Synthesis and binding studies of carboxylate binding pocket analogs of vancomycin

Article abstract of DOI:10.1016/S0040-4039(00)01270-3

Synthetic receptors based on the carboxylate binding pocket of vancomycin were synthesized. Using a UV binding assay, affinities of various cell wall related carboxylates were measured in organic solvents. Affinities in the 10³-10⁴ M^-1 range were determined as well as almost equal affinities for acylated alanine and lactate. Introduction of additional hydrogen bonding donors or positive charges into the receptors led to increased affinities for the carboxylates. (C) 2000 Published by Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01270-3

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 7541–7545
Synthesis and binding studies of carboxylate binding pocket
analogs of vancomycin
Roland J. Pieters*
Department of Medicinal Chemistry, Utrecht Institute for Pharmaceutical Sciences, Utrecht University,
PO Box 80082, NL-3508 TB Utrecht, The Netherlands
Received 9 June 2000; accepted 26 July 2000
Abstract
Synthetic receptors based on the carboxylate binding pocket of vancomycin were synthesized. Using a
UV binding assay, affinities of various cell wall related carboxylates were measured in organic solvents.
Affinities in the 103–104 M−1 range were determined as well as almost equal affinities for acylated alanine
and lactate. Introduction of additional hydrogen bonding donors or positive charges into the receptors led
to increased affinities for the carboxylates. © 2000 Published by Elsevier Science Ltd.
In the battle against pathogenic bacteria vancomycin is an important player as the drug of last
resort against Gram-positive pathogens such as methicillin-resistant S. aureus. Considering its
clinical importance it is worrisome that vancomycin-resistant S. aureus have emerged1,2 and are
likely to spread. In susceptible bacteria the drug binds to the
D
-Ala- -Ala dipeptide sequence of
D
the peptidoglycan cell-wall precursors and this prevents proper cell-wall construction, resulting
in bacterial lysis. A common pattern with resistant bacteria is the occurrence of weakened
binding due to replacement of the terminal
D-Ala with a
D
-lactate residue, which constitutes a
net replacement of an NH with an oxygen. This subtle replacement turns an attractive hydrogen
bond into a dipolar repulsive interaction and results in a 1000-fold lower affinity.3,4 Despite
major efforts towards the synthesis of vancomycin5 and vancomycin derivatization,6 the minimal
structural features needed for binding and selectivity are not known. Many compounds which
represent part of the vancomycin structure have been prepared over the years7 but their binding
properties have not been systematically studied.8 Considerable effort has also been devoted to
creating synthetic receptors9 for various targets including
D
-Ala-
-Ala and related peptides.10
D
These have been highly instructive with respect to molecular recognition phenomena but the
creation of selective high-affinity receptors for cell-wall peptides still remains an important goal.
This report describes synthetic receptors that mimic part of the vancomycin structure and their
binding to acylated alanine and lactate.
* Fax: +31-30-2536655; e-mail: r.j.pieters@pharm.uu.nl
0040-4039/00/$ - see front matter © 2000 Published by Elsevier Science Ltd.
PII: S0040-4039(00)01270-3

