3-Fluoro-N-methyl-D-aspartic acid (3F-NMDA) stereoisomers as conformational probes for exploring agonist binding at NMDA receptors

Article abstract of DOI:10.1002/chem.201200071

N-Methyl-D-aspartate (NMDA) is the prototypical agonist of the NMDA receptor subtype of ionotropic glutamate receptors. Stereogenic placement of a C-F bond at the 3-position of (S)-NMDA generates either the (2S,3S)- or (2S,3R)- diastereoisomers of 3F-NMDA. The individual diastereoisomers were prepared by synthesis in enantiomerically pure forms and it was found that (2S,3S)-3F-NMDA is an agonist with a comparable potency to NMDA itself, whereas the (2S,3R)-diastereoisomer has negligible potency. The difference in potency of these stereoisomers is attributed to a preference of the C-F bond (2S,3S)-3F-NMDA to adopt a gauche conformation to the C-N⁺ bond in the binding conformation, whereas the (2S,3R)-3F-NMDA forces these bonds anti, losing electrostatic stabilisation, to achieve the required binding conformation. These observations illustrate the utility of stereoselective fluorination in influencing the molecular conformation of β-fluorinated amino acids and thus probing the active conformations of bioactive compounds at receptors. Copyright

Full text of DOI:10.1002/chem.201200071

FULL PAPER
DOI: 10.1002/chem.201200071
3-Fluoro-N-methyl-d-aspartic acid (3F-NMDA) Stereoisomers as
Conformational Probes for Exploring Agonist Binding at NMDA Receptors
Poh Wai Chia,[a] Matthew R. Livesey,[b] Alexandra M. Z. Slawin,[a] Tanja van Mourik,[a]
David J. A. Wyllie,[b] and David OꢀHagan*[a]
Abstract:
N-Methyl-d-aspartate
NMDA itself, whereas the (2S,3R)-dia-
stereoisomer has negligible potency.
The difference in potency of these ste-
reoisomers is attributed to a preference
whereas the (2S,3R)-3F-NMDA forces
these bonds anti, losing electrostatic
stabilisation, to achieve the required
binding conformation. These observa-
tions illustrate the utility of stereoselec-
tive fluorination in influencing the mo-
lecular conformation of b-fluorinated
amino acids and thus probing the
active conformations of bioactive com-
pounds at receptors.
(NMDA) is the prototypical agonist of
the NMDA receptor subtype of iono-
tropic glutamate receptors. Stereogenic
À
À
placement of a C F bond at the 3-posi-
of the C F bond (2S,3S)-3F-NMDA to
À
tion of (S)-NMDA generates either the
(2S,3S)- or (2S,3R)- diastereoisomers of
3F-NMDA. The individual diastereo-
isomers were prepared by synthesis in
enantiomerically pure forms and it was
found that (2S,3S)-3F-NMDA is an ag-
onist with a comparable potency to
adopt a gauche conformation to the C
N+ bond in the binding conformation,
Keywords: fluorine · glutamate re-
ceptors · molecular conformation ·
neurotransmitters · NMDA
Introduction
Glutamate (1) is the principal excitatory neurotransmitter of
the mammalian central nervous system where it acts on sev-
eral subtypes of ligand-gated ion channels. N-Methyl-d-as-
partate (NMDA) (2) is a synthetic analogue of aspartate[1]
and is a prototypical agonist and widely used pharmacologi-
cal tool that defines one of these subclasses. N-Methyl-d-as-
partate receptors (NMDARs) mediate the slow component
of glutamatergic synaptic currents but play key roles in
brain development, learning and memory and are implicated
in a range of neuro-developmental/degenerative diseases
such as schizophrenia, Alzheimerꢀs, Parkinsonꢀs, Hunting-
tonꢀs and ischemia.[2] NMDARs are tetrameric proteins, the
majority of which contain two GluN1 and two GluN2
NMDAR subunits. Four subtypes of the GluN2 subunit exist
(A–D) and it is these that confer the majority of the sub-
type-dependent pharmacological and biophysical properties
of NMDARs.[2–4] Activation of NMDARs requires glycine
(or d-serine) binding at the GluN1 subunit and glutamate
binding at the GluN2 subunit.
Structural studies of the NMDAR by Furukawa et al.
have resulted in co-crystals of glutamate (1) and most re-
cently with NMDA (2) bound in the ligand binding domain
of the GluN2D NMDAR subunit.[7,8] This has revealed
a binding mode in which the carboxylate groups of NMDA
(2) arrange gauche to each other. The current study set out
to explore the relative efficacies of the two 3-fluoro NMDA
diastereoisomers (2S,3S)-3 and (2S,3R)-4 as glutamate ago-
nists and as analogues of NMDA (2) and assays have been
carried out on GluN2A and GluN2B NMDARs. Fluorine is
the smallest atom after hydrogen that can form a covalent
bond to carbon. It is also the most polar bond in organic
chemistry due to the electronegativity of fluorine, the high-
est on the Pauling scale.[12,13] Thus, fluorine can replace hy-
drogen, with minimum steric impact, but the dipole of the
[a] P. W. Chia, Prof. Dr. A. M. Z. Slawin, Dr. T. v. Mourik,
Prof. Dr. D. OꢀHagan
Department of Chemistry
Biomolecular Sciences Building, University of St Andrews
North Haugh, St Andrews, Fife, KY16 9ST (UK)
Fax : (+44)1334-462595
E-mail: do1@st-andrews.ac.uk
[b] Dr. M. R. Livesey, Prof. Dr. D. J. A. Wyllie
Centre for Integrative Physiology, University of Edinburgh
Hugh Robson Building, 15 George Square, Edinburgh, EH8 9XD
(UK)
À
C F bond has a significant electronic influence.
