The synthesis of enantiopure α-fluoro and α,α-difluoro- β3-arginine derivatives

Article abstract of DOI:10.1071/CH13590

The synthesis of a set of monofluorinated, difluorinated, and non-fluorinated N-acetylated-β3-arginine esters, potential inhibitors of trypsin-like proteases, is described. Elaboration to the target compounds from previously reported enantiopure precursors derived from 3-hydroxypropanal involved 6-7 steps and was achieved in 48-65% overall yield. The α,α-difluoro-β3-arginine derivative was found to be particularly prone to hydrolysis. Three β3-arginine derivatives were tested for their ability to inhibit trypsin, the α,α-difluoro compound being assayed in the form of a carboxylate zwitterion. CSIRO 2014.

Full text of DOI:10.1071/CH13590

RESEARCH FRONT
CSIRO PUBLISHING
Aust. J. Chem. 2014, 67, 997–1004
Full Paper
http://dx.doi.org/10.1071/CH13590
The Synthesis of Enantiopure a-Fluoro and
a,a-Difluoro-b3-Arginine Derivatives
A
B
B
Taryn L. March, Jamie A. Freemont, Geoff Dumsday,
A
A B C
, ,
Martin R. Johnston, and Peter J. Duggan
A
School of Chemical and Physical Sciences, Flinders University,
Adelaide, SA 5042, Australia.
B
CSIRO Materials Science and Engineering, Bag 10, Clayton South, Vic. 3169, Australia.
C
Corresponding author. Email: peter.duggan@csiro.au
The synthesis of a set of monofluorinated, difluorinated, and non-fluorinated N-acetylated-b3-arginine esters, potential
inhibitors of trypsin-like proteases, is described. Elaboration to the target compounds from previously reported
enantiopure precursors derived from 3-hydroxypropanal involved 6–7 steps and was achieved in 48–65 % overall yield.
The a,a-difluoro-b3-arginine derivative was found to be particularly prone to hydrolysis. Three b3-arginine derivatives
were tested for their ability to inhibit trypsin, the a,a-difluoro compound being assayed in the form of a carboxylate
zwitterion.
Manuscript received: 31 October 2013.
Manuscript accepted: 3 December 2013.
Published online: 20 January 2014.
Introduction
measure of complexity. We recently published reports describ-
ing tandem conjugate addition–fluorination[17] and new sono-
catalysed Reformatsky[18] methodologies for the preparation of
a-fluoro- and a,a-difluoro-b3-amino acids, respectively, that
are compatible with more complex side chains. As these
methods do not rely on natural amino acids as precursors, they
allow the synthesis of fluorinated b-amino acids with a wide
range of natural and non-natural side chains. Conveniently, both
methods utilise the same achiral aldehyde as starting material,
which can be varied in order to incorporate the desired b3-amino
acid side chain.
a-Fluorinated b-amino acids are an emerging class of amino
acids that are attracting attention because of their biological
activity and their interesting conformational properties. The
various stereoelectronic effects that influence the structure of
a-fluorinated b-amino acid derivatives and peptides containing
this motif have recently been reviewed in detail.[1] Non-peptidic
derivatives of a-fluorinated b-amino acids have also been
investigated for their anti-cancer,[2] anti-fungal,[3] and anti-
insomnia effects,[4] and their ability to inhibit proteases[5] and
2,3-aminomutase[6] has also been examined. These enzymatic
studies are generally performed with either aliphatic or aromatic
a-fluorinated b-amino acids, with trifunctional analogues that
possess functional groups on the amino acid side chain only
rarely reported in the literature. This is partly because the ste-
reoselective synthesis of a-fluorinated b-amino acids bearing
substituted side chains has a much greater degree of difficulty
relative to the synthesis of similar compounds with purely
hydrocarbon side chains.[1] As a result, further investigations
into their inhibitory properties is hindered by a lack of adequate
methods for the preparation of these more highly functionalised
substrates.
Within the class of fluorinated b-amino acids, over the past
decade, a,a-difluoro-b3-amino acids have attracted by far the
most attention from the synthetic community.[1,7,8] This is
presumably because the achiral gem-difluoromethylene moiety
is easily introduced by several building block methods.[1,9–12]
Most of these approaches do, however, focus solely on simple
aromatic or aliphatic compounds. In contrast, there have been
few literature reports describing the stereoselective synthesis
of the monofluorinated analogues, the a-fluoro-b3-amino
acids,[1,13–16] whose additional stereocentre introduces an extra
With the ability of a-fluorinated b3-phenylalanine deriva-
tives (i.e. compounds 1–3, Fig. 1) to inhibit the serine protease a-
chymotrypsin established,[5b,c] it was of interest to learn if the
same functionality would also inhibit serine proteases that
cleave proteins adjacent to basic side chains such as lysine
and arginine. Important enzymes in this class, known as trypsin-
like proteases, are involved in digestion, fertilisation, and blood
coagulation. This investigation would require the synthesis of
trifunctional a-fluorinated b3-amino acids 6 and 7, as well as the
non-fluorinated analogue 5 as a control (Fig. 2). These acet-
amido-methyl esters are direct structural and stereochemical
analogues of those tested with a-chymotrypsin.
