Strategies for the synthesis of HBGl3, a glutamic acid derived ligand bearing phenolic and azacarboxylate donor groups at the nitrogen atom

Article abstract of DOI:10.1016/j.tet.2017.09.032

The development of a route applicable to the preparation of acyclic glutamic acid-based chelating ligands bearing two different auxiliary donor groups linked to the nitrogen atom by methylene spacers is described and applied to the synthesis of the new polydentate ligand HBGl₃, the first example of such a structure. The synthesis is accomplished using a strategy employing reductive amination and t-butyl ester protected intermediates. The most basic pK_a values for the HBGl₃ ligand have been estimated via potentiometric and UV–Visible titration techniques.

Full text of DOI:10.1016/j.tet.2017.09.032

Accepted Manuscript
Strategies for the synthesis of HBGl , a glutamic acid derived ligand bearing phenolic
3
and azacarboxylate donor groups at the nitrogen atom
Raminder S. Mulla, Melissa T. Walden, Dmitry S. Yufit, Tanya Desa, Elena Lurie-
Luke, J.A. Gareth Williams
PII:
S0040-4020(17)30961-4
10.1016/j.tet.2017.09.032
TET 28983
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 6 June 2017
Revised Date: 5 September 2017
Accepted Date: 15 September 2017
Please cite this article as: Mulla RS, Walden MT, Yufit DS, Desa T, Lurie-Luke E, Williams
JAG, Strategies for the synthesis of HBGl , a glutamic acid derived ligand bearing phenolic and
3
azacarboxylate donor groups at the nitrogen atom, Tetrahedron (2017), doi: 10.1016/j.tet.2017.09.032.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Strategies for the synthesis of HBGl3, a glutamic acid derived ligand
bearing phenolic and azacarboxylate donor groups at the nitrogen atom
Raminder S. Mulla(a)*, Melissa T. Walden(a), Dmitry S. Yufit(a), Tanya Desa(b),
Elena Lurie-Luke(b) and J. A. Gareth Williams(a)*
(a) Department of Chemistry, Durham University, Durham, DH1 3LE, U.K.
(b) Procter and Gamble Technical Centres Limited, Rusham Park, Whitehall Lane,
Egham, Surrey, TW2 9NW.
Abstract
The development of a route applicable to the preparation of acyclic glutamic acid-
based chelating ligands bearing two different auxiliary donor groups linked to the
nitrogen atom by methylene spacers is described and applied to the synthesis of the
new polydentate ligand HBGl3, the first example of such a structure. The synthesis is
accomplished using a strategy employing reductive amination and t-butyl ester
protected intermediates. The most basic pKa values for the HBGl3 ligand have been
estimated via potentiometric and UV-Visible titration techniques.
Keywords: polydentate ligand, polyaminocarboxylate ligand, phenolate, reductive
amination, lactam, crystal structure
* E-mails: raminder.mulla@durham.ac.uk j.a.g.williams@durham.ac.uk Fax: +44 191 334 2051
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1. Introduction
The naturally occurring glutamic acid motif (Fig. 1) has often been used as a
coordinating unit, binding to metal ions via the amine and α-carboxylate to form a 5-
membered chelate. Such a coordination mode leaves the ε-carboxylate non-
coordinating and thus available, for example, to suppress the interaction of complexes
of these ligands with anionic species1,2 or to serve as a point of conjugation to other
molecules through amide or ester formation.3
Fig. 1. The structure of L-glutamic acid
Another incentive for its incorporation into a ligand is that the presence of naturally
occurring fragments in organic molecules can improve their biodegradability.4 One
such example is the well-known ligand GLDA {N,N-bis(carboxymethyl)glutamic
acid or L-glutamic acid diacetic acid}, which has been shown to be readily
biodegradable and is produced on a commercial scale (Fig. 2A).5
However, to the best of our knowledge, there has been no previous work on the
preparation of acyclic glutamic acid-based chelating agents related to GLDA in which
the donor groups pendent from the tertiary amine group are (i) different from one
another, and (ii) linked to the amine group via methylene spacers. Owing to our
interest in the development of biodegradable ligands that target both main group and
first-row transition metal ions, we sought to prepare such a chelating agent that
incorporates both aminocarboxylate and phenolate donors. We therefore chose the
phenolic derivative HBGl3 as a synthetic target in the pursuit of a strategy towards the
preparation of differentially N-substituted glutamic acid-based ligands; HBCl3 = (S)-
2-{(carboxymethyl)[(o-hydroxyphenyl)methyl]amino}glutaric acid.
In this
compound, introduction of the phenol may be expected to increase the selectivity of
the system for Fe3+ compared to GLDA, as well as adding a useful UV/Vis
spectroscopic handle.
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Fig. 2. Structures of the GLDA, HBGl3 and HBIDA ligands, with some potential
disconnection points towards HBGl3 shown. (HBIDA = hydroxybenzyliminodiacetic
acid).
2. Results and Discussion
2.1 Synthetic strategies
From the disconnections that would lead us back to L-glutamic acid as a starting
material (Fig. 2B), we first investigated the introduction of the amine as a separate
fragment along disconnection A, which could be reacted with an α-haloglutarate
alkylating agent. In this strategy, the use of separate amine fragments was considered
advantageous since the fragments could also be used in other syntheses.
As such, the racemic α-haloglutarate alkylating compound 1a was easily accessed
from glutamic acid via diazotisation,6 and could be subject to Finkelstein conditions
to afford the more reactive iodo-compound 1b (Scheme 1). So in order to evaluate
the feasibility of a route based on disconnection A, 1a was subjected to a test reaction
with dibenzylamine which gave 2 in modest yields. Buoyed by this result, we
prepared amine 3a via reductive amination, for subsequent reaction with 1a, but
observed no consumption of starting materials.
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Scheme 1. Intermediates used towards the preparation of HBGl3 via the use of an
α-haloglutarate electrophile
Using the corresponding benzyl ether of 3a, namely 3b, we were able to obtain key
intermediate 4 after a prolonged reaction period with the reactive electrophile 1b (6
days). At this point, the low yields and racemisation along the route from the
preparation of 1a, together with the need for multiple deprotection steps, prompted us
to abandon an approach based on disconnection A.
We then looked to reductive amination chemistry to introduce the salicylaldehyde unit
onto glutamic acid (disconnection B) via intermediate 5 (Scheme 2),7 since a route
along this disconnection would involve fewer steps and retain chirality. Although 5
was easily accessible due to it being isolable through a simple precipitation-filtration
purification procedure, N-alkylations of 5 attempted with either chloroacetic acid8 or
t-butyl bromoacetate under basic conditions (NaOH, KHCO3 or Et3N) in water,
acetonitrile or DMF were all unsuccessful, giving recovered starting material only.
