Stable triazolylphosphonate analogues of phosphohistidine

Article abstract of DOI:10.1007/s00726-011-1145-2

Histidine-phosphorylated proteins and the corresponding kinases are important components of bacterial and eukaryotic cell-signalling pathways, and are therefore potential drug targets. The study of these biomolecules has been hampered by the lability of the phosphoramidate functional group in the phosphohistidines and the lack of generic antibodies. Herein, the design and concise synthesis of stable triazolylphosphonate analogues of N1-and N3-phosphohistidine, and derivatives suitable for bioconjugation, are described. Springer-Verlag 2011.

Full text of DOI:10.1007/s00726-011-1145-2

Amino Acids (2012) 43:857–874
DOI 10.1007/s00726-011-1145-2
ORIGINAL ARTICLE
Stable triazolylphosphonate analogues of phosphohistidine
•
Shin Mukai Gavin R. Flematti Lindsay T. Byrne
•
•
•
•
Paul G. Besant Paul V. Attwood Matthew J. Piggott
Received: 12 April 2011 / Accepted: 31 October 2011 / Published online: 22 November 2011
Ó Springer-Verlag 2011
Abstract Histidine-phosphorylated proteins and the cor-
responding kinases are important components of bacterial
and eukaryotic cell-signalling pathways, and are therefore
potential drug targets. The study of these biomolecules has
been hampered by the lability of the phosphoramidate
functional group in the phosphohistidines and the lack of
generic antibodies. Herein, the design and concise syn-
thesis of stable triazolylphosphonate analogues of N1- and
N3-phosphohistidine, and derivatives suitable for biocon-
jugation, are described.
Bu
Butyl
Cp*
DCM
d
Pentamethylcyclopentadienyl
Dichloromethane
Doublet
dd
Doublet of doublets
DEAD Diethyl azodicarboxylate
DEPT
Distortionless enhancement by polarisation
transfer
DIAD
Diisopropyl azodicarboxylate
DIPEA Diisopropylethylamine
DMF
E1cb
ESI
N,N-Dimethylformamide
Elimination unimolecular conjugate base
Electrospray ionisation
Ethyl
Keywords Phosphohistidine ꢀ Azide-alkyne
cycloaddition ꢀ Triazolylalanine ꢀ Stable analogues ꢀ
Synthesis ꢀ Haptens
Et
Fmoc
9-Fluorenylmethoxycarbonyl
Fourier transform
Abbreviations
FT
Ac
All
aq.
Ar
Acetyl
HCTU O-(6-Chlorobenzotriazol-1-yl)-N,N,N0,N0-
tetramethyluronium hexafluorophosphate
Allyl
Aqueous
Aryl
HMBC Heteronuclear multiple bond correlation
HHK
HPLC
HR
Histone H4 histidine kinase
High-performance liquid chromatography
High-resolution (MS)
Internal diameter
Isopropyl
Bn
Boc
br
Benzyl
tert-Butoxycarbonyl
Broad
i.d.
i-Pr
IR
Infrared
lit.
Literature
S. Mukai ꢀ G. R. Flematti ꢀ L. T. Byrne ꢀ
P. G. Besant ꢀ P. V. Attwood ꢀ M. J. Piggott (&)
School of Biomedical, Biomolecular and Chemical Sciences,
The University of Western Australia, Crawley, WA 6009,
Australia
M
Multiplet
Me
Methyl
Mp
Melting point
MS
MWD
m/z
Mass spectrum
e-mail: matthew.piggott@uwa.edu.au
Multiple-wavelength detector
Mass:charge ratio
Present Address:
L. T. Byrne
Centre for Microscopy, Characterisation and Analysis,
The University of Western Australia, Crawley,
WA 6009, Australia
NDPK Nucleoside diphosphate kinase
NMR
Ph
Nuclear magnetic resonance
Phenyl
123

858
S. Mukai et al.
O
O
q
Quartet
H
N
H
N
protein
protein
Rf
Retention factor
Serine
protein
3 N
N
H
protein
N 1
N
H
Ser
sat.
t
Saturated
N1
Triplet
O
N3
O
O
O
P
P
t
Tertiary
O
O
TFA
THF
TIPS
TLC
Tza
UV
Trifluoroacetic acid
Tetrahydrofuran
Triisopropylsilyl
Thin layer chromatography
Triazolylalanine
Ultraviolet
1
2
O
O
O
O
O
H3N
O1
H3N
O
H
N 1
3
3
O
O
O
O
P
P
O
O
3
4
Introduction
O
Histidine kinases are a family of enzymes that catalyse the
phosphorylation of the imidazole N1 (1) or N3 (2) of spe-
cific histidine residues in proteins (Fig. 1) (Janiak-Spens
and West 2004). Their better-known cousins, the serine/
threonine and tyrosine kinases, have been implicated in the
regulation of almost all eukaryotic cellular processes. In
prokaryotes, fungi and plants, histidine kinases play critical
roles in the response to environmental stimuli (Stock et al.
2000). There is also evidence that histidine kinases and their
substrates are important components of mammalian cell-
signalling pathways (Besant and Attwood 2005; Besant
et al. 2003; Steeg et al. 2003). However, the only mam-
malian histidine kinase that has been well characterised is
nucleoside diphosphate kinase (NDPK), which catalyses the
interconversion of nucleoside di- and triphosphates via a
phosphohistidyl-enzyme intermediate, as well as phos-
phorylating a number of proteins, including the beta subunit
of some trimeric G-proteins (Attwood et al. 2007).
H3N
H3N
N
H
N
H
N
N 1
N
1
N
3 N
N
P
O
3
P
O
O
O
O
O
5
6
O
O
H
AcN
H
AcN
N 1
O
N
N
1
N
3 N
N
P
O
3
O
O
P
O
O
O
7
8
Fig. 1 N1- (1) and N3-phosphohistine (2) residues and stable
phosphohistidine analogues, 3–8, shown here in their predominant
protonation state at physiological pH. The numbering shown reflects
that used by biochemists for histidine
One mammalian histidine kinase that is of particular
interest as a potential therapeutic target is histone H4 his-
tidine kinase (HHK). The role of histone H4 phosphory-
lation is uncertain, but HHK activity is elevated in foetal,
regenerating, and cancerous liver cells (Tan et al. 2004).
Conversely, the enzyme activity is low in normal adult
liver cells. The obvious inference is that HHK is intimately
involved in liver cell proliferation.
3-thiophosphohistidine (Pirrung et al. 2000), has been used
to generate immunogens for this purpose, the failure has been
attributed to rapid hydrolysis of the N–P bond in vivo (Besant
PG and Attwood PV, Unpublished work).
Stable phosphonate analogues of N3-phosphohistidine,
specifically furan (3) (Schenkels et al. 1999) and pyrrole
(4) (Tan E, Pirrung MC, Attwood PV, Unpublished work)
congeners, have been synthesised previously. Antibodies
raised using 4 as a hapten recognised the immunogen and
the hapten but had no affinity for phosphohistidine residues
in a protein context (Tan E, Pirrung MC, Attwood PV,
Unpublished work). One possible explanation for this
failure is that the pyrrole analogue 4 lacks the hydrogen
bond-accepting nitrogen atom present in phosphohistidine
residues (red in Fig. 1). It is likely that this nitrogen would
In contrast to the robust serine, threonine and tyrosine
phosphates, the hydrolytically labile phosphoramidate
functional group in the phosphohistidines makes their
identification, purification, and study challenging (Attwood
et al. 2007). In particular, the availability of generic phos-
photyrosine and phosphoserine/threonine antibodies has
greatly facilitated the study of processes involving protein O-
phosphorylation, but all attempts to generate an equivalent
phosphohistidine antibody have thus far been unsuccessful.
Where phosphohistidine itself, or the more acid-stable
123

