Partitioning the loss in vancomycin binding affinity for D-Ala-D-Lac into lost H-bond and repulsive lone pair contributions

Article abstract of DOI:10.1021/ja035901x

The binding affinity of 4, which incorporates a methylene (CH₂) in place of the key linking amide of Ac₂-L-Lys-d-Ala-d-Ala, for vancomycin was compared with that of Ac₂-L-Lys-d-Ala-d-Ala (3) and Ac₂-L-Lys-d-Ala-

Full text of DOI:10.1021/ja035901x

Published on Web 07/11/2003
Partitioning the Loss in Vancomycin Binding Affinity for D-Ala-D-Lac into Lost
H-Bond and Repulsive Lone Pair Contributions
Casey C. McComas, Brendan M. Crowley, and Dale L. Boger*
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, California 92037
Received May 1, 2003; E-mail: boger@scripps.edu
Vancomycin (1) is the parent member of the class of clinically
important glycopeptide antibiotics (Figure 1).1 It is used with
patients allergic to â-lactam antibiotics, and it is now a frontline
therapy for endocarditis and for many bacterial infections in patients
undergoing cycles of cancer chemotherapy.2 Most importantly, it
has become the drug of last resort for the treatment of methicillin-
resistant Staphylococcus aureus (MRSA).3
Following studies which indicated that the glycopeptide antibiot-
ics inhibit bacterial cell wall biosynthesis,4 Perkins demonstrated
that vancomycin selectively binds the precursor peptidoglycan
peptide terminus N-acyl-D-Ala-D-Ala, preventing bacterial cell wall
maturation.5 Shortly after the disclosure of the structure of
vancomycin,6 Williams provided the structure of the N-acyl-D-Ala-
D-Ala complex with the antibiotic which was found to be stabilized
by an extensive array of van der Waals contacts (hydrophobic
contacts) within the vancomycin binding pocket and five key
H-bonds lining the pocket (Figure 1A).7,8
With its more frequent use, vancomycin-resistant Gram-positive
bacteria have emerged, including vancomycin-resistant enterococci
(VRE).2 The elucidation of the origin of the VanA and VanB
bacterial resistance and its structural basis were described by Walsh,
Courvalin, and co-workers.9,10 It entails the reprogramming of the
peptidoglycan termini from D-Ala-D-Ala to D-Ala-D-Lac. This
simple substitution of a linking ester for an amide with the exchange
of a single atom (NHfO) reduces the binding to vancomycin 1000-
fold and accounts fully for the 3 orders of magnitude higher MICs
seen in VRE clinical isolates.9 The complex of vancomycin with
N-acyl-D-Ala-D-Lac lacks the central H-bond characteristic of the
D-Ala-D-Ala complex and has been suspected to suffer from a
repulsive lone pair interaction between the vancomycin residue 4
carbonyl and the D-Ala-D-Lac ester oxygen (Figure 1B). Herein,
we provide the first experimental estimation of the magnitude of
these two effects which suggests that it is the repulsive lone pair
interactions (2.6 kcal/mol), not the H-bond loss (1.5 kcal/mol), that
Figure 1.
is responsible for the larger share of the reduced binding affinity
(4.1 kcal/mol).
The estimation is derived from a comparison of the vancomycin
and vancomycin aglycon binding affinity for 411 (Scheme 1) versus
that for Ac2-L-Lys-D-Ala-D-Ala (3) and Ac2-L-Lys-D-Ala-D-Lac (5)
measured by titration of the antibiotic hosts with the ligand (UV),
Figure 2.12 The structure of 4 incorporates a methylene (CH2) in
place of the amide NH of 3 and the ester O of 5. Thus, 4 lacks the
Figure 2.
capabilities for forming the H-bond of 3 and does not suffer the
repulsive lone pair/lone pair destabilization of 5. To a first
approximation, the comparison of 4 with 3 provides an estimation
of the H-bond contribution to binding, whereas its comparison with
5 provides an estimation of the repulsive lone pair binding
destabilization of 5. In the case of both vancomycin (1) and the
vancomycin aglycon (2), the affinity for 4 was roughly 10-fold less
than that of Ac2-L-Lys-D-Ala-D-Ala (3), but 100-fold greater than
that of Ac2-L-Lys-D-Ala-D-Lac (5), Table 1. In the absence of
compensating or detrimental effects of the methylene substitution
in 4, this suggests that the reduced binding affinity of 5 (4.1 kcal/
mol) may be attributed to both the loss of the H-bond of 3 (1.5-
1.8 kcal/mol) and the destabilizing lone pair/lone pair interaction
introduced with 5 (2.6 kcal/mol) with the latter, not the lost H-bond,
being responsible for the largest share of the reduction.
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10.1021/ja035901x CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