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Scheme 2. Reactions of glutamic acid derivatives leading to cyclisation
We therefore opted to protect the carboxyl groups of 5, using a Fisher-type
esterification, since many reported ligand syntheses involving N-alkylation utilise
protected carboxyl intermediates to prevent the possible detrimental influence of the
carboxylate groups in subsequent reactions. In the first instance then, we chose to
convert the carboxyl groups to their ethyl esters using thionyl chloride, in the hope
that competing lactam formation would be suppressed by the protonation of the amine
group in 5 under these conditions. This proved not to be the case. Indeed, lactam 6a
was the sole product isolated. It may be noted that the cyclisation proceeded with the
preservation of the S-stereocentre of 5, as verified by single-crystal X-ray
crystallography (Fig. 3).
Fig. 3. Molecular structure in the crystals of 6a. The stereocentre at C4 is shown for
clarity. Thermal ellipsoids are shown at the 50% probability level.
We then looked to prepare 7, the di-isopropyl ester of L-glutamic acid as a reductive
amination substrate, in the hope that the bulkier isopropyl esters would be base-labile
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but also large enough to inhibit lactam formation. Unfortunately, cyclisation was also
observed using this approach, giving lactam 6b as the main product.
Finally, we turned our attention to intermediates bearing the bulky t-butyl ester
protecting group, which has shown the ability to prevent the attack of some strong
nucleophiles on a given carboxyl group,9 in the hope of preventing the cyclisation
seen earlier (Scheme 2). This change to t-butyl esters allowed HBGl3 to be obtained
according to Scheme 3.
Scheme 3 Successful preparation of HBGl3 via t-butyl ester intermediates
.
Di-t-butyl-glutamate, 8, is commercially available, and could be converted into
protected intermediate 10 using reductive amination in either a one-pot process using
NaBH(OAc)3,10 or a two-pot procedure via the isolated imine 9. In our hands, the use
of the two-pot procedure, using NaBH4 as the reducing agent, offered much better
reproducibility.
Alkylation of amine 10 with t-butyl bromoacetate to form HBGl3-tBu was slow (> 1
week for the disappearance of starting material by TLC), but afforded the compound
in reasonable yields. Unfortunately, attempts to shorten the reaction time (e.g. using
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microwave irradiation and high boiling solvents like DMF) failed, often leading to
complex mixtures even after repeated chromatographic purification.
HBGl3-tBu was then treated with a mixture of TFA/DCM in the presence of anisole
as a t-butyl cation scavenger to give HBGl3 in 15–18% cumulative yield, which is
fairly typical for the synthesis of polyaminocarboxylate ligands.11,12
The low reaction rate for the alkylation of 10 to HBGl3-tBu might be attributable
either to steric hindrance of the amine group in 10, or to strong intramolecular
hydrogen bonding, and to determine the root cause we sought to investigate both of
these possibilities. First, we investigated the role of sterics via the synthesis of the
less hindered, benzylated intermediate 11. The alkylation of 11 to form 12 proceeded
readily within a 24-hour period (Scheme 4), a much faster alkylation than that to form
HBGl3-tBu.
Scheme 4. Synthesis of the alkylation test substrate 11 and alternative HBGl3
precursor
We also speculated as to whether an intramolecular hydrogen bond in 10 could be a
contributing factor accounting for the slow rate of alkylation. The question was posed
on the basis of the single crystal X-ray structure of 9, which shows the presence of a
strong intramolecular O–H···N bond, d(H···N) = 1.723(3) Å, see Fig. 4. We
reasoned that the structure of 9 could help us predict the hydrogen bonding behaviour
of 10 because the hydroxybenzyl group is unchanged in going from 9 to 10 and
intramolecular hydrogen bonding in other hydroxybenzylamines is known.13,14 As a
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control, we therefore prepared a TBDMS-ether of 10, that is 10a (Scheme 3), which
would be unable to form an intramolecular hydrogen bond like that seen in 9.
Preliminary alkylation studies on 10a were then performed and showed very slow
conversion (10a was not fully consumed even after 7 days), indicating that the reason
behind the slow alkylation of 10 is likely to be the presence of the ortho-substituted
aromatic group hindering attack of an incoming electrophile as opposed to an
intramolecular hydrogen bond.
Fig. 4. Molecular structure in the crystal of 9. The N-H distance is shown in Å.
Thermal ellipsoids are shown at the 50% probability level
To assess whether introduction of the salicylaldehyde moiety further along the route
to HBGl3-tBu would give improved yields and reaction times, 12 was debenzylated
via catalytic hydrogenation to form tri-ester 13 (also accessible via 8 – see
Experimental Section), which was in turn subjected to reductive amination with
salicylaldehyde in attempts to form HBGl3-tBu. However these attempts were
unsuccessful and only starting materials were recovered.
2.2 Spectroscopic properties and pKa
With HBGl3 in hand, we were able to estimate the pKa values for the amine group and
the phenol as being around 8 and 13 respectively. The value for the amine (R2NH+
⇌R2NH) equilibrium was determined from the pH titration curve and could also be
followed by UV-Vis spectroscopy.
The largest bathochromic shift (15 nm) that occurs in the UV-Vis spectrum of HBGl3
as a function of increasing pH was used to determine the pKa of the phenolic group by
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taking the ratio of absorbances at 277 and 291 nm. This bathochromic shift is
assignable to the presence of smaller energy gaps between the molecular orbitals
involved in the transitions of interest, evidenced by TD-DFT studies at the B3LYP/ 6-
31G++ level of theory. Indeed, seen in Fig. 5C, there is a lower energy feature for the
calculated spectrum of the phenoxide form of HBGl3 that is not present in the
calculated spectrum of the phenolic form. For both the phenolic and phenoxide forms
of HBGl3, the molecular orbitals with the greatest contribution to the lowest energy
bands (λmax 277 and 291 nm for the phenolic and phenoxide forms respectively) are
the HOMO and LUMO+1.
It is also noteworthy that the localisation of the HOMOs in the phenolic and
phenoxide forms of HBGl3 are different: the phenolic form can be considered to have
predominantly n character whereas the phenoxide HOMO possesses π character. It is
clear that destabilisation of the HOMO, more so than the stabilisation of the
LUMO+1, is responsible for the reduced energy gap between these two orbitals in the
phenoxide form of HBGl3 (Fig. 5D).
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Fig. 5. (A) UV-Visible spectra for HBGl3 as a function of pH (T = 25oC, I = 0.1M
KCl). (B) Plot of the ratio of absorbance values at 291 and 277nm (A291/A277) as a
function of pH. (C) Comparison of the experimental spectra (solid lines) of the
phenolic (predominant at pH 10-12, black) and phenoxide (predominant at pH >13,
blue) forms of HBGl3 with those calculated (dashed lines) from TD-DFT calculations.
(D) Main orbitals involved in transitions, HOMO and LUMO+1 for the HBGl3 ligand
in the phenolic and phenoxide forms.
The amine dissociation event is also observed when spectra obtained between pH 5
and 11 are considered in isolation and an absorbance ratio plotted from the pH
titration data in (Fig. 5A). A sigmoid curve results with a midpoint at pH = 8.2,
which is in reasonable agreement with the value obtained from the potentiometric data
(Fig. 6B).
