Synthesis and evaluation of ω-borono-α-amino acids as active-site probes of arginase and nitric oxide synthases

Article abstract of DOI:10.1039/a908140b

Enantiomerically pure ω-borono-α-amino acids of various chain lengths have been synthesized according to a general methodology involving condensation of alkenyl and alkynyl bromides with Ni^II complex of the Schiff base derived from glycine and (S)-2-[N'-(N-benzylprolyl)amino]benzophenone, hydroboration of the intermediate ω-unsaturated α-amino acids with diisopinocampheylborane, and oxidation with acetaldehyde. Some of these compounds act as potent inhibitors of rat liver and murine macrophage arginases, demonstrating that distance between the B(OH)₂ and α-amino acid groups is a key determinant for their interaction with arginase. In contrast, they are without effect on neuronal and inducible NO synthases.

Full text of DOI:10.1039/a908140b

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Synthesis and evaluation of ꢀ-borono-ꢁ-amino acids† as active-site
probes of arginase and nitric oxide synthases
Sylvain Collet,a François Carreaux,a Jean-Luc Boucher,b Stéphanie Pethe,b Michel Lepoivre,c
Renée Danion-Bougota and Daniel Danion*a
1
a Synthèse et Electrosynthèse organiques, UMR 6510 CNRS, Université Rennes I,
Campus de Beaulieu, 35042 Rennes Cedex, France
b Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques,
UMR 8601 CNRS, Université Paris V, 45 rue des Saints Pères, 75270 Paris Cedex 06, France
c URA 1116 CNRS, Université Paris Sud-Orsay, Bat. 430, 91405 Orsay Cedex, France
Received (in Cambridge, UK) 11th October 1999, Accepted 15th November 1999
Enantiomerically pure ω-borono-α-amino acids of various chain lengths have been synthesized according to a
general methodology involving condensation of alkenyl and alkynyl bromides with NiII complex of the Schiff
base derived from glycine and (S)-2-[NЈ-(N-benzylprolyl)amino]benzophenone, hydroboration of the intermediate
ω-unsaturated α-amino acids with diisopinocampheylborane, and oxidation with acetaldehyde. Some of these
compounds act as potent inhibitors of rat liver and murine macrophage arginases, demonstrating that distance
between the B(OH)2 and α-amino acid groups is a key determinant for their interaction with arginase. In contrast,
they are without effect on neuronal and inducible NO synthases.
In mammalian cells, -arginine is metabolized by two major
pathways: arginase catalyses its hydrolysis to -ornithine and
urea in the first step of the urea cycle, whereas NO synthases
(NOS) catalyse its oxidation to -citrulline and nitric oxide.1
The general biological importance of arginase lies in its roles in
controlling nitrogen excretion and cellular levels of -arginine
and -ornithine involved in protein synthesis, as well as produc-
tion of creatine, proline and polyamines.2,3 NO synthases
are flavohaemoproteins that require NADPH, O2, tetrahydro-
biopterin and calmodulin for the stepwise oxidation of
-arginine to initially produce Nω-hydroxy--arginine (NOHA,
Chart 1) and, in a second step, to form -citrulline and NO.4,5
the same substrate, they differ in their affinity for -arginine
with Km-values of 1–10 mM and 1–15 µM respectively.3,4 The
two enzymes also have different patterns of inhibition. Most of
the enzymology of NOSs and the pharmacological roles of
NO have been determined by experiments using Nω-substituted
analogues of -arginine like Nω-methyl--arginine and Nω-nitro-
-arginine.4,8 In contrast, few compounds are known as potent
arginase inhibitors.3
Since the two enzymes can be found in similar tissues and
cells, and because their expression may be regulated in response
to the same stimuli (cytokines, endotoxines),9–12 the regulation
of arginases and NOSs by selective and potent inhibitors may
have considerable importance in therapy and could allow clear
estimation of their roles in tumour growth or rejection.13 Many
studies now support the hypothesis that arginase may be essen-
tial in the regulation of NOS activity by modulating local
-arginine concentration.14–16 It recently appeared that the
NOS’s intermediate NOHA is a potent inhibitor of rat liver and
murine macrophage arginases.15,17,18 Several studies confirmed
the importance of NOHA as an endogenous arginase inhibitor
increasing -arginine availability for NO-biosynthesis.14,15 Some
analogues of NOHA have been synthesized and evaluated as
inhibitors of NOSs and arginases. This has led to the discovery
of the new α-amino acid Nω-hydroxy-nor--arginine (nor-
NOHA) as one of the most potent arginase inhibitors (Ki
0.5 0.1 µM).19,20 A possible explanation for the specific effects
of this molecule is that it could act by replacement of the H2O
Chart 1 Structure of Nω-hydroxy--arginine (NOHA), Nω-hydroxy-
nor--arginine (nor-NOHA), ω-borono-α-amino acids 1a–e, and
decarboxylated analogue 1f.
(or OH) bridging ligand of the MnII -cluster by its N-hydroxy
2
function.2,19,20 Almost simultaneously, the proposal of a tetra-
hedral transition state in -arginine hydrolysis resulted in the
evaluation of borate anion as arginase inhibitor and in the
synthesis of the first boronic analogue of -arginine, (S)-2-
amino-6-(dihydroxyboryl)hexanoic acid.21,22 The high affinity
of this compound for arginase (IC50 0.8 µM) corroborates the
postulated mechanism for inhibition.