7542
The syntheses started with 111 derived from
D-p-hydroxyphenylglycine. The amino group of
1 was deprotected with TFA and coupled to Boc-protected alanine by means of the coupling
reagent HATU, after which the Boc group was removed, again with TFA to give 2. The
dipeptide was coupled to N-Boc-4-fluoro-3-nitro phenyl alanine (3) with HATU, to give
tripeptide 4. Amino acid 3 was obtained via diastereoselective alkylation of Scho¨llkopf’s chiral
bislactim ether.12 Tripeptide 4 was cyclized using cesium fluoride in dimethylformamide. This
reagent not only deprotects the silyl protected aromatic hydroxyl group but is also basic enough
to act as a base in the SNAr macrocyclization.13 The cyclization results in an inseparable 3:1
mixture of two atropisomers (5) with respect to the position of the nitro group. All further
reactions and binding studies were conducted with this mixture of atropisomers. Deprotection of
5 by TFA resulted in the ammonium receptor 6, which was then further functionalized. Reaction
of 6 with tert-butyl isocyanate resulted in the urea compound 7. Coupling of 6 to Boc-
D-Ala led
to receptor 8 (Scheme 1).
F
NO2
Oallyl
F
Oallyl
Oallyl
Br
Br
Br
OTBDMS
O
OTBDMS
OTBDMS
3
NO2
HO
c,
NHBoc
O
H
O
a,b,a
H
H
H
N
N
N
NH2.TFA
N
Boc
H3C
H3C
H3C
N
H
N
H
N
H
NHBoc
O
O
O
O
4
2
1
5
6
R = NHBoc
R = NH3+ CF3COO-
a
e
Oallyl
NO2
Br
O
O
R =
R =
N
d
7
H
O
H
N
H
N
f
N
t-Bu
H
H3C
R
N
H
O
O
O
N
H
3:1 mixture of atropisomers
8
HN
Boc
Scheme 1. Reagents and conditions: (a) TFA/CH2Cl2 1:2, 2 h, quant.; (b) N-Boc-Ala, HATU, NiPr2Et, CH2Cl2, DMF,
14 h, 51%; (c) HATU, NiPr2Et, CH2Cl2, DMF, 14 h, 76%; (d) CsF, DMF, 14 h, 85%; (e) tert-butyl isocyanate,
NiPr2Et, CH2Cl2, 14 h, 71%; (f)
D
-Boc-Ala, HATU, NiPr2Et, CH2Cl2, DMF, 14 h, 99%
Indications that the prepared molecules were indeed capable of binding peptidic cell-wall
precursor ligands came from NMR spectroscopy. Addition of the tetra-n-butyl salt of N-Ac-
D
-
Ala (0.7–8 mM) to a solution of receptor 7 in CD3CN (0.7 mM) resulted in downfield shifts of
at least two of the NH resonances to 8.5 and 9.0 ppm.14 These NH resonances were broad in
the spectrum of the free receptor and located between 6.6 and 7.3 ppm. Furthermore, upfield
and downfield shifts of all the aromatic protons were observed of ca. 0.1 ppm.15 In organic
media the association is mostly based on hydrogen bonding. Hydrophobic effects, which
contribute significantly to vancomycin’s binding free energy in water, are not addressed.
However, since hydrogen bonding is of great importance for biological activity, or the lack
thereof (see
D
-lactate replacement of
D
-Ala mentioned above), studies in apolar media may
provide valuable insights into the nature of the receptor–ligand interactions and may suggest
solutions for their optimization.

7543
UV spectroscopy proved to be valuable for the evaluation of the binding strength of the
receptors. Titration of a dilute solution of 5 (1×10−4 M in CHCl3) with a solution of the
tetra-n-butyl ammonium salt of N-Ac- -Ala (while maintaining a constant receptor concentra-
D
tion), resulted in an increased absorption around 270–280 nm (Fig. 1). This increase could be
fitted to a 1:1 binding isotherm and indicated an association constant of 1.9×104 M−1, or 5.8 kcal
mol−1 of binding free energy. Very similar observations were made in the more polar solvent
CH3CN, although the affinity was somewhat lower and the absorbance differences upon
complexation somewhat smaller (DAbs.=0.03–0.07 for all systems studied here at 270 or 280
nm).
Figure 1. Shifts in the UV spectrum upon addition of the carboxylate of N-Ac-D-Ala in CHCl3 to 5 [0.1 mM]. Inset:
Binding curve of a titration experiment based on absorbance changes at 280 nm, association constant Ka=
1.9×104 M−1
In CH3CN several titrations were run and the results are summarized in Table 1.16 These
titrations indicated that the affinity of 5 for N-Ac-
than that for the -enantiomer (entry 1). Vancomycin itself shows significant preference for
-Ala-
-Ala over its enantiomer,17 which is either caused by hydrophobic effects in water,
L-Ala (entry 2) was only marginally weaker
D
D
D
and/or by interactions which also involve the second alanine residue. Interestingly, in our
titrations, changing the alanine residue for a lactate (entry 3) results in only a small drop in
Table 1
Binding affinities of receptor 5–8 [0.1 mM] to the tetra-n-butyl ammonium salts of various guests, expressed as
association constants (Ka, M−1) and free energies (kcal mol−1, 1 cal=4.184 J), determined by UV titrations
monitoring the absorbance at 270 nm, at 296 K in CH3CN
Entry
Receptor
Guest
Ka (M−1
)
−DG (kcal mol−1 a
)
1
2
3
4
5
6
7
5
5
5
5
6
7
8
N-Ac-
D
-Ala
9.7×103
6.9×103
5.8×103
3.8×102
3.2×104
3.7×104
2.3×104
5.4
5.2
5.1
3.5
6.1
6.2
5.9
N-Ac-
L
-Ala
Ac-
L
-Lac
OAc−
N-Ac-
N-Ac-
N-Ac-
D
D
D
-Ala
-Ala
-Ala
a Estimated error in DG: 90.2 kcal mol−1
.