À
The stereospecific incorporation of the C F bond to gen-
erate (2S,3S)-3 and (2S,3R)-4 is anticipated to introduce
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201200071.
a conformational bias in solution due to a stabilising
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À À
charge–dipole interaction ( C
+
[9,10,11]
À
F·· NH3 ).
This electro-
static interaction has been cal-
culated in other systems to con-
tribute up to 5.00 kcalmolÀ1 sta-
bilisation energy, similar in
magnitude to a good hydrogen
bond, when the substituents are
À
gauche relative to when the C
+
À
F and C N are antiperiplanar
to each other. Thus, the two
diastereoisomers (2S,3S)-3 and
(2S,3R)-4 are predicted to more
easily access different confor-
mations on the receptor.
Scheme 1. Synthesis of erythro (2S,3S)-3F-NMDA HCl (3).
Previously, we have prepared
separate enantiomers of 3-
fluoro-g-aminobutyric acid (3F-GABA) (6) to probe the
chirality of g-aminobutyric acid (GABA) (5) binding to
both GABAA and GABAC receptors.[14,15,16] In those studies
we observed significantly different efficacies for the two
enantiomers (R)-6 and (S)-6 suggesting that the GABAA
and GABAC receptors bind g-aminobutyric acid in different
conformations. The conformational bias was attributed to
by treatment of the resultant amino alcohol 8 with [bis(2-
methoxyethyl)amino]sulfur trifluoride (Deoxo-Fluor) to
generate a single diastereoisomer of the dehydroxyfluorinat-
ed product 10. This fluorination reaction occurs with com-
plete stereoselectivity, as evinced by production of a single
diastereoisomer, and with an overall retention of configura-
tion, proceeding by double inversion after fluoride ion
the electrostatic charge–dipole interaction between the C F
attack of an aziridinium intermediate 9.[14,18,19] With the C F
À
À
+
À
and C N bonds as illustrated in Figure 1, thus the different
bond now located, N-debenzylation by hydrogenation result-
ed in diester 11 and then acidic hydrolysis gave (2S,3S)-3F-
NMDA (3) as a single diastereoisomer (see the Supporting
Information). The enantiomeric purity of (2S,3S)-3F-NMDA
(3) is reasonably assumed based on its origin from tartarate
7 and the stereointegrity of the epoxide ring opening and
fluorination steps, each of which gave rise to single diaste-
reoisomers.
Figure 1. GABA (5) and 3-F-GABA enantiomers (6). For enantiomers
(6) their solution conformation is influenced by a charge dipole interac-
tion indicated by the dashed bonds.
The relative stereochemistry required for the synthesis of
(2S,3R)-3F-NMDA (4) necessitated that the route start from
the achiral meso-epoxide 12.[20] Thus a resolution, to access
the individual enantiomers, is required. It was envisaged
that ring opening of meso-epoxide 12 with either (S)-N or
(R)-N-a-dimethylbenzylamine (13) would generate a set of
diastereoisomers that could be separated into single enantio-
mers by chromatography. In the reaction, epoxide ring
opening with the enantiomerically pure amines was relative-
ly straightforward, however the diastereoisomeric products
14 and 15 could not be easily separated by chromatography.
Instead the product mixture was treated with Deoxo-Fluor
in a dehydroxyfluorination reaction, which following prece-
dent,[14,18,19] is anticipated to proceed with an overall reten-
tion of configuration. This gave a product mixture of diaste-
reoisomers 16 or 17. The fluorohydrins could now be sepa-
rated by careful chromatography. For practical purposes
each enantiomer was secured by reaction of either (S)-N or
(R)-N-a-dimethylbenzylamine (13), since the slower eluting
diastereoisomer on chromatography could be cleanly sepa-
rated, whereas the faster eluting diastereoisomer was always
contaminated with the slower eluting isomer. Only the route
using (S)-13 is shown in Scheme 2. Each stereoisomer, 16 or
17, which was enantiomerically pure as a consequence of
enantiomers of 3F-GABA can adopt preferred twisted con-
formations of the opposite chiral sense, and a favoured bind-
ing mode was deduced by comparing the efficacy of the two
enantiomers of 3F-GABA (6). In this research programme
À
the C F bond is incorporated by synthesis into the two dia-
stereotopic locations at C-3 of NMDA (2) to generate dia-
stereoisomers (2S,3S)-3F-NMDA (3) and (2S,3R)-3F-
NMDA (4). These diastereoisomers were then assayed in
a glutamate receptor assay to compare their relative effica-
cies as agonists.
Results and Discussion
Synthesis and analysis of 3F-NMDA diastereoisomers: The
(2S,3S)-3F-NMDA stereoisomer (3) was synthesised as illus-
trated in Scheme 1. The synthesis started from (S,S)-epoxy
succinic ester (7), which was prepared from diethyl (À) d-
tartrate as previously described.[17] This then involved ring
opening of epoxide 7 with N-benzylmethylamine, followed
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3F-NMDA Stereoisomers as Conformational Probes
FULL PAPER
Figure 3. Calculated minimum energy DFT structures for diastereoiso-
mers 3 (left) and 4 (right). Each of the DFT calculated structures is con-
sistent with the solution conformations (boxed Newman projections
Figure 4).