NO thus produced is a key biological molecule involved in
vasodilation, neurotransmission and cytotoxicity.6
Arginine hydrolysis by mammalian arginases is achieved by
a metal-activated water molecule that bridges a MnII cluster
2
at the active site.2,7 The hydrolysis is postulated to proceed
through a tetrahedral intermediate resulting from the nucleo-
philic attack of metal-bridging hydroxide ion at the guanidin-
ium carbon of -arginine.7 Although arginases and NOSs share
Boronic acid analogues have been previously found to be
very potent inhibitors of serine proteases, dipeptidyl peptidases
and dihydroorotase.23–25 The rationale for the effects of such
compounds was the well characterized hydration of the
electron-deficient boron atom to a stable tetrahedral borate
† In this paper, ‘borono’ is used as a term for ‘dihydroxyboryl’.
J. Chem. Soc., Perkin Trans. 1, 2000, 177–182
This journal is © The Royal Society of Chemistry 2000
177

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complex which is very close to the intermediate involved in the
hydrolysis. Unlike the unstable sp3 carbon species, the analo-
gous tetrahedral boronic structures are stable and thus inhibit
the target enzyme.26
Protection of amino and carboxylic acid functions was per-
formed under standard procedures as the NHBoc and methyl
carboxylic ester derivatives 4a–e in a one-pot procedure.27
Protected amino acids 4a–e were hydroborated with diisopino-
campheylborane (Ipc2BH) which allows a complete chemo- and
regioselectivity. The resulting boranes were oxidized in situ by
an excess of acetaldehyde32 and the intermediate diethylboro-
nates were converted to isolable and thoroughly characterized
pinanediol boronates 5a–e.27 Total deprotection of 5a–e was
performed in an almost quantitative manner by refluxing with
6 M or 12 M HCl followed by chromatography, and afforded
free ω-borono-α-amino acids 1a–e as hygroscopic white pow-
ders. Enantiomeric excesses were measured by HPLC after
derivatization and synthesis of samples of racemic acids 1a–e.
Compounds 1a and 1d have been previously prepared follow-
ing our general methodology27 whereas 1f has already been
described.30 Previously obtained in 1.3% overall yield pro-
tected -glutamic acid,22 compound 1b was obtained in 24%
overall yield from 4-bromobut-1-ene following our general
methodology.
We recently described a new stereoselective synthesis of
ω-borono-α-amino acids based on hydroboration of ω-unsatur-
ated, non-racemic, α-amino acids obtained through a general
methodology.27 The present work describes the synthesis of
ω-borono-α-amino acids of various chain lengths 1a–c (Chart
1). As amino boronic acids and related oligopeptides are very
prone to give internal complexes,28,29 we also synthesized con-
formationaly restricted unsaturated analogues 1d,e. The
importance of an intact α-amino acid function for arginase
inhibition was evaluated with the decarboxylated analogue 1f
synthesized according to a previously described procedure.30
This paper reports a study of the effects of compounds 1a–e
and of nor-NOHA as arginase inhibitors of purified rate liver
arginase (RLA) and of arginase contained in murine macro-
phages. In addition, compounds 1a–f were also studied as pos-
sible inhibitors of purified recombinant neuronal and inducible
NOSs.
Biological results and discussion
Results and discussion
Three ω-borono-α-amino acids 1b, 1c and 1e led to a clear
concentration-dependent inhibition of purified RLA activity.
Compound 1b was as potent as nor-NOHA to inhibit RLA
activity (IC50-values of 1.6 0.5 and 1.3 0.4 µM, respect-
ively, at physiological pH) (Table 1). This result is in good
accord with the previously published IC50-value for 1b (0.8
µM)22 and shows that differences in enzyme preparation or
conditions of incubation weakly affect its potent inhibitory
effect. The shorter analogues of 1b, 1a and 1d, were almost
without effect at the highest concentration used (5 mM)
whereas the longer analogue, 1c, displayed an IC50-value 150-
fold higher than 1b. Interestingly, the unsaturated analogue
of 1b, compound 1e, displayed an IC50-value 10-fold higher
than 1b, and the decarboxylated analogue, 1f, was almost
without effect (IC50-value close to 3 mM). These results
indicate that changing chain length by the addition or loss
of only one methylene group strongly affects the affinity for
the active site of RLA. Similar effects have been observed
when we compared α-amino acids bearing either an N-
hydroxyguanidino group, such as nor-NOHA, NOHA and
homo-NOHA,20 or an N-hydroxylamino function, Nδ-hydroxy-
-ornithine and Nε-hydroxy--lysine.19 Furthermore, introduc-
tion of a double bond in compound 1b restricts chain flexibility
and modifies the distance between the α-amino acid and the
B(OH)2 groups. Removal of the carboxylic acid group (compar-
ing 1b and 1f in Table 1), or modifications of the α-amino acid
function,18,19 give almost inactive products. These results
demonstrate that the presence of a free α-amino acid is crucial
for RLA recognition and reinforce the proposal that there must
be an optimal distance between the α-amino acid function and
the OH group of the studied compounds to achieve good
inhibition.19,20
Synthesis of ꢀ-borono-ꢁ-amino acids
The synthesis of the ω-borono-α-amino acids was performed as
outlined in Scheme 1 for the unsaturated analogue 1e. Enantio-
Scheme 1 Reagents and conditions: a, NaOH, DMF, r.t., 10 min; b,
2 M HCl, 60 ЊC, 1 h; c, Dowex (Hϩ); d, SOCl2, MeOH, reflux, 3 h; e,
Boc2O, NEt3, CH3CN, r.t., 1.5 h; f, Ipc2BH, THF, Ϫ20 ЊC, 1 h and
r.t., 5 h; g, CH3CHO, 0 ЊC, 1 h and r.t. 24 h; h, H2O, r.t., 1 h; i, (ϩ)-
pinanediol, Et2O, r.t., 15 h; j, 6 M HCl, 70 ЊC, 5 h; k, SiO2 (EtOH–14 M
NH3).