7544
affinity. However, acetate (entry 4) binds much more weakly to receptor 5 with a Ka of 380 M−1.
These results are in contrast to binding studies with vancomycin in aqueous buffer where acetate
and Ac-
D
-Lac bind similarly weakly (30 and 40 M−1)18 and N-Ac-
-Ala binds an order of
D
magnitude stronger, presumably due to the extra hydrogen bond that can be formed to its NH.
Our observations may be due to the following: the system has the possibility to present either
a hydrogen bond donor or an acceptor, depending on the nature of the guest, by rotating the
CONHMe group. This would explain the similar affinities for the alanine and lactate containing
guests and the drop in affinity of acetate.19
In order to increase binding affinities, the carboxylate binding pocket of 5 was modified by
introducing additional hydrogen bonding donors or positive charges.20 Indeed, the ammonium
compound 6 binds the carboxylate ligand significantly stronger (entry 5), presumably due to
additional electrostatic interactions. Introduction of a urea functional group in 7 (entry 6),
which constitutes an O to NH mutation, led to an increased binding free energy of 0.8 kcal
mol−1 compared to 5. The protected alanine compound 8 (entry 7) also results in binding
enhancement, possible due to additional interactions of the carboxylate ligand with the Boc NH.
In summary, vancomycin binding pocket model systems have been prepared that are capable
of binding N-Ac-Ala carboxylates and acylated lactate. The use of organic solvents was required
here to observe binding. However, it provides a magnifying glass for the important hydrogen
bonds, which are weak in water, and led to affinities in the 103–104 M−1 range. For comparison,
in aqueous solution, even vancomycin binds to acylated
D-Ala and
D-lactate only weakly (Ka
values are 300 and 40 M−1, respectively).18 With our receptors a relatively high affinity for
acylated lactate versus alanine was measured. This interesting phenomenon is possibly due to the
freely rotatable C-terminal CONHMe group and underscores the promise of creating van-
comycin analogs with altered hydrogen bonding characteristics. Modification of receptor 5 led
to increased affinities with the introduction of additional hydrogen bond donors or positive
charges. This augurs well for a combinatorial approach of further structural optimization, which
is currently in progress.
Acknowledgements
The Royal Netherlands Academy of Arts and Sciences (KNAW) is gratefully acknowledged
for financial support. Professor R.M.J. Liskamp is acknowledged for support, helpful discus-
sions and encouragement.
References
1. Williams, D. H.; Bardsley, B. Angew. Chem., Int. Ed. 1999, 38, 2700.
2. Walsh, C. Science 1999, 284, 442.
3. Bugg, T. D.; Wright, G. D.; Dutka-Malen, S; Arthur, M.; Courvalin, P.; Walsh, C. T. Biochemistry 1991, 30,
10408.
4. Walsh, C. T.; Fisher, S. L.; Park, I.-S.; Prahalad, M.; Wu, Z. Chem. Biol. 1996, 3, 21.
5. Nicolaou, K. C.; Boddy, C. N. C.; Bra¨se, S.; Winssinger, N. Angew. Chem., Int. Ed. 1999, 38, 2097.
6. Malabarba, A.; Nicas, T. I.; Thompson, R. C. Med. Res. Rev. 1997, 17, 69.
7. Rao, A. V. R.; Gurjar, M. K.; Reddy, K. L.; Rao, A. S. Chem. Rev. 1995, 95, 2135.