The elaboration of an enantiopure a-fluorinated b3-amino
acid derivative, itself obtained from the tandem conjugate
addition–fluorination method, to
a protected form of
a-fluorinated b3-lysine was recently demonstrated,[17] and it
was envisaged that similar methods could be used to prepare the
a-fluorinated b3-arginine 6. The preparation of the correspond-
ing difluoride 7, however, presents a more interesting synthetic
challenge because of the influence of the geminal difluoro
substituents on the compound’s reactivity. The enhancement
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www.publish.csiro.au/journals/ajc

998
T. L. March et al.
O
O
O
fluoride (TBAF) at 408C to give 10a and 10b, after which the
free hydroxy groups of these compounds were transformed to
protected guanidines under Mitsunobu conditions. The use of
di-carboxybenzyl (Cbz) protected guanidine as the guanidiny-
lating agent ensured that the intermediates could be easily
handled during subsequent synthetic steps. The non-fluorinated
b3-arginine derivative 11a was obtained in an excellent 86 %
yield while the yield of a-fluorinated 11b was slightly lower
(68 %). The unusual Cbz substitution pattern of the guanidine
moiety was established by 2D HMBC NMR experiments that
showed a correlation between the d-carbon protons and only one
of the two Cbz groups, an observation supported by other
literature reports.[19,20] Trifluoracetic acid (TFA)-mediated
hydrolysis of the tert-butyl ester proceeded cleanly in each case
without concomitant cleavage of the Cbz groups, and the result-
ing carboxylic acids were then esterified in dry methanolic HCl –
generated by addition of thionyl chloride to dry MeOH – to give
the methyl esters 12a and 12b. The last step in the sequence
involved removal of the two Cbz groups, which was accom-
plished in quantitative yield under standard hydrogenolysis
conditions to furnish the target b3-arginine derivatives 5 and 6.
The synthesis of the a,a-difluoro analogue 7 was initially
expected to be relatively straightforward given the similarities in
the structures of conjugate adducts 8 and b-amino ester 13. This
difluorinated intermediate 13 was prepared from 4 by first
forming an imine with ethyl R-phenylglycine, and then treating
this with BrZnCF2CO2Et, according to the recently published
sonocatalysed procedure (Scheme 2).[18] Initial attempts to
liberate the free amine derived from 13 under various hydro-
genolysis conditions, however, resulted only in unreacted start-
ing material, a result at odds with the high yields obtained
previously for the concomitant hydrogenolysis-Boc protection
of these substrates.[18] In light of this, a one-pot hydrogenolysis–
acetylation technique was employed in which the in situ Ac2O
was observed to help facilitate cleavage of the sterically hin-
dered phenylglycine moiety. The use of dry 10 % AcOH/EtOAc
as the reaction medium was found to be optimal for reducing
unwanted by-products and minimising hydrolysis of the highly
electrophilic ethyl ester. The silyl ether also remained intact
under these conditions, providing 14 in 78 % yield.
O
NH
F
O
O
NH
NH
Ph
Ph
OMe
OMe
Ph
OMe
R1
R1
R2
R2
F
1a R1 ꢀ H, R2 ꢀ F
1b R1 ꢀ F, R2 ꢀ H
3a R1 ꢀ H, R2 ꢀ F
3b R1 ꢀ F, R2 ꢀ H
2
Fig. 1. a-Fluorinated and a,a-difluorinated b-phenylalanine derivatives
previously examined for their ability to inhibit a-chymotrypsin.[5b,c]
O
O
TBSO
OtBu
H
TBSO
Ph
4
O
Ph
ꢂ
Ph
OEt
N
ꢁ
Li
NH2
then BrZnCF2CO2Et
then ꢃXꢁ
ꢃ
O
O
NH2
NH
O
NH2
NH
F
O
HN
N
H
OMe
HN
N
H
OMe
F
X
7
5
6
X ꢀ H
X ꢀ F
Fig. 2. Overall synthetic approach to a-fluorinated and a,a-difluorinated
b3-arginine derivatives.
of the electrophilicity of the ester carbonyl could potentially
make the compound more potent in biological assays, but might
also promote degradation reactions involving nucleophilic
attack at the carbonyl group.
Results and Discussion
Syntheses
The installation of the guanidinium substituent first required
the cleavage of the tert-butyldimethylsilyl (TBS) ether. The
highly electron withdrawing CF2 moiety appeared to promote
hydrolysis of the ethyl ester during TBAF-mediated desilylation
as a result of residual H2O commonly found in commercial
TBAF solutions; storage of the TBAF solution over molecular
sieves failed to resolve the problem. Anhydrous ethanol solu-
tions of both p-toluenesulfonic acid and acetyl chloride gave
only moderate yields of the desired hydroxy compound. The
optimum deprotection conditions employed a slight excess of
pyridinium p-toluenesulfonate (PPTS) in dry EtOH, which
cleanly furnished the alcohol with the ester intact. The product
was immediately subjected to guanidinylation to minimise
intramolecular lactonisation. The guanidinylated b-amino ester
15 was subsequently isolated in 79 % yield over two steps. As
mentioned above, the aim of this investigation was to develop
synthetic methods for the preparation of a consistent set of
arginine-type analogues similar to the b-phenylalanine deriva-
tives (1–3) previously shown to be inhibitors of a-chymotrypsin.
Thus it was necessary to convert the ethyl ester 15 into the
methyl ester 16. The sensitivity of the functional groups present
in 15 meant that direct transesterification was not feasible.
Instead a stepwise approach was used. Hydrolysis of the ethyl
The approach used to prepare compounds 5–7 is illustrated
in Fig. 2. An important step in the preparation of the non-
fluorinated and monofluorinated analogues 5 and 6 was
the conjugate addition of a chiral lithium amide to an a,b-
unsaturated ester, with the intermediate enolate being quenched
with either a proton source or an electrophilic fluorinating
agent – in this case N-fluorobenzenesulfonimide.[17] The
aldehyde precursor possessed a silyl ether ‘handle’ from which
the requisite guanidinium moiety could be elaborated. As our
previous attempts to prepare a,a-difluoro-b3-amino esters by
deprotonation–fluorination of an intermediate monofluoride
were only moderately successful,[17] synthesis of the difluoro
analogue 7 proceeded by the aforementioned[18] sonocatalysed
diastereoselective Reformatsky reaction.
Beginning with the non- and monofluorinated substrates,
conjugate adducts 8a and 8b were prepared as described
previously[17] (Scheme 1). N-Debenzylation of each analogue
was accomplished in less than 3 h using Pearlman’s catalyst
under an atmosphere of H2, with each crude amine immediately
acetylated to give derivatives 9a and 9b in 98 % and quantitative
yield respectively over two steps. Silyl ether cleavage was
effected in a solution of AcOH-buffered tetrabutylammonium

a-Fluoro and a,a-Difluoro-b3-Arginine Derivatives
999
Ph
X
O
Ph
N
O
Ref. [17]
a, b
OtBu
TBSO
OtBu
TBSO
a
b
series: X ꢀ H
series: X ꢀ F
8a
8b
O
O
NH
O
NH
O
d
c
OtBu
HO
OtBu
TBSO
X
X
9a 98 %
9b
10a 92 %
10b 77 %
quant.