merically pure ω-unsaturated α-amino acids were synthesized
through alkylation of the NiII complex 2 of the Schiff base
derived from glycine and (S)-2-[NЈ-(N-benzylprolyl)amino]-
benzophenone (BPB) with an ω-bromoalkene or ω-bromo-
alkyne.27 This general methodology, developed by Belokon,31
affords very good diastereoisomeric excess (de) under
thermodynamic equilibrating conditions and the major
diastereoisomers are readily isolated, after crystallization or
chromatography, with yields in the 60–80% range. Decomplex-
ation of the free amino acids 6a–e occurs without racemization
under mild conditions with recovery of the chiral auxiliary.
RLA shows highest activity at pH 9.0,2,3 and the inhibitory
effects of 1a–f were then studied at this pH. By comparison to
experiments performed at pH 7.4, IC50-values for 1a–f signifi-
cantly increased (5- to 10-fold) at pH 9.0 (Table 1). Similar
results have been observed with compounds bearing an N–OH
function.19 It was postulated that RLA strongly recognizes
positively charged compounds (-arginine, NOHA or nor-
NOHA) but weakly interacts with non-protonated hydroxyl-
amines or amidoximes.19 In the case of boronic analogues
1a–e, the strongest interaction is observed at pH 7.4, a pH-
value where the tricoordinated boron atom predominates. At
pH 9.0, the boron atom should be mainly tetracoordinated and
could less strongly interact with the OH group bridging the two
MnII atoms at the RLA active site.
The ability of boronic compounds 1a–f and nor-NOHA to
178
J. Chem. Soc., Perkin Trans. 1, 2000, 177–182

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Table 1 Inhibition of rat liver and murine macrophage arginases by
ω-borono-α-amino acids 1a–f
cological studies that require selective and potent inhibition of
arginase.
IC50 (µM)
Experimental
Mps were determined with a Köfler apparatus and are uncor-
RLAa
Compound
pH 7.4
5000
pH 9.0
mMAb
1000
rected. NMR spectra were recorded on a Bruker AM WB 300
1
(300 MHz, 75 MHz and 96 MHz for H, 13C and 11B, respect-
1
ively) and ARX 200 (200 MHz and 50 MHz for H and 13C,
1a
>5000
(10%)c
respectively) spectrometers, using CDCl3 as a solvent, unless
otherwise stated. Chemical shifts (δ) are reported in ppm
[relative to internal TMS (1H, 13C) or external BF3ؒOEt2 (11B)]
and coupling constants (J) in Hz. If necessary, assignments
were determined after selective decoupling and two-dimen-
sional experiments. J Values are only given for the first
described protons in coupled systems. Multiplicities of the 13C
NMR spectra were assigned using DEPT sequence. Optical
rotations were measured on the sodium -line (589.3 nm) using
a Perkin-Elmer 341 polarimeter and were recorded in units of
10Ϫ1 deg cm2 gϪ1. The enantiomeric excesses (ees) were deter-
mined by HPLC on a Perkin-Elmer chromatograph 250. Mass
spectra were recorded on Varian MAT 311 (electron impact,
EI) or Micromass ZABSpec TOF [LSIMS or electrospray
(CH3CN–H2O), as stated] spectrometers by the ‘Centre
Régional de Mesures Physiques de l’Ouest’ (Rennes, France).
Elemental analyses were performed by the ‘Service de micro-
analyse du CNRS’ (Gif-sur-Yvette, France). Column chrom-
atography was carried out using Merck silica gel 60 (40–63 µm).
NiII complex 2 of the Schiff base derived from glycine and BPB
was prepared according to the literature method.35
1b
1c
1d
1.6 0.8
300 40
>5000
18
8
5
5000
>10000
(20%)d
100 20
5000
1
350 50
>5000
(30%)c
(10%)c
65 15
>5000
1e
1f
15
5
3000
(40%)c
a Assays quantitated the [14C]urea produced from -[G-14C]Arg accord-
ing to a previously described method.19 A typical assay was performed
in 0.1 cm3 of 0.2 M Tris-HCl (pH 7.4) containing 10 mM -Arg, 0.05
µCi of -[G-14C]Arg and variable concentrations of inhibitors. The
reactions were initiated by the addition of purified RLA.19 Protein
amounts were adjusted to yield less than 15% substrate conversion.
After 10 min at 37 ЊC, 0.15 cm3 of cold stop buffer (0.25 M acetic acid
and 7 M urea) was added and [14C]urea was separated from unchanged
-[G-14C]Arg by mixing with 0.25 cm3 of a 1:1 slurry of Dowex AG-
50W-X8 (Hϩ-form) in stop buffer, and centrifugation. Aliquots of the
supernatant were counted after addition of Pico-Fluor 40. IC50 mean
values SD from 3 to 5 independent experiments. b Murine macro-
phages (mMA) were obtained from C3H/HeN mice injected intraperi-
toneally with thioglycolate broth (Institut Pasteur, France) 3 days
before the cells were harvested.18 Macrophages were washed and resus-
pended (2 × 106 cmϪ3) in RPMI medium containing 600 µM -Arg, 0.05
µCi -[G-14C]Arg and appropriate concentration of inhibitors. Cells
were incubated 2 h at 37 ЊC under a 5% CO2 atmosphere. Arginase
activity was measured as above after addition of 0.15 cm3 of cold stop
buffer, 0.25 cm3 of a slurry of Hϩ Dowex, centrifugation, and counting
of [14C]urea contained in aliquots of the supernatant. IC50 mean
values SD from 3 independent experiments. c Highest concentration
5 mM. d Highest concentration 10 mM.