7545
8. For a related system and an early exception see: Pant, N.; Hamilton, A. D. J. Am. Chem. Soc. 1988, 110, 2002.
This system has affinities for simple carboxylates that are of the same order of magnitude (Ka cyanoacetate 580
M−1 in CDCl3 versus Ka of OAc− for 5 is 380 M−1 in CH3CN, Table 1, entry 4) as those measured here, but
included a benzyl amine instead of the phenyl glycine unit and therefore lacked the additional hydrogen bonding
group responsible for the high
D-Ala affinity in the present system.
9. See e.g. Still, W. C. Acc. Chem. Res. 1996, 29, 155 and references cited therein.
10. Henly, P. D.; Waymark, C. P.; Gillies, I.; Kilburn, J. D. J. Chem. Soc., Perkin Trans. 1 2000, 1021.
11. This is an intermediate in the synthesis of an amino acid used in the vancomycin aglycon synthesis see: Evans,
D. A.; Wood, M. R.; Trotter, B. W.; Richardson, T. I.; Barrow, J. C.; Katz, J. L. Angew. Chem., Int. Ed. 1998,
37, 2700; We thank Professor D. A. Evans (Harvard University) for making detailed synthetic procedures
available to us.
12. Vegne, C.; Bois-Choussy, M.; Ouazzani, J.; Beugelmans, R.; Zhu, J. Tetrahedron: Asymmetry 1997, 8, 391 and
references cited therein.
13. Zhu, J. Synlett 1997, 133.
14. Other signals are obscured due to peak overlap.
15. Downfield NH shifts are also observed for the NBu4+ salts of OAc− and Ac-
L-Lac and distinct variations are
observed in the complexation induced shifts of the resonances of the receptor for each guest.
16. Both receptor and carboxylate were thoroughly dried under high vacuum. A 0.1 mM solution of the receptor was
prepared in CH3CN and a carboxylate solution was prepared using the receptor solution. Carboxylate
concentrations ranged from 2 to 40 mM depending on the affinity. In a double beam spectrophotometer spectra
were taken after incremental additions (up to 400 mL in total) of the carboxylate solution to a receptor solution
(1.0 mL) in a Teflon stoppered cuvette. The cuvette was briefly vortexed after each addition. A cuvette containing
CH3CN was used in the reference beam. From the collected data the absorbance changes at 270 or 280 nm were
fitted to a 1:1 binding isotherm using a nonlinear least squares regression in ASSOCIATE 1.6 (Peterson, B. K.;
Ph.D. Dissertation, UCLA, 1994). The carboxylates do not absorb at this wavelength. Experiments were run in
duplicate or triplicate and results were averaged using material from different synthetic batches. HPLC grade
CH3CN (Biosolve Ltd., water <0.02%) was used and experiments were run at 296 K. In this study atropisomeric
mixtures were used, which will similarly be obtained in our planned combinatorial synthesis and screening on
solid phase. Although this fact does not seem to preclude structure–affinity studies we will pursue binding studies
of single isomers as well as detailed studies into the binding mode.
17. Chu, Y.-H.; Whitesides, G. M. J. Org. Chem. 1992, 57, 3524.
18. Loll, P. J.; Kaplan, J.; Selinsky, B. S.; Axelsen, P. H. J. Med. Chem. 1999, 42, 4714.
19. A similar rationale has recently been used to explain enhanced
D-Ala-D-lactate binding of members of a
combinatorial library which all contain the vancomycin carboxylate binding pocket tetra peptide: Xu, R.;
Greiveldinger, G.; Marenus, L. E.; Cooper, A.; Ellman, J. A. J. Am. Chem. Soc. 1999, 121, 4898. The different
basicities of the carboxylates would make acetate a stronger binder than N-Ac-Ala, as can for example be seen
in the following carboxylate receptor system: Schmuck, C. Chem. Eur. J. 2000, 6, 709; pKa acetic acid: 4.8, pKa
N-Ac-L-Ala: 3.8 (King, E. J.; King, G. W. J. Am. Chem. Soc. 1956, 78, 1089).
20. For related studies with vancomycin itself see: Go¨rlitzer, J.; Gale, T. F.; Williams, D. H. J. Chem. Soc., Perkin
Trans. 1 1999, 3253.
.