Fig. 6. (A) UV-Visible spectra (between pH 5 and 11) of HBGl3 (T = 25oC, I = 0.1M
KCl) used to confirm the amine pKa value obtained by pH-metric titration. (B) Plot of
the ratio of absorbance values at 267 and 273nm (A267/A273) as a function of pH
At a first glance the value for the amine pKa might appear rather low compared to
other amine-bearing ligands like IDA or NTA (iminodiacetic acid and nitrilotriacetic
acid respectively). Martell and others have reported similarly low pKa values for the
amino group of HBIDA (hydroxybenzyl-iminodiacetic acid) and related
ligands,15,16,17 and these values have usually been assigned to the formation of an
intramolecular hydrogen bond between the free amine and the un-ionised phenol.
This may also be the reason behind the relatively high phenol pKa observed for
HBGl3 compared to phenol itself, in that ionisation of the phenolic group to
phenoxide in HBGl3 is penalised by loss of this intramolecular hydrogen bond.
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3. Conclusions
A route has been devised to prepare the first acyclic glutamic acid based ligand that
bears different chelating groups attached to the nitrogen atom via a methylene bridge,
HBGl3. The ligand is prepared using stepwise functionalisation at the nitrogen of a
glutamic acid equivalent, as opposed to pre-forming an amine fragment for alkylation
onto an α-haloglutarate type intermediate. t-Butyl ester protection is employed to
prevent unwanted cyclisation reactions that occur when smaller ethyl or isopropyl
protecting groups are used to mask the glutamic carboxyl groups on the synthetic
intermediates. It is envisaged that the adoption of this route will facilitate the
straightforward synthesis of other glutamic acid based chelating ligands.
4. Experimental
Materials. Reagents were obtained from commercial sources and used without further
purification unless otherwise stated. Solvent extractions were performed in a 250 mL
separating funnel with ca. 50 mL for each phase, unless otherwise stated. For
procedures involving dry solvent, glassware was oven-dried for at least 8 h prior to
use. Dedicated oxygen-free nitrogen or argon cylinders (BOC, UK) were used to
provide an inert atmosphere.
Instrumentation. NMR: Routine 1H (400MHz) and 13C NMR (101MHz) spectra were
acquired on Bruker Avance 400, or Varian Mercury 400 NMR spectrometers. Two-
1
13
dimensional NMR (COSY, HSQC and HMBC), and certain H / C NMR spectra,
were acquired by the solution state NMR service at Durham University on Varian
VNMRS-600 (600 MHz) or VNMRS-700 (700 MHz) instruments. Where visible,
1
signals corresponding to CDCl3 were referenced to δ =7.26 ppm in H NMR spectra
and δ =77.2 ppm for 13C spectra.
Mass spectrometry. ES-MS data (positive and negative ionisation modes) were
obtained on a Waters TQD mass spectrometer interfaced with an Acquity UPLC
system. GC-MS (EI ionisation) was performed on an Agilent instrument equipped
with a 5973 model quadrupole mass spectrometer.
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FT-IR. All infra-red spectra were recorded on a Perkin-Elmer Spectrum 90
spectrometer equipped with an ATR stage. Substances for analysis were used neat
unless otherwise indicated.
Chromatography. TLC was performed on silica using Merck foil-backed TLC plates.
Column chromatography on silica was undertaken in one of two ways. Method i)
involved the use of a standard glass column and silica sourced from Fluorochem
Limited and elution was performed according to the method of Clark Still.18 Method
ii) involved the use of a Teledyne ISCO CombiFlash instrument equipped with
RediSep Rf silica cartridges to perform automated gradient elution. Analytical and
preparative HPLC was performed by the chromatography service at Durham
University.
Elemental Analysis. Elemental analyses were performed using an Exeter CE-440
Elemental Analyser device.
X-Ray crystallography. The X-ray single crystal data for compounds 6a and 9 have
been collected on a Bruker D8Venture (λCuKα radiation, λ = 1.54178 Å, Photon100
CMOS detector, IꢀS-microsource, focusing mirrors, ω- and φ-scan, 1.0°/frame) and
Bruker SMART CCD 6000 (λMoKα radiation, λ = 0.71073 Å, fine-focus sealed tube,
graphite monochromator, MonoCap optics, ω-scan, 0.3°/frame) diffractometers
equipped with a Cryostream (Oxford Cryosystems) open-flow nitrogen cryostats at
temperatures 120.0(2)K. All structures were solved by direct method and refined by
full-matrix least squares on F2 for all data using Olex219 and SHELXTL20 software.
All non-hydrogen atoms were refined anisotropically, hydrogen atoms in structures 3a
and 9 were refined isotropically, the hydrogen atoms in structure 6a were placed in
the calculated positions and refined in riding mode.
The compounds 1a, 5 and 7 were prepared using previously reported conditions, as
summarised in the Supporting Information.
Dimethyl 2-iodoglutarate, 1b
Sodium iodide (2.22 g, 13.4 mmol) was partially dissolved in acetone (12 mL) and
ester 4a (0.66 g, 2.76 mmol) was added at once forming a yellow-orange solution that
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was refluxed for 2 h. Volatiles were then removed in vacuo and the resulting brown
solid was triturated in diethyl ether and then filtered. Concentration of the filtrate in
vacuo afforded the product as a brown oil (0.59 g, 2.06 mmol, 75%). δH (400 MHz,
CDCl3) 4.46 (1H, dd, J 8.5, 6.0 Hz, H3), 3.75 (3H, s, H1), 3.68 (3H, s, H7), 2.47 – 2.43
(2H, m, H5), 2.29 – 2.25 (2H, m, H4); δC (101 MHz, CDCl3) 172.1 (C6), 171.3 (C2),
52.8 (C1), 51.7 (C7), 33.2 (C3), 30.8 (C5), 18.9 (C4); m/z (GC-EI+) 286.0 (M•+, M=
C7H11IO4), 256.0 (M–2Me•+).
Dimethyl 2-[bis(benzyl)amino]glutarate, 2
A solution of haloester 1a (400 mg, 1.69 mmol) in acetonitrile (4 mL, dried over
molecular sieves) was prepared and allowed to stir at room temperature under
nitrogen in a Schlenk tube prior to the addition of triethylamine (0.5 mL, 3.61 mmol)
at once followed by the dropwise addition of dibenzylamine over 15 minutes (0.39
mL, 2.03 mmol). The brown solution was heated at 60 oC for 40 h under nitrogen
leading to the formation of a precipitate that was filtered off after cooling to room
temperature. The filtrate was concentrated in vacuo and partitioned between diethyl
ether and water; the organic layer was washed with brine, dried over magnesium
sulphate and concentrated in vacuo prior to purification of the yellow residue via
column chromatography (method ii, dichloromethane:methanol 100:0 → 90:10) to
afford the product as a yellow oil (140 mg, 0.394 mmol, 23 %). δH (600 MHz, CDCl3)
7.37 – 7.27 (8H, m, H6, H7, H9, H10), 7.21 – 7.13 (2H, m, H8), 3.89 (2H, d, J 14.0 Hz,
H4), 3.77 (3H, s, H1), 3.54 (3H, s, H14), 3.52 (2H, d, J 14.0 Hz, H4), 3.38 – 3.33 (1H,
m, H3), 2.47 – 2.41 (1H, m, H11), 2.34 – 2.27 (1H, m, H11), 2.05 – 2.00 (2H, m, H12);
δC (151 MHz, CDCl3) 173.5 (C13), 172.9 (C2), 139.4 (C5), 129.1, 128.4, 127.2 (C6, C7,
C8, C9), 59.8 (C3), 54.6 (C14), 51.6 (C4), 51.3 (C1), 30.5 (C11), 24.5 (C12); m/z (ES-
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MS+) 356.2 (100 %, [M+H]+); HRMS (ES-MS+) MH+ found 356.1854, [C21H26NO4]+
requires 356.1862.