Alkylation of complex 2 with 4-bromobut-1-ene: NiII complex of
the Schiff base derived from BPB and but-3-enylglycine, 3b
Finely powdered NaOH (1 g, 25 mmol) and 4-bromobut-1-ene
(1.5 cm3, 1.5 equiv.) were added, under N2, to a stirred mixture
of 2 (5 g, 10 mmol) in dry CH3CN (45 cm3). After 3 h, the
reaction mixture was treated by 150 cm3 of 0.1 M HCl. The red
product was then extracted with CH2Cl2 (4 × 100 cm3), dried
(MgSO4), and the solvent was removed in vacuo. The two
diastereoisomers (98:2) were separated by chromatography on
silica gel (CH2Cl2–Me2CO, 3:1) and 3b was obtained as a red
solid (3.4 g, 62%), mp 210 ЊC (from AcOEt) (Found: C, 67.2; H,
5.8; N, 7.6. C31H31N3NiO3 requires C, 67.4; H, 5.65; N, 7.6%);
δH 2.00–2.10 (1 H, m, 1δ-Hpro), 2.10–2.23 (1 H, m, 1γ-Hpro),
2.46–2.60 (1 H, m, 1β-Hpro), 2.66–2.80 (1 H, m, 1β-Hpro), 3.48
(1 H, dd, J 11.2 and 5.8, α-Hpro), 3.43–3.62 (2 H, m, 1γ-,
1δ-Hpro), 3.56 and 4.43 (2 H, AB system, J 12.6, CH2Ph),
ethylenic chain 1.64–1.75 (1 H, m, 3-H), 2.10–2.23 (1 H, m,
3-HЈ), 2.23–2.34 (1 H, m, 4-H), 2.66–2.80 (1 H, m, 4-HЈ), 3.91
(1 H, dd, J 8.5 and 3.5, 2-H), 4.87 (1 H, dm, J6,5 10.2, J6,6Ј 1.7,
J6,4 1.3, 6-H), 4.96 (1 H, dq, J6Ј,5 17.1, J6Ј,4 1.5, 6-HЈ), 5.53 (1 H,
ddt, J5,4 6.5, 5-H), 6.63–8.12 (14 H, m, ArH); δC 23.8 (γ-Cpro),
29.5 (C-4), 30.8 (β-Cpro), 35.1 (C-3), 57.1 (δ-Cpro), 63.1
(CH2Ph), 69.8 (C-2), 70.3 (αCpro), 115.8 (C-6), 120.8–142.2
inhibit arginase activity contained in murine macrophages was
also investigated (Table 1). Compounds 1b and 1e demon-
strated the highest potency to inhibit [14C]urea formation with
IC50 of 5 1 and 100 20 µM, respectively, whereas nor-
NOHA displayed an IC50-value of 3.0 1.0 µM. Compounds
1a, 1c, 1d and 1f were far less active, with IC50-values in the mM
range. Although the experimental conditions were different
[substrate concentration 10 mM in RLA assays but only 0.6
mM in RPMI (cell culture medium) containing macrophages],
almost identical IC50-values were measured for the two argin-
ases. These results suggest that compounds 1b and 1e which
display similar potency against the macrophage and the rat liver
enzymes penetrate into the intracellular medium.
When tested on recombinant purified neuronal and inducible
NOSs, compounds 1a–f exhibited very weak effects and failed
to inhibit (less than 10% inhibition at 5 mM) the oxidation of
-[14C]arginine to -[14C]citrulline and NO when followed by
two classical methods used to measure NOS activity.33,34 In
addition, compounds 1a–f did not significantly modify the
haem absorption spectra of the oxygenase domain of inducible
NOS.4,5
(18 × Ar-C), 136.6 (C-5), 170.4 (C᎐N), 179.2 (NC᎐O), 180.4
᎐
᎐
(C-1); m/z (LSIMS) 552.179 [(Mϩ ϩ H). C31H32N3NiO3 requires
m/z, 552.1797].