1
Comparison of the H and 19F NMR spectra of the diaste-
reoisomer
(2S,3S)-3F-NMDA
(3)
prepared
above
(Scheme 1), showed very clear differences in chemical shifts
and coupling constants consistent with their diastereoiso-
meric relationship (see the Supporting Information). The
NMR analysis provides some insight into the solution con-
formations of both 3F-NMDAs (3) and (2S,3R)-3F-NMDA
3
3
Scheme 2. Synthesis of 3F-NMDA diastereoisomers (2S,3R)-4 and
(2R,3S)-4.
(4). Both the JHH the JHF coupling constants have an angu-
lar dependence. Similar to the known Karplus relationship
for vicinal hydrogen couplings, syn- and antiperiplanar vici-
nal H-C-C-F relationships are typically about 30 Hz, where-
as gauche relationships are around 10–15 Hz.[23,24,25] For
diastereoisomer separation, was
separately submitted to depro-
tection by hydrogenation. This
gave (2R,3S)-F-NMDA (4) or
(2S,3R)-3F-NMDA (4), respec-
tively. A suitable crystal of 4
was subjected to X-ray crystal-
3
isomer (2S,3S)-3F-NMDA (3) the JHH value is small at
À
about 2.0 Hz suggesting vicinal C H bonds a little larger
than 608 and no significant contribution from conformer B,
3
in which the vicinal hydrogen atoms are anti. The JHF cou-
pling constant of 29 Hz suggests either an anti or syn H to F
relationship (conformer A syn or conformer C anti). DFT
calculations identify conformer A as the lowest in energy
(see below) and thus it appears the more likely solution con-
lography, and the resultant
structure (Figure 2) had the ex-
pected relative stereochemistry,
confirming that the fluorination
step has occurred with a reten-
tion of configuration. Notably,
there is a very clear gauche re-
lationship between the vicinal
Figure 2. X-ray crystal struc-
ture of either (2S,3R)- or
(2R,3S)-4. The carboxylate
former for this isomer. Isomer (2S,3R)-3F-NMDA (4) has
groups are antiperiplanar to
3
À
a zero value for JHH =0.0 Hz, suggesting vicinal C H bonds
+
À
À À
each other and C F·· N C
dihedral angle is 62.9(6)8 high-
lighting the expected gauche
relationship.
at 908 and thus conformer C is not significant in solution. It
is less easy to unambiguously distinguish between A and B
on coupling constant values, however DFT identified confor-
mer B as the lowest in energy and conformer B is similar to
that observed in the X-ray structure of 4 (Figure 2). Confor-
+
À
À
C F and C N bonds (torsion angle=62.78) consistent with
the expected charge–dipole interaction, and as a conse-
quence the carboxylate groups are antiperiplanar to each
other in this diastereoisomer.
mer A is anticipated to be relatively high in energy because
+
À
À
the C F and C N bonds are anti and thus lose electrostatic
stabilisation, thus conformer B is most likely to be the domi-
nant one in solution. The structural biology study of Furuka-
wa et al. indicates that conformer A (Figure 4) most closely
represents the NMDA binding conformation for the
GluN2D NMDAR.[8]
The two enantiomers of 4, as prepared in Scheme 2, have
identical NMR spectra and differ only in optical rotation, as
expected.
A DFT study was undertaken to explore the relative ener-
gies of conformers of 3 and 4 (see the Supporting Informa-
tion). Geometry optimisations using the M06-2X function-
al[21] and the PCM continuum solvation model[22] identified
the lowest energy conformers in each case for both (2S,3S)-3
and (2S,3R)-4 (Figure 3). The calculated structure for 4 is
almost identical to the X-ray determined structure shown in
Figure 2.
Assessment of 3F-NMDA isomers at GluN2A- and
GluN2B-containing NMDARs: The synthesis provided the
(2S,3S)-3 isomer and both the (2S,3R)-4 and (2R,3S)-4 iso-
mers of 3F-NMDA. We assessed the activity of (2S,3S)-3,
(2S,3R)-4 and (2R,3S)-4 at recombinant NMDARs ex-
pressed in Xenopus laevis oocytes comparing their potencies
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D. OꢀHagan et al.
unable to detect responses to
(À)-4 or (+)-4 at GluN2B
NMDARs. We further quanti-
fied the potency of (2S,3S)-3
relative to that of NMDA-2 by
constructing concentration-re-
sponse curves. The concentra-
tions of (2S,3S)-3 or NMDA (2)
required to produce half-maxi-
mal responses at GluN2A
NMDARs were (150Æ2) and
(105Æ2) mm,
respectively,
whereas the corresponding
values at GluN2B NMDARs
were (55Æ1) and (48Æ3) mm.