Methyl {[(o-hydroxyphenyl)methyl]amino}acetate, 3a
Glycine methyl ester hydrochloride (1.01 g, 8.01 mmol) was added to a solution of
triethylamine (1.2 mL, 8.60 mmol) in 1,2-dichloroethane (30 mL) followed by the
addition of salicylaldehyde (0.87 mL, 8.89 mmol). The reaction mixture was
subsequently stirred under nitrogen for 10 minutes prior to the addition of sodium
triacetoxyborohydride (2.10 g, 9.91 mmol) and monitored via TLC (90:10
dichloromethane:methanol) and ES-MS techniques until the absence of starting
material and derivative imines were observed (< 4 h). The mixture was then quenched
with aqueous, saturated potassium bicarbonate (30 mL) and the biphasic mixture was
stirred for a few minutes prior to extraction of the organic layer with dichloromethane
(2 × 30 mL). The combined extracts were dried with magnesium sulphate and
concentrated in vacuo to a yellow solid which was purified by column
chromatography (method ii, dichloromethane:methanol, compound eluted in 90:10
v/v dichloromethane:methanol) to give the title compound as a crystalline sold (0.36
g, 1.84 mmol, 23%). Slow evaporation of a sample from acetonitrile gave crystals
suitable for analysis by X-ray crystallography. δH (400 MHz, CDCl3) 7.22 – 6.75 (4H,
m, H7, H8, H9, H10), 4.00 (2H, s, H4), 3.76 (3H, s, H1), 3.45 (2H, s, H3); δC (101 MHz,
CDCl3) 172 (C2), 158 (C5), 130, 129, 122, 120, 117 (C6, C7, C8, C9, C10), 53 (C4),
52.5 (C1), 49 (C3); m/z (ES-MS+) 196 (100 %, [M+H]+);
Crystal data for 3a: C10H13NO3, M = 195.21, orthorhombic, space group P bca, a =
9.2513(8), b = 9.0842(8), c = 22.8517(19) Å, U = 1920.5(3) Å3, F(000) = 832, Z = 8,
Dc = 1.350 mg m-3, µ = 0.10 mm-1. 18663 reflections were collected (2Θ 3.56- 60°)
yielding 2799 unique data (Rmerg = 0.0546). Final wR2(F2) = 0.1146 for all data (179
refined parameters), conventional R1 (F) = 0.0404 for 1966 reflections with I ≥ 2σ,
GOF = 0.987. CCDC-1551895
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Methyl ({[o-(benzyloxy)phenyl]methyl}amino)acetate, 3b
o-(Benzyloxy)benzaldehyde (510 mg, 2.41 mmol) was dissolved in 1,2-
dichloroethane (10 mL) and methyl glycinate hydrochloride (330 mg, 2.63 mmol) was
suspended in the mixture prior to the addition of triethylamine (0.5 mL, 3.6 mmol)
and sodium triacetoxyborohydride (1.01 g, 4.77 mmol). The reaction mixture was
then stirred at room temperature under nitrogen for 17 h prior to the addition of a
solution of saturated sodium carbonate. Dichloromethane was then added to the
biphasic mixture and the organic layer extracted, dried over magnesium sulphate and
the solvent removed in vacuo to afford the title compound (0.50 g, 1.75 mmol, 74 %)
as a colourless oil. δH (700 MHz, CDCl3) 7.47 – 7.42 (2H, m, H14), 7.43 – 7.34 (2H,
m, H15), 7.36 – 7.28 (1H, m, H16), 7.28 (1H, dd, J 7.5, 2.0 Hz, H7), 7.22 (1H, dd, J 8.0,
2.0 Hz, H9), 6.98 – 6.88 (2H, m, H8, H10), 5.08 (2H, s, H12), 3.89 (2H, s, H5), 3.63
(3H, s, H1), 3.40 (2H, s, H3), 2.64 (1H, s, H4); δC (176 MHz, CDCl3) 172.7 (C2), 156.8
(C11), 137.1 (C13), 130.0 (C7), 128.6 (C15), 128.5 (C9), 127.9 (C16), 127.9 (C6), 127.2
(C14), 120.8 (C8), 111.8 (C10), 69.9 (C12), 51.6 (C1), 50.0 (C3), 48.5 (C5).
Dimethyl-2-({[o-(benzyloxy)phenyl]methyl}(methoxycarbonylmethyl)amino)
glutarate, 4
Alkyl iodide 4b (181 mg, 0.625 mmol) and amine 6a (170 mg, 0.596 mmol) were
dissolved in acetonitrile (3 mL, dried over molecular sieves) and potassium carbonate
o
(0.17 g, 1.23 mmol) added. The reaction mixture was then heated at 60 C under
nitrogen for 6 days. Once cooled to room temperature, solids were filtered off and the
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solvent removed from the filtrate in vacuo. The residue was then purified by column
chromatography (method ii, ethyl acetate:hexanes 0:100 → 100:0 compound eluted in
ca. 30:70 v/v ethyl acetate:hexanes) to afford the title compound (47.0 mg, 0.108
mmol, 18 %) as a colourless oil. νmax (ATR) 1730 (ester C=O st), 1200 (ester C–O st);
δH (600 MHz, CDCl3) 7.46 (1H, dd, J 7.5, 2.0 Hz H11), 7.42 (2H, dd, J 7.5, 1.5 Hz,
H19), 7.38 (2H, dd, J 7.5, 1.0 Hz, H18), 7.33 – 7.28 (1H, m, H20), 7.23 – 7.15 (1H, m,
H13), 6.95 (1H, dd, J 7.5, 1.0 Hz, H12), 6.89 (1H, dd, J 8.0, 1.0 Hz, H14), 5.07 (2H, s,
H16), 4.04 (1H, d, J 14.0 Hz, H9), 3.84 (1H, d, J 14.0 Hz, H9), 3.62 (3H, s) and 3.61
(3H, s, H8, H22), 3.59 (3H, s, H1), 3.56 (1H, s H6), 3.51-3.44 (2H, m, H5, H6), 2.65-
2.36 (2H, m, H3), 2.15-1.86 (2H, m, H4); δC (151 MHz, CDCl3) 173.9 (C2), 173.3,
172.3 (C7, C21), 157.1 (C15), 137.7 (C17), 130.8 (C11), 128.6 (C18), 128.4 (C13), 127.9
(C20), 127.3 (C19), 127.2 (C10), 120.9 (C12), 111.9 (C14), 70.1 (C16), 62.1 (C5), 51.9
(C6), 51.5 (C8, C22), 51.5 (C1), 51.3 (C8, C22), 50.0 (C9), 30.3 (C3), 25.0 (C4); m/z (ES-
MS+) 444.5 (100 %, [M+H]+); HRMS (ES-MS+) MH+ found 444.2026, [C24H30NO7]+
requires 444.2022.