Alkylation of complex 2 with 5-bromopent-1-ene: NiII complex of
Further studies are necessary to understand the mechanisms
of inhibition of RLA by compounds 1b and 1e. However,
our results show a very different behaviour for 1b and 1e
towards RLA and NOSs. Compound 1b is a strong inhibitor of
RLA and of murine macrophage arginase, as potent as nor-
NOHA, but without effects towards NOSs. It does not bear an
N-hydroxyguanidino function and should be less sensitive to
oxidation mediated by superoxide ions or haem proteins than
are nor-NOHA and NOHA. Compound 1b is a useful tool for
future comparisons of the active sites of these two classes of
enzymes that use -arginine as substrate as well as for pharma-
the Schiff base derived from BPB and pent-4-enylglycine, 3c
The reaction was carried out as above with 2.9 g (5.8 mmol)
of 2. The two diastereoisomers (96:4) were separated by
chromatography on silica gel (CH2Cl2–Me2CO, 2:1) and 3c was
obtained as a red solid (1.7 g, 52%), mp 192 ЊC (from AcOEt)
(Found: C, 67.9; H, 5.9; N, 7.35. C32H33N3NiO3 requires C,
67.85; H, 5.85; N, 7.4%); δH 1.93–2.07 (1 H, m, 1δ-Hpro), 2.08–
2.29 (1 H, m, 1γ-Hpro), 2.45–2.59 (1 H, m, 1β-Hpro), 2.72–2.81
(1 H, m, 1β-Hpro), 3.47 (1 H, dd, J 11.0 and 5.8, α-Hpro), 3.44–
3.62 (2 H, m, 1γ-, 1δ-Hpro), 3.59 and 4.45 (2 H, AB system,
J. Chem. Soc., Perkin Trans. 1, 2000, 177–182
179

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J 12.7, CH2Ph), ethylenic chain 1.59–1.74 (2 H, m, 3-, 4-H),
1.86–2.07 (3 H, m, 3-HЈ, 5-H2), 2.08–2.29 (1 H, m, 4-HЈ), 3.91
(1 H, dd, J 8.1 and 3.5, 2-H), 4.96 (1 H, dm, J7.6 10.3, 7-H), 4.98
(1 H, dm, J7Ј,6 17.0, 7-HЈ), 5.73 (1 H, ddt, J6,5 6.6, 6-H), 6.63–
8.15 (14 H, m, ArH); δC 23.6 (γ-Cpro), 24.6 (C-4), 30.7
(β-Cpro), 33.3 (C-5), 34.8 (C-3), 57.0 (δ-Cpro), 63.1 (CH2Ph),
70.3 (α-Cpro), 70.4 (C-2), 115.2 (C-7), 120.7–142.2 (18 × Ar-C),
(2 H, m, 4-H2), 2.39 (1 H, s, 6-H), 3.82 (1 H, dd, J2,3 7.2, J2,3Ј 5.5,
2-H); δC (D2O) 17.0 (C-4), 31.8 (C-3), 56.5 (C-2), 73.5 (C-6),
85.6 (C-5), 176.8 (C-1).
Protected amino acids 4
Amino acids 6 were protected as methyl ester and tert-butyl
carbamate, as previously described.27
137.7 (C-6), 170.3 (C᎐N), 179.3 (NC᎐O), 180.4 (C-1); m/z
᎐
᎐
(LSIMS) 566.195 [(Mϩ ϩ H). C32H34N3NiO3 requires m/z,
566.1954].
(S)-2-(tert-Butoxycarbonylamino)hex-5-enoic acid methyl
ester 4b.—Oil (83%); [α]1D8 Ϫ15.2 (c 1.2 in CHCl3) (lit.,40 [α]2D0
Ϫ17) (Found: C, 59.15; H, 8.55; N, 5.7. C12H21NO4 requires C,
59.25; H, 8.7; N, 5.75%); δH 1.45 [9 H, s, (CH3)3], 1.63–1.76
(1 H, m, 3-H), 1.83–1.95 (1 H, m, 3-HЈ), 2.06–2.13 (2 H, m,
4-H2), 3.72 (3 H, s, OCH3), 4.27–4.34 (1 H, m, 2-H), 4.99 (1 H,
dm, J6,5 10.1, J6,6Ј 1.8, 6-H), 5.01 (1 H, br d, NH), 5.03 (1 H, dm,
J6Ј,5 17.1, 6-HЈ), 5.76 (1 H, ddt, J5,4 6.6, 5-H); δC 28.3 [(CH3)3],
29.5 (C-4), 32.0 (C-3), 52.2 (OCH3), 53.0 (C-2), 79.9 [C(CH3)3],
Alkylation of complex 2 with 4-bromobut-1-yne: NiII complex of
the Schiff base derived from BPB and but-3-ynylglycine, 3e
4-Bromobut-1-yne was prepared from but-3-ynyl toluene-p-
sulfonate36 according to the literature method.37
The reaction with 2 (1 mmol) was carried out as above but in
dry DMF (1.2 cm3) with 3 equiv. of NaOH and 1.1 equiv.
(0.15 g) of 4-bromobut-1-yne. After 10 min, the mixture
was neutralized with AcOH (0.25 cm3) and poured into water
(25 cm3). Crude 3e was extracted with CH2Cl2 and the two
diastereoisomers (95:5) were separated by chromatography on
silica gel (CH2Cl2–Me2CO, 4:1) to give pure 3e as red crystals
(330 mg, 60%), mp 218 ЊC (from AcOEt) (Found: C, 66.4; H,
5.4; N, 7.5. C31H29N3NiO3ؒ0.5 H2O requires C, 66.55; H, 5.4; N,
7.5%); δH 2.03–2.12 (1 H, m, 1δ-Hpro), 2.15–2.24 (1 H, m, 1γ-
Hpro), 2.45–2.59 (1 H, 1β-Hpro), 2.69–2.84 (1 H, m, 1β-Hpro),
3.48 (1 H, dd, J 10.8 and 5.6, α-Hpro), 3.46–3.70 (2 H, m, 1γ-,
1δ-Hpro), 3.58 and 4.43 (2 H, AB system, J 12.6, CH2Ph),
ethylenic chain 1.80 (1 H, t, J6,4 2.5, 6-H), 1.83–1.92 (1 H, m,
3-H), 2.27–2.36 (1 H, m, 3-HЈ), 2.35–2.44 (1 H, m, 4-H), 2.69–
2.84 (1 H, m, 4-HЈ), 3.99 (1 H, dd, J2,3 8.8, J2,3Ј 3.5, 2-H),
6.61–8.13 (14 H, m, ArH); δC 15.1 (C-4), 23.9 (γ-Cpro), 30.7
(β-Cpro), 34.1 (C-3), 57.1 (δ-Cpro), 63.1 (CH2Ph), 69.4
(C-2), 69.6 (C-6), 70.2 (α-Cpro), 82.7 (C-5), 120.8–142.3
(18 × Ar-C), 171.0 (C᎐N), 178.8 (NC᎐O), 180.4 (C-1); m/z
115.7 (C-6), 137.0 (C-5), 155.3 (NC᎐O), 173.3 (C-1); m/z (EI)
᎐
187.084 [(Mϩ Ϫ C4H8). C8H13NO4 requires m/z, 187.0844].