Stable triazolylphosphonate analogues of phosphohistidine
859
be critical for molecular recognition in a phosphohistidine
antibody–antigen complex.
then filtered. Solvents were evaporated under vacuum or a
stream of N2.
Accordingly, we set out to design and synthesise better
phosphohistidine analogues and apply them to the gener-
ation of generic phosphohistidine antibodies. Triazolyla-
laninephosphonates 5–8 (Fig. 1) were immediately
appealing as the C–P bond is non-hydrolysable and they
retain the spatial orientation of key functional groups likely
to be important for molecular recognition, in particular, the
hydrogen-bond-accepting ring nitrogen. The N-methylam-
ide and acetamide functional groups in 5/6 and 7/8,
respectively, mimic the peptide bonds of native phos-
phohistidine residues, with the free amino and carboxyl
groups ready for bioconjugation to a carrier protein through
amide linkages with aspartate/glutamate and lysine resi-
dues, respectively. In addition, the robust azide-alkyne
dipolar cycloaddition (Meldal and Tornoe 2008; Rostovt-
sev et al. 2002; Wu and Fokin 2007) offered the potential
to rapidly access enantiopure analogues of both the N1- and
N3-regioisomers of phosphohistidine, both of which are
biologically relevant (Attwood et al. 2007; Besant and
Attwood 2009).
Flash chromatography was conducted with Merck silica
gel 60. Analytical TLC was performed on Whatman flex-
ible plates (250 lm layer, silica gel 60 F254). Spots were
visualised under UV light and by staining with ammonium
molybdate or ninhydrin.
Melting points were measured on a Kofler hot-stage
melting point apparatus. IR spectra were acquired using a
Perkin-Elmer Spectrum One FTIR spectrometer. Mass
spectra were acquired on an Applied Biosystems QSTAR
pulsar I quadruple time-of-flight instrument. NMR spectra
were acquired on Varian Inova 300 (300 MHz, 1H;
75.5 MHz, 13C; 120 MHz, 31P), Bruker Avance 500
(500 MHz, 1H; 125 MHz, 13C) or Bruker AV600
1
(600 MHz, H; 150 MHz, 13C; 240 MHz, 31P) spectrom-
eters, as indicated. All spectra were recorded in CDCl3
unless otherwise indicated. Chemical shifts are expressed
in ppm, relative to CHCl3 (1H, 7.26 ppm), CDCl3 (13C,
77.0 ppm), CHD2OD (1H, 3.30 ppm), CD3OD (13C,
49.0 ppm), and 85% H3PO4 (external capillary, 31P,
0 ppm), as appropriate. Routine assignments of 13C NMR
spectra were made with the assistance of DEPT 135 and
DEPT 90 experiments.
Independently, the Muir group has recently adopted an
almost identical core strategy and applied it to the solid
phase synthesis of peptides containing phosphono-
triazolylalanine residues (Kee et al. 2010). In a break-
through for phosphohistidine research, they have used this
approach to successfully generate antibodies specific for
histidine-phosphorylated histone H4. Using native chemical
ligation, they have also incorporated the phosphohistidine
analogue into histone H4.
HPLC
HPLC was conducted using a Hewlett Packard 1050 HPLC
system equipped with a multiple wavelength detector
(MWD) and a 250 9 10 mm i.d., 5 lm, Apollo C18
reversed-phase column (Grace-Davison), with a 33 mm 9
7 mm guard column of the same material. The samples
were eluted at 4 mL/min with 30% (v/v) MeCN–water. UV
absorbance was measured at 220 nm.
Similarly, Webb and co-workers have recently reported
the synthesis of an Fmoc-protected triazolylalanine phos-
phonate analogue of N3-phosphohistidine, and its use in the
solid phase synthesis of a heptapeptide based on the
phosphocarrier domain of pyruvate, phosphate dikinase
(McAllister et al. 2011).
Enantioselective chromatography of the carboxylic
acids 31 and 32 was carried out using a 250 9 4.6 mm i.d.,
5 lm Chiracel OD-H column (Diacel), eluted at 1.0 mL/
min with 8% i-PrOH/hexanes, and detection at 220 nm.
Despite these recent advances, antibodies that recognise
the phosphohistidine epitope in a broad protein context are
yet to be discovered. Herein we describe the design and
synthesis of haptens that may prove useful in achieving this
goal.
Synthesis
General procedure for the synthesis of azidoalanine
derivatives
Materials and methods
A stirred solution of the serine derivative (5–15 mmol) and
triphenylphosphine (1.2 eqv.) in anhydrous THF
(20–50 mL) at -78°C, under N2, was treated with a 2.5 M
solution of HN3 in toluene (Yeager and Finney 2004)
(1.2 eqv.) and DEAD (1.2 eqv.). The resulting solution was
allowed to warm to room temperature. After 5 h the
solution was diluted with water (100 mL), the layers were
separated, and the aqueous phase was extracted with Et2O
(3 9 100 mL). The combined organic phase was washed
General
All solvents were distilled prior to use; anhydrous solvents
and reagents were distilled under N2. THF for organome-
tallic reactions was distilled from sodium benzophenone
ketyl. All reaction temperatures refer to bath temperatures.
Organic extracts were dried over anhydrous MgSO4 and
123

860
S. Mukai et al.
(S)-Benzyl 2-acetamido-3-azidopropanoate (9d)
with brine (50 mL), dried and evaporated, and the residue
was subjected to flash chromatography.
Following the general procedure with Ac-Ser-OBn (23)
(2.00 g, 12.4 mmol), and DIAD (2.00 mL, 10.2 mmol)
instead of DEAD, flash chromatography and elution with
1:1 Et2O/hexanes gave 9d as a yellow oil (1.35 g, 61%).
Rf = 0.45 (Et2O). IR (thin film) cm-1: 3,293 (NH), 2,106
(N3), 1,743 (OC=O), 1,655 (HNC=O). 1H NMR
(500 MHz, CDCl3): d 7.26 (m, 5H, Ar), 7.15 (br d,
J = 7.5 Hz, NH), 5.11 (d, 2H, OCH2), 4.76 (m, 1H, a-H),
3.63 (dd, 2H, J = 4.0 Hz, 1.5 Hz, b-H), 1.95 (s, 3H, CH3).
13C NMR (125 MHz, CDCl3): d 170.1 (C=O), 169.2
(C=O), 134.5 (Ar), 128.2 (ArH), 128.1 (ArH), 127.8 (ArH),
67.2 (OCH2), 51.9, 51.8, 22.2 (CH3). MS (ESI) m/z: 263
[M?H]?, 285 [M?Na]?. HRMS (ESI): observed,
(S)-Methyl 3-azido-2-(t-butoxycarbonylamino)propanoate
(9a)
This compound was synthesised according to the method of
Boger (Boger et al. 1994) with slight modifications. Fol-
lowing the general procedure with Boc-Ser-OMe (3.61 g,
16.5 mmol), flash chromatography and elution with 1:4
Et2O/hexanes gave 9a as a colourless oil (2.90 g, 72%). 1H
NMR (600 MHz, CD3OD): d 5.61 (br d, 1H, NH), 4.28 (m,
1H, a-CH), 3.55 (s, 3H, OCH3), 3.49 (m [apparent br d],
2H, b-CH2), 1.23 (s, 9H, t-Bu). 13C NMR (150 MHz) d
169.7 (CO2), 154.6 (HNCO2), 79.5, 77.2, 53.0, 51.9, 27.6
[(CH3)3]; MS (ESI) m/z: 267 [M?Na]?. [a]D = ?36.4°
(c 2.0, CHCl3) [lit. (Dondoni et al. 2004) ?36.0° (c 0.6,
263.1133,
[a]D = ?22.8° (c 1.0, CHCl3).
[C12H14N4O3?H]?
requires
263.1139.
1
CHCl3)]. The H NMR data are similar to those reported
(S)-Benzyl 3-azido-2-(t-butoxycarbonylamino)propanoate
(9e)
(Dondoni et al. 2004).
(S)-Methyl 2-acetamido-3-azidopropanoate (9b)
This compound has been prepared previously by a similar
method (Kogan and Rawson 1992). Following the general
procedure with Boc-Ser-OBn (1.50 g, 5.08 mmol), flash
chromatography and elution with 1:4 Et2O/hexanes gave 9e
as a colourless oil (1.18 g, 72%). Rf = 0.20 (1:4 Et2O/
hexanes). 1H NMR (400 MHz, CDCl3): d 7.34 (m, 5H, Ar),
5.53 (br d, J = 7.6 Hz, 1H, NH), 5.19 (d, J = 2.8 Hz, 2H,
OCH2) 4.50 (m, 1H, a-H), 3.71 (d, J = 3.6 Hz, 2H, b-H),
1.44 (s, 9H, t-Bu). 13C NMR (100 MHz, CDCl3): d 169.3
(CO2), 154.7 (NCO2), 134.6 (ArC), 128.24 (ArH), 128.17
(ArH), 127.9 (ArH), 80.0 [OC(CH3)3], 67.2 (OCH2), 53.3
(a), 52.2 (b), 27.8 [(CH3)3]. MS (ESI) m/z: 267 [M?Na]?.
[a]D = ?8.2° (c 1.0, CHCl3) [lit. (Kee et al. 2010) ?8.0°
Following the general procedure with Ac-Ser-OMe (20)
(2.00 g, 12.4 mmol) and using DIAD (3.00 mL,
15.3 mmol) instead of DEAD, flash chromatography and
elution with 1:1 Et2O/hexanes gave 9b as a yellow oil
1
(1.39 g, 60%). H NMR (300 MHz, CDCl3): d 6.51 (br d,
1H, J = 5.4 Hz, NH), 4.74 (m, 1H, a-H), 3.79 (s, 1H,
OCH3, 3H), 3.73 (dd, 2H, J = 3.6, 1.2 Hz, b-H), 2.05 (s,
3H, OCCH3). 13C NMR (75 MHz, CDCl3): d 170.1 (C=O),
169.5 (C=O), 53.8, 52.9, 52.2, 22.9 (COCH3). MS (ESI) m/
z: 187 [M?H]?, 209 [M?H]?. [a]D = ?79.3° (c 1.0,
CHCl3). [lit. (Davoli et al. 1995) ?74.2° (c 1.4, CHCl3)].
1
The H NMR data are similar to those reported (Davoli
1
(c 1.0, CHCl3)]. The H and 13C NMR data are similar to
et al. 1995).
those reported (Kee et al. 2010).
(S)-Allyl 2-acetamido-3-azidopropanoate (9c)
(S)-Benzyl 2-(((9H-fluoren-9-yl)methoxy)
carbonylamino)-3-azidopropanoate (9f)
Following the general procedure with Ac-Ser-OAll (22)
(2.00 g, 10.7 mmol), flash chromatography and elution
with 1:1 Et2O/hexanes gave 9c as a brown oil (1.38 g,
61%). Rf = 0.40 (Et2O); IR (thin film) cm-1: 3,274 (NH),
2,107 (N3) 1,743 (OC=O), 1,660 (HNC=O). 1H NMR
(500 MHz, CDCl3): d 6.84 (d, 1H, J = 7.5 Hz, NH), 5.83
(m, 1H, HC=CH2), 5.27 (dd, 1H, J = 17.0, 1.5 Hz,
HC=CH2 trans), 5.20 (dd, 1H, J = 10.5, 1.0 Hz, HC=CH2
cis), 4.72 (m, 1H, a-H), 4.60 (dd, 2H, J = 4.5, 1.5 Hz,
OCH2), 3.68 (d, 2H, b-H), 1.99 (s, 3H, CH3). 13C NMR
(125 MHz, CDCl3): d 170.2 (C=O), 169.2 (C=O), 130.9
(HC=CH2), 119.0 (HC=CH2), 66.3 (OCH2), 52.1, 52.0,
22.6 (CH3). MS (ESI) m/z: 235 [M?Na]?. HRMS (ESI):
observed, 235.0808, [C8H13NO4?H]? requires 235.0802.
[a]D = ?16.3° (c 1.0, CH3Cl).
Following the general procedure with Fmoc-Ser-OBn (36)
(1.51 g, 5.76 mmol), flash chromatography and elution
with 1:4 Et2O/hexanes gave 9f as a white solid (1.38 g,
65%). mp 70–72°C. Rf = 0.15 (1:1 Et2O/hexanes). IR (thin
film) cm-1: 3,337 (NH), 2,107 (N3) 1,650–1,750 (br,
1
C=O ? NC=O). H NMR (500 MHz, CDCl3): d 7.80 (d,
J = 7.5 Hz, 2H, Ar), 7.64 (br d, J = 7.0 Hz, 2H, Ar),
7.33–7.46 (m, 9H, Ar), 5.85 (br d, J = 7.5 Hz, 1H, NH),
5.26 (d, J = 4.5 Hz, 2H, OCH2Ph), 4.64 (m, 1H, a-H), 4.45
(m, 2H, NCO2CH2), 4.26 (t, J = 7.5 Hz, 1H, H90), 3.79 (br
s, 2H, b-H). 13C NMR (125 MHz, CDCl3): d 169.2 (CO2),
155.6 (NCO2), 143.6, 143.5, 141.2, 134.8 (ArC), 128.6
(ArH), 128.2 (ArH), 127.6 (ArH), 127.0, 125.0, 119.9, 67.8
123