Ethyl (2S)-1-[(o-hydroxyphenyl)methyl]-5-oxo-2-pyrrolidinecarboxylate, 6a
Amino acid 5 (420 mg, 1.66 mmol) was suspended in ethanol (7 mL) and thionyl
chloride (0.6 mL, 8.27 mmol) was added dropwise. The suspension was then refluxed
for 48 h under argon, cooled and then filtered. The filtrate was then concentrated in
vacuo to give a white solid that was triturated with ethanol using ultrasound (3 × 5
mL), and then diethyl ether (3 × 5 mL), the solvent being removed in vacuo between
cycles. Dichloromethane was used to suspend the resulting solid which was filtered
through Celite® and the solvent removed from the pink filtrate to afford the title
compound (270 mg, 1.03 mmol, 62 %) as a pink crystalline solid. Single crystals
suitable for structural and stereochemical determination via X-ray crystallography
o
were grown from this solid via slow evaporation from acetone. mp 111–112 C; νmax
(ATR) 3069 (br, phenol O–H st), 2978 (sp3 C–H st), 1739 (ester C=O st), 1660
16

ACCEPTED MANUSCRIPT
(amide C=O st), 1203 (C–O st); δH (600 MHz, CDCl3) 8.76 (1H, s, H8), 7.22 (1H, dd,
J 7.5, 1.5 Hz, H5), 7.02 (1H, dd, J 7.5, 1.5 Hz, H3), 6.94 (1H, d J 7.5 Hz, H6), 6.79
(1H, dd, J 7.5, 1.5 Hz, H4), 4.64 (1H, d, J 15.0 Hz, H10), 4.26 – 4.28 (1H, m, H13),
4.18–4.11 (m, 2H, H13, H14), 4.06 (1H, dq, J 11.0, 7.0 Hz, H14), 2.65 – 2.52 (1H, m,
H1), 2.49 – 2.37 (1H, m, H1), 2.35 – 2.25 (1H, m, H12), 2.17 – 2.06 (1H, m, H12), 1.22
(3H, t, J 7.0 Hz, H15); δC (151 MHz, CDCl3) 177.5 (C2), 171.3 (C13), 155.9 (C7), 131.0
(C3), 130.3 (C5), 121.3 (C9), 119.6 (C4) 117.8 (C6), 61.8 (C14), 60.4 (C11), 43.0 (C10),
29.2 (C1), 23.0 (C12),14.0 (C15); m/z (ES-MS+) 264.0 (100%, [M+H]+); HRMS (ES-
MS+) MH+ found 264.1243, [C14H18O4]+ requires 264.1236;
Crystal data for 6a: C14H17NO4, M = 263.29, triclinic, space group P 1, a = 6.8353(3),
b = 7.5669(3), c = 7.7552(3) Å, α = 96.5965(19), β = 112.7236(19); γ = 106.441(2)°,
U = 343.27(2) Å3, F(000) = 140, Z = 1, Dc = 1.274 mg m-3, µ = 0.775 mm-1. 2382
reflections were collected (2Θ 12.6 - 139.9°) yielding 1722 unique data (Rmerg
=
0.0554). Final wR2(F2) = 0.2218 for all data (174 refined parameters), conventional
R1 (F) = 0.0767 for 1666 reflections with I ≥ 2σ, GOF = 1.086, Hooft -0.1(3). CCDC-
1551896.
Isopropyl 1-[(o-hydroxyphenyl)methyl]-5-oxo-2-pyrrolidinecarboxylate, 6b
Diisopropyl glutamate hydrochloride 7 (610 mg, 2.28 mmol ) and salicylaldehyde
(280 mg, 2.28 mmol) were dissolved in methanol (6 mL) and stirred under nitrogen at
o
40 C for 1 h, and then at room temperature overnight. Then, sodium borohydride
(410 g, 10.9 mmol) was then added portionwise and the mixture stirred at room
temperature for 6 h. Solids were then filtered off and the filtrate concentrated in vacuo
prior to re-dissolution in dichloromethane and the removal of precipitates via
filtration. The filtrate was then washed with aqueous hydrochloric acid, extracted,
dried using magnesium sulphate and the solvent removed in vacuo to provide an oil
that was purified via column chromatography (method ii, ethyl acetate:hexanes
compound eluted in 30:70 v/v ethyl acetate:hexanes) to afford the title compound
17

ACCEPTED MANUSCRIPT
(87.0 mg, 0.31 mmol, 11%) as a pink oil. νmax (ATR) 3182 (br, phenol O–H st), 1735
(ester C=O st), 1660 (amide C=O st), 1103 (C–O st); δH (700 MHz, CDCl3) 8.79 (1H,
s, H8), 7.25 – 7.18 (1H, m, H5), 7.04 – 6.97 (1H, m, H3), 6.96 – 6.91 (1H, m, H6),
6.83 – 6.77 (1H, m, H4), 5.05 – 4.97 (1H, m, J 6.5 Hz, H14), 4.68 (1H, d, J 15.0 Hz,
H10), 4.17 (1H, dd, J 9.3, 3.3 Hz, H11), 4.10 (1H, d, J 15.0 Hz, H10), 2.65 – 2.54 (1H,
m, H1), 2.43 – 2.36 (1H, m, H1), 2.36 – 2.38 (1H, m, H12), 2.13 – 2.03 (1H, m, H12),
1.28 (3H, d, J 6.5 Hz, H15), 1.16 (3H, d, J 6.5 Hz, H15); δC (176 MHz, CDCl3) 178
(C2), 171 (C13), 156 (C7), 131 (C3), 130.5 (C5), 121 (C9), 120 (C4), 118 (C6), 70 (C14),
61 (C11), 43 (C10), 29 (C1), 23 (C12), 22 (C15), 21.6 (C15); m/z (ES-MS+) 278.0 (100%,
[M+H]+); HRMS (ES-MS+) MH+found 278.1412, [C15H20NO4]+ requires 278.1392.