(S)-2-(tert-Butoxycarbonylamino)hept-6-enoic acid methyl
ester 4c.—Oil (83%); [α]2D0 14.1 (c 1.17 in CHCl3) (lit.,41 [α]2D4
13.16) (Found: C, 60.8; H, 9.0; N, 5.35. C13H23NO4 requires C,
60.7; H, 9.0; N, 5.45%); δH 1.37–1.52 (2 H, m, 4-H2), 1.45 [9 H,
s, (CH3)3], 1.57–1.69 (1 H, m, J3,3Ј 12.8, J3,2 6.8, 3-H), 1.75–1.87
(1 H, m, J3Ј,2 5.0, 3-HЈ), 2.07 (2 H, qdd, J5,4 = J5,6 = 6.7, J5,7 1.3,
J5,7Ј 2.7, 5-H2), 3.74 (3 H, s, OCH3), 4.27–4.34 (1 H, m, 2-H),
4.97 (1 H, dm, J7,6 10.3, J7,7Ј 2.0, 7-H), 5.03 (1 H, dm, J7Ј,6 17.0,
7-HЈ), 5.05 (1 H, br d, NH), 5.77 (1 H, ddt, J6,5 6.6, 6-H);
δC 24.6 (C-4), 28.3 [(CH3)3], 32.2 (C-5), 33.2 (C-3), 52.2
(OCH3), 53.3 (C-2), 79.8 [C(CH3)3], 115.1 (C-7), 138.0 (C-6),
ϩ
ؒ
155.4 (NC᎐O), 173.4 (C-1); m/z (EI) 198.150 [(M Ϫ CO CH ).
᎐
2
3
C11H20NO2 requires m/z, 198.1494].
(S)-2-(tert-Butoxycarbonylamino)hex-5-ynoic acid methyl
ester 4e.—Oil (85%); [α]D20 17 (c 1.2 in CHCl3) (Found: C, 59.5;
H, 7.75; N, 5.5. C12H19NO4 requires C, 59.75; H, 7.95; N, 5.8%);
δH 1.45 [9 H, s, (CH3)3], 1.89 (1 H, m, J3,3Ј 13.7, J3,2 7.6,
J3,4 ≈ J3,4Ј ≈ 7.0, 3-H), 2.00 (1 H, t, J6,4 2.6, 6-H), 2.09 (1 H, m,
J3Ј,4Ј ≈ J3Ј,4 ≈ 7.1, J3Ј,2 5.3, 3-HЈ), 2.26–2.32 (2 H, m, 4-H2), 3.76
(3 H, s, OCH3), 4.39 (1 H, m, J2,NH 6.7, 2-H), 5.15 (1 H, d, NH);
δC 14.9 (C-4), 28.3 [(CH3)3], 31.5 (C-3), 52.4 (OCH3), 52.7 (C-2),
᎐
᎐
(LSIMS) 550.164 [(Mϩ ϩ H). C31H30N3NiO3 requires m/z,
550.1641].
Hydrolysis of 3b, 3c, 3e and recovery of BPB
Hydrolysis of the complexes was performed as previously
described.27 A solution of a complex 3 (6 mmol) in MeOH (100
cm3) was added to warm 2 M HCl (70 cm3). The mixture was
refluxed for 1 h, then cooled to room temperature, and conc.
NH3 was added up to pH 9–10. BPB was quantitatively
recovered by extraction with CH2Cl2. The aqueous layer was
concentrated to dryness and the residue was chromatographed
with a cation-exchange resin (Dowex 50x8, Hϩ) to obtain the
amino acids 6.
69.3 (C-6), 80.0 [C(CH ) ], 82.8 (C-5), 155.3 (NC᎐O), 172.7
᎐
3
3
ϩ
ؒ
(C-1); mz (EI) 182.118 [(M Ϫ CO2CH3). C10H16NO2 requires
m/z, 182.1180].
Hydroboration of compounds 4: boronic esters 5
The ω-unsaturated amino esters 4 were first hydroborated with
Ipc2BH (2 equiv. for 4b, 4c and 1.2 equiv. for 4e). The resulting
boranes were added to an excess of acetaldehyde (20 equiv. for
4b, 4c and 12 equiv. for 4e), affording boronic acids after
hydrolysis, which were converted to pinanediol boronates 5 by
reaction with (ϩ)-pinanediol (1 equiv.). Compounds 5 were
purified by chromatography on silica gel (heptane–ethyl acetate,
8:2).27
(S)-2-Aminohex-5-enoic acid 6b.—White powder (96%), mp
248 ЊC (decomp.); [α]D20 13.6 (c 0.9 in H2O) (lit.,38 13.5); δH [D2O,
δ(H2O) 4.80] 1.80–2.00 (2 H, m, 3-H2), 2.09–2.16 (2 H, m,
4-H2), 3.68 (1 H, dd, J 6.8 and 5.4, 2-H), 5.04 (1 H, dm, J6,5 10.2,
J6,6Ј 1.9, 6-H), 5.09 (1 H, dm, J6Ј,5 17.2, 6-HЈ), 5.84 (1 H, ddt,
J5,4 6.5, 5-H); δC (D2O) 31.3 (C-4), 32.5 (C-3), 57.0 (C-2), 118.5
(C-6), 139.7 (C-5), 177.6 (C-1).