O
O
NH2
O
NH2
NH
O
NH
e, f
(Z)
OtBu
CbzN
N
Cbz
CbzN
N
Cbz
OMe
X
X
11a 86 %
11b 68 %
12a 84 %
12b 94 %
O
O
NH
NH2
g
N
H
HN
OMe
X
quant.
quant.
5
6
Scheme 1. Reagents and conditions: (a) Pd(OH)2/C, H2, MeOH/EtOAc (2 : 1), AcOH (cat.); (b) Et3N, Ac2O,
4-dimethylaminopyridine (DMAP), dichloromethane (DCM); (c) tetrabutylammonium fluoride (TBAF), AcOH,
THF, 408C; (d) PPh3, di-carboxybenzyl (Cbz) guanidine, diisopropylazodicarboxylate (DIAD), THF, 08C to
room temperature (RT); (e) TFA/DCM (1 : 2); (f) SOCl2, MeOH, 08C to RT; (g) Pd/C, H2, MeOH.
ester was accomplished at room temperature with careful
treatment with aqueous HCl in methanol for 2.5 h; higher
temperatures appeared to lead to a loss of one or more Cbz
groups and longer reaction times resulted in hydrolysis of the
guanidinium group to give the corresponding urea. The crude
carboxylic acid was then scrupulously dried and esterified
according to the SOCl2/MeOH method to give 16 in 78 % yield.
The Cbz protecting groups were subsequently cleaved
under hydrogenolysis conditions to furnish the a,a-difluoro-
b3-arginine derivative 7 in quantitative yield.
After the successful preparation of the target a,a-difluoro
acetamido ester, it was disappointing to find that this compound
was highly susceptible to hydrolysis. An NMR sample prepared
in D2O immediately showed signs of ester hydrolysis and after a
small number of cycles of dissolution in D2O and concentration
to dryness, a clean sample of the hydrolysis product 17
(Scheme 3) was obtained. The presence of the guanidine
functionality in 7 appears to significantly accelerate the
hydrolysis of the methyl ester. Indeed, solutions of methyl
propionate and methyl a,a-difluoropropionate dissolved in
10 % D2O/d6-DMSO showed a much lower rate of hydrolysis:
While methyl a,a-difluoropropionate hydrolysed more rapidly
than methyl propionate, almost 40 % remained intact after
7 days at room temperature.
Enzyme Studies
The ability of compounds 5, 6, and 17 to inhibit bovine trypsin
was examined at concentrations up to 8 mM using N-a-benzoyl-
D,L-arginyl p-nitroanilide hydrochloride as the substrate. While
no evidence for trypsin inhibition could be seen with the non-
fluorinated 5 and monofluorinated 6 b3-arginine analogues,
the difluorinated compound 17 appeared to show weak mixed
inhibition, involving competitive inhibition at lower substrate
and inhibitor concentrations and uncompetitive inhibition at
higher concentrations. These effects were weak, however, and
firm conclusions about the trypsin inhibitory abilities of 17
based on the obtained data cannot be drawn.
While the lack of inhibition shown by the unfluorinated
5 was expected, it was disappointing that the monofluoride 6
was also inactive at the concentrations examined. Takei

1000
T. L. March et al.
O
O
OEt
NH
Ph
Ph
H
OEt
O
Ref. [18]
Ref. [18]
OEt
NH2
Ph
O
N
ꢁ
O
TBSO
OEt
TBSO
F
F
TBSO
H
13
O
O
O
NH
NH2
NH
F
O
a
b, c
79 %
N
Cbz
TBSO
OEt
78 %
CbzN
OEt
F
F
F
14
15
O
O
NH2
NH2
NH
NH
O
O
d, e
f
quant.
CbzN
N
OMe
HN
N
OMe
78 %
H
Cbz
F
F
F
F
16
7
Scheme 2. Reagents and conditions: (a) Pd(OH)2/C, H2, Ac2O, EtOAc/AcOH (9 : 1); (b) pyridinium p-toluene-
sulfonate (PPTS), EtOH, 408C, 24 h; (c) PPh3, di-carboxylbenzyl (Cbz) guanidine, diisopropylazodicarboxylate
(DIAD), THF, 08C to room temperature (RT); (d) aq. HCl, MeOH, 2.5 h; (e) SOCl2, MeOH, 08C to RT; (f) Pd/C, H2,
MeOH.
O
O
aldehyde and the use of orthogonal protecting groups. Thus,
concise and high-yielding stereoselective routes to both a-fluoro
and a,a-difluoro N-acetylated b3-arginine derivatives have been
developed using a common aldehyde as the starting material.
a-Fluoro-b3-arginine ester 6 was prepared from conjugate adduct
8b in seven steps in 49% overall yield, while the corresponding
non-fluorinated analogue was prepared from 8a in 65% yield.
Synthesis of a,a-difluoro analogue 7 in only six steps proceeded
in 48% overall yield from 13. The a,a-difluoro analogue was
found to be highly susceptible to hydrolysis, which appears to be
promoted by the guanidine functional group.
NH2
NH2
NH
F
O
F
NH
F
O
D2O/H2O
RT
ꢁ
H2N
ꢂ
O
N
N
H
HN
OMe
H
F
7
17
Scheme 3. Facile hydrolysis of a,a-difluoro-b3-arginine methyl ester in
D2O/water (RT: room temperature).
and co-workers reported that the two epimers of 1 showed
quite different inhibition of a-chymotrypsin (Ki 1a ¼
19.1 mM, Ki 1b ¼ 6.44 mM)[5b] and it may be that a similar
phenomenon occurs with trypsin. Methods for the stereose-
lective synthesis of the 2R-isomers of protected b3-amino
esters have been investigated but so far these have seen
limited success, but will be the subject of further work, with
the aim of accessing the 2R-isomer of 6. As was seen
by Takei with the a,a-difluoro ester 2 (Ki ¼ 0.034 mM,
a-chymotrypsin), the difluoride 17 showed the most promise
as a trypsin inhibitor. In this case the fact that 17 is a
carboxylate rather than an ester most likely limits its effec-
tiveness, as the negatively charged carboxylate may weaken
its affinity for the protease active site and preclude any
possible covalent interactions with the active site serine.