Thus, although (2S,3S)-3 produ-
ces a lower maximum response
than NMDA (2) at both
Figure 4. The favoured solution conformers of 3F-NMDA isomers (2S,3S)-3F 3 and (2S,3R)-3F 4 as deduced
1
by H and 19F NMR analysis are highlighted in the boxed Newman projections.
to that of NMDA (2). Isomer (2S,3S)-3 reliably evoked cur-
Table 1. NMDA (2) and 3F-NMDA stereoisomer relative efficacies at
rents in oocytes expressing either GluN2A or GluN2B
NMDARs although the observed responses were less than
those obtained with the equivalent concentration of NMDA
(2) (Figure 5 and Table 1). It was anticipated that (2R,3S)-4
would be significantly less active than (2S,3R)-4, because
NMDA is an order of magnitude more potent an agonist
than the l-configured isomer. In the assay however it was
found that neither of the enantiomers of 4 was very active
and currents, if present, could only be detected when the
gain of the amplifier was increased by a factor of 100–the
lower traces in Figure 5 c and d and illustrated on the ex-
panded ordinate axis in Figure 6b. Furthermore, we were
GluN2A and GluN2B NMDARs.
GluN2A
GluN2B
NMDA (2)
(2S,3S)-3
1.0
1.0
G
0.72Æ0.03
0.009Æ0.0009
0.001Æ0.0005
0.45Æ0.01
0.0031Æ0.0009
not detected
(+)-(2S,3R)-4 or (2R,3S)-4
(À)-(2R,3S)-4 or (2S,3R)-4
Figure 6. a) Summary of the relative efficacies for NMDA (2), (2S,3S)-3,
(2S,3R)-4 and (2R,3S)-4. Currents have been normalized to the response
produced by NMDA (2). b) Same data as illustrated in (a) but on an ex-
panded scale to indicate the small responses evoked by(+)-(2S,3R)-4 or
(2R,3S)-4 and (À)-(2R,3S)-4 or (2S,3R)-4.
GluN2A and GluN2B NMDARs the concentrations with
which (2S,3S)-3 and NMDA (2) produce their respective
half maximal responses are similar. Isomer (2S,3S)-3 can
thus be considered to act as a partial agonist at both
GluN2A and GluN2B NMDARs. These findings suggest
that (2S,3S)-3 is the sole stereoisomer that can access a bind-
ing conformation similar to NMDA (2) to act as an agonist
at GluN2A and GluN2B NMDARs.
Figure 5. Agonist activities of the NMDA (2), (2S,3S)-3, (2S,3R)-4 and
(2R,3S)-4 stereoisomers at NMDARs. Example TEVC currents recorded
from an oocyte expressing GluN2A NMDARs. Note that both NMDA
(2) (a) and (2S,3S)-3 (b) each elicit large inward currents. However
(+)-(2S,3R)-4 or (2R,3S)-4 (c) and (À)-(2R,3S)-4 or (2S,3R)-4 (d) evoke
only modest currents which are only resolvable on a higher gain setting
(light grey traces).
The data also indicated that neither of the enantiomers
(2S,3R)-4 and (2R,3S)-4 are effective agonists at the concen-
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3F-NMDA Stereoisomers as Conformational Probes
FULL PAPER
trations tested. These observations are consistent with the
solution conformation of 3 (conformer A in Figure 4), most
closely representing the receptor binding conformation. Ste-
reoisomers (2S,3R)-4 and (2R,3S)-4 would have to adopt
be similar to that required for binding to the receptor. On
the other hand the (2S,3R)-4 isomer would have to orientate
+
À
À
the C F and C N bonds antiperiplanar in a less favoured
orientation, to adopt the same agonist binding mode, thus as
predicted this isomer is a very poor agonist. The deduced
binding mode of NMDA to GluN2A and GluN2B subunits
gets some support from a recent structural biology study on
the GluN2D receptor subunit. A referee raised the reasona-
À
a higher energy conformation in which the C F bond is ori-
+
À
ented anti to the C N bond, losing electrostatic stabilisa-
tion. These observations are consistent with the recent X-
ray crystallography study by Furukawa et al., in which
NMDA (2) was bound in a co-crystal with the GluN2D pro-
tein as illustrated in Figure 7, although the assays were car-
ried out, in our study, on GluN2A and GluN2B and not
GluN2D NMDARs. There is however high homology be-
tween these receptors, with all binding residues conserved,
suggesting similar binding modes.
À
ble possibility that the C F bond in 4 might interact in a re-
pulsive manner with the surface of the agonist binding site,
and that the poorer agonist activity could be due to that.
This cannot be excluded per se from our data, although
careful examination of the co-crystal structure of NMDA
and the GluN2D NMDAR subunit does not reveal a candi-
date residue for such a repulsive interaction if (4) is bound
into that structure.
Experimental Section
Methods: NMR spectra were recorded on either Bruker AV-300 (1H at
300.06 MHz, 13C at 75.45 MHz), 19F at 282.34 MHz), or Bruker AV-300
(1H at 300.13 MHz, 13C at 75.48 MHz) or Bruker AV-500 (1H at
499.90 MHz, 19F at 470.33 MHz). Samples were prepared either in deuter-
ated chlorofrom, deuterated methanol or deuterium oxide. Chemical
shifts d are reported in parts per millions (ppm) and quoted relative to
internal standard Me4Si for 1H and 13C and external standard CFCl3 for
19F. Coupling constants (J) are given in Hertz (Hz) and the splitting pat-
terns described as: singlet-s, doublet-d, triplet-t, multiplet-m, doublet of
doublets-dd or a broad doublet-brd. Spectroscopic data were assigned
based on the combination of one- and two-dimensional experiments
(COSY, HSQC and HMBC). IR experiments were recorded by the Nico-
let Avatar 360 FT-IR by preparing the sample in nujol method. Optical
rotations determination was performed using a Perkin–Elmer Model 341
polarimeter, [a]D values are determined at 589 nm and given in 10À1
degcm2 gÀ1. Single X-ray diffraction analyses was carried out by Prof.