(S)-2-(tert-Butoxycarbonylamino)-6-[(1S,2S,3R,5S)-(؉)-
pinanyl-2,3-dioxyboryl]hexanoic acid methyl ester 5b.—Oil
(51%); [α]2D0 23.6 (c 3 in CHCl3) (Found: C, 61.6; H, 8.9; N, 3.1.
C22H38BNO6ؒ0.33 H2O requires C, 61.55; H, 9.1; N, 3.25%);
δH 0.79 (2 H, J 7.6, 6-H2), 0.82 (3 H, s), 1.26 (3 H, s) and 1.35
(3 H, s) (together 3 × CH3), 1.06 (1 H, d, J 10.9, 7Ј-H), 1.41
[9 H, s, (CH3)3], 1.25–1.47 (4 H, m, 5-, 4-H2), 1.53–1.65 (1 H, m)
and 1.70–1.80 (1 H, m) (together 3-H2), 1.80 (1 H, ddd, 4Ј-H),
1.85–1.91 (1 H, m, 5Ј-H), 2.01 (1 H, t, J ≈ 5.5, 1Ј-H), 2.15–2.23
(1 H, m, 7Ј-H), 2.30 (1 H, ddt, 4Ј-H), 3.72 (3 H, s, OCH3), 4.20–
4.28 (1 H, m, 2-H), 4.22 (1 H, dd, J 8.8 and 1.8, 3Ј-H), 4.98 (1 H,
d, J2,NH 7.9, NH); δC 10.4 (C-6), 23.8 (C-5), 24.0, 27.1 and 28.7
(3 × CH3), 26.5 (C-7Ј), 27.9 (C-4), 28.3 [(CH3)3], 32.4 (C-3), 35.5
(C-4Ј), 38.1 (C-6Ј), 39.5 (C-5Ј), 51.2 (C-1Ј), 52.1 (OCH3), 53.4
(S)-2-Aminohept-6-enoic acid 6c.—White powder (70%), mp
224 ЊC (decomp.); [α]D20 8.4 (c 0.5 in H2O); δH (D2O) 1.26–1.40
(2 H, m, 4-H2), 1.58–1.71 (2 H, m, 3-H2), 1.98 (2 H, br q,
J5,4 = J5,6 ≈ 7.0, 5-H2), 3.43 (1 H, t, J 6.2, 2-H), 4.89 (1 H, dm,
J7,6 10.2, 7-H), 4.96 (1 H, dm, J7Ј,6 17.3, 7-HЈ), 5.77 (1 H, ddt,
J6,5 6.7, 6-H); δC (D2O) 24.2 (C-4), 31.8 (C-5), 33.0 (C-3), 55.5
(C-2), 115.2 (C-7), 139.4 (C-6), 178.5 (C-1).
(S)-2-Aminohex-5-ynoic acid 6e.—White powder (84%), mp
244 ЊC (decomp.); [α]D18 3.7 (c 1 in H2O) (lit.,39 3.8); δH (D2O)
1.97 (1 H, m, J3,3Ј 14.3, 3-H), 2.07 (1 H, m, 3-HЈ), 2.34–2.37
180
J. Chem. Soc., Perkin Trans. 1, 2000, 177–182

View Article Online
(C-2), 77.6 (C-3Ј), 79.7 [C(CH ) ], 85.4 (C-2Ј), 155.4 (NC᎐O),
173.5 (C-1); δB 33.4; m/z (EI) 366.210 [(M Ϫ C4H9). C18H29-
BNO6 requires m/z, 366.2088].
(1 H, dt, J5,4 6.2, 5-H); δC (D2O) 32.1 and 33.0 (C-3 and -4), 57.2
᎐
3
3
ϩ
ؒ
(C-2), 126.0 (C-6), 152.7 (C-5), 177.4 (C-1); δB (H2O) 27.9; m/z
(Electrospray) 174.093 [(Mϩ ϩ H). C6H13BNO4 requires m/z,
174.0938].
(S)-2-(tert-Butoxycarbonylamino)-7-[(1S,2S,3R,5S)-(؉)-
pinanyl-2,3-dioxyboryl]heptanoic acid methyl ester 5c.—Oil
(60%); [α]2D0 22.1 (c 1 in CHCl3) (Found: C, 63.45; H, 9.3; N,
3.15. C23H40BNO6 requires C, 63.15; H, 9.2; N, 3.2%); δH 0.80 (2
H, t, J 7.6, 7-H2), 0.84 (3 H, s), 1.29 (3 H, s) and 1.38 (3 H, s)
(together 3 × CH3), 1.10 (1 H, d, J 10.8, 7Ј-H), 1.44 [9 H, s,
(CH3)3], 1.27–1.47 (6 H, m, 6-, 5-, 4-H2), 1.55–1.67 (1 H, m) and
1.73–1.83 (1 H, m) (together 3-H2), 1.83 (1 H, ddd, 4Ј-H),
1.88–1.94 (1 H, m, 5Ј-H), 2.04 (1 H, t, J ≈ 5.5, 1Ј-H), 2.17–
2.26 (1 H, m, 7Ј-H), 2.33 (1 H, ddt, 4Ј-H), 3.73 (3 H, s,
OCH3), 4.23–4.30 (1 H, m, 2-H), 4.24 (1 H, dd, J 8.7 and 1.9,
3Ј-H), 4.99 (1 H, d, J2,NH 8.2, NH); δC 10.6 (C-7), 23.9 (C-6),
24.0, 27.1 and 28.7 (3 × CH3), 25.1 (C-4), 26.5 (C-7Ј), 28.3
[(CH3)3], 31.9 (C-5), 32.7 (C-3), 35.5 (C-4Ј), 38.1 (C-6Ј), 39.5
(C-5Ј), 51.3 (C-1Ј), 52.1 (OCH3), 53.5 (C-2), 77.6 (C-3Ј), 79.8
Measurement of optical purity
The ees of the protected boronates 5 derivatized as benzamides
were measured by HPLC using a PIRKLE covalent (S,S)
whelk-01 column.27 Elution was performed with a mixture of
hexane–propan-2-ol (9:1), flow rate was 1 cm3 minϪ1, and the
UV detector was set at 225 nm. Samples of racemic derivatives
were obtained through a subsequent deprotection of the carb-
oxylic group with sodium hydroxide, oxazolone formation with
DCC,42 and opening of the heterocycle with methanol.27 Ees
were found to be superior to 98% for the three derivatives.