Amide analogues of 7 have the potential to be more effective
inhibitors of trypsin-like proteases and will be the focus of
future investigations.
The three N-acetyl-b3-arginine esters 5, 6, and 17 were tested
for their ability to inhibit trypsin. The non-fluorinated ‘control’ 5
was not found to be active, nor was the 2S-mono-fluoride 6 up
to 8 mM. The analogous 2S-N-acetyl-b3-phenylalanine ester 1a
has previously been shown to be a weaker inhibitor of
a-chymotrypsin than the corresponding 2R-N-acetyl-b3-
phenylalanine ester 1b.[5b] The difference in activity of
these two stereoisomers may reflect the influence of the
b-fluoroethylamide effect on inhibitor conformation.[1] Future
work will focus on the development of methods for the stereo-
selective synthesis of 2R-analogues of compounds like 6. The a,
a-difluoride 17 showed the most promise as a trypsin inhibitor
but the fact that it is a carboxylate rather than a methyl ester most
likely limited its effectiveness. The preparation of amide analo-
gues of 7 will be the subject of future investigations.
Experimental
General Remarks
Conclusions
All chemicals were obtained from Sigma–Aldrich. Dichloro-
methane (DCM) was distilled from CaH and THF was distilled
from sodium benzophenone ketyl immediately before use. All
other reagents were purified according to Perrin.[21] Thin-layer
chromatography (TLC) was performed on pre-coated sheets of
Merck silica gel 60 and visualised under UV light (254 nm) or
using ninhydrin or KMnO4 stains. Column chromatography was
Extending the asymmetric conjugate addition-fluorination and
Reformatsky methodologies to the preparation of trifunctiona-
lised b3-amino acids provides new access to a biologically
significant class of compound rarely reported in the literature
because of the inherent difficulties associated with their syn-
thesis. These new techniques rely only upon judicious choice of

a-Fluoro and a,a-Difluoro-b3-Arginine Derivatives
1001
carried out at atmospheric pressure using Davisil silica gel
˚
tert-Butyl (3S)-5-(Nd,Nv-Dibenzyloxycarbonylguanidino)-
3-N-acetylpentanoate (11a)
(LC60A, 40–63 mm). Melting points were obtained using a
Reichert hot-stage microscope. Optical rotations were recorded
on a PoIA AR21 polarimeter referenced to the sodium D line
(589 nm) at the temperature specified. 1H and 13C NMR spectra
were recorded on a Bruker Avance III 400 spectrometer at 400
(1H) and 100 MHz (13C), and a Bruker Avance III 600 spec-
trometer at 600 MHz and 150 MHz (13C). 19F NMR spectra were
recorded on a Bruker AV 200 spectrometer at 188 MHz (proton-
decoupled) or Varian Gemini spectrometer at 282 MHz (proton-
coupled). All spectra were recorded at 293 K unless otherwise
stated, and in deuterated solvents as indicated. Chemical shifts
(d) are reported as parts per million (ppm) and referenced to
residual solvent peaks, except in the case of D2O, which was
referenced to internal 1,4-dioxan. Peaks were assigned with the
aid of homonuclear (1H–1H) correlation spectroscopy (COSY)
and heteronuclear (1H–13C) correlation spectroscopy (HMQC
and HMBC) when required. High-resolution mass spectrometry
(HRMS) data was obtained using positive ion electrospray
ionisation (ESIþ) at CSIRO Materials Science and Engineering,
Melbourne, Australia; and Otago University, Dunedin, New
Zealand.
A solution of 10b (55 mg, 0.24 mmol), PPh3 (81 mg,
0.31 mmol), and di-Cbz guanidine[19] (195 mg, 0.59 mmol) in
THF (3 mL) was cooled to 08C and diisopropylazodicarboxylate
(DIAD) (0.060 mL, 0.31 mmol) was added dropwise. The mix-
ture was allowed to warm to room temperature overnight and
then concentrated under vacuum. Column chromatography
(1 : 1 to 3 : 1 EtOAc/hexane) afforded 11a contaminated with a
minor amount of di-Cbz guanidine. The mixture was dissolved
in hot MeOH and allowed to cool overnight to crystallise out the
unwanted guanidine, which was then filtered off. The filtrate
was concentrated under vacuum to afford pure amino ester 11a
as a white solid (112 mg, 86 %), mp 54–568C; [a]2D0 þ5 (c 1.0,
CHCl3). dH (400 MHz, CDCl3) 9.42 (br s, 2H), 7.39–7.30
(m, 10H), 6.82 (br s, 1H), 5.26 (d, J 16.8, 1H), 5.23 (d, J 16.8,
1H), 5.13 (s, 2H), 4.08–4.00 (m, 1H), 4.00–3.89 (m, 2H), 2.56
(dd, J 15.6, 4.8, 1H), 2.32 (dd, J 15.2, 7.6, 1H), 1.95–1.78
(m, 2H), 1.70 (s, 3H), 1.39 (s, 9H). dC (100 MHz, CDCl3) 170.6,
170.5, 162.7, 160.1, 155.6, 136.2, 134.4, 129.0, 128.9, 128.5,
128.41, 128.38, 128.2, 80.6, 69.3, 67.3, 45.0, 42.3, 40.2, 32.0,
28.0, 22.9. m/z 541.2651 [M þ H]þ. HRMS Anal. Calc. for
C28H37N4O7 541.2657.
