free amino groups, whereas eremo-mycin has three amino groups, which is in agreementwith the data on the structures of these antibiotics.

ACKNOWLEDGMENTSThis work was carried out within the framework of

the Agreement on Scientific and Technical Cooperationof the Gauze Research Institute of New Antibiotics,

Russian Academy of Medical Sciences, and the Zelin-skii Institute of Organic Chemistry, Russian Academyof Sciences. This work was partially supported by theRussian Foundation for Basic Research (project no. 06-03-33120a).

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9. Bérdy, J., Proceedings of International Conference onMicrobial Secondary Metabolism, Interlaken, Suisse,1994, p. 2.

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pp. 1360–1368.12. Dawson, R., Elliott, D., Elliott, W., and Jones, K., Data

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