THEO CHEM

ELSEVIER Journal of Molecular Structure (Theochem) 4 I8 (1997) 93-98

Computational study of novel catechol-type siderophore analogs

Michael Meyera’*, Wolfram Trowitzsch-Kienastb

“A&. Molekulare Strukturforschung. GBF (Gesellschajfir Bioterhnologische Forschung). Mascheroder We8 I,

D-38124 Braunschweig Germany

hFuchbereich 3, Chemie und Biotechnik, Tet-hnische Fachhochschule Berlin, Luremburger Str. IO, D-13353 Berlin Germany

Received 2 December 1996; accepted 10 February I997

Abstract

A series of catechol-type siderophores has been proposed using the ligand design program LUDI. Semiempirical PM3 calculations have been carried out for gallium siderophore complexes including enterobactin, TRENCAM and the myxochelin series with an alkyl chain backbone. The tris(catechol) gallium group of both diasteromeric [Ga(ent)]s- complexes can be overlaid closely with [Ga(cat)3J3-. This indicates an energetically favourable conformation without any strain. The energy difference between both [Ga(ent)] ‘- diastereomers having a A- and A-configuration is very small. A similar optimal geometry of the chelathgand is only possible for special chain lengths of the alkyl backbone. Short chains connecting the catechol groups lead to serious deformations of the complex structure and therefore it is impossible that metal ions are ligated by the six catechol oxygens of a single siderophore with a short alkyl chain backbone. 0 1997 Elsevier Science B.V.

Keywords: Enterobactin; Myxochelin; Gallium; Ligand design; PM3

1. introduction

Siderophores are low molecular weight ligands produced by bacteria and fungi for iron uptake [ 11. The potential use of siderophores for antibiotics has motivated us to propose novel siderophores with ligand design methods using enterobactin (Fig. 1) as

a lead and to carry out semiempirical studies for the elucidation of structural and energetic properties of siderophores. Besides Fe”‘, the siderophores can form complexes with Ga”‘, Cr”’ or V’“, which have been used often for experimental studies of model

* Corresponding author. Present address: Biocomputing, Institut fiir Molekulare Biotechnologie, Postfach 100 8 13, D-07708 Jena,

Germany, Tel.: +49-364-1656203; fax: +49-364-1656203; e-mail:

mmeyer@imb-jena.de

systems, because the ionic radii of these species are close to each other (Gal” 0.62, Cr”’ 0.63, V” 0.63, Fe”’ 0.64 A). The affinity of siderophores to other metal ions like Mg” or Ca” is much lower. A very stable complex [Fe(ent)] ‘- can be formed between the catechol-type siderophore enterobactin and iron. The most interesting structural aspect of this complex is the absolute configuration of the chelation, which might be important for the recognition at the receptor for iron uptake by microorganisms. The species with the A- (right-handed propeller) and A-configuration (left-handed propeller) of the tris(catecho1) ligand are diasteromers in consequence of the additional chiral serine residues forming the trilactone backbone. A A-configuration of the catecholat ligands has been derived from CD spectra for [Went)]‘- and [Fe(ent)13- [2] and the crystal structure of [V(ent)]‘-

0 166.1280/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved PII SOl66- 1280(97)00067-5

94 M. Meyer. W. Trowitzsch-Kienaslurnal of Molrcular Structure (Theorhem) 41X (1997) 93-98

HO 0 co .co OH

‘N NH HO OH

NH

co

b

OH

0 OH

Fig. 1. Enterobactin Hb

[3,4] shows clearly the same absolute configuration. An NMR-study of [Ga(ent)] ‘- confirmed again the A- configuration of enterobactin complexes [5]. We

investigated the energy difference between both [Ga(ent)13- diastereomers and compared the com- puted structure with the experimental one of the related K,[V(ent)]. The structural analysis of [Ga(TRENCAM)13- (Fig. 3) [6] completes the studies of known ligands.

Furthermore, we wanted to find siderophores with a

novel backbone. A new chiral centre can be generated for alkyl backbone siderophores shown in Fig. 2 if the numbers of methylene groups from the central carbon to the catecholat groups are different (i # j # k). This is a new feature of synthetic siderophores resembling enterobactin, which is absent in ligands

HO CO /CO OH

-NH HO

.NH ‘(CH&,(CHA

OH

I

p2 NH I co

b OH

0 OH

Fig. 2. TRENCAM H6.

like TRENCAM, but already known from the natural siderophores myxocheline A and B isolated from

myxobacteria [7,8]. We proceeded in two steps to propose new siderophore backbones. First, we com- puted from small molecular fragments new backbone structures, which were required to fit into a pseudo-

receptor, since an experimental receptor structure is not available. Then, we employed semiempirical methods for a more detailed investigation of selected

complexes and compared them with know sidero-

phores and [Ga(cat)3)] 3m. There are no chains connect- ing the catechol rings of this anion, which might force these rings in an unfavourable conformation.