Synthesis of Unfluorinated N-Acetylated
b3-Arginine Ester (5)
Methyl (3S)-5-(Nd,Nv-Dibenzyloxycarbonylguanidino)-
3-N-acetylpentanoate (12a)
tert-Butyl (3S)-5-(tert-Butyldimethylsilyloxy)-
3-N-acetyl-pentanoate (9a)
The tert-butyl ester 11a (121mg, 0.22mmol) was stirred in a
solution of DCM/TFA (2: 1, 1.5 mL) for 3 h and then concentrat-
ed under vacuum. Excess TFA was removed by co-evaporation
with CHCl3 (2 ꢁ 5 mL). The residue was redissolved in dry
MeOH (2.5 mL), cooled to 08C, and then SOCl2 (0.024 mL,
0.34 mmol) was added dropwise and the solution was allowed to
warm to room temperature overnight. After removal of the
solvent CHCl3 (20 mL) was added and the solution washed with
saturated aq. NaHCO3 (20 mL) and H2O (20 mL). The aqueous
layer was extracted with CHCl3 (10 mL) and the combined
organic layers washed with brine (20 mL), dried (Na2SO4) and
concentrated under vacuum. Flash chromatography over a plug
of silica gel (1 : 1 EtOAc/hexane to 100 % EtOAc) gave the
methyl ester 12a as a colourless oil that solidified on standing
(92 mg, 84 %), mp 1068C; [a]2D0 þ8 (c 1.0, CHCl3). dH
(400 MHz, CDCl3) 9.41 (br s, 1H), 9.24 (br s, 1H), 7.40–7.29
(m, 10H), 6.89 (d, J 6.4, 1H), 5.28–5.20 (m, 2H), 5.12 (s, 2H),
4.09–4.00 (m, 1H), 3.95–3.91 (m, 2H), 3.59 (s, 3H), 2.69 (dd, J
15.6, 4.8, 1H), 2.42 (dd, J 15.6, 7.6, 1H), 1.95–1.79 (m, 2H), 1.69
(s, 3H). dC (100 MHz, CDCl3) 171.7, 170.6, 163.3, 160.3, 155.5,
136.3, 134.4, 128.84, 128.77, 128.4, 128.27, 128.26, 128.1,
69.1, 67.1, 51.5, 44.9, 41.9, 38.6, 32.0, 22.7. m/z 521.1996
[M þ Na]þ. HRMS Anal. Calc. for C25H30N4O7Na 521.2012.
To a solution of 8a[17] (244 mg, 0.49 mmol) in MeOH/EtOAc
(2 : 1, 5 mL) was added Pd(OH)2/C (20 wt-%, 98 mg) and AcOH
(5 drops). The mixture was stirred under 1 atm of H2 for 2.5 h
and then filtered through Celite, and concentrated under vacuum.
The residue was redissolved in dry DCM (4 mL) followed by
Et3N (0.14 mL, 0.98 mmol), Ac2O (0.055 mL, 0.59 mmol), and
4-dimethylaminopyridine (DMAP, 6 mg) were added. After
stirring for 1 h the solution was concentrated and the residue
purified by column chromatography (1 : 1 EtOAc/hexane) to
give the N-acetyl amino ester 9a as a colourless oil (166 mg,
98 %); [a]2D0 ꢀ22 (c 1.0, CHCl3). dH (400 MHz, CDCl3) 6.44
(d, J 7.9, 1H), 4.34–4.26 (m, 1H), 3.77–3.64 (m, 2H), 2.55
(dd, J 15.6, 5.2, 1H), 2.42 (dd, J 15.6, 6.4, 1H), 1.91 (s, 3H),
1.79–1.76 (m, 2H), 1.42 (s, 9H), 0.88 (s, 9H), 0.04 (s, 3H), 0.03
(s, 3H). dC (100 MHz, CDCl3) 171.1, 169.1, 80.8, 60.4, 44.9,
39.3, 35.7, 28.0, 25.9, 23.4, 18.1, ꢀ5.5. m/z 346.2414 [M þ H]þ.
HRMS Anal. Calc. for C17H36NO4Si 346.2408.
tert-Butyl (3S)-5-Hydroxy-3-N-acetylpentanoate (10a)
Tetra-n-butyl ammonium fluoride (1.0 M in THF, 1.26 mL,
1.26 mmol) and AcOH (0.072 mL, 1.26 mmol) were added to a
solution of 9b (217 mg, 0.63 mmol) in THF (2 mL). After stirring
at 408C for 6 h the solvent was removed under vacuum and the
residue redissolved in CHCl3 (50 mL). This solution was poured
into saturated aq. NaHCO3 (20 mL) and the layers were separat-
ed. The organic layer was washed with brine (20 mL), dried
(Na2SO4), and concentrated under vacuum. Passage through a
plug of silica with EtOAc afforded the hydroxy ester 10a as a
colourless gum (134 mg, 92 %); [a][D20] ꢀ5 (c 1.0, CHCl3). dH
(400 MHz, CDCl3) 6.69 (d, J 8.4, 1H), 4.40–4.32 (m, 1H), 3.63–
3.58 (m, 1H), 3.55–3.48 (m, 1H), 2.51 (dd, J 15.6, 5.2, 1H), 2.40
(dd, J 15.6, 5.2, 1H), 2.00 (s, 3H), 1.77–1.72 (m, 1H), 1.54–1.45
(m, 1H), 1.43 (s, 9H). dC (100 MHz, CDCl3) 171.5, 161.0, 81.6,
58.4, 43.3, 39.7, 37.0, 31.1, 28.0, 23.1. m/z 232.1544 [M þ H]þ.
HRMS Anal. Calc. for C11H22NO4 232.1543.