A.M.Z. Slawin on a Robotic Diffractometer consisting of a Mo sealed
tube X-Ray system. Column chromatography was performed using
Apollo Scientific silica gel 60 (43–63 micron). Thin layer chromatography
(TLC) was performed using Macherey–Nagel Polygram Sil G/UV254
plastic and aluminium plates. Visualization was achieved by inspection
under UV light (255 nm) or by use of potassium permanganate stain or
molybdenum-based stain. High resolution and low resolution mass spec-
tra were recorded using a Micromass LCT-TOF mass spectrometer using
ES ionization in positive and negative mode, sometimes the CI mode was
used if they were inadequate. Dry tetrahydrofuran, dichloromethane, tol-
uene and diethylether were obtained from the Solvent Purification
System MB SPS-800.
Figure 7. The X-ray structure of Furukawa et al., showing NMDA (2)
bound to the GluN2D subunit, and a flattened distorted view of key hy-
drogen bonding interactions. The lower structures illustrate the bound
conformation of NMDA (2) as a Newman projection and then as it is ori-
À
entated on the receptor and the relationship between the C F bond and
+
À
the C N bonds in the stereoisomers of (2S,3S)-3 and (2S,3R)-4. Isomer
(2S,3S)-3 bound in this conformation would have a stabilising charge–
+
À
À
dipole gauche relationship between the C F and C N bonds, whereas
as for (2S,3R)-4 there is no such stabilising interaction as these bonds are
antiperiplanar to each other.
Synthetic methods
Conclusion
Diethyl (2R,3S)-2-(N-benzyl-N-methyl amino)-3-hydroxy succinate (8): A
saturated solution of N-benzylmethylamine in EtOH (7.74 mL, 0.06 mol)
was added dropwise to a suspension of diethyl (2S, 3S)-epoxysuccinate
(7; 9.7 g, 0.05 mol) in EtOH (60 mL). The solution was stirred at reflux
for 15 h, and then the excess of benzylmethylamine and EtOH were re-
moved under reduced pressure. The residue was purified with silica gel
(hexane/EtOAc; 6:4) to give the title compound 8 (9.5 g, 61%) as a col-
An electrostatic effect of a fluorine atom, stereospecifically
located at the C-3 position of the glutamate receptor agonist
NMDA has been used to influence binding efficiency to the
À
glutamate receptor. Stereogenic placement of the C F bond
to generate (2S,3S)-3 and (2S,3R)-4 3F-NMDAs gave rise to
very different levels of agonist activity on glutamate recep-
tors. Indeed, (2S,3S)-3 showed similar activity to NMDA
(2), whereas (2S,3R)-4 was almost inactive. NMR, X-ray
structural analysis and DFT theory analyses was used to
assess the favoured conformations of these diastereoisomers.
For (2S,3S)-3 the favoured conformer in solution appears to
1
ourless oil. [a]D = +50.4 (c=0.9 in CHCl3); H NMR (400 MHz, CDCl3):
d=7.24–7.22 (5H, m, Ar-H), 4.51 (1H, d, J=6.6 Hz, CHOH), 4.16 (4H,
m, OCH2), 3.75 (1H, d, J=6.6 Hz, CHN), 3.74 (2H, s, CH2), 2.32 (3H, s,
NCH3),1.23 ppm (6H, t, J=7.2 Hz, 2ꢂCH3); 13C NMR (100 MHz,
CDCl3): d=172.8 (C=O), 169.9 (C=O), 139.0 (Ar C), 128.7 (Ar CH),
128.7 (Ar CH), 127.1 (Ar CH), 127.1 (Ar CH), 70.9 (CHOH), 68.6
(CHN), 61.8 (OCH2), 60.8 (OCH2), 59.4 (CH2), 39.0 (NCH3), 14.4 ppm
(2ꢂCH3); IR (nujol mull) n˜ =3472, 1732, 1572, 1458, 1372, 1258, 1151,
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D. OꢀHagan et al.
1098, 1025 cmÀ1; MS (ESI+): m/z (%): 332 [M+Na+] (100); HRMS (ES+
): m/z calcd for C16H23NO5Na: 332.1474 [M+Na]+; found: 332.1474.
(S)-14 and (S)-15 (26.5 g, 85%) as a pale yellow oil. Data for the mixture
of diastereoisomers (S)-14 and (S)-15: 1H NMR (300 MHz, CDCl3): d=
7.41–7.21 (30H, m), 5.33–5.00 (8H, m, OCH2), 4.63 (1H, d, J=8.3 Hz,
CHOH), 4.52 (1H, d, J=8.6 Hz, CHOH), 4.16 (1H, d, J=8.4 Hz, CHN),
3.98 (2H, q, J=6.7 Hz, CH), 3.91 (2H,q, J=6.4 Hz, CH), 3.69 (1H, d,
J=8.5 Hz, CHN), 2.43 (3H, s, NCH3), 2.21 ppm (3H, s, NCH3);
13C NMR (75 MHz, CDCl3): d=171.3 (C=O), 169.4 (C=O), 143.1 (Ar C),
128.7 (Ar CH), 128.6 (Ar CH), 128.5 (Ar CH), 128.3 (Ar CH), 128.2 (Ar
CH), 127.6 (Ar CH), 127.3 (Ar CH), 127.1 (Ar CH), 68.8 (CHOH),68.5
(CHOH), 67.3 (CHN), 67.0 (CHN), 66.7 (2ꢂOCH2), 66.6 (2ꢂOCH2),
63.0 (NCH), 62.6 (NCH), 35.7 (NCH3), 33.7 (NCH3), 21.1 (CH3),
20.5 ppm (CH3Ph); IR (nujol mull) n˜ =3470, 1731, 1572, 1448, 1362, 1253,
1150, 1097, 1020 cmÀ1; MS (ESI+): m/z (%): 470.1 [M+Na+] (100);
HRMS (ES+): m/z calcd for C27H29NO5Na: 470.1938 [M+Na]+; found
470.1943.