Up to now, force field calculations have been

carried out to determine structural parameters of side- rophores and to explain the preference of enterobactin for the A-configuration [9,10]. The criticism of some aspects of these studies [4,1 l] and the availability of semiempirical parameters for gallium caused us to use

semiempirical methods.

2. Methods

The crystal structure of [V(ent)3)] ‘- [4] has been

used to generate a pseudo receptor for catechol-type siderophores using LUDI [12] in the active analog

mode. Siderophore backbones have been constructed at an rms tolerance of 0.4 A using [V(ent)3] 2- with the trilactone ring removed. Additional conformers of the molecules proposed by LUDI have been generated to check the relative energies with semiempirical methods after substituting vanadium by gallium.

The semiempirical calculations have been carried

out with the PM3 method [13,14] of MOPAC 6.0 [15]. The calculations have been carried out with the mole- cular mechanics correction for amide groups. An initial structure of [Ga(ent)3)]3- with a A-configura- tion has been generated from the coordinates of KJV(ent) 3)] [4]. For the diastereomeric A-configura- tion we started with the optimized structure of the corresponding tris(catecho1) complex and constructed the corresponding trilactone ring starting from L- serines corresponding to a P-sheet conformation. An initial structure for the gallium complex with TRENCAM has been generated from the experi- mental structure of Kz[V(TRENCAM)] [6] by substi- tuting vanadium and adding hydrogen atoms. Initial

M. Meyer, W. Trowitzsch-KienasVJoumal of Molecular Structure (Theochem) 418 (1997) 93-98 95

+

NH

I co

b OH

0 OH

Fig. 3. Novel alkyl chain siderophores.

geometries for the alkyl backbone siderophores have been constructed from the tris(catecho1) complexes adding first the CONH groups and then the appropri- ate number of methylene groups. Initially, a series of different backbone conformations has been generated

manually. Then, the systematic conformational search method implemented in SPARTAN [16] has been used to generate different alkyl chain conformations for each of the three chains shown in Fig. 3. Finally, the

three alkyl chains of suitable conformers were con- nected, optimized with the SYBYL forcefield at a fixed tris(catecho1) geometry and submitted to a complete structure optimization at semiempirical level. Local energy minima have been verified by a frequency calculation.

3. Results

3.1. L&and design

We searched for two types of siderophore back- bones, cyclic ones like the trilactone ring of entero-

bactin (Fig. 1) and non-cyclic ones corresponding to TRENCAM (Fig. 3). One of the two cyclic structures proposed by LUDI, was a reconstruction of the original enterobactin trilactone ring. According to

the second structure, the three > CH-CH2-O- CO- ring fragments in enterobactin (Fig. 1) can be

replaced by a > CH-CH+ZC- group. For the non-cyclic structures (Fig. 3) LUDI

proposed an alkyl backbone with a symmetric number

of methylene groups i,j,k = 2,2,2 (C = 6). Similar to the conformations of the pyramidal nitrogen atom in

the gallium TRENCAM complex ‘in’ and ‘out’ con-

formations of the CH group connecting the three chains are possible. The conformations arise from the possibility that the hydrogen at the CH group may point towards the gallium or in the opposite direction prior to the binding of the other catechol groups. LUDI generates an ‘in’-conformer for the

2,2,2 alkyl backbone siderophore. This is analogue

to the experimental TRENCAM structure (see Section 3.2.3). For chain length 2,2,2 the > C=O group may

be replaced by a > CH2 group and the -CONH- group may be substituted by a -CH2-CH2- group. In the latter case, intramolecular hydrogen bonds are absent. Furthermore, a backbone with i,j,k = 1,3,3 (c = 7) and an ‘out’-conformation has been

proposed.

3.2. Semiempirical calculations

3.2.1. Tris-(catechol) complexes

For [Ga(cat)3]“- the GaO bond length was cal- culated to be 1.85 A at a heat of formation AHr = -266.5 kcal mall’. This bond length is somewhat

shorter than the experimental distance r(Cr0) = 1.986(4) A and r(Fe0) = 2.015(6) A of the corre- sponding catechol complexes [17]. The computed angle Q(OGa0) = 90” corresponds to an octahedral coordination whereas the corresponding angles Q(OCr0) = 83.56(14)” and Q(OFe0) = 81.26(14)” indicate a trigonal distortion, which is absent in gallium complexes.