Methyl (3S)-5-Guanidino-3-N-acetylpentanoate (5)
The benzylcarbamate 12a (42 mg, 0.084 mmol) was dis-
solved in dry MeOH (1.5 mL) and Pd/C (10 wt-%, 6 mg) was
added. After stirring under 1 atm of H2 for 2 h the mixture was
filtered through Celite, washing through with dry MeOH, and
the filtrate concentrated under vacuum to give the b3-arginine
derivative 1 as a glassy solid (19 mg, quant.), with no further
purification required ($95 % pure by 1H NMR analysis); [a]2D0
ꢀ41 (c 1.0, MeOH). dH (400 MHz, CD3OD) 4.31–4.23 (m, 1H),
3.67 (s, 0.9 H), 3.35 (s, 2.1H, CH3OH from ester exchange with
CD3OD), 3.28–3.12 (m, 2H), 2.61–2.50 (m, 2H), 1.95 (s, 3H),
1.90–1.80 (m, 1H), 1.77–1.67 (m, 1H). dC (100 MHz, CD3OD)

1002
T. L. March et al.
173.3, 173.0, 158.9, 49.9, 45.4, 39.5, 39.4, 34.7, 22.7. m/z
231.1441 [M þ H]þ. HRMS Anal. Calc. for C9H19N4O3
231.1452.
581.2392 [M þ Na]þ. HRMS Anal. Calc. for C28H35FN4O7Na
581.2382.
Methyl (2S,3S)-5-(Nd,Nv-
Dibenzyloxycarbonylguanidino)-3-N-
acetyl-2-fluoropentanoate (12b)
Synthesis of Monofluorinated N-Acetylated
b3-Arginine Ester (6)
tert-Butyl (2S,3S)-5-(tert-Butyldimethylsilyloxy)-3-N-
acetyl-2-fluoropentanoate (9b)
The methyl ester 12b was prepared from the t-butyl ester 11b
(52 mg, 0.093 mmol) by the same two step process as that
described for the preparation of 12a. The title compound was
obtained as a colourless oil that solidified on standing (45 mg,
94 %), mp 126–1278C. [a]2D0 þ22 (c 1.0, CHCl3). dH (400 MHz,
CDCl3) 9.41 (br s, 1H), 9.20 (br s, 1H), 7.42–7.30 (m, 10H), 7.00
(d, J 6.4, 1H), 5.25–5.10 (m, 5H), 4.27–4.14 (m, 1H), 4.00–3.83
(m, 2H), 3.67 (s, 3H), 1.90–1.74 (m, 2H), 1.71 (s, 3H). dC
(100 MHz, CDCl3) 171.2, 168.2 (d, J 23.6), 163.3, 160.2, 155.4,
136.2, 134.4, 128.9, 128.8, 128.5, 128.4, 128.3, 128.2, 88.8 (d, J
187.4), 69.2, 67.1, 52.3, 49.3 (d, J 20.3), 41.5, 26.8 (d, J 3.5),
22.5. dF (188 MHz, CDCl3) ꢀ206.3 (br s). m/z 539.1906 [M þ
Na]þ. HRMS Anal. Calc. for C25H29FN4O7Na 539.1918.
To a solution of 8b[17] (240 mg, 0.46 mmol) in MeOH/EtOAc
(1 : 1, 4 mL) was added Pd(OH)2/C (20 wt-%) (50 mg) and
AcOH (10 drops). The mixture was stirred under 1 atm of H2
for 3 h, filtered through Celite, and then concentrated under
vacuum. The residue was redissolved in dry DCM (2 mL), and
Ac2O (0.066 mL, 0.70 mmol) and DMAP (5 mg) were added.
After cooling to 08C, Et3N (0.065 mL, 0.46 mmol) was added
and stirring continued for 1 h before the solution was concen-
trated under vacuum. Column chromatography (2 : 3 EtOAc/
hexane) afforded the amino ester 9b as a colourless oil (169 mg,
quant.); [a]2D0 ꢀ2 (c 1.0, CHCl3). dH (400 MHz, CDCl3) 6.33
(d, J 7.6, 1H), 5.02 (dd, J 49.2, 3.2, 1H), 4.56–4.43 (m, 1H),
4.83–3.78 (m, 1H), 3.71–3.67 (m, 1H), 1.98 (s, 3H), 1.75–1.71
(m, 2H), 1.49 (s, 9H), 0.89 (s, 9H), 0.06 (s, 3H), 0.04 (s, 3H). dC
(100 MHz, CDCl3) 169.7, 166.8 (d, J 24.1), 89.4 (d, J 185.6),
83.2, 60.2, 49.1 (d, J 19.7), 30.8 (d, J 2.9), 28.0, 25.8, 23.3, 18.1,
ꢀ5.56, ꢀ5.60. dF (282 MHz, CDCl3) ꢀ204.4 (dd, J 50.4, 27.2).
m/z 364.2319 [M þ H]þ. HRMS Anal. Calc. for C17H35FNO4Si
364.2314.
Methyl (2S,3S)-5-Guanidino-3-N-acetyl-2-
fluoropentanoate (6)
The guanidine 6 was prepared from 12b (32 mg, 0.062 mmol)
by hydrogenation, as described for the preparation of 5 to give
the b3-arginine derivative as a glassy solid (15 mg, quant.); [a]2D0
ꢀ19 (c 1.0, MeOH). dH (400 MHz, CD3OD) 5.02 (dd, J 48.6,
3.8, 1H), 4.47–4.36 (m, 1H), 3.35 (s, 3H, CH3OH from ester
exchange with CD3OD), 3.33–3.27 (m, 1H), 3.21–3.14 (m, 1H),
2.01 (s, 3H), 1.81 (m, 2H). dC (100 MHz, CD3OD) 173.6, 169.6
(d, J 23.8), 158.7, 91.1 (d, J 187.2), 49.8, 49.4 (d, J 21.2), 39.1,
28.2 (d, J 4.0), 22.5. dF (188 MHz, CD3OD–CDCl3) ꢀ203.13
(s). m/z 249.1341 [M þ H]þ. HRMS Anal. Calc. for
C9H18FN4O3 249.1357.