Di-tert-butyl (2S)-2-{[(o-hydroxyphenyl)methyl]amino}glutarate, 10
One-pot method:
l-Glutamic acid di-tert butyl ester hydrochloride, 8 (500 mg, 1.69 mmol) was
suspended in 1,2-dichloroethane (10 mL) and triethylamine (0.26 mL, 1.86 mmol)
was added to the stirred mixture under nitrogen prior to the addition of
salicylaldehyde (0.175 mL, 1.65 mmol). The resulting pale yellow suspension was
stirred under nitrogen for 20 minutes and sodium triacetoxyborohydride (780 mg,
3.68 mmol) was added. After 3 h of stirring at room temperature, a further equivalent
of sodium triacetoxyborohydride (800 mg, 3.77 mmol) was added and the reaction
mixture was allowed to stir for a further 36 h under nitrogen, at which point the
reaction was judged to be complete by TLC. A saturated aqueous potassium
bicarbonate solution (20 mL) was then used to quench the reaction mixture, and the
resulting mixture extracted into dichloromethane (3 × 50 mL). The organic extracts
were recombined and washed with brine prior to solvent removal in vacuo.
Purification of the resulting oil via column chromatography (silica, 0 → 20% ethyl
acetate:hexanes) led to the isolation of the title molecule 10 as a colourless oil (450
mg, 1.23 mmol, 75%).
18

ACCEPTED MANUSCRIPT
Via imine 9:
l-Glutamic acid di-tert butyl ester hydrochloride, 8 (1.00 g, 3.38 mmol) was
suspended in chloroform (10 mL) and triethylamine (0.94 mL, 6.74 mmol) added.
The reaction mixture was then heated at 50 °C for 90 min and then transferred into a
separating funnel and washed with water and then brine. The yellow organic layer
was then dried over magnesium sulphate and concentrated to afford 9 as a yellow
solid (1.10 g, 3.03 mmol, 89%). Recrystallisation of a sample of 9 from hot ethanol
gave single crystals suitable for analysis via X-ray crystallography. Crystal data for
9: C14H17NO4, M = 363.44, orthorhombic , space group P212121, a = 5.8887(2), b =
10.4967(4), c = 32.3124(13) Å, U = 1997.3(1) Å3, F(000) = 784, Z = 4, Dc = 1.209
mg m-3, µ = 0.703 mm-1. 22828 reflections were collected (2Θ 5.47 – 141.89°)
yielding 3816 unique data (Rmerg = 0.0286). Final wR2(F2) = 0.0628 for all data (351
refined parameters), conventional R1 (F) = 0.0247 for 3752 reflections with I ≥ 2σ,
GOF = 1.092, Hooft = 0.05(4). CCDC-1551897.
Imine 9 (1.10 g, 3.03 mmol) was then dissolved in methanol (12 mL) and sodium
borohydride (230 mg, 6.05 mmol) was added at once CAUTION- gas evolution. The
reaction mixture was stirred at room temperature for 4 h, after which the solvent was
removed in vacuo and the residue taken up in chloroform (50 mL) and washed in
saturated ammonium chloride (50 mL) to give an emulsion. The emulsion was
extracted, dried over magnesium sulphate and concentrated in vacuo.
Dichloromethane (50 mL) was added to the residue, which was then washed in brine
(50 mL). The organic layer was extracted, dried over magnesium sulphate and
concentrated in vacuo to afford 10 (775 mg, 2.11 mmol, 70%) as a yellow oil. [ꢀ]ꢂꢁꢃ=
–6.4 (c = 0.28, DCM); νmax (ATR) 2976 (sp3 C–H), 1724 (ester C=O st), 1149 (ester
C–O st); δH (700 MHz, CDCl3) 7.18 (1H, dd, J 8.0, 1.5 Hz, H8), 6.96 (1H, d, J 7.5, 1.5
Hz, H10), 6.85 (1H, dd, J 8.0, 1.0 Hz, H7), 6.78 (1H, dd, J 7.5, 1.0 Hz, H9), 4.04 (1H,
d, J 13.5 Hz, H5), 3.74 (1H, d, J 13.5 Hz, H5), 3.24 (1H, dd, J 6.0, 1.5 Hz H4), 2.34 –
2.28 (2H, m, H12), 2.03 – 1.91 (1H, m, H13), 1.92 – 1.81 (1H, m, H13), 1.50 (9H, s,
H1), 1.42 (9H, s, H16); δC (176 MHz, CDCl3) 173.0 (C3), 172.0 (C14), 157.9 (C6),
129.2 (C8), 128.8 (C10), 122.3 (C11), 119.4 (C8), 116.7 (C7), 82.3 (C2), 80.9 (C15), 60.0
(C4), 51.1 (C5), 31.9 (C13), 28.5 (C12), 28.3 (C1), 28.2 (C16); m/z (ES-MS–) 366.2 (100
%, [M–H]–); HRMS MH– found 366.2288, [C20H32NO5]– requires 366.2280.
19

ACCEPTED MANUSCRIPT
Di-tert-butyl (2S)-2-{[(o-hydroxyphenyl)methyl] (tert-butoxycarbonylmethyl)
amino}glutarate, HBGl3-tBu
Aminoester 10, (775 mg, 2.11 mmol) was dissolved in acetonitrile (10 mL, dried over
molecular sieves) and potassium hydrogen carbonate (0.43 g, 4.30 mmol) was
suspended in the stirred solution. A catalytic amount of potassium iodide (35.0 mg,
0.211 mmol) was then added followed by t-butyl bromoacetate (0.34 mL, 3.00 mmol).
The mixture was then heated to 55°C under argon for 12 days. Upon cooling to room
temperature, the reaction mixture was filtered, concentrated in vacuo and the target
compound isolated by column chromatography (ethyl acetate:hexanes 0:100 → 30:70
compound eluted in ca. 15:85 v/v ethyl acetate:hexanes) as a colourless oil (470 mg,
0.981 mmol, 46%). [ꢀ]ꢂꢁꢃ= –46.7 (c = 0.094, DCM); νmax (ATR) 3340 (O–H st), 2977
(sp3 C–H st), 1722 (ester C=O st); 1137 (C–O st); δH (700 MHz, CDCl3) 9.12 (1H, s
H16), 7.17 (1H, dd, J 7.5, 2.0 Hz, H13), 6.96 (1H, dd, J 7.5, 2.0 Hz, H11), 6.86 (1H, dd,
J 7.5, 1.0 Hz, H14), 6.75 (1H, ddd, J 7.5, 1.0 Hz H12), 3.91 (1H, d, J 13.5 Hz, H9), 3.73
(1H, d, J 13.5 Hz, H9), 3.59 (1H , d, J 18.0 Hz, H5), 3.26 (1H, d, J 18.0 Hz, H5), 3.22
(1H, dd, J 9.0, 6.0 Hz, H4), 2.37 – 2.19 (1H, m, H18), 2.17 – 2.02 (1H, m, H18), 2.02 –
1.84 (1H, m, H17), 1.84 – 1.68 (1H, m, H17), 1.48 (9H, s, H21), 1.47 (9H, s, H8), 1.35
(9H, s H1); δC (176 MHz, CDCl3) 172.3 (C19), 172.0 (C6), 171.2 (C3), 157.3 (C15),
130.2 (C11), 129.5 (C13), 121.6 (C10), 119.3 (C12), 116.6 (C14), 82.4 (C20), 81.8 (C7),
80.4 (C2), 60.5 (C4), 55.9 (C9), 51.5 (C5), 31.8 (C18), 28.3 (C21), 28.1 (C8), 28.0 (C1),
24.7 (C18); m/z (ES-MS+) 480.5 (100 %, [M+H]+); HRMS (ES-MS+) MH+ found
480.2948, [C26H42NO7]+ requires 480.2961.