3.2.2. Enterobactin complex

The optimized complex with an L-serin trilactone

ring and a A-configuration is only 0.2 kcal mall’ more

stable than the diastereomer with the A-configuration. The computed heats of formation are AHf = -673.7 kcal mall’ and AH,= -673.5 kcal mol-‘, respectively. Selected structural parameters are given in Table 1. The bond lengths between Ga and the catechol oxygen are close to the ones of [Ga(cat)3]‘-. Similar to the

experimental structure of K2[V(ent)i)]‘-, the distance between gallium and the ortho-catechol oxygen 0 1 is slightly longer (1.85 A) than the corresponding dis- tance between gallium and the meta-catechol oxygen 02 (1.84 A). The computed distances are 0.1 A

96 M. Meyer, W. Trowirzvch-KienasdJournal of Molecular Structure (Theochem) 418 (1997) 93-98

Table I Heats of formation AHr (kcal mol-‘) and structural parameters for gallium (X = Ga) and vanadium“ (X = V) complexes with enterobactin and

TRENCAM

AHrh r(XG1) r(XO2) r(NH...Ol) <(0X0) rms’

A-[Ga(ent)]‘- -673.7 1.84 1.85 1.79 90 0.08

A-[Ga(ent)]‘- -673.5 1.84 1.85 1.80 90 0.10

[Ga(TRENCAM)] ‘- -438.6 1.84 1.85 1.79 90 0.11

A-[V(ent)] ‘- 1.939 1.946 1.86 79.8

[V(TRENCAM)] ‘- 1.985 2.007 80.75

A Ref. [4,6].

h Molecular mechanics correction applied.

’ rms deviation of the volume of the tris(catecho1) subunits relative to [Ga(cat)?)] ‘-,

shorter than the experimental ones of the vanadium

complex. The mean value of the distances between the amide hydrogen and the orrho-catechol oxygen is 1.79 A compared to 1.86 A determined experimentally for the vanadium complex. These computed structural parameters for the A-[Ga(cat)3]3- are practically the same as for the A-configuration.

with a sixfold coordination of gallium with catechol oxygens. The entries are labelled by the number of methylene groups i,j and k. All molecules in a section having the same sum c of i,j and k are isomers. If the three indices are different from each other two backbone configurations R and S arise in addition

to the A- and A-configuration of the tris-(catechol) moiety.

3.2.3. TRENCAM

The refined structure of the catechol subunit is in close agreement with the previously determined structures for the unsubstituted tris-(catechol) com- plex and the enterobactin complex (Table 1). A varia- tion of the initial backbone geometry shows that the ‘in’-conformation with the tertiary nitrogen directed

towards the gallium is 6.7 kcal mol-’ more stable than a conformation with the pyramidal nitrogen in the

opposite direction.

3.2.4. Alkyl backbone siderophores

The heats of formation for the gallium complexes with alkyl backbone siderophores are given in Table 2. We have only taken into account those structures

Table 2 suggests the following general conclusions

for the heats of formation. The ‘in’-conformation is energetically unfavourable relative to the ‘out’ con- formation if one backbone chain contains no methy-

lene group (i = 0). In this case, the CH hydrogen pointing inward to gallium is eclipsed with the amide hydrogen of the corresponding chain. The ‘in’-conformation of the symmetric 2,2,2 backbone is energetically more favourable than the ‘out’-con- formation, which resembles the structure of the

TRENCAM complex. The energy difference 8.7 kcal mall’ between the ‘in’- and ‘out’-conformations is somewhat higher than the one of TRENCAM. For the 1,3,3 backbone, the ‘out’-conformation is slightly more stable.

Table 2

Heats of formation for gallium complexes with alkyl backbone siderophores

i j k Configuration AHLout) AHdin)

3 0 1 2 AR

AS

4 0 I 3 AR

AS

5 0 I 4 AR

AS

6 2 2 2

7 1 3 3

-423.3 -409.8

-423.8 -411.9

-432. I -430.9

-435. I -425.5

-441.4 -434.0

-444.4 -432.7

-451.8 -460.5

-462.1 -461.9

M. Meyer, W. Trowitzsch-Kienast/Journal of Molecular Structure (Theochem) 418 (1997) 93-98 91

4. Discussion

The semiempirical PM3 method has been selected

because of its parametrisation for gallium. To our knowledge detailed comparisons between experimen- tal and PM3 calculations for gallium have not been

reported. Previously, its ability for the description of hydrogen bonds, which is important for this type of

siderophores, has been reported [ 181. However, PM3 has a tendency towards pyramidal amide nitrogens, which must be corrected using the molecular

mechanics correction. Some examples have shown previously that

gallium and iron catechol-type complexes are iso- structural [ 17,191. Thus, our results should hold for iron complexes of siderophores as well. A small characteristic difference is the trigonal distortion of the tris-(catechol) moiety in Cr, V and Fe complexes,