tert-Butyl (2S,3S)-5-Hydroxy-3-N-acetyl-2-
fluoropentanoate (10b)
To a solution of 9b (50 mg, 0.14 mmol) in THF (1.5 mL) was
added TBAF (1.0 M in THF, 0.27 mL, 0.27 mmol) and AcOH
(0.016 mL, 0.27 mmol). After stirring at 408C for 4 h the mixture
was diluted with CHCl3 (15 mL) and then washed with saturated
aq. NaHCO3 (15 mL) and brine (15 mL), dried (Na2SO4), and
concentrated under vacuum. The residue was passed through a
plug of silica gel with EtOAc to give the hydroxy ester 10b as a
colourless gum (27 mg, 77 %). [a]2D0 þ2 (c 1.0, CHCl3). dH
(400 MHz, CDCl3) 6.25 (d, J 8.8, 1H), 4.91 (dd, J 49.4, 3.0, 1H),
4.65–4.51 (m, 1H), 3.70–3.65 (m, 1H), 3.58–3.52 (m, 1H), 3.33
(br s, 1H), 2.06 (s, 3H), 1.75–1.66 (m, 1H), 1.59–1.51 (m, 1H),
1.50 (s, 9H). dC (100 MHz, CDCl3) 171.3, 166.2 (d, J 23.9), 90.0
(d, J 185.4), 83.9, 58.1, 47.8 (d, J 19.3), 31.4 (d, J 3.1), 27.9,
23.1. dF (282 MHz, CDCl3) ꢀ201.5 (dd, J 48.4, 27.0). m/z
272.1270 [M þ Na]þ. HRMS Anal. Calc. for C11H20FNO4Na
272.1274.
Synthesis of a,a-Difluorinated N-Acetylated
b3-Arginine Ester (7)
Ethyl (3S)-5-(tert-Butyldimethylsilyloxy)-3-N-acetyl-
2,2-difluoropentanoate (14)
To a solution of 13[18] (286 mg, 0.60 mmol) in EtOAc (4 mL)
was added Ac2O (0.085 mL, 0.91 mmol), anhydrous AcOH
(0.4 mL), and Pd(OH)2/C (144 mg). After stirring under 1 atm
of H2 for 24 h the reaction mixture was filtered through Celite.
The filtrate was diluted with EtOAc (20 mL), then washed with
saturated aq. NaHCO3 (2 ꢁ 20 mL) and brine (20 mL), and then
dried (Na2SO4) and concentrated under vacuum. Purification of
the residue by column chromatography (1 : 9 to 2 : 1 EtOAc/
hexane) afforded the N-acetyl amino ester 14 as a colourless oil
that crystallised on standing (166 mg, 78 %), mp 50–528C; [a]D20
ꢀ3 (c 1.0, CHCl3). dH (400 MHz, CDCl3) 6.11 (d, J 8.8, 1H),
4.81–4.68 (m, 1H), 4.28 (q, J 7.2, 2H), 3.80–3.66 (m, 2H), 2.03–
1.94 (m, 1H), 1.96 (s, 3H), 1.75–1.67 (m, 1H), 1.32 (t, J 7.2, 3),
0.88 (s, 9H), 0.04 (s, 3H), 0.03 (s, 3H). dC (100 MHz, CDCl3)
169.8, 163.2 (dd, J 33.0, 30.8), 114.3 (t, J 253.9), 63.2, 59.6, 49.0
(dd, J 27.7, 23.2), 30.4 (t, J 1.7), 25.8, 23.0, 18.1, 13.8, ꢀ5.6. dF
(188 MHz, CDCl3) ꢀ112.9 (d, J 259.8), ꢀ117.3 (d, J 261.7). m/z
376.1725 [M þ Na]þ. HRMS Anal. Calc. for C15H29F2NO4SiNa
376.1726.
tert-Butyl (2S,3S)-5-(Nd,Nv-
Dibenzyloxycarbonylguanidino)-3-N-acetyl-2-
fluoropentanoate (11b)
The Cbz-protected guanidino compound 11b was prepared
from the alcohol 10b (36 mg, 0.14 mmol) by a Mitsunobu
reaction as described for the preparation of 11a, affording the
product as a colourless oil (53 mg, 68 %); [a]2D0 þ18 (c 1.0,
CHCl3). dH (400 MHz, CDCl3) 9.42 (br s, 1H), 9.23 (br s, 1H),
7.28–7.33 (m, 10H), 6.89 (d, J 6.4, 1H), 5.24 (d, J 18.0, 1H), 5.21
(d, J 18.0, 1H), 5.13 (s, 2H), 5.05 (dd, J 49.6, 3.2, 1H), 4.27–4.14
(m, 1H), 4.00–3.88 (m, 2H), 1.92–1.75 (m, 2H), 1.71 (s, 3H),
1.39 (s, 9H). dC (100 MHz, CDCl3) 171.1, 166.8 (d, J 23.7),
163.4, 160.2, 155.5, 136.3, 134.3, 129.0, 128.9, 128.5, 128.44,
128.36, 128.2, 88.6 (d, J 186.2), 83.0, 69.3, 67.2, 49.3 (d, J 19.9),
41.6, 27.9, 26.7, 22.6. dF (188 MHz, CDCl3) ꢀ205.7 (br s). m/z
Ethyl (3S)-5-(Nd,Nv-Dibenzyloxycarbonylguanidino)-
3-N-acetyl-2,2-difluoropentanoate (15)
The b-amino ester 11 (84 mg, 0.24 mmol) was dissolved in
dry EtOH (1 mL) and PPTS (90 mg, 0.36 mmol) was added.