Stable triazolylphosphonate analogues of phosphohistidine
861
(OCH2), 67.2 (OCH2), 53.9 (a), 52.4 (b), 46.9 (C90). MS
(ESI) m/z: 443 [M?H]?, 465 [M?Na]?. HRMS (ESI):
NMR (150 MHz, CDCl3): d 106.4 (d, JC,P = 37.6 Hz, C2),
96.3 (d, JC,P = 269.4 Hz, C1), 63.0 (d JC,P = 5.4 Hz),
18.3 (i-Pr CH3), 15.9 (d, JC,P = 6.8 Hz, OCH2CH3), 10.7
(CSi). 31P NMR (120 MHz): d -7.38; MS (ESI) m/z: 319
[M?H]?, 341 [M?Na]?. This compound has been repor-
ted (Lecercle et al. 2006) but no characterisation data were
given.
observed,
465.1539,
[C25H22N4O4?Na]? requires
465.1533. [a]D = ?25.7° (c 1.0, CHCl3).
(S)-Benzyl 3-hydroxy-2-(t-butoxycarbonylamino)
propanoate [Boc-Ser-OBn]
Benzyl bromide (1.80 mL, 15.2 mmol) was added to a
solution of Boc-Ser-OH (3.00 g, 14.6 mmol) and DIPEA
(1.30 mL, 15.2 mmol) in DMF (20 mL) under N2 at 0°C.
The resulting solution was allowed to warm to room tem-
perature, stirred for 18 h, diluted with sat. aq. NH4Cl
(30 mL), and then extracted with EtOAc (3 9 50 mL). The
organic extract was washed with brine (20 mL), dried and
evaporated, and the residue was subjected to flash chro-
matography. Elution with 1:4 EtOAc/hexanes gave Boc-
Ser-OBn as a colourless oil (3.66 g, 85%). 1H NMR
(500 MHz, CDCl3): d 7.34–7.27 (m, 5H, Ar), 5.71 (d,
J = 8.0 Hz, 1H, NH), 5.15 (d, J = 2.5 Hz, 2H, OCH2Ph),
4.39 (m, 1H, a-H), 3.95 (m [apparent br d], 1H, b-Ha), 3.84
(dd, J = 11.0, 3.5 Hz, 1H, b-Hb), 3.50 (br s, 1H OH), 1.42
(s, 9H, t-Bu); 13C NMR (125 MHz, CDCl3): d 170.8 (CO2),
155.7 (NCO2), 135.1 (ArC), 128.4 (ArH), 128.2 (ArH),
127.9 (ArH), 80.0 [C(CH3)3], 67.1 (OCH2Ph), 62.9 (b),
55.7 (a), 28.1 [C(CH3)3]. MS (ESI) m/z: 296 [M?H]?, 318
[M?Na]?. [a]D = -19.6° (c 1.0, MeOH) [lit. (Nakata
et al. 1996) -19.0° (c 1.0, CHCl3)]. The NMR data are
similar to those reported (Tummatorn et al. 2007).
Diethyl ethynylphosphonate (10)
A solution of diethyl (triisopropylsilyl)ethynylphosphonate
(1.00 g, 3.14 mmol) and KF (0.38 g, 6.5 mmol) in MeOH
(10 mL) was stirred for 2 h and then concentrated under
reduced pressure. The residue was diluted with water and
extracted with Et2O (3 9 40 mL). The organic extract was
washed with brine (30 mL), dried, and concentrated under
reduced pressure. The residue was subjected to flash
chromatography. Elution with 1:1 Et2O/hexane gave 8 as a
pale yellow oil (310 mg, 61%). 1H NMR (500 MHz,
CDCl3) d: 4.04 (m, 4H, CH2), 3.00 (d, J = 13.5 Hz 1H,
CH), 1.24 (dt, JH,P = 0.5 J = 7.0 sHz, 6H, 2 9 CH3). 13C
NMR (125 MHz, CDCl3): d 88.1 (d, J = 50.6 Hz, CH),
73.8 (d, J = 289 Hz, CP), 63.1 (d, J = 5.5 Hz, OCH2CH3),
15.7 (d, J = 7.0 Hz, OCH2CH3). MS (ESI) m/z: 163
1
[M?H]?. The H NMR data are similar to those reported
(McAllister et al. 2011). This compound was also prepared
using a published method (Vuilhorgne et al. 2003).
General procedure for thermal Huisgen cycloadditions
Diethyl (triisopropylsilyl)ethynylphosphonate
A stirred solution of the azide 9 and diethyl eth-
ynylphosphonate 10 (1 eqv.) in toluene (5–10 mL), under
N2, was heated under reflux for 5 h. The toluene was
evaporated and the residue was subjected to flash chro-
matography (details below).
Ethyl bromide (1.00 mL, 13.7 mmol) was added dropwise
to
a suspension of stirred Mg turnings (416 mg,
17.0 mmol) in THF (10 mL) at 0°C under N2 and the
resulting mixture was heated under reflux under N2 for
30 min. The ethynylmagnesium bromide solution thus
prepared was added dropwise via canula to a solution of
TIPS-acetylene (3.00 mL, 13.4 mmol) in THF (10 mL) at
0°C under N2. The reaction mixture was allowed to warm
to room temperature and stirring was continued for 1 h
before being cooled to 0°C and treated dropwise with
diethyl chlorophosphite (2.20 mL, 15.2 mmol). The
resulting solution was allowed to warm to room tempera-
ture and after 1 h was diluted with sat. aq. NH4Cl (30 mL)
and extracted with Et2O (3 9 50 mL). The organic extract
was washed with brine (30 mL), dried and concentrated
under reduced pressure, and the residue was subjected to
flash chromatography. Elution with 1:4 EtOAc/hexane
gave the title phosphonate as pale yellow oil (3.49 g, 80%).
Rf = 0.80 (1:1 EtOAc/hexanes). 1H NMR (600 MHz,
CDCl3) d: 4.11 (m, 4H, OCH2CH3), 1.32 (dt, JH,P = 0.6,
Thermal Huisgen cycloaddition of 9a
Following the general procedure with azide 9a (0.150 g,
0.614 mmol), flash chromatography and elution with (1:1
EtOAc/hexanes) gave 11a as pale yellow oil (53 mg, 21%),
identical with the material described below. Further elution
with EtOAc gave 12a as pale yellow oil (144 mg, 58%),
identical with the material described below.
Thermal Huisgen cycloaddition of 9b
Following the general procedure with azide 9b (1.00 g,
5.37 mmol), flash chromatography and elution with (7:3
EtOAc/hexanes) gave 11b as pale yellow oil (486 mg,
26%), identical with the material described below. Further
elution with EtOAc gave 12b as pale yellow oil (1.12 g,
60%), identical with the material described below.
J = 7.1 Hz, 6H, OCH2CH3), 1.03–1.10 (m, 21H, i-Pr). 13
C
123