which is absent in the computed Ga complexes. The PM3 calculations for [Ga(ent)3)] 3- predict that the A- configuration is 0.2 kcal mol-’ more stable than the A- configuration. In Cr”‘, V” and Fe”’ complexes with

enterobactin, the same configuration has been observed. The predicted preference of the A-config- uration is in agreement with the experiment [5] and the energy difference is comparable to the 0.5 kcal mol-’ determined with force field calculations for [Fe”‘(ent)13- [lo]. Nevertheless, one should not over- value the calculated energy difference, because the errors of the relative energies are larger at semi- empirical level. The small magnitude of the energy difference supports also the opinion that the entero- bactin backbone conformation prior to the metal bind-

ing induces a stereospecific binding of the metal. We predict hydrogen bonds between the orrho-catechol oxygen and the amid hydrogen for [Ga(ent) j)]‘-. This is also an important structural feature in other

chiral siderophor analogs [20,2 11. The structures show practically no significant differences which might explain an energy difference between both dia- steromers. There are no obvious differences in amid hydrogen bonding or in the structural parameters of the Ga-0 moiety between both configurations. Furthermore, the tris(catecho1) subunits are almost

identical with the one of [Ga(cat)3]“-. The rms deviation of the superimposed volumes of the Ga

and the catechol rings of [Ga(cat)3]3- and [Ga(ent)] j- is 0.08 A”. This nearly perfect

arrangement without unfavourable steric interactions in enterobactin is one reason for the high stability of

the enterobactin complexes. But, it cannot explain the

preference of the A-configuration relative to the A- configuration of enterobactin since the RMS deviation of the energetically unfavourable configuration is only slightly higher. A superposition of both trilactone rings reveals also no major structural difference which might explain the preference of the A-configuration.

The ligand design has given some hints for poten- tial siderophores. In addition to a new siderophor with

a cyclic backbone non-cyclic structures have been proposed. It appears that the optimum number of methylene groups is six or seven. No alkyl chain back- bone with less than six methylene groups (Z < 6) has been proposed. Furthermore, an undistorted tris-(cate- chol) vanadium moiety seems to require that the sum of the methylene groups of two chains is at least 4. This is also fulfilled the structure of the natural side- rophore myxochelin A (i = Oj = 4) having only two catechol groups [7]. Semiempirical calculations for

the corresponding gallium complexes support this stereochemical requirement, too. For complex forma- tions, those modifications of the backbone are allowed, which make a minimum perturbation of the

tris(catecho1) subunit and the CONH group of the complex. We have superimposed this computed sub- unit of the gallium enterobactin complex with the corresponding ones of Table 2. The molecules with a 2,2,2 and with a 1,3,3, backbone are favourable

candidates. On the other hand, the chain length 0,1,2 (myxo-

chelin E) is much too short and leads to severe distor- tions of the gallium tris(catecho1) subunits having non-planar Ga-O-C-C-O five rings. This holds for 0,1,3 (myxochelin D) and 0,1,4 (myxochelin C) to a

smaller extent. Therefore, iron complexes with a six- fold catechol ligation of a single siderophor cannot exist in solution if the backbone chains are too short. Water molecules may replace the ligand with the shortest chain or complexes with two catechol groups from one siderophore and one catechol group from another siderophore molecule may exist. This may lead to an association of siderophores and metal ions to chain-type structures. The myxochelin siderophores have been synthesized recently [21], but experimental structures are not available. The enormous effect of myxochelin siderophorev in

98 M. Meyer, W. Trowitzsch-Kienast/Journal of Molecular Structure (Theochem) 418 (1997) 93-98

growth promotion experiments might indicate that a sixfold catechol ligation by a single siderophore is not necessary for the siderophore receptor interaction.

Growth promotion experiments with a variety of bacteria showed a dependency of the iron transport

capacity of the new siderophores not only on the chain lengths but also on the R- or S-configuration of the backbone indicating quite a different receptor

backbone interaction among the various bacteria tested.

5. Conclusion

An alkyl chain backbone with the number of methylene groups 2,2,2 or 1,3,3 is optimal for the ligation of metal ions with three catechol groups simi- lar to enterobactin (Fig. 3). For the 2,2,2 backbone the ‘in’-conformation is 8.7 kcal mol-’ more stable than

the ‘out’-conformation, whereas the energy difference is small for the 1,3,3 backbone. The binding of one metal ion to three catecholat groups connected by short alkyl chains (e.g., 0,1,2 in myxochelin E), leads to severe distortions of the tris(catecho1) gallium subunits. Therefore such structures are unlikely to exist in a solution of siderophores.

Acknowledgements

We would like to thank K.D. Aumann and G.

Wohlfahrt for support of our study.

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