Diethyl (2S,3S)-2-(N-benzyl-N-methylamino)-3-fluoro succinate (10):
Deoxo-Fluor (6.44 mL, 0.03 mol) was added into a solution of (2R,3S)-di-
ethyl 2-(benzylACHTUNGTRENNUNG(methyl)amino)-3-hydroxysuccinate (8; 4 g, 0.01 mol) in
dry CH2Cl2 (20 mL) at RT. The solution was stirred for 15 h at RT, and
then the excess of Deoxo-Fluor was quenched with sat. NaHCO3 sodium
hydrogen carbonate to pH 7. The reaction mixture was extracted into
EtOAc (3ꢂ50 cmÀ3) and dried with MgSO4. The residue was purified
with silica gel (hexane/EtOAc; 9:1) to give the title compound 10 (2.8 g,
90%) as a pale yellow oil. [a]D = +4.64 (c=0.2 in CHCl3); H NMR
(300 MHz, CDCl3): d=7.24–7.19 (5H, m, Ar-H), 5.20 (1H, dd, J=5.8,
48.2 Hz, CHF), 4.21–4.19 (4H, m, CH2), 3.88 (1H, dd, J=5.9, 21.6 Hz,
CHN), 3.71 (2H, s, CH2), 2.29 (3H, s, NCH3), 1.28 ppm (6H, m, J=
7.2 Hz, 2ꢂCH3); 13C NMR (75 MHz, CDCl3): d=172.8 (C=O), 169.9 (C=
O), 137.4 (Ar C), 127.7 (Ar CH), 127.7 (Ar CH), 126.3 (Ar CH), 126.3
(Ar CH), 88.4 (d, J=189.0 Hz, CHF), 65.7 (d, J=24.0, CHN), 60.7
(OCH2), 60.1 (OCH2), 58.7 (CH2), 37.9 (NCH3), 13.3 (CH3), 13.0 ppm
(CH3); 19F{1H} NMR (280 MHz, CDCl3): d=À195.85 ppm (1F, s, J=5.8,
5.9, 21.6, 48.2 Hz, C(3)HF,); IR (nujol mull) n˜ =1744, 1691, 1576, 1458,
1376, 1270, 1141, 1094, 1037, 796, 739 ppm; MS (ESI+): m/z (%): 334
[M+Na+] (100); HRMS (ES+): m/z calcd for C16H22NO4 FNa: 334.1434
[M+Na]+; found: 334.1431.
Mixture of (2R,3R)- and (2S,3S)-dibenzyl 2-hydroxy-3-(N-methyl((R)-1’-
N-phenylethyl)amino) succinate (R)-15 and (R)-14: Mixture of diastereo-
isomer (R)-15 and (R)-14 was synthesized as described for (S)-14 and
(S)-15 but using the (R)-N-a-dimethylbenzylamine enantiomer. Products
(R)-15 and (R)-14 (27.8 g, 89%) were isolated as a pale yellow oil. Data
for the mixture of diastereoisomer was identical to that using (S)-N-a-di-
methylbenzylamine.
AHCTUNGTERG(NNUN 2R,3S)- Or (2S,3R)-Dibenzyl 3-fluoro-2- (N-methyl ((S)-1’-N-phenylethy-
Diethyl-(2S, 3S)-2-(N-methylamino)-3-fluoro succinate (11): Pd/C
(10 mol%) was added into a solution of diethyl (2S,3S)-fluoro benzylme-
thylamine succinate (10; 3.2 g, 0.01 mol) in dry ethanol (20 mL) and
stirred at RT in the presence of hydrogen gas. The reaction mixture was
followed by TLC until the reaction had gone completion and filtered
through celite. The reaction mixture was extracted into EtOAc (3ꢂ
50 cmÀ3) and dried with MgSO4. Concentration of the reaction crude to
give the title compound 11 (2.1 g, 97%) as a colourless oil. [a]D =À3.79
(c=0.1 in CHCl3); 1H NMR (400 MHz, CDCl3): d=5.13 (1H, dd, J=
2.79, 47.7 Hz, CHF), 4.22–4.14 (4H, m, CH2), 3.65 (1H, dd, J=2.9,
24.2 Hz, CHN), 2.43 (3H, s, NCH3), 1.28 ppm (6H, m, 2ꢂCH3);
13C NMR (100 MHz, CDCl3): d=169.4 (C=O), 167.3 (C=O), 90.6 (d, J=
191.6, CHF), 64.6 (d, J=21.1, CHN), 61.7 (OCH2), 61.7 (OCH2), 35.4
(NCH3), 14.0 ppm (2ꢂCH3); 19F{1H} NMR (376 MHz, CDCl3): d=