2-Benzoylamino-6-[(1S,2S,3R,5S)-(؉)-pinanyl-2,3-dioxy-
boryl]hexanoic acid methyl ester from 5b.—(S): tR 24.8 min;
(R,S): tR1 22.6 min; tR2 25.0 min.
[C(CH ) ], 85.3 (C-2Ј), 155.4 (NC᎐O), 173.5 (C-1); δ 33.6;
᎐
3
3
B
ϩ
ؒ
m/z (EI) 378.283 [(M Ϫ CO2CH3). C21H37BNO4 requires
m/z, 378.2815].
2-Benzoylamino-7-[(1S,2S,3R,5S)-(؉)-pinanyl-2,3-dioxy-
boryl]heptanoic acid methyl ester from 5c.—(S): tR 28.0 min;
(R,S): tR1 26.3 min; tR2 29.0 min.
(S)-2-(tert-Butoxycarbonylamino)-6-[(1S,2S,3R,5S)-(؉)-
pinanyl-2,3-dioxyboryl]hex-5-enoic acid methyl ester 5e.—Oil
(50%); [α]2D0 27.7 (c 1.5 in CHCl3) (Found: C, 61.2; H, 8.45; N,
2.9. C22H36BNO6ؒ0.5 H2O requires C, 61.4; H, 8.65; N, 3.25%);
δH 0.85 (3 H, s), 1.29 (3 H, s) and 1.40 (3 H, s) (together
3 × CH3), 1.13 (1 H, d, J 10.9, 7Ј-H), 1.44 [9 H, s, (CH3)3], 1.69–
1.80 (1 H, m, 3-H), 1.84 (1 H, ddd, 4Ј-H), 1.87–1.99 (2 H, m, 3-,
5Ј-H), 2.04 (1 H, t, J 5.5, 1Ј-H), 2.12–2.25 (3 H, m, 4-H2, 7Ј-H),
2.32 (1 H, ddt, 4Ј-H), 3.73 (3 H, s, OCH3), 4.27 (1 H, dd, J 8.5
and 1.8, 3Ј-H), 4.23–4.34 (1 H, m, 2-H), 5.02 (1 H, d, J2,NH 8.1,
NH), 5.47 (1 H, dt, J6,5 18.0, J6,4 1.5, 6-H), 6.56 (1 H, dt, J5,4 6.3,
5-H); δC 24.0, 27.1 and 28.6 (3 × CH3), 26.4 (C-7Ј), 28.3
[(CH3)3], 31.25 and 31.35 (C-3 and -4), 35.5 (C-4Ј), 38.1 (C-6Ј),
39.5 (C-5Ј), 51.3 (C-1Ј), 52.3 (OCH3), 53.1 (C-2), 77.7 (C-3Ј),
79.9 [C(CH3)3], 85.6 (C-2Ј), 119.5 (C-6), 152.0 (C-5), 155.3
2-Benzoylamino-6-[(1S,2S,3R,5S)-(؉)-pinanyl-2,3-dioxy-
boryl]hex-5-enoic acid methyl ester from 5e.—(S): tR 27.8 min;
(R,S): tR1 25.9 min; tR2 28.1 min.
Acknowledgements
We thank Dr Marie-Agnes Sari (UMR 8601 CNRS, Paris,
France) for the preparation of neuronal NOS and Dr Dennis
Stuehr (Cleveland Clinic Foundation, Cleveland, USA) for gifts
of purified inducible NOS.
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70 ЊC (12 M HCl; 2 h for 5b or 5c; 6 M HCl; 5 h for 5e).27
Compounds 1 were purified by chromatography on silica gel
(EtOH–14 M NH3; 2:1).
(S)-2-Amino-6-(dihydroxyboryl)hexanoic acid 1b.—White
powder (79%), mp 261 ЊC (decomp.); δH (D2O) 0.80 (2 H, t,
J6,5 7.6, 6-H2), 1.26–1.47 (4 H, m, 4-, 5-H2), 1.74–1.92 (2 H,
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(S)-2-Amino-7-(dihydroxyboryl)heptanoic acid 1c.—White
powder (91%) mp 246 ЊC (decomp.); δH (H2O/ext DSS) 0.76
(2 H, t, J7,6 7.4, 7-H2), 1.27–1.44 (6 H, m, 4-, 5-, 6-H2), 1.77–1.88
(2 H, m, 3-H2), 3.71 (1 H, dd, J2,3 6.4, J2,3Ј 5.8, 2-H); δC (D2O)
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2.07 (2 H, m, 3-H2), 2.20–2.30 (2 H, m, 4-H2), 3.73 (1 H, dd, J2,3
6.7, J2,3Ј 5.6, 2-H), 5.51 (1 H, dt, J6,5 18.1, J6,4 1.5, 6-H), 6.51
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