20

ACCEPTED MANUSCRIPT
(S)-2-{(Carboxymethyl)[(o-hydroxyphenyl)methyl]amino}glutaric acid, HBGl3
HBGl3-tBu (110 mg, 0.229 mmol) was dissolved in dichloromethane (1 mL) and
anisole (0.05 mL, 0.462 mmol) was added, followed by trifluoroacetic acid (1 mL).
The mixture was stirred at room temperature for 24 h and the solvent removed in
vacuo. The residue was then taken up in dichloromethane (ca. 5 mL) and the solvent
was then removed in vacuo. The residue was triturated with hexane (ca. 25 mL) using
ultrasonic agitation to afford a white precipitate that was collected via centrifugation
(4000 rpm, 5 minutes). This solid was then dissolved in aqueous hydrochloric acid (1
M, 10 mL), transferred to a round bottom flask and the solvent removed in vacuo to
give HBGl3 as an off-white solid (37.9 mg, 0.122 mmol, 53%). [ꢀ]ꢂꢁꢃ= –14.3
(c = 0.028, MeOH); tR (H2O:MeOH, 95:5 H2O:MeOH to 5:95 H2O:MeOH = 5 mins,
MS detection); 1.8 min; νmax (ATR) 2960 (O–H st), 1730 (acid C=O st), 1220 (acid
C–O st); δH (700 MHz, D2O, pD ~ 2); 7.40 (1H, dd, J 8.0, 1.5 Hz, H12), 7.36 (1H, dd,
J 8.0, 1.5 Hz, H14), 7.00 (1H, dd, J 8.0, 1.5 Hz, H13), 6.98 (1H, dd, J 8.0, 1.5 Hz, H11),
4.61 (1H, d, J 13.0 Hz, H8), 4.51 (1H, d, J 13.0 Hz, H8), 4.11 (1H, t, J 7.0 Hz, H4),
4.09 (2H, s, H2), 2.81 – 2.69 (1H, m, H6), 2.67 – 2.61 (1H, m, H6), 2.35 – 2.23 (2H,
m, H5); δC (176 MHz, D2O, pD ~ 2) 176.1 (C7), 170.7 (C3), 169.4 (C1), 155.4 (C10),
132.5 (C14), 132.2 (C12), 120.7 (C9), 115.6 (C13), 115.5 (C11), 64.4 (C4), 54.7 (C8),
52.6 (C2), 30.2 (C6), 21.25 (C5); m/z (ES-MS+) 312.1 (100 %, [M+H]+); HRMS (ES-
MS+) MH+ found 312.1079, [C14H18NO7]+ requires 312.1083.
Di-tert-butyl (2S)-2-{[(o-tertbutyldimethylsiloxyphenyl)methyl]amino}glutarate,
10a
21

ACCEPTED MANUSCRIPT
Aminoester 10 (0.283 g, 0.775 mmol) was dissolved in anhydrous dichloromethane (4
mL) and cooled in an ice bath prior to the addition of imidazole (160 mg, 2.35 mmol)
and TBDMSCl (150 mg, 0.995 mmol) in rapid succession. The ice bath was then
removed and the reaction mixture allowed to stir for 6 h at room temperature. The
reaction mixture was then poured into a separating funnel and washed with water, and
the organic layer was then extracted, dried over magnesium sulphate. Purification of
the residue via column chromatography (method i, diethyl ether:hexanes 0:100 →
20:80 compound eluted in ca. 20:80 v/v diethyl ether:hexanes) gave 10a as a
colourless oil (190 mg, 0.396 mmol, 51 %). δH (600 MHz, CDCl3) 7.33 (1H, d, J 7.5,
1.5 Hz, H11), 7.09 (1H, ddd, J 7.5, 1.5 Hz, H9), 6.90 (1H, ddd, J 7.5, 1.0 Hz H10), 6.76
(1H, dd, J 7.5, 1.0 Hz, H8), 3.78 (1H, d, J 14.0 Hz, H6), 3.65 (1H, d, J 14.0 Hz, H6),
3.21 – 3.05 (1H, m, H4), 2.41 – 2.31 (2H, m, H17), 1.95 – 1.77 (3H, m, H5, H16), 1.44
(9H, s, H1), 1.42 (9H, s, H20), 1.00 (9H, s, H15), 0.23 (6H, s, H13); δC (151 MHz,
CDCl3) 174.4 (C3), 172.7 (C18), 153.6 (C12), 130.8 (C7), 129.4 (C11), 127.7 (C9), 121.1
(C10), 118.5 (C8), 81.0 (C2), 80.2 (C19), 61.0 (C4), 47.1 (C6), 32.1 (C17), 28.9 (C16),
28.2 (C1), 25.9 (C20), 18.2 (C15), –4.05 (C13, C14) ; m/z (ES-MS+) 480.1 (100 %,
[M+H]+); HRMS (ES-MS+) MH+ found 480.3143, [C26H46NO5Si]+ requires 480.3145.
Di-tert-butyl (2S)-2-benzylaminoglutarate, hydrochloride salt, 11
l-Glutamic acid di-tert butyl ester hydrochloride, 8 (500 mg, 1.69 mmol) and
triethylamine (0.28 mL, 3.82 mmol) were added to methanol (8 mL), resulting in the
formation a white precipitate. Benzaldehyde (0.18 mL, 1.78 mmol) was then added
and the mixture stirred under nitrogen for 2.5 h. Sodium borohydride (230 mg, 6.07
mmol) was subsequently added portionwise CAUTION- gas evolution and the
mixture was allowed to stir under nitrogen for a further 3 h. Volatiles were then
removed in vacuo and the residue partitioned between dichloromethane and aqueous
hydrochloric acid (1 mol dm-3) and the organic layer extracted, washed with brine,
dried with magnesium sulphate and the solvent removed in vacuo. The resulting white
22

ACCEPTED MANUSCRIPT
solid was triturated in hexane to afford the title compound (290 mg, 0.751 mmol
(based on molecular weight of the monohydrochloride salt), 45 %) as a white powder.
mp 140-142 oC; νmax (ATR); 2982 (sp3 C–H st), 2631 (N+–H st), 1725 (ester C=O st),
1149 (C–O st); δH (600 MHz, CDCl3) 10.81 (1H, s, H5), 9.84 (1H, s, H5), 7.63 (2H, d,
J 7.0 Hz, H8), 7.42 – 7.31 (3H, m, H9 ,H10), 4.24 (2H, dd, J 22.0, 11.5 Hz, H6), 3.62
(1H, s, H4), 2.63 – 2.22 (4H, m, H11, H12), 1.51 (9H, s, H15), 1.35 (9H, s, H1); δC (151
MHz, CDCl3) 171.3 (C13), 166.7 (C3), 131.0 (C8), 130.0 (C7), 129.7, 129.3 (C9, C10),
84.9 (C14), 81.3 (C2), 57.5 (C4), 49.7 (C6), 31.3, 28.2 (C15), 28.1 (C1) 25.2 (C11, C12);
m/z (ES-MS+) 350.2 (100 %, [M+H]+); HRMS (ES-MS+) MH+ found 350.2330 ,
[C20H32NO4]+ requires 350.2331.