À199.63 ppm (1F, s, J=2.79, 2.9, 24.4, 47.7 Hz, C(3)HF); IR (nujol mull)
l)amino) succinate (S)-16 or (S)-17: Deoxo-Fluor (5.76 mL, 26.8ꢂ
10À3 mol) was added into a solution of (S)-14 and (S)-15 (4 g, 8.94ꢂ10À3
mol) in dry CH2Cl2 (20 mL) at RT. The solution was stirred for 15 h at
RT, and then the excess of Deoxo-Fluor was quenched with sodium hy-
drogen carbonate to pH 7. The reaction mixture was extracted into
EtOAc (3ꢂ50 cmÀ3) and dried with MgSO4. The residue was purified
with silica gel (hexane/EtOAc; 9:1) to give either one of the stereoisomer
of (S)-(16) or (S)-(17) as a pale yellow oil (3.37 g, 43%). Data for either
one of the stereoisomer of (S)-(16) or (S)-(17): [a]D =À69.5 (c=0.6 in
D2O); 1H NMR (400 MHz, CDCl3): d=7.38–7.05 (15, m, Ar-H), 5.40
(1H, dd, J=4.6 Hz, 48.4 Hz, CHF), 5.27 (4H, m, OCH2), 3.90 (1H, dd,
J=4.3, 32.4 Hz, CHN), 3.95 (1H, q, J=6.3 Hz, CH), 2.66 (3H, s, NCH3),
1.58 ppm (3H, s, CH3); 13C NMR (100 MHz, CDCl3): d=129.6 (Ar CH),
128.6 (Ar CH), 128.4 (Ar CH), 128.3 (Ar CH), 127.5 (Ar CH), 91.5 (d,
J=231.8 Hz, CHF), 67.2 (d, J=49.9 Hz, CHN), 63.1 (CH), 35.2 (NCH3),
21.3 ppm (CH3); 19F{1H} NMR (376 MHz, CDCl3): d=À195.7 ppm (1F, s,
J=4.3, 4.6, 32.4, 48.4 Hz, C(3)HF); IR (nujol mull) n˜ =2986, 1609, 1451,
1327, 1276, 1163, 1096, 1073, 800, 751 cmÀ1; MS (ESI+): m/z (%): 472.1
[M+Na+] (30); HRMS (ES+): m/z calcd for C27H28NO4 FNa: 458.1895
[M+Na]+; found: 472.1900.
n˜ =1744, 1691, 1576, 1458, 1376, 1270, 1141, 1094, 1037, 796, 739 cmÀ1
;
MS (ESI+): m/z (%): 222 [M+H+] (40); HRMS (ES+): m/z calcd for
C9H17NO4 F: 222.1142 [M+H]+; found: 222.1137.
ACHTUNGTRENNUNG(2S,3S)-3-Fluoro NMDA HCl (3): A few drops of ethanol were added
into a solution of (2S,3S)-3-fluoro amino alcohol methylamine (11) (1.6 g,
7.23ꢂ10À3 mol) until complete dissolution. HCl (4m, 4 mL) was added
into the reaction mixture. The solution was heated at reflux for 48 h and
diluted with CH2Cl2 (30 mL) and concentrated under reduced pressure to
give the title compound (2S,3S)-3 as a pale yellow oil (597 mg, 50%).
[a]D =À7.28 (c=4.1 in D2O); 1H NMR (400 MHz, CDCl3): d=5.57 (1H,
d, J=48.0 Hz, CHF), 4.70 (1H, d, J=29.0 Hz, CHN), 2.74 ppm (3H, s,
NCH3); 13C NMR (100MHz, CDCl3) d=169.4 (d, J=22.2, C=O), 86.4 (d,
J=189.0, CHF), 62.3 (d, J=22.7, CHN), 31.8 ppm (NCH3); 19F{1H} NMR
(376 MHz, CDCl3): d=À198.6 ppm (1F, s, J=48.0 Hz, C(3)HF); MS
(ESIÀ): m/z (%): 164 [MÀH+] (100); HRMS (ESÀ): m/z calcd for
C5H7NO4 F: 164.0365 [MÀH]À; found: 164.0359.
meso-Dibenzyl (2S,3R)-oxirane-2,3-dicarboxylate (12): Compound 12 was
synthesized as previously described.[20] 1H NMR (CDCl3): d=7.40–7.34
(10H, m, Ar-H), 5.13 (2H, s, OCH2), 3.77 (s, 2H, CH); dc (CDCl3)
128.7–128.6 (Ar-C), 67.6 (CH), 52.6 ppm (OCH2); MS (ESI+): m/z (%):
334.9 [M+Na+] (100); HRMS (ES+): m/z calcd for C18H16O5Na:
335.0890 [M+Na+]; found: 335.0896.