energy15 of an amide H-bond (0.0-1.5 kcal/mol) and that resulting
from a destabilizing lone pair/lone pair interaction (1.6-2.7 kcal/
mol).15
Table 1. Association Constants (K) and Binding Free Energy
(-∆G°, 25 °C)a
-1
-1
vancomycin (1) K, M
vancomycin aglycon (2) K, M
(−∆G°, kcal mol-1
ligand
(−∆G°, kcal mol-1
)
)
More importantly and independent of the origin of the effects,
these observations have significant ramifications in the reengineer-
ing of the vancomycin structure to bind D-Ala-D-Lac, suggesting
that the design could focus principally on removing the destabilizing
lone pair interaction rather than reintroduction of a H-bond and
that this may be sufficient to compensate for 2 of the 3 orders of
magnitude in binding affinity lost with D-Ala-D-Lac. Such efforts
are underway and will be reported in due course.
3 (X ) NH)
4 (X ) CH2)
5 (X ) O)
4.4 × 105 (7.7)
3.3 × 104 (6.2)
4.3 × 102 (3.6)
5.8 × 105 (7.8)
2.5 × 104 (6.0)
3.1 × 102 (3.4)
a 25 °C, 0.00011 M vancomycin in 0.02 M sodium citrate, pH 5.1,
observed at 279 nm, ref 12.
Scheme 1
Acknowledgment. We gratefully acknowledge the financial
support of the National Institutes of Health (CA 41101) and the
Skaggs Institute for Chemical Biology. B.M.C. is a Skaggs Fellow.
Supporting Information Available: Experimental details and
characterization data for the synthesis of 4 (PDF). This material is
available free of charge via the Internet at http://pubs.acs.org.
References
(1) Glycopeptide Antibiotics; Nagarajan, R., Ed.; Marcel Dekker: New York,
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Chem., Int. Ed. 1999, 38, 2096.
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Notably, this substitution in 4 can have several effects that would
impact binding beyond those which we purport to be estimating.
In addition to removing the repulsive lone pair destabilization of
the ester O of 5, it increases the hydrophobic character of the ligand
favoring binding in a hydrophobic pocket and alters the solvation
characteristics of the ligand in a way that may favor binding relative
to 5. In this case, the difference in the 4 versus 5 binding would
overestimate the lone pair electrostatic destabilization with 5. In
addition to removing the H-bond of 3, the methylene substitution
in 4 increases its conformational flexibility and reduces its
conformational preference relative to both 3 and 5 (rigidity: 3 (trans
amide) > 5 (syn eclipsed ester) > 4) and could introduce
unfavorable steric interactions,13 both of which would disfavor
binding relative to 3. This would result in the comparison of the 4
versus 3 binding overestimating the H-bond contribution to the
binding of 3. Moreover, the simplistic partitioning of the effects
into a lost H-bond versus introduction of repulsive lone pair
interactions does not take into account cooperative enthalpic or
entropic binding enhancements attributable to adjacent binding
interactions.14 Nonetheless, the estimates are consistent with intui-
tive expectations resulting from the stabilizing differential binding
(7) Williams, D. H.; Williamson, M. P.; Butcher, D. W.; Hammond, S. J. J.
Am. Chem. Soc. 1983, 105, 1332.
(8) Review: Williams, D. H.; Bardsley, B. Angew. Chem., Int. Ed. 1999, 38,
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(9) Bugg, T. D. H.; Wright, G. D.; Dutka-Malen, S.; Arthur, M.; Courvalin,
P.; Walsh, C. T. Biochemistry 1991, 30, 10408.
(10) Reviews: Walsh, C. T.; Fisher, S. L.; Park, I.-S.; Prahalad, M.; Wu, Z.
Chem. Biol. 1996, 3, 21. Walsh, C. T. Science 1993, 261, 308.
(11) The synthesis and characterization of 4 is provided in Supporting
Information.
(12) Perkins, H. R. Biochem. J. 1969, 111, 195. Nieto, M.; Perkins, H. R.
Biochem. J. 1971, 123, 773.
(13) Simple modeling of 3-5 bound to the vancomycin aglycon did not reveal
a newly introduced destabilizing steric interaction with 4 that would result
in the overestimation of the H-bond of 3. Moreover, simple interaction
energies of the three modeled complexes (Amber, Macromodel) closely
followed the trends experimentally observed herein.
(14) Searle, M. S.; Sharman, G. J.; Groves, P.; Benhamu, B.; Beauregard, D.
A.; Westwell, M. S.; Dancer, R. J.; Maguire, A. J.; Try, A. C.; Williams,
D. H. J. Chem. Soc., Perkin Trans. 1 1996, 2781.
(15) Morgan, B. P.; Scholtz, J. M.; Ballinger, M. D.; Zipkin, I. D.; Bartlett, P.
A. J. Am. Chem. Soc. 1991, 113, 297 and references therein.
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