Di-tert-butyl (2S)-2-[(1R)-(benzyl)(tert-butoxycarbonylmethyl)amino]glutarate,
12
Aminoester 11 (400 mg, 1.04 mmol) was added to acetonitrile (3.5 mL, Aldrich
anhydrous) and potassium carbonate (570 mg, 4.13 mmol), potassium iodide (30.0
mg, 0.181 mmol) salts were added along with t-butyl bromoacetate (0.23 mL, 1.57
mmol) and the mixture was heated at 60 oC under nitrogen. TLC (ethyl
acetate:hexanes 20:80 v/v) indicated that consumption of starting material was
achieved within 6 h, but heating was maintained for a further 16 h prior to cooling to
room temperature and removal of solids by filtration. The filtrate was concentrated in
vacuo and subjected to column chromatography (method ii, diethyl ether:hexanes
0:100 → 40:60 compound eluted in ca. 15:85 v/v diethyl ether:hexanes) to afford the
title compound (240 mg, 0.518 mmol, 50 %) as a colourless oil. νmax (ATR); 2974
(sp3 C–H st), 1719 (ester C=O st), 1137 (ester C–O st); δH (700 MHz, CDCl3) ; 7.40-
7.22 (5H, m, H11, H12, H13) , 3.95 (1H, d, J 13.5 Hz, H5), 3.74 (1H, d, J 13.5 Hz, H5),
3.43 (1H, d, J 17.0 Hz, H9), 3.34 – 3.20 (2H, m, H9, H4 ), 2.48 (1H, ddd, J 16.5, 9.5,
6.0 Hz, H15), 2.26 (1H, ddd, J 16.5, 9.5, 6.0 Hz, H15), 1.96 – 1.85 (2H, m, H14), 1.50
23

ACCEPTED MANUSCRIPT
(9H, s, H8), 1.43 (9H, s, H18), 1.40 (9H, s, H1); δC (176 MHz, CDCl3); 172.9 (C16),
171.9 (C6), 170.9 (C3), 129.3, 128.4, 127.4 (C10, C11, C12, C13), 81.5 (C17), 80.9 (C7),
80.2 (C2), 62.4 (C4), 56.7 (C5), 52.7 (C9), 32.0 (C15), 28.5 (C18), 28.3 (C8), 28.2 (C1),
25.2 (C14); m/z (ES-MS+) 464.3 (100 %, [M+H]+); HRMS (ES-MS+) MH+ found
464.3009, [C26H42NO6]+ requires 464.3012.
Di-tert-butyl (2S)-2-[(tert-butoxycarbonylmethyl)amino]glutarate, 13
Via intermediate 12:
Ethanol (25 mL) was used to dissolve 12 (80.0 mg, 0.172 mmol) and the colourless
solution was evacuated and back-filled with nitrogen 3 times prior to the addition of
5% palladium on charcoal (7 mg, 3 ꢀmol with respect to Pd). Once the catalyst was
added, the reaction mixture was evacuated and back-filled with hydrogen gas from a
balloon three times prior to stirring at room temperature. Reaction progress was
1
monitored by the use of H NMR which indicated that debenzylation was achieved
within 4 h. The reaction mixture was subsequently allowed to stand for a few minutes
under a flow of nitrogen prior to catalyst removal via filtration over Celite®.
Concentration of the filtrate in vacuo, followed by re-dissolution of the residue in
chloroform and a second solvent removal in vacuo led to isolation of the title
molecule (51 mg, 0.144 mmol, 84 %) as a colourless oil.
Direct synthesis from commercially available starting materials:
l-Glutamic acid di-tert butyl ester hydrochloride, 8 (1.50 g, 5.07 mmol) was stirred at
room temperature in chloroform under argon in the presence of activated zinc dust
(3.00 g, 46.1 mmol) for 1 h. The zinc dust was then filtered off, and the solvent
removed from the filtrate in vacuo to afford the free base. The resulting oil was
dissolved in acetonitrile (28 mL, dried over molecular sieves). When the solution was
nearly homogeneous, anhydrous triethylamine (1.21 mL, 8.68 mmol) was added at
once, followed by the dropwise addition of t-butyl bromoacetate (0.89 mL, 6.07
mmol). Finally, a catalytic amount of potassium iodide (ca. 0.1 g) was added and the
resulting mixture stirred at room temperature under argon for 24 h. After the solvent
24

ACCEPTED MANUSCRIPT
was removed in vacuo, the oily residue was partitioned between water and
dichloromethane (50 mL of each) and the combined organic extracts were
subsequently washed in brine prior to being dried with magnesium sulphate and the
solvent being removed in vacuo. The resulting oil was dissolved in the minimum
volume of dichloromethane and subjected to column chromatography (method i,
silica, ethyl acetate:hexanes 30:70 → 60:40) to afford the pure compound (0.44 g,
1.18 mmol, 23 %) as a colourless oil.
νmax (ATR) 2970 (N–H st), 2930 (sp3 C–H), 1731 (ester C=O st), 1149 (ester C–O st);
δH (700 MHz, CDCl3) 3.29 (1H, d, J 17.0 Hz, H6), 3.22 (1H, d, J 17.0 Hz, H6), 3.14
(1H, dd, J 7.5, 6.0 Hz, H4), 2.34 (2H, t, J 7.5 Hz, H11), 1.92 – 1.89 (2H, m, H5, H10),
1.88-1.76 (1H, m, H10), 1.46 (9H, s, H1), 1.44 (9H, s, H14), 1.43 (9H, s, H9); δC (176
MHz, CDCl3) 173.8 (C3), 172.8 (C12), 171.3 (C7), 81.7 (C2), 81.5 (C13), 80.5 (C8),
60.8 (C4), 50.2 (C6), 32.2 (C11), 28.6 (C10), 28.4 (C1, C9, C14); m/z (ES-MS+) 374.2
(100 %, [M+H]+); HRMS (ES-MS+) MH+ found 374.2538, [C19H36NO6]+ requires
374.2543.
5. Acknowledgements
We thank P&G and Durham University for support of this work.
6. Notes and references
Crystallographic data for the structures in this paper have been deposited with the
Cambridge Crystallographic Data Centre as supplementary publication numbers
15518951 – 1551897.
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