a-Fluoro and a,a-Difluoro-b3-Arginine Derivatives
1003
After stirring at 408C for 12 h the solution was diluted with
EtOAc (30 mL) and then washed with saturated aq. NaHCO3
(10 mL), H2O (10 mL), and brine (20 mL). After drying
(Na2SO4) and concentration under vacuum the residue was
redissolved in THF (6 mL). PPh3 (81 mg, 0.31 mmol) and di-Cbz
guanidine (234 mg, 0.71 mmol) were added, the solution was
cooled to 08C and DIAD (0.06 mL, 0.31 mmol) was added
dropwise. After warming to room temperature overnight the
reaction mixture was concentrated and the residue purified by
column chromatography (1 : 1 to 2 : 1 EtOAc/hexane), which
afforded 15 as a viscous oil (103 mg, 79 %). [a]2D0 þ4 (c 1.0,
CHCl3). dH (400 MHz, CDCl3) 9.37 (br s, 1H), 9.20 (br s, 1H),
7.43–7.28 (m, 10H), 6.53 (d, J 8.4, 1H), 5.26 (d, J 18.0, 1H), 5.23
(d, J 18.0, 1H), 5.14 (d, J 14.8, 1H), 5.11 (d, J 14.8, 1H), 4.57–
4.45 (m, 1H), 4.24 (q, J 7.2, 2H), 3.40–3.87 (m, 2H), 2.16–2.07
(m, 1H), 1.89–1.79 (m, 1H), 1.72 (s, 3H), 1.28 (t, J 7.2, 3H). dC
(100 MHz, CDCl3) 171.1, 163.3, 163.0 (dd, J 32.8, 30.8), 160.0,
155.4, 136.3, 134.4, 129.0, 128.9, 128.5, 128.32, 128.26, 128.1,
113.7 (t, J 254.2), 69.2, 67.1, 63.1, 49.4 (dd, J 28.3, 23.5),
41.2, 25.6 (t, J 2.2), 22.4, 13.8. dF (188 MHz, CDCl3) ꢀ113.0
(d, J 253.8), ꢀ119.9 (d, J 251.9). m/z 549.2160 [M þ H]þ.
HRMS Anal. Calc. for C26H31F2N4O7 549.2155.
3.89 (s, 3H), 3.38–3.18 (m, 2H), 2.15–1.93 (m, 1H), 2.03 (s, 3H),
1.88–1.75 (m, 1H). dC (100 MHz, D2O) 175.1, 169.4 (t, J 27.2),
157.4, 116.5 (t, J 253.5), 54.9, 49.6 (t, J 25.2), 37.7, 25.7, 22.2.
dF (188 MHz, D2O) ꢀ112.2 (d, J 242.9), ꢀ115.2 (d, J 242.9).
m/z 267.1260 [M þ H]þ. HRMS Anal. Calc. for C9H17F2N4O3
267.1263.
(3S)-5-Guanidino-3-N-acetyl-2,2-difluoropentanoic
Acid (17)
After three cycles of dissolution of the methyl ester 7 in D2O
and then concentration to dryness, hydrolysis to the carboxylic
acid 17 was complete. nmax/cmꢀ1 3266, 3156 (br s), 1613, 1549,
1412, 1197, 1092, 1062, 741, 612. dH (400 MHz, D2O) 4.60–
4.46 (m, 1H), 3.37–3.25 (m, 1H), 3.25–3.17 (m, 1H), 2.05
(s, 3H), 2.01–1.98 (m, 1H), 1.80 ꢀ1.68 (m, 1H). dC
(100 MHz, D2O, 13C–19F decoupled) 175.1, 169.4, 157.4,
116.5, 49.5, 37.9, 27.1, 22.5. dF (376.5 MHz, D2O) ꢀ113.1
(d, J 244.9), ꢀ115.8 (d, J 240.9). m/z 253.1107 [M þ H]þ;
275.0926 [M þ Na]þ. HRMS Anal. Calc. for C8H15F2N4O3
253.1107; C8H14F2N4O3Na 275.0926.
Supplementary Material
Copies of 1H and 13C NMR spectra for all new compounds and
experimental details of ester hydrolysis and trypsin inhibition
studies are available on the Journal’s website.
Methyl (3S)-5-(Nd,Nv-Dibenzyloxycarbonylguanidino)-
3-N-acetyl-2,2-difluoropentanoate (16)
The ethyl ester 15 (103 mg, 0.19 mmol) was dissolved in
MeOH (2 mL) and 4 M HCl (0.5 mL) was added. After stirring
for 2 h the solvent was evaporated and the residue concentrated
under high vacuum to remove residual water. The foamy residue
was redissolved in dry MeOH (2 mL) and cooled to 08C before
the dropwise addition of SOCl2 (0.021 mL, 0.28 mmol). After
warming to room temperature overnight the MeOH was
removed under vacuum, EtOAc (20 mL) was added and the
solution was poured into saturated aq. NaHCO3 (20 mL). The
layers were separated and the organic layer was washed with
brine (20 mL), dried (Na2SO4), and concentrated under vacuum.
Column chromatography (1 : 1 EtOAc/hexane) afforded methyl
ester 16 as a colourless oil that solidified on standing (78 mg,
78 %), mp 119–1208C. [a]2D0 þ5 (c 1.0, CHCl3). dH (400 MHz,
CDCl3) 9.38 (br s, 1H), 9.20 (br s, 1H), 7.44–7.29 (m, 10H), 6.62
(d, J 8.0, 1H), 5.27 (d, J 16.6, 1H), 5.24 (d, J 16.6, 1H), 5.14 (d, J
14.4, 1H), 5.11 (d, J 14.4, 1H), 4.54–4.42 (m, 1H), 4.00–3.86 (m,
2H), 3.79 (s, 3H), 2.17–2.07 (m, 1H), 1.90–1.79 (m, 1H), 1.72 (s,
3H). dC (100 MHz, CDCl3) 171.3, 163.5 (dd, J 33.6, 30.1),
163.3, 160.0, 155.4, 136.3, 134.4, 129.0, 128.9, 128.5, 128.33,
128.29, 128.2, 113.6 (t, J 254.0), 69.3, 67.1, 53.5, 49.6 (t,
J 28.6, 23.6), 41.2, 25.4, 22.3. dF (282 MHz, CDCl3) ꢀ113.6
(d, J 254.2), ꢀ120.0 (d, J 254.2). m/z 535.2007 [M þ H]þ.
HRMS Anal. Calc. for C25H29F2N4O7 535.1999.
Acknowledgements
The authors are grateful to Flinders University for providing a Research
Scholarship to T.M. and CSIRO for providing a scholarship top-up for T.M.
and research funding. The Australian Research Council is acknowledged for
LIEF funding (LEO668489) for NMR spectrometers.
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