AHCTUNGTERG(NNUN 2R,3S)- or (2S,3R)-Dibenzyl 3-fluoro-2-(N-methyl((R)-1’-N-phenylethy-
l)amino)succinate (R)-17 or (R)-16: Compounds (R)-17 and (R)-16 were
synthesized as described for (S)-16 or (S)-17. Data for either one of the
stereoisomer (R)-16 or (R)-17: [a]D = +52.2 (c=0.6 in D2O); 1H NMR
(300 MHz, CDCl3): d=7.38–7.05 (15, m, Ar H), 5.40 (1H, dd, J=4.6,
48.4 Hz, CHF), 5.27 (4H, m, OCH2), 3.90 (1H, dd, J=4.3, 32.4 Hz,
CHN), 3.95 (1H, q, J=6.8 Hz, CH), 2.66 (3H, s, NCH3), 1.58 ppm (3H, s,
CH3); 13C NMR (75 MHz, CDCl3): d=129.0 (Ar C), 128.6 (Ar CH),
128.4 (Ar CH), 128.3 (Ar CH), 127.5 (Ar CH), 91.5 (d, J=231.8 Hz,
CHF), 67.2 (d, J=49.9 Hz, CHN), 63.1 (CH), 35.2 (NCH3), 21.3 ppm
(CH3Ph); 19F{1H} NMR (376 MHz, CDCl3): À195.7 (1F, s, J=4.3, 4.6,
32.4, 48.4 Hz, C(3)HF); IR (nujol mull) n˜ =2986, 1609, 1451, 1327, 1276,
1163, 1096, 1073, 800, 751; MS (ESI+): m/z (%): 472.1 [M+Na+] (30);
HRMS (ES+): m/z calcd for C27H28NO4FNa: 458.1895 [M+Na]+; found
472.1900.
N-Methyl aspartates (2R,3S)-4 or (2S,3R)-4: Pd/C (10 mol%) was added
into a solution of (S)-16 or (S)-17 (1.6 g, 3.67ꢂ10À3 mol) in ethyl acetate
(20 mL) and stirred at RT in the presence of hydrogen gas. The reaction
mixture was followed by TLC until the reaction had gone completion
and filtered through celite. The reaction mixture was extracted into
EtOAc (3ꢂ50 cmÀ3). Concentration of the reaction crude and HCl (1m,
0.1 mL) was added to give either one of the stereoisomer d-4 or l-4
(1.6 g, 72%) as a pale yellow oil (517 mg, 85%). Data for (2S,3R) (4) or
(2R,3S) (4) : [a]D =À2.1 (c=1.0 in D2O); 1H NMR (500 MHz, CDCl3):
d=5.39 (1H, dd, J=45.1 Hz, CHF), 4.26 (1H, dd, J=27.4 Hz, CHN),
Mixture of (2S,3S)- and (2R,3R)-dibenzyl 2-hydroxy-3-(N-methyl-(S)-1’-
N-phenylethyl)amino) succinate (S)-14 and (S)-15: A saturated solution
of and (S)-N-a-dimethylbenzylamine (19.70 mL) was added dropwise to
a suspension of (12; 10.0 g, 0.07 mol) in DMF (10 mL) and the resulting
solution was stirred at reflux for 4 h. The reaction mixture was worked
up with sodium hydrogen carbonate (3ꢂ50 cmÀ3) and the aqueous layer
was washed with ethyl acetate. Solvent evaporation and purification with
silica gel (hexane/EtOAc; 8:2) to afford the mixture of diastereoisomers
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3F-NMDA Stereoisomers as Conformational Probes
FULL PAPER
2.69 ppm (3H, NCH3); 13C NMR (75, CDCl3): d=171.0 (C=O), 168.6
(C=O), 87.9 (d, J=189.5 Hz, CHF), 62.5 (d, J=21.7 Hz, CHN), 32.3 ppm
(NCH3); 19F{1H} NMR (376 MHz, CDCl3): d=À196.38 ppm (1F, s, J=
27.4, 45.1 Hz C(3)HF); MS (ESIÀ): m/z (%): 164 [MÀH+] (100); HRMS
(ESÀ): m/z calcd for C5H7NO4F: 164.0369 [MÀH]À; found 164.0359.
Starting from a solution of (R)-(17) or (R)-(16) (as above) gave l-4 or d-
4 (1.6 g, 72%) as a pale yellow oil (517 mg, 85%). Data for (2R,3S)-4 or
(2S,3R)-4: [a]D = +2.9
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Acknowledgements
We are grateful to the University of Malaysia Terengganu and the Minis-
try of Higher Education of Malaysia for a PhD Studentship and we
thank Scott Badin and Kin Chow for performing initial experiments with
3F-NMDA isomers. We also thank EaStCHEM via the EaStCHEM Re-
search Computing Facility and DOꢀH acknowledges an ERC Advanced
Investigator grant.
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Received: January 8, 2012
Published online: && &&, 2012
Chem. Eur. J. 2012, 00, 0 – 0
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www.chemeurj.org
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D. OꢀHagan et al.
Two stereoisomeric analogues of the
glutamate receptor agonist N-methyl-
d-aspartate (NMDA), display very dif-
ferent activities as a result of the dia-
stereotopic replacement of each of the
C-3 hydrogens for fluorine. The differ-
ence in agonist activity is attributed to
Conformation Analysis
P. W. Chia, M. R. Livesey,
A. M. Z. Slawin, T. v. Mourik,
D. J. A. Wyllie,
D. OꢀHagan* .................. &&&& — &&&&
3-Fluoro-N-methyl-d-aspartic acid (3F-
NMDA) Stereoisomers as Conforma-
tional Probes for Exploring Agonist
Binding at NMDA Receptors
À
the C F bond inducing a different con-
formational bias in each case. The
study illustrates the power of using the
À
C F bond to influence the conforma-
tion of bioactive amines and explore
preferred binding modes to large pro-
tein receptors.
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Chem. Eur. J. 0000, 00, 0 – 0
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These are not the final page numbers!