469VOL. XXXIV NO.4 THE JOURNAL OF ANTIBIOTICS

COMPLEXATION OF ACETYL-D-ALANYL-

D-ALANINE BY ANTIBIOTIC A35512B

ANN H. HUNT & PAUL D. VERNON

Lilly Research Laboratories, Indianapolis, Indiana 46285, U.S.A.

(Received for publication February 5, 1981)

Antibiotic A3 5512B1.2) is a member of the

glycopeptide family of antibiotics; complete structures have been reported for two members of this group, vancomycin3' and ristocetin A4' a) . Both vancomycin and ristocetin have been shown to form complexes with mucopeptides containing the terminal dipeptide D-alanyl-D-alaninee) ; this interaction within the bacterial cell inhibits cross-linking of the cell wall and leads to eventual lysis of the organism. Chemical attack on A35512B2)

produces degradation products which are closely related to those obtained from ristocetin A7), suggesting structural similarities between the two antibiotics. Thus, A35512B might also be ex-

pected to form complexes with mucopeptide analogs. This paper reports the observation of such a complex with acetyl-D-alanyl-D-alanine

(Ac-D-Ala-D-Ala). The interactions of glycopeptide antibiotics

with acetyl-D-alanyl-D-alanine and related mole-cules have been examined by NMR methods

(both vancomycin8-11) and ristocetinl2'18)) and by UV difference spectroscopy (chiefly vanco-mycin6'14-18)). The UV difference method was used to examine complex formation between Ac-D-Ala-D-Ala and A35512B in the work report-ed here. In order to compare the binding results for A35512B with those for a more thoroughly characterized glycopeptide, an identical experi-ment was conducted using vancomycin.

A tandem-cell arrangementa.la) was used for titration of Ac-D-Ala-D-Ala into a solution of the antibiotic (A35512B or vancomycin). Initial concentrations were 2.00x10-1m Ac-D-Ala-D-Ala (0.404 mg/ml), 1.60 x 10-4 M A3 5512B (0.313 mg/ml), and 1.72 x 10-4 M vancomycin hydro-chloride (0.256 mg/ml). The solvent for all solutions was 0.02m sodium citrate buffer, pH 5.115). The total lightpath of each cell was 2 cm, and the initial volume of antibiotic solution was 0.8 ml in each case. Examples of the UV differ-ence spectra produced by the addition of Ac-D-Ala-n-Ala to A35512B or to vancomycin are shown in Fig. 1.

Fig. 1. UV difference spectra induced by Ac-D-Ala- D-Ala by complex formation with A35512B or with vancomycin.

For A35512B, Ao'=1.33 x 10-4 M; [peptide]/

[antibiotic] =2.50. For vancomycin, Ao' =1.43 x 10-4M; [peptide]/[antibiotic] =2.33.

A

+0.04

0

-0 .04

-0 .08

-0 .12

260 280 300 320 nm

Vancomycin

A35512B

Fig. 2. Magnitude of the absorbance difference between the positive (2=294.5 nm) and negative (2=281 nm) peaks in the difference UV spectra of vancomycin plus Ac-D-Ala-D-Ala.

Experimental points are plotted as a function of the molar ratio [peptide]/[vancomycin] ; the solid line is the behavior of d predicted by equation (3) for values of V ranging from 0 to 0.908.

(e)0.12

0.10

0.08

0.06

0.04

0.02

0.0010 2 4 6 8 10 12

[Peptide]/[Antibiotic]

Vancomycin

K=1.4x104M-1

D = 0.1755

470 THE JOURNAL OF ANTIBIOTICS APR. 1981

For both antibiotics both positive and negative difference peaks are produced. The experimental

property used in the calculation of equilibrium constants for peptide-glycopeptide complexation is the difference, d, between these positive and negative peaks. In Fig. 1, for example, d =

(A2e8 . b -A288) =0.0988 for A35512B, and d = (A294 . b -A281) =0.1138 for vancomycin. The ob-served values of d are plotted as a function of the molar ratios of Ac-D-AIa-D-Ala to glyco-

peptide in Figs. 2 and 3. The equilibrium constant for the complexation

reaction A+PFC is given by

K= C _ C

(A)(P) (A0'-C)(P0'-C)

where C=concentration of the complex, A0'= total concentration of free and complexed anti-biotic, and Po'=total concentration of free and complexed peptide.

(If the initial concentrations of antibiotic and peptide are A0 and Pa and the initial antibiotic volume is V0 ml, then after the addition of V ml of peptide solution A0' =A0 (V0)/(V0 + V) and P0' =P0 (V)/(V0 +V)•) The equilibrium constant expression can be solved for C to give expression (1) :

(1) C=

[A0'+Po' +(1 /K)] - [[A0' + Po'+(1 /K)]2-4(Ao')(Pa')]"2 2

If the value of d which would be observed for a totally-complexed antibiotic at concentration Aa is defined as D, then at any point in the titration the fraction of antibiotic complexed is given by :

Q(V0+V)/Vo C D A0'

which can be simplified to equation (2) :

(2) 4 = C(D/A o).

Substitution of (1) into (2) produces an expres-sion for d (the observed UV difference) as a func-tion of V (the volume of peptide added), with D and K as adjustable parameters :

(3) dD

2A0 [Ao'+P0'+(1/K)

- v'[A 0' + Po' + (1 /K)]2 - 4(A o')(Po')]

(Note that both A0' and Pa' are defined in terms of V.)

The solid lines in Figs. 2 and 3 demonstrate the least-squares fit of equation (3) to the values of d shown in the figures. For vancomycin, K = 1.4 x 104 M-1, which is identical with the result obtained from an NMR dilution study8~ . For A35512B, K=7.3 x 104 M-1, indicating an even higher affinity for Ac-D-AIa-D-Ala than that ex-hibited by vancomycin. These data imply both a structural and a biological similarity between A35512B and other members of the glycopeptide antibiotics class. Structure elucidation studies on A35512B are in progress.

Acknowledgement

We thank Mrs. MARY ANN BOGAN for synthesis of

the Ac-D-Ala-n-Ala and Dr. D.E. DORMAN for instruc-

tion in computer usage.

References

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Fig. 3. Magnitude of the absorbance difference between the positive (2=298.5 nm) and negative

(2=288 nm) peaks in the difference UV spectra of A35512B plus Ac-n-Ala-n-Ala. Experimental points are plotted as a function of

the molar ratio [peptide]/[A35512B]; the solid line is the behavior of d predicted by equation (3) for values of V ranging from 0 to 0.845.

0.12

0.10

0.08

0.06

0.04

0.02 0

.000 2 4 6 8 10 12

[Peptide]/[Antibiotic]

A355128

K = 7.3 x 104M-1

0=0.1284

471VOL. XXXIV NO. 4 THE JOURNAL OF ANTIBIOTICS

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