862
S. Mukai et al.
Thermal Huisgen cycloaddition of 9c
EtOAc/hexanes gave 11a as pale yellow oil (93 mg, 56%).
Rf = 0.15 (EtOAc/hexanes 1:1); IR (thin film) cm-1: 3,306
(NH), 1,747 (m, OC=O), 1,715 (s, NC=O); 1H NMR
(600 MHz, CDCl3): d 7.95 (s, 1H, triazolyl), 5.69 (br d,
J = 6.0 Hz, 1H, NH), 4.88–5.00 (m, 3H, a-CH and b-
CH2), 4.17 (m, 4H, OCH2CH3), 3.76 (s, 3H, OCH3), 1.36
(s, 9H, t-Bu), 1.35 (apparent q J = 7.2 Hz, 6H, OCH2CH3).
13C NMR (125 MHz, CDCl3): d 169.5 (CO2), 154.8
(NCO2), 140.0 (d, JC,P = 20.4 Hz, triazolyl CH), 126.7 (d,
JC,P = 219.6 Hz, CP), 79.8 [OCCH3)3], 63.4 (d,
JC,P = 5.9 Hz, OCH2CH3a), 63.3 (d, JC,P = 5.8 Hz,
OCH2CH3b), 53.2 (a), 52.5 (OCH3), 50.4 (b), 27.8
[CCH3)3], 15.9 (d, JC,P = 4.4 Hz, OCH2CH3a), 15.8 (d,
JC,P = 4.5 Hz, OCH2CH3b); 31P NMR (240 MHz): d 4.03.
MS (ESI) m/z: 407 [M?H]?, 429 [M?Na]?; HRMS (ESI):
observed, 407.1688, [C15H27N4O7P?H]? requires
407.1690. [a]D = -31.0° (c 1.7, EtOAc). Further elution
with EtOAc gave 12a as pale yellow oil (35 mg, 21%),
identical with the material described below.
Following the general procedure with azide 9c (1.00 g,
4.71 mmol), flash chromatography and elution with (7:3
EtOAc/hexanes) gave 11c as pale yellow oil (722 mg,
41%), identical with the material described below. Further
elution with EtOAc gave 12c as pale yellow oil (774 mg,
44%), identical with the material described below.
Thermal Huisgen cycloaddition of 9d
Following the general procedure with azide 9d (1.00 g,
3.81 mmol), flash chromatography and elution with 1:1
EtOAc/hexanes gave 11d as pale yellow oil (697 mg,
43%), identical with the material described below. Further
elution with EtOAc gave 12d as a pale yellow oil (729 mg,
45%), identical with the material below.
Thermal Huisgen cycloaddition of 9e
Following the general procedure with azide 9e (0.200 g,
0.624 mmol), flash chromatography and elution with (1:1
EtOAc/hexanes) gave 11e as pale yellow oil (123 mg,
41%), identical with the material described below. Further
elution with EtOAc gave 12e as pale yellow oil (129 mg,
42%), identical with the material described below.
(S)-Methyl 2-acetamido-3-(5-(diethoxyphosphoryl)-1H-
1,2,3-triazol-1-yl)propanoate [Ac-5-PO(OEt)2Tza-OMe]
(11b)
Following the general procedure with azide 9b (0.200 g,
1.07 mmol), flash chromatography and elution with 7:3
EtOAc/hexanes gave 11b as pale yellow oil (213 mg,
57%). Rf = 0.35 (EtOAc). IR (thin film) cm-1: 3,283
(NH), 1,748 (OC=O), 1,671 (NC=O). 1H NMR (500 MHz,
CDCl3): d 7.81 (s, 1H, triazolyl), 7.51 (br d, 1H,
J = 8.0 Hz, NH), 4.99 (dd, 1H, J = 8.0, 4.0 Hz, a-H), 4.88
(dd, 1H, J = 14.0, 4.5 Hz, b-Ha), 4.75 (dd, 1H, J = 14.0,
8.0 Hz, b-Hb), 4.05 (m, 4H, 2 9 OCH2), 3.58 (s, 3H,
OCH3) 1.79 (s, 3H, NCOCH3), 1.20 (m, 6H,
2 9 CH2CH3). 13C NMR (125 MHz, CDCl3): d 170.1
(C=O), 169.1 (C=O), 139.9 (d, JC,P = 20.4 Hz, triazolyl
CH), 126.7 (JC,P = 220.4 Hz, CP), 63.6 (d, JC,P = 5.9 Hz,
OCH2), 52.5, 51.8, 50.0, 22.2 (acetyl CH3), 15.81
(JC,P = 3.6 Hz, OCH2CH3a), 15.76 (JC,P = 3.8 Hz,
OCH2CH3b). 31P NMR (120 MHz, CDCl3): d 4.36. MS
(ESI) m/z: 349 [M?H]?, 371 [M?Na]?. HRMS (ESI):
observed, 371.1097, [C12H21N4O6P?Na]? requires
371.1091. [a]D = ?15.7° (c 1.0, CH3Cl). Further elution
with EtOAc gave 12b as pale yellow oil (71 mg, 19%),
identical with the material described below.
Thermal Huisgen cycloaddition of 9f
Following the general procedure with azide 9f (0.100 g,
0.226 mmol), flash chromatography and elution with 2:3
EtOAc/hexanes gave the 11f as a pale yellow oil (53 mg,
39%), identical with the material described below. Further
elution with EtOAc gave the 12f as pale yellow oil (56 mg,
41%), identical with the material described below.
General procedure for Ru(II)-catalysed azide-alkyne
cycloadditions
Cp*RuCl(PPh3)2 (1.5 mol%) was added to a solution of the
azide 9 (0.2–1.2 mmol) and 10 (1 eqv.) in toluene (5–20 mL)
under N2. The resulting solution was stirred at 60°C for 24 h,
then diluted with water (10–20 mL), and extracted with
EtOAc (3 9 30–100 mL). The extract was washed with
brine (10–20 mL), dried and evaporated, and the residue was
subjected to flash chromatography (details) below.
(S)-Allyl 2-acetamido-3-(5-(diethoxyphosphoryl)-1H-1,2,3-
triazol-1-yl)propanoate [Ac-5-PO(OEt)2Tza-OAll] (11c)
(S)-Methyl 2-(t-butoxycarbonylamino)-3-(5-
(diethoxyphosphoryl)-1H-1,2,3-triazol-1-yl)propanoate
[Boc-5-PO(OEt)2Tza-OMe] (11a)
Following the general procedure with azide 9c (250 mg,
1.2 mmol), flash chromatography and elution with 1:1
EtOAc/hexanes gave 11c as pale yellow oil (323 mg,
73%). Rf = 0.30 (EtOAc); IR (thin film) cm-1: 3,284
Following the general procedure with azide 9a (0.10 g,
0.41 mmol), flash chromatography and elution with 1:1
123

Stable triazolylphosphonate analogues of phosphohistidine
863
(NH), 1,747 (OC=O), 1,682 (NC=O). 1H NMR (500 MHz,
CDCl3): d 7.88 (s, 1H, triazolyl), 7.19 (br d, 1H,
J = 8.0 Hz, NH), 5.78 (m, 1H, HC=CH2), 5.23 (ddd, 1H,
J = 17.0, 2.5, 1.0 Hz HC=CH2 trans), 5.15 (ddd, 1H,
J = 10.5, 2.5, 1.0 Hz, HC=CH2 cis), 5.11 (m, 1H, a-H)
4.95 (dd, 1H, J = 14.0, 4.5 Hz, b-Ha), 4.86 (dd, 1H,
J = 14.5, 8.0 Hz, b-Hb), 4.56 (m, 2H, allyl OCH2), 4.13
(m, 4H, 2 9 OCH2CH3), 1.88 (s, 1H, NCOCH3), 1.29 (m,
6H, 2 9 OCH2CH3). 13C NMR (125 MHz, CDCl3): d
170.1 (C=O), 168.5 (C=O), 140.1 (d, JC,P = 20.4 Hz,
triazolyl CH), 131.0, 126.9 (d, JC,P = 220.4 Hz, CP),
118.8, 66.3, 63.73 (JC,P = 4.5 Hz, OCH2CH3a), 63.69
(JC,P = 4.7 Hz, OCH2CH3b), 52.1, 50.2, 22.5 (acetyl
CH3), 16.01 (d, JC,P = 3.6 Hz, OCH2CH3a), 15.96 (d,
JC,P = 3.8 Hz, OCH2CH3b). 31P NMR (120 MHz, CDCl3):
d 4.48. MS (ESI) m/z: 375 [M?H]?, 397 [M?Na]?.
HRMS (ESI): observed, 375.1422, [C14H23N4O6P?H]?
requires 375.1428. [a]D = ?21.0° (c 1.0, CH3Cl). Further
elution with EtOAc gave 12c as pale yellow oil (44 mg,
10%), identical with the material described below.
EtOAc/hexanes gave 11e as pale yellow oil (241 mg,
80%). Rf = 0.20 (1:1 EtOAc/hexanes). IR (thin film)
1
cm-1: 3,305 (NH), 1,747 (m, C=O), 1,716 (s, NC=O). H
NMR (500 MHz, CDCl3): d 7.94 (s, 1H, triazolyl), 7.29
(m, 5H, Ar), 5.75 (br s, 1H, NH), 5.15 (s, 2H, OCH2Ph),
4.88–5.00 (m, 3H, a-CH ? b-CH2), 4.14 (m, 4H,
2 9 OCH2CH3), 1.34 (s, 9H, t-Bu), 1.27–1.36, (m, 6H,
2 9 OCH2CH3). 13C NMR (125 MHz, CDCl3): d 169.1
(CO2), 155.0 (NCO2), 140.3 (d, JC,P = 20.4 Hz, triazolyl
CH), 134.8 (ArC), 128.5 (ArH), 128.4 (ArH), 128.2
(ArH), 127.0 (d, JC,P = 219.6 Hz, CP), 80.1 [OC(CH3)3],
67.6 (OCH2Ph), 63.64 (d, JC,P = 5.7 Hz, OCH2CH3a),
62.58 (d, JC,P = 5.8 Hz, OCH2CH3b), 53.5 (a), 50.6 (b),
28.1 [C(CH3)3], 16.09 (d, JC,P = 4.8 Hz, OCH2CH3a),
16.09 (d, JC,P = 4.9 Hz, OCH2CH3b). 31P NMR
(120 MHz, CDCl3): d 4.60. MS (ESI) m/z: 483 [M?H]?,
505 [M?Na]?. HRMS (ESI): observed, 505.1820,
[C21H31N4O7P?Na]? requires 505.1823. [a]D = ?9.2°
(c 1.0, CHCl3).
(S)-Benzyl 2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-
3-(5-(diethoxyphosphoryl)-1H-1,2,3-triazol-1-
yl)propanoate [Fmoc-5-PO(OEt)2Tza-OBn] (11f)
(S)-Benzyl 2-acetamido-3-(5-(diethoxyphosphoryl)-1H-
1,2,3-triazol-1-yl)propanoate [Ac-5-PO(OEt)2Tza-OBn]
(11d)
Following the general procedure with azide 9f (0.100 g,
0.226 mmol), flash chromatography and elution with 2:3
EtOAc/hexanes gave 11f as pale yellow oil (107 mg,
78%). IR (thin film) cm-1: 3,272 (NH), 1,700–1,750 (br,
OC=O ? NC=O). The NMR spectra showed the presence
Following the general procedure with azide 9d (0.200 g,
0.763 mmol), flash chromatography and elution with 3:2
EtOAc/hexanes gave 11d as pale yellow oil (262 mg,
81%). Rf = 0.40 (EtOAc). IR (thin film) cm-1: 3,290
(NH), 1,747 (OC=O), 1,683 (NC=O). 1H NMR (500 MHz,
CDCl3): d 7.83 (s, 1H, triazolyl), 7.44 (br d, 1H,
J = 8.5 Hz, NH), 7.16 (m, 5H, Ar), 5.09 (dd, 1H, J = 8.0,
4.5 Hz, a-H), 5.02 (d, 2H, J = 6.5 Hz, OCH2Ph), 4.90 (dd,
1H, J = 14.0, 4.5 Hz, b-Ha), 4.81 (dd, 1H, J = 14.0,
8.0 Hz, b-Hb), 4.00 (m, 4H, OCH2CH3), 1.81 (s, 1H,
NCOCH3), 1.18 (m, 6H, OCH2CH3) 13C NMR (125 MHz,
1
of two rotamers. H NMR (500 MHz, CDCl3): d [7.99,
7.98 (2 s, 1H, triazolyl)], 7.74 (d, J = 7.5 Hz, 2H, Ar) 7.54
(d, J = 7.5 Hz, 2H, Ar), 7.23–7.43 (m, 9H, Ar), [6.42,
6.26 (2 br d, J = 7.5 Hz, 1H, NH)], 5.25 (d, J = 5.0 Hz,
2H, OCH2Ph), 4.98–5.21 (m, 3H, a-CH ? b-CH2),
4.07–4.70 (m, 7H, NCO2CH2 ? H90 ? 2 9 OCH2CH3),
1.33 (t, J = 7.0 Hz, 3H, OCH2CH3a), 1.31 (t, J = 9.5 Hz,
3H, OCH2CH3b). 13C NMR (125 MHz, CDCl3): d [168.7,
168.4 (CO2)], [156.9, 155.8, (NCO2)], 143.7, 143.5,
141.44, 141.39, 141.15, 141.14, 140.59, 140.56, [140.34,
140.27 (2 9 d, JC,P = 20.3 Hz, triazolyl CH)], [134.7,
134.6 (ArC)], 130.3, 128.3, 128.6 (ArH), 128.53 (ArH),
128.47 (ArH), 127.7, 126.63, 126.62, [127.3, 127.2 (2 d,
JC,P = 221.4 Hz, CP)], 127.01, 126.99, 126.5, 126.4,
120.0, 119.9, 67.9, 67.4, 65.0, 64.0, [63.99, 63.92 (2 d,
CDCl3):
d 170.1 (C=O), 168.5 (C=O), 140.0 (d,
JC,P = 20.6 Hz, triazolyl CH), 134.5, 128.1, 128.0, 127.8,
126.6 (d, JC,P = 220.4 Hz, CP), 67.2 (OCH2Ph), 63.45 (d,
JC,P = 6.2 Hz, OCH2CH3a), 63.40 (d, JC,P = 6.0 Hz,
OCH2CH3b), 52.8, 50.0, 22.1 (acetyl CH3), 15.70 (d,
JC,P = 4.2 Hz, OCH2CH3a), 15.65 (d, JC,P = 4.2 Hz,
OCH2CH3b). 31P NMR (120 MHz, CDCl3): d 4.35. MS
(ESI) m/z: 425 [M?H]?, 447 [M?Na]?. HRMS (ESI):
observed, 425.1590, [C18H25N4O6P?H]? requires
425.1584 [a]D = ?14.0° (c 1.0, CH3Cl).
JC,P = 5.9 Hz,
OCH2CH3a)],
[63.8,
63.7
(2 d,
JC,P = 5.5 Hz, OCH2CH3b)], [54.3, 54.0 (a)], [50.5, 50.4
(b)], 46.9 (C90), 16.11 (d, JC,P = 6.0 Hz, OCH2CH3a),
16.07 (d, JC,P = 5.2 Hz, OCH2CH3b). 31P NMR
(120 MHz, CDCl3): d 4.54. MS (ESI) m/z: 605 [M?H]?,
627 [M?Na]?. HRMS (ESI): observed, 605.2168,
[C31H33N4O7P?H]? requires 605.2160. [a]D = ?2.3°
(c 1.0, CHCl3).
(S)-Benzyl 2-(t-butoxycarbonylamino)-3-(5-
(diethoxyphosphoryl)-1H-1,2,3-triazol-1-yl)propanoate
[Boc-5-PO(OEt)2Tza-OBn] (11e)
Following the general procedure with azide 9e (0.200 g,
0.624 mmol), flash chromatography and elution with 3:2
123

864
S. Mukai et al.
General procedure for Cu(I)-catalysed azide-alkyne
cycloadditions
OCH2CH3), 52.4, 52.0, 49.8, 22.0 (acetyl CH3), 15.70 (d,
JC,P = 0.6 Hz, OCH2CH3a), 15.65 (JC,P = 0.75 Hz,
OCH2CH3b). 31P NMR (120 MHz, CDCl3): d 7.71. MS
(ESI) m/z: 349 [M?H]?, 371 [M?Na]?. HRMS (ESI):
observed, 371.1086, [C12H21N4O6P?Na]? requires
371.1091. [a]D = ?62.0° (c 1.0, CH3Cl).
CuSO4.5H2O (10 mol %) was added to a solution of the
azide 9 (0.6–1.3 mmol), 10 (1 eqv.) and sodium ascorbate
(10 mol %) in 1:1 t-BuOH/water (5–15 mL). The resulting
solution was stirred for 24 h, diluted with water
(10–20 mL), and extracted with DCM or EtOAc
(3 9 30–100 mL). The organic extract was washed with
brine (10–20 mL), dried and evaporated, and the residue
was subjected to flash chromatography (details below).
(S)-Allyl 2-acetamido-3-(4-(diethoxyphosphoryl)-1H-1,2,3-
triazol-1-yl)propanoate [Ac-4-PO(OEt)2Tza-OAll] (12c)
Following the general procedure with 9c (250 mg,
1.2 mmol), flash chromatography and elution with EtOAc
gave 12c as pale yellow oil (371 mg, 84%). Rf = 0.15
(EtOAc); IR (thin film) cm-1: 3,271 (NH), 1,745 (OC=O),
1,674 (NC=O). 1H NMR (500 MHz, CDCl3): d 8.10 (s, 1H
triazolyl), 7.21 (br d, 1H, J = 7.5 Hz, NH), 5.80 (m, 1H,
HC=CH2), 5.25 (ddd, 1H, J = 17.5, 3.0, 1.5 Hz HC=CH2
trans), 5.19 (ddd, 1H, J = 10.5, 2.5, 1.5 Hz HC=CH2 cis),
4.93 (m, 1H, a-H), 4.85 (m, 2H, b-H), 4.57 (apparent dt,
2H, J = 5.5 Hz, 1.0 Hz, allylic CH2), 4.09 (m, 4H,
OCH2CH3), 1.92 (s, 1H, NCOCH3), 1.251 (t, 3H,
J = 7.5 Hz, OCH2CH3a), 1.248 (t, 3H, J = 7.5 Hz,
OCH2CH3b). 13C NMR (125 MHz, CDCl3): d 170.6
(C=O), 168.3 (C=O), 136.9 (d, JC,P = 239.8 Hz, CH),
131.8 (d, JC,P = 33.1 Hz, triazolyl CH), 130.9 (HC=CH2),
119.2 (HC=CH2), 66.6 (allylic), 63.0 (d, JC,P = 0.9 Hz,
OCH2CH3a), 62.9 (d, JC,P = 0.8 Hz, OCH2CH3b), 52.4,
50.2, 22.5 (acetyl CH3), 16.02 (d, JC,P = 1.3 Hz, OCH2-
CH3a), 15.96 (d, JC,P = 1.1 Hz, OCH2CH3b). 31P NMR
(120 MHz, CDCl3): d 7.52. MS (ESI) m/z: 375 [M?H]?.
HRMS (ESI): observed, 375.1433, [C14H23N4O6P?H]?
requires 375.1428. [a]D = ?25.3° (c 1.0, CH3Cl).
(S)-Methyl 2-(t-butoxycarbonylamino)-3-(4-
(diethoxyphosphoryl)-1H-1,2,3-triazol-1-yl)
propanoate [Boc-4-PO(OEt)2Tza-OMe] (12a)
Following the general procedure with 9a (0.200 g,
0.819 mmol), flash chromatography and elution with
EtOAc gave 12a as pale yellow oil (316 mg, 95%).
Rf = 0.10 (EtOAc/hexanes 1:1); IR (thin film) cm-1: 3,293
(NH), 1,744 (m, C=O), 1,716 (s, NC=O).1H NMR
(600 MHz, CDCl3): d 8.03 (s, 1H, triazolyl), 5.78 (br d,
J = 7.7 Hz, 1H, NH), 4.76 (dd, J = 13.5, 4.5 Hz, 1H,
b-Ha), 4.70 (dd, J = 14.0, 6.5 Hz, 1H, b-Hb), 4.55 (m, 1H,
a-H), 3.99 (m, 4H, OCH2CH3), 3.59 (s, 3H, OCH3), 1.21 (s,
9H, t-Bu), 1.144, (t, J = 7.2 Hz, 3H, OCH2CH3a), 1.140,
(t, J = 7.2 Hz, 3H, OCH2CH3b); 13C NMR (150 MHz,
CDCl3):
d 169.1 (CO2), 154.8 (NCO2), 136.7 (d,
JC,P = 239 Hz, CP), 131.6 (d, JC,P = 32 Hz, triazolyl CH),
80.0 [OCCH3)3], 62.59 (d, JC,P = 5.7 Hz, OCH2CH3a),
62.58 (d, JC,P = 5.9 Hz, OCH2CH3b), 53.3 (a), 52.5
(OCH3), 50.1 (b), 27.7 [CCH3)3], 15.8 (d, JC,P = 6.5 Hz,
OCH2CH3); 31P NMR (240 MHz): d 6.97. MS (ESI) m/z:
407 ([M?H]?; HRMS (ESI): observed, 407.1696,
[C15H27N4O7P?H]? requires 407.1690. [a]D = -15.9°
(c 2.2, EtOAc).
(S)-Benzyl 2-acetamido-3-(4-(diethoxyphosphoryl)-1H-
1,2,3-triazol-1-yl)propanoate [Ac-4-PO(OEt)2Tza-OBn]
(12d)
(S)-Methyl 2-acetamido-3-(4-(diethoxyphosphoryl)-1H-
1,2,3-triazol-1-yl)propanoate [Ac-4-PO(OEt)2Tza-OMe]
(12b)
Following the general procedure with 9d (0.200 mg,
0.763 mmol), flash chromatography and elution with
EtOAc gave 12d as pale yellow oil (282 mg, 87%).
Rf = 0.20 (EtOAc). IR (thin film) cm-1: 3,271 (NH), 1,744
1
Following the general procedure with 9b (0.250 g,
1.34 mmol), flash chromatography and elution with EtOAc
gave 12b as pale yellow oil (425 mg, 91%). Rf = 0.10
(EtOAc). IR (thin film) cm-1: 3,272 (NH), 1,747 (OC=O),
1,673 (NC=O). 1H NMR (500 MHz, CDCl3): d 8.11 (s, 1H,
triazolyl), 7.57 (br d, 1H, J = 7.5 Hz, NH), 4.78 (m, 2H,
b-H), 4.69 (dd, 1H, J = 14.0, 7.0 Hz, a-H), 3.96 (m, 4H,
2 9 OCH2CH3), 3.55 (s, 3H, OCH3) 1.78 (s, 3H, acetyl
CH3), 1.121 (t, 3H, J = 7.0 Hz, OCH2CH3a), 1.118 (t, 3H,
J = 7.0 Hz, OCH2CH3b). 13C NMR (125 MHz,): d 170.5
(C=O), 168.9 (C=O), 136.3 (d, JC,P = 240.2 Hz, CP), 131.6
(d, JC,P = 33.1 Hz, triazolyl CH), 62.7 (d, JC,P = 5.8 Hz,
(OC=O), 1,675 (NC=O). H NMR (500 MHz, CDCl3): d
8.10 (s, 1H, triazolyl), 7.67 (br d, 1H, J = 8.0 Hz, NH),
7.15 (m, 5H, Ar), 5.00 (s, 2H, OCH2Ph), 4.88 (dd, 1H,
J = 7.0, 4.5 Hz, a-H), 4.81 (dd, 1H, J = 14.0, 4.5 Hz,
b-Ha), 4.71 (dd, 1H, J = 14.0, 7.5 Hz, b-Hb), 3.98 (m, 4H,
OCH2CH3) 1.79 (s, 1H, NCOCH3), 1.13 (t, 6H,
J = 7.0 Hz, OCH2CH3) 13C NMR (125 MHz, CDCl3): d
170.3 (C=O), 168.3 (C=O), 136.3 (d, JC,P = 239.8 Hz,
CP), 134.4 (ArC), 131.5 (d, JC,P = 33.1 Hz, triazolyl CH),
128.0 (ArH), 127.9 (ArH), 127.7 (ArH), 67.1 (benzylic),
62.5, (d, JC,P = 5.7 Hz, OCH2CH3) 52.1, 49.7, 22.0 (acetyl
CH3), 15.6 (d, JC,P = 6.4 Hz, OCH2CH3). 31P NMR
123

Stable triazolylphosphonate analogues of phosphohistidine
865
(120 MHz, CDCl3): d 7.73. MS (ESI) m/z: 425 [M?H]?,
447 [M?Na]?. HRMS (ESI): observed, 447.1410,
[C18H25N4O6P?Na]? requires 447.1404. [a]D = ?13.9°
(c 1.0, CH3Cl).
OCH2CH3a), 15.98 (d, JC,P = 1.0 Hz, OCH2CH3b). 31P
NMR (120 MHz, CDCl3): d 7.56. MS (ESI) m/z: 605
[M?H]?, 627 [M?Na]?. HRMS (ESI): observed,
627.1975, [C31H33N4O7P?Na]? requires 627.1979.
[a]D = ?10.4° (c 1.0, CHCl3).
(S)-Benzyl 2-(t-butoxycarbonylamino)-3-(4-
(diethoxyphosphoryl)-1H-1,2,3-triazol-1-yl)
propanoate [Boc-4-PO(OEt)2Tza-OBn] (12e)
(S)-t-butyl 3-(5-(diethoxyphosphoryl)-1H-1,2,3-triazol-1-
yl)-1-(methylamino)-1-oxopropan-2-ylcarbamate [Boc-5-
PO(OEt)2Tza-NHMe] (13)
Following the general procedure with 9e (0.200 mg,
0.624 mmol), flash chromatography and elution with
EtOAc gave 12e as pale yellow oil (265 mg, 88%).
Rf = 0.10 (1:1 EtOAc/hexanes). IR (thin film) cm-1: 3,293
(NH), 1,744 (m, C=O), 1,716 (s, NC=O).1H NMR
(500 MHz, CDCl3): d 8.03 (s, 1H, triazolyl), 7.27 (5H, m,
Ar) 5.71 (br d, J = 7.5 Hz, 1H, NH), 5.12 (d, J = 2.0 Hz,
2H, OCH2Ph), 4.84 (m, 2H, b-H), 4.68 (m, 1H, a-H), 4.10
(m, 4H, OCH2CH3), 1.33 (s, 9H, t-Bu), 1.261, (t,
J = 7.0 Hz, 3H, OCH2CH3a), 1.259, (t, J = 7.0 Hz, 3H,
OCH2CH3b); 13C NMR (125 MHz, CDCl3): d 168.6
(CO2), 154.9 (NCO2), 137.0 (d, JC,P = 239.4 Hz, CP),
134.4 (ArC) 131.2 (d, JC,P = 33.1 Hz, triazolyl CH), 128.4
(ArH), 128.4 (ArH), 128.2 (ArH), 80.4 [OC(CH3)3], 67.8
(OCH2Ph), 62.8 (d, JC,P = 5.8 Hz, OCH2CH3), 53.5 (a),
50.5 (b), 27.9 [CCH3)3], 15.9 (d, JC,P = 6.5 Hz,
OCH2CH3). 31P NMR (120 MHz, CDCl3): d 7.52. MS
(ESI) m/z: 483 [M?H]?, 505 [M?Na]?. HRMS (ESI):
observed, 505.1825, [C21H31N4O7P?Na]? requires
505.1823. [a]D = ?11.1° (c 1.0, CHCl3).
Thirty-three percentage aqueous methylamine (2.4 mL,
25 mmol) was added to a solution of 11a (240 mg,
0.60 mmol) in MeOH (5 mL). The solution was stirred at
room temperature for 4 h, the volatiles were evaporated,
and the residue was subjected to flash chromatography.
Elution with MeOH/DCM (1:20) gave 13 as pale yellow oil
(207 mg, 85%). Rf = 0.20 (1:20 MeOH/DCM). IR (thin
film) cm-1: 3,307 (NH), 1,716 (OC=O), 1,672 (NC=O). 1H
NMR (500 MHz, CD3OD): d 8.11 (s, 1H, triazolyl), 5.12
(br d, 1H, J = 11.0 Hz, a-H), 4.74 (m, 2H, b-H), 4.25 (m,
4H, 2 9 OCH2CH3), 2.75 (s, 1H, NCH3), 1.38, (t, 6H,
J = 7.5 Hz, 2 9 OCH2CH3), 1.33 (s, 9H, t-Bu). 13C NMR
(125 MHz, CD3OD): d 171.5 (CO2), 157.2 (NCO2), 141.6
(d, JC,P = 20.8 Hz, triazolyl CH), 127.9 (d, JC,P
=
222 Hz, CP), 80.9 [OC(CH3)3], 65.5 (d, JC,P = 5.8 Hz,
OCH2CH3a), 65.4 (d, JC,P = 5.9 Hz, OCH2CH3b), 55.7,
52.3, 28.6, 26.5, 16.54 (d, JC,P = 2.9 Hz, OCH2CH3a),
16.49 (d, JC,P = 3.0 Hz, OCH2CH3b). 31P NMR
(120 MHz, CD3OD): d 4.74. MS (ESI) m/z: 406 [M?H]?,
428 [M?Na]?. HRMS (ESI): observed, 428.1673,
[C15H28N5O6P?Na]? requires 428.1669. [a]D = -33.8°
(c 1.0, MeOH).
(S)-Benzyl 2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-
3-(4-(diethoxyphosphoryl)-1H-1,2,3-triazol-1-
yl)propanoate [Fmoc-4-PO(OEt)2Tza-OBn] (12f)
(S)-t-Butyl 3-(4-(diethoxyphosphoryl)-1H-1,2,3-triazol-1-
yl)-1-(methylamino)-1-oxopropan-2-ylcarbamate [Boc-4-
PO(OEt)2Tza-NHMe] (14)
Following the general procedure with 9f (0.100 mg,
0.226 mmol), flash chromatography and elution with
EtOAc gave 12f as a white solid (116 mg, 85%). mp
100–101°C Rf = 0.10 (1:1 EtOAc/hexanes). IR (thin film)
Thirty-three percentage aqueous methylamine (3.0 mL,
31 mmol) was added to a solution of triazole 12a (0.30 g,
0.74 mmol) in MeOH (5 mL). The solution was stirred at
room temperature for 4 h, then the volatiles were evapo-
rated, and the residue was subjected to flash chromatogra-
phy. Elution with MeOH/DCM (1:10) gave 14 as pale yellow
oil (270 mg, 90%). Rf = 0.20 (MeOH/DCM 1:9). IR (thin
film) cm-1: 3,305 (NH), 1,712 (OC=O), 1,666 (NC=O). 1H
NMR (500 MHz, CD3OD): d 8.39 (s, 1H, triazolyl), 4.92
(dd, 1H, J = 12.5 Hz, 3.0 Hz, a-H), 4.63 (m, 2H, b-H), 4.14
(m, 4H, 2 9 OCH2CH3), 2.70 (s, 1H, CONHCH3), 1.32
(s, 9H, t-Bu), 1.288 (t, 3H, J = 7.0 Hz, OCH2CH3a), 1.287
(t, 3H, J = 7.0 Hz, OCH2CH3b). 13C NMR (125 MHz,
1
cm-1: 3,271 (NH), 1,700–1,750 (br, C=O ? NC=O). H
NMR (500 MHz, CDCl3): d 8.10 (s, 1H, triazolyl), 7.73 (d,
J = 7.5 Hz, 2H, Ar) 7.54 (d, J = 7.0 Hz, 2H, Ar),
7.22–7.39 (m, 9H, Ar), 6.31 (br d, J = 7.5 Hz, 1H, NH),
5.17 (br s, 2H, OCH2Ph), 4.90 (m [apparent br d], 2H, b-H),
4.83 (m, 1H, a-H), 4.33 (m [apparent t], 2H, NCO2CH2),
4.09–4.22 (m, 5H, H90 ? 2 9 OCH2CH3), 1.26–1.32 (m,
6H, 2 9 OCH2CH3). The 13C NMR spectra showed the
presence of two rotamers. 13C NMR (125 MHz, CDCl3): d
168.3 (CO2), 155.7 (NCO2), 143.3, 141.0, 137.2 (d,
JC,P = 239.1 Hz, CP), 134.4 (ArC), 131.8 (d,
JC,P = 33.5 Hz, triazolyl CH), 128.5 (ArH), 128.46 (ArH),
128.3 (ArH), 127.6, 126.92, 126.88, 124.93, 124.85, 119.8,
[68.0, 67.2 (benzylic CH2)], 62.9 (d, JC,P = 5.7 Hz,
OCH2CH3), 54.0 (a), 50.4 (b), 46.8 (C90) 16.03 (br s,
CD3OD): d 171.3 (CO2), 157.1 (NCO2), 137.4 (d, JC,P
=
242.2 Hz, CP), 133.5 (d, JC,P = 33.2 Hz, triazolyl
CH), 81.0, 64.45 (d, JC,P = 4.2 Hz, OCH2CH3a), 64.50
123

866
S. Mukai et al.
(d, JC,P = 4.2 Hz, OCH2CH3b), 55.7, 52.3, 28.6, 26.5, 16.6
(d, JC,P = 6.5 Hz, OCH2CH3). 31P NMR (120 MHz,
CD3OD): d 8.73. MS (ESI)m/z: 406 [M?H]?, 428 [M?Na]?.
HRMS (ESI): observed, 406.1855, [C15H28N5O6P?H]?
requires 406.1850. [a]D = -5.4° (c 1.0, MeOH).
room temperature and stirred for 5 h; then the volatiles were
evaporated. The residue was diluted with sat. aq. NaHCO3
(30 mL) and extracted with EtOAc (3 9 50 mL). The
organic extract was washed with brine (30 mL), dried and
evaporated, and the residue was subjected to flash chroma-
tography. Elution with 1:10 MeOH/DCM gave 17 as pale
yellow oil (302 mg, 80%). Rf = 0.35 (1:9 MeOH/DCM). IR
(thin film) cm-1: 3,392 (NH), 1,741 (C=O). 1H NMR
(500 MHz, CDCl3): d 8.19 (s, 1H, triazolyl), 4.75 (dd,
J = 13.5, 4.0 Hz, 1H, b-Ha), 4.54 (dd, J = 13.5, 7.0 Hz, 1H,
b-Hb), 4.17 (m, 4H, 2 9 OCH2CH3), 3.97 (br s, 1H, a), 3.74
(s, 3H, OCH3), 1.312, (t, J = 7.0 Hz, 3H, OCH2CH3a), 1.311,
(t, J = 7.0 Hz, 3H, OCH2CH3b). 13C NMR (150 MHz,
CDCl3): d 172.3 (CO2), 136.9 (d, JC,P = 239.6 Hz, CP),
132.0 (d, JC,P = 33.3 Hz, triazolyl CH), 63.0 (d,
JC,P = 5.8 Hz, OCH2CH3), 54.2 (a), 53.3 (OMe), 52.6 (b),
16.0 (d, JC,P = 6.5 Hz, OCH2CH3). 31P NMR (120 MHz,
CDCl3): d 7.73. MS (ESI) m/z: 307 [M?H]?. HRMS (ESI):
observed, 307.1160, [C10H19N4O5P?H]? requires 307.1166.
[a]D = -4.9° (c 1.0, CHCl3).
(S)-1-(Methylamino)-1-oxo-3-(5-phosphono-1H-1,2,3-
triazol-1-yl)propan-2-aminium bromide [H2N-5-
PO(OH)2Tza-NHMe.HBr] (15)
A solution of 123 (0.10 g, 0.25 mmol) in 33% HBr in AcOH
(2.0 mL, 8.1 mmol) was stirred for 48 h. The volatiles were
evaporated and the residue was purified by Dowex cation-
exchange chromatography. Elution with water gave 15 as a
white solid (54 mg, 71%). Rf = 1.0 on reverse-phase TLC
(H2O), mp 141–143°C. IR (KBr) cm-1: 3,000–3,500 (br,
1
NH3 ? OHs), 1,685 (C=O). H NMR (600 MHz, D2O): d
7.73 (s, 1H, triazolyl), 4.89 (dd, J = 6.6, 2.4 Hz, 2H, b-H),
4.37 (apparent t, J = 6.0 Hz, 1H, a-H), 2.51 (s, 1H, CH3).
13C NMR (125 MHz, D2O): d 166.4 (C=O), 138.4 (br s,
triazolyl CH), 134.6 (d, JC,P = 192.1 Hz, CP), 52.5, 49.0,
25.9 (CH3). 31P NMR (120 MHz, D2O): d -3.48 MS (ESI)
m/z: 250 [M?H]? (where M = free base); HRMS (ESI):
observed, 250.0703, [C6H11N5NaO4P?H]? requires
250.0700. [a]D = ?38.4° (c 1.0, H2O).
(S)-Allyl 2-acetamido-3-hydroxypropanoate [Ac-Ser-OAll]
(22); (S)-Allyl 2-acetamido-3-acetoxypropanoate
[Ac-Ser(OAc)-OAll] (24)
Acetic anhydride (6.30 mL, 67.0 mmol) was added to a
suspension of L-serine (5.90 g, 56.1 mmol) in AcOH
(25 mL). The mixture was stirred for 18 h, and then the
volatiles were evaporated. The residue was dissolved in
DMF (25 mL), and allyl bromide (4.9 mL 56 mmol) and
DIPEA (9.8 mL, 56 mmol) were added. The resulting
solution was stirred for 18 h, diluted with sat. aq. NH4Cl,
and extracted with EtOAc (3 9 50 mL). The organic
extract was washed with brine, dried and evaporated, and
the residue was subjected to flash chromatography. Elution
with EtOAc/hexanes (1:1) gave 24 as a white solid (5.79 g,
45%). mp 81–82°C Rf = 0.40 (EtOAc); IR (thin film)
cm-1: 3,294 (NH), 1,747 (OC=O), 1,661 (NC=O). 1H
NMR (500 MHz, CDCl3): d 7.27 (d, 1H, J = 8.0 Hz, NH),
5.56 (m, 1H, HC = CH2), 4.98 (dd, 1H, J = 17.0, 1.5 Hz,
HC = CH2 trans), 4.89 (dd, 1H, J = 10.0, 1.0 Hz,
HC = CH2 cis), 4.53 (1H, m, a-H), 4.30 (m, 2H, allylic),
4.04 (dd, 2H, J = 6.5, 5.0 Hz, b-H), 1.69 (s, 3H, OCH3),
1.68 (s, 3H, acetyl CH3). 13C NMR (125 MHz, CDCl3): d
169.9 (C=O), 169.6 (C=O), 168.5 (C=O), 130.8 (vinylic
CH), 117.7 (vinylic CH2), 65.2, 62.9, 50.9, 21.7 (CH3),
20.1 (CH3); MS (ESI) m/z: 230 [M?H]?, 252 [M?Na]?.
HRMS (ESI): observed, 230.1020, [C10H15NO5?H]?
requires 230.1023. [a]D = ?58.8° (c 1.0, CH3Cl).
(S)-1-(Methylamino)-1-oxo-3-(4-phosphono-1H-1,2,3-
triazol-1-yl)propan-2-aminium bromide [H2N-4-
PO(OH)2Tza-NHMe.HBr] (16)
A solution of 14 (0.10 g, 0.25 mmol) in 33% HBr in AcOH
(2.00 mL, 8.1 mmol) was stirred for 48 h. The volatiles
were evaporated and the residue was purified by Dowex
cation-exchange chromatography. Elution with water gave
16 as a white solid (56 mg, 73%). Rf = 1.0 on reverse-
phase TLC (H2O). mp 145–146°C. IR (KBr disk) cm-1
:
3,100–3,500 (br, NH3 ? OHs), 1,646 (C=O). 1H NMR
(500 MHz, D2O): d 8.14 (s, 1H, triazolyl), 4.97 (d,
J = 5.5 Hz, 2H, b-H), 4.54 (apparent t, J = 5.0 Hz, 1H, a-
H), 2.69 (s, 1H, CH3). 13C NMR (125 MHz, D2O): d 167.0
(C=O), 144.5 (d, JC,P = 214.7 Hz, CP), 129.7 (d,
JC,P = 28.9 Hz, triazolyl CH), 52.9, 49.8, 26.3 (CH3). 31P
NMR (120 MHz, D2O): d 1.55 MS (ESI) m/z: 272
[M?Na]?, 294 [M?2Na-H]? (where M = free base);
HRMS (ESI): observed, 272.0522, [C6H12N5NaO4P?Na]?
requires 272.0519. [a]D = ?27.2° (c 1.0, H2O).
(S)-Methyl 2-amino-3-(4-(diethoxyphosphoryl)-1H-1,2,3-
triazol-1-yl)propanoate [H2N-4-PO(OH)2Tza-OMe.HBr]
(17)
Further elution with EtOAc gave 22 as brown oil
(2.21 g, 21%). Rf = 0.15 (EtOAc); IR (thin film) cm-1
:
TFA (1.0 mL, 13 mmol) was added to a solution of 12a in
DCM (10 mL) at 0°C. The solution was allowed to warm to
3,366 (NH), 1,741 (OC=O), 1,655 (NC=O). 1H NMR
(400 MHz, CDCl3): d 7.28 (d, 1H, J = 7.6 Hz, NH), 5.77
123

Stable triazolylphosphonate analogues of phosphohistidine
867
(S)-2-Acetamido-3-(4-(diethoxyphosphoryl)-1H-1,2,3-
triazol-1-yl)propanoic acid [Ac-4-PO(OEt)2Tza-OH] (27)
(m, 1H, HC=CH2), 5.20 (ddd, 1H, J = 17.6, 3.2, 1.6 Hz,
HC=CH2 trans), 5.11 (ddd, 1H, J = 10.4, 2.4, 1.2 Hz,
HC=CH2 cis), 4.51 (2H, dt, J = 6.0 Hz, 1.2 Hz, allylic
CH2), 4.47 (dd, 1H, J = 8.0, 4.0 Hz, a-H), 3.83 (dd, 1H,
J = 11.2, 4.0 Hz, b-Ha), 3.71 (dd, 1H, J = 11.2, 3.2 Hz,
b-Hb), 1.90 (s, 1H, acetyl CH3). 13C NMR (100 MHz,
CDCl3): d 171.2 (C=O), 170.1 (C=O), 131.2 (vinylic CH),
118.3 (vinylic CH2), 65.8, 62.0, 54.5, 22.3 (acetyl CH3).
MS (ESI) m/z: 188 [M?H]?, 210 [M?Na]?. HRMS (ESI):
observed, 188.0914, [C8H13NO4?H]? requires 188.0917.
[a]D = ?33.6° (c 1.0, CH3Cl).
Method A, saponification using LiOH: LiOH.H2O
(60 mg, 1.44 mmol) was added to a solution of 12b
(0.500 g, 1.44 mmol) in 3:1 MeOH/water (15 mL) at 0°C.
After 2 h, the reaction mixture was acidified to pH 4–5
with sat. aq. KHSO4 and then extracted with EtOAc
(3 9 50 mL). The organic extract was washed with brine
(10 mL), dried and evaporated, and the residue was sub-
jected to flash chromatography. Elution with 1:4 MeOH/
DCM gave 27 as colourless oil (144 mg, 30%). Identical in
every respect except optical activity with the material
described below. [a]D = ?5.1° (c 1.0, MeOH).
(S)-Benzyl 2-acetamido-3-hydroxypropanoate [Ac-Ser-
OBn] (23); (S)-Benzyl 2-acetamido-3-acetoxypropanoate
[Ac-Ser(OAc)-OBn] (25)
Method B, saponification using K2CO3: K2CO3 (0.200 g,
1.44 mmol) was added to a solution of 12b (0.500 g,
1.44 mmol) in 3:1 MeOH/water (15 mL) at 0°C. The
solution was allowed to warm to room temperature and
stirred for 2 h, then acidified to pH 4 with sat. aq. KHSO4,
and extracted with EtOAc (3 9 50 mL). The organic
extract was washed with brine (10 mL), dried and evapo-
rated, and the residue was subjected to flash chromatog-
raphy. Elution with 1:4 MeOH/DCM gave 27 as colourless
oil (226 mg, 47%). Rf = 0.10 (1:4 MeOH/DCM). IR (thin
film) cm-1: 3,293 (NH), 1,720 (C=O), 1,618 (NC=O).1H
NMR (500 MHz, CD3OD): d 8.47 (s, 1H, triazolyl), 5.06
(m, 1H, b-Ha), 4.78–4.86 (m, 2H, b-Hb?a-H), 4.20 (m,
4H, OCH2CH3), 1.96 (s, 3H, NCH3), 1.361 (t, J = 7.0 Hz,
3H, OCH2CH3a), 1.360 (t, J = 7.2 Hz, 3H, OCH2CH3b);
13C NMR (125 MHz, CD3OD): d 175.7 (CO), 173.9 (CO),
138.0 (d, JC,P = 242.3 Hz, CP), 134.3 (d, JC,P = 33.3 Hz,
triazolyl CH), 65.47 (d, JC,P = 0.9 Hz, OCH2CH3a), 65.43
(d, JC,P = 0.7 Hz, OCH2CH3b), 57.0 (a), 53.8 (b), 23.5
(CH3), 17.4 (d, JC,P = 6.5 Hz, OCH2CH3). 31P NMR
(120 MHz, CD3OD): d 8.90. MS (ESI) m/z: 335 [M?H]?,
357 [M?Na]. HRMS (ESI): observed, 357.1046,
[C11H19N4O6P?Na]? requires 357.0934. [a]D = ?8.8°
(c 1.0, MeOH).
Acetic anhydride (6.30 mL, 67.0 mmol) was added to a
suspension of L-serine (5.90 g, 56.1 mmol) in AcOH
(25 mL). The mixture was stirred for 18 h, and then the
volatiles were evaporated. The residue was dissolved in
DMF (25 mL), and benzyl bromide (6.70 mL 56.4 mmol)
and DIPEA (9.80 mL, 56.3 mmol) were added. The
resulting solution was stirred for 18 h, diluted with sat. aq.
NH4Cl, and then extracted with EtOAc (3 9 50 mL). The
organic extract was washed with brine (30 mL), dried and
evaporated, and the residue was subjected to flash chro-
matography. Elution with EtOAc/hexanes (1:1) gave 25 as
a white solid (7.05 g, 45%). Rf = 0.50 (EtOAc). IR (thin
film) cm-1: 3,293 (NH), 1,746 (OC=O), 1,661 (NC=O). 1H
NMR (400 MHz, CDCl3): d 7.28 (m, 5H, Ar), 7.00 (br d,
1H, J = 8.0 Hz, NH), 5.13 (dd, 2H, J = 31.6 Hz, 12.4 Hz,
OCH2Ph), 4.87 (m, 1H, a-H), 4.42 (dd, 1H, J = 11.2 Hz,
4.0 Hz, b-Ha), 4.29 (dd, 1H, J = 11.2 Hz, 3.6 Hz, b-Hb),
1.97 (s, 1H, NCOCH3), 1.87 (s, 1H, COCH3). 13C NMR
(100 MHz, CDCl3): d 170.1 (C=O), 169.9 (C=O), 169.1
(C=O), 134.7 (ArC), 128.2 (ArH), 128.1 (ArH), 127.9
(ArH), 67.0, 63.5, 51.3, 22.4 (CH3), 20.0 (CH3). MS (ESI)
m/z: 280 [M?H]?, 302 [M?Na]?. HRMS (ESI): observed,
302.1005,
[C14H17NO5?Na]?
requires
302.0999.
[a]D = ?24.5° (c 1.0, CH3Cl).
(S)-2-Acetamido-3-(5-phosphono-1H-1,2,3-triazol-1-
yl)propanoic acid [Ac-4-PO(OH)2Tza-OH] (29)
Further elution with EtOAc gave 23 as a white solid
(4.15 g, 31%). mp 60–62°C. Rf = 0.20 (EtOAc); IR (thin
film) cm-1: 3,313 (NH), 1,742 (OC=O), 1,657 (NC=O). 1H
NMR (400 MHz, CDCl3): d; 7.30 (m, 5H, Ar), 7.14 (br d,
1H, J = 7.6 Hz, NH), 5.14 (d, 2H, J = 1.2 Hz, OCH2Ph),
4.63 (m, 1H, a-H), 3.95 (dd, 1H, J = 11.6 Hz, 4.0 Hz,
b-Ha), 3.81 (dd, 1H, J = 11.2 Hz, 3.2 Hz, b-Hb), 1.97 (s,
3H, CH3). 13C NMR (100 MHz, CDCl3): d 171.1 (C=O),
170.3 (C=O), 135.0 (ArC), 128.4 (ArH), 128.2 (ArH),
127.8 (ArH), 67.1 (OCH2Ph), 62.4, 54.7, 22.6 (CH3). MS
(ESI) m/z: 238 [M?H]?, 260 [M?Na]?. HRMS (ESI):
observed, 238.1077, [C12H16NO?H]? requires 238.1074.
[a]D = ?14.3° (c 1.0, CH3Cl).
A solution of 11d (0.20 g, 0.47 mmol) in 33% HBr in
AcOH (4.0 mL, 16 mmol) was stirred for 48 h. The vola-
tiles were evaporated and the residue was purified by
Dowex anion exchange chromatography. Elution with
water gave 29 as a white solid (90 mg, 69%). mp
146–148°C. IR (KBr disk) cm-1: 3,000–3,500 (br,
NH3 ? OHs), 1,717 (C=O), 1,650 (NC=O). 1H NMR
(500 MHz, D2O): d 7.83 (s, 1H, triazolyl), 4.71 (dd, 1H,
J = 14.0, 3.5 Hz, a-H), 4.54 (dd, 1H, J = 9.0, 3.5 Hz,
b-Ha), 4.46 (dd, 1H, J = 14.0, 9.5 Hz, b-Hb), 1.38 (s, 3H,
123

868
S. Mukai et al.
CH3). 13C NMR (125 MHz, D2O): d 173.8 (C=O), 170.5
(C=O), 136.1 (d, JC,P = 197.9 Hz, CP), 134.5 (br s, triaz-
olyl), 51.6, 51.1, 21.5 (CH3). 31P NMR (120 MHz, D2O): d
-2.65. MS (ESI) m/z: 279 [M?H]?, 301 [M?Na]?.
HRMS (ESI): observed, 301.0305 [C7H11N4O6P?Na]?
requires 301.0308; [a]D = -23.8° (c 1.0, H2O).
JC,P = 20.6 Hz, triazolyl CH), 128.4 (d, JC,P = 222.5 Hz,
CP), 81.1 [OC(CH3)3], 66.3 (d, JC,P = 5.5 Hz, OCH2-
CH3a), 66.2 (d, JC,P = 5.5 Hz, OCH2CH3b), 57.1 (a), 54.5
(b), 29.5 (CH3)3, 17.4 (d, JC,P = 6.3 Hz, OCH2CH3a), 17.3
(d, JC,P = 5.9 Hz, OCH2CH3b). MS (ESI) m/z: 393
[M?H]?, 415 [M?Na]?.
A sample that had partially degraded was further puri-
fied by HPLC to compare with the reported spectra in
(S)-2-Acetamido-3-(4-phosphono-1H-1,2,3-triazol-1-
yl)propanoic acid [Ac-5-PO(OH)2Tza-OH] (30)
1
CDCl3. H NMR (500 MHz, CDCl3): d 8.00 (br s, 1H,
triazolyl), 5.90 (br s, 1H, NH) 5.07 (br m, 1H, a-CH), 4.91
(br m, 2H, b-CH2), 4.22 (m, 4H, OCH2CH3), 1.35–1.40 (m,
6H, OCH2CH3), 1.36 (s, 9H, t-Bu). 13C NMR (125 MHz,
CDCl3): d 171.1 (br, CO2), 155.8 (NCO2), 140.5 (br s,
triazolyl CH), 126.7 (br d, JC,P = 231.5 Hz, CP), 80.8
[OCCH3)3], 64.3 (br s, OCH2CH3), 53.8 (a), 51.1 (b), 28.2
[CCH3)3], 16.1 (br s, OCH2CH3); [a]D = ?1.2° (c 0.5,
CDCl3). The NMR data were similar to those reported (Kee
et al. 2010). See Table 3 for enantiopurity.
Method A, from the benzyl ester 12d: A solution of 12d
(200 mg, 0.47 mmol) in 33% HBr in AcOH (4.0 mL,
16 mmol) was stirred for 48 h. The volatiles were evapo-
rated and the residue was subjected to Dowex anion-
exchange chromatography. Elution with water gave 30 as a
white solid (88 mg, 67%). mp 135–137°C. IR (KBr disk)
cm-1: 3,100–3,500 (br, NH3 ? OHs), 1,732 (C=O), 1,637
1
(NC=O). H NMR (500 MHz, D2O): d 8.10 (s, 1H, triaz-
olyl), 4.71 (dd, 2H, J = 12.5, 5.5 Hz, b-H), 4.60 (dd, 1H,
J = 13.5, 7.0 Hz, a-H), 1.67 (s, 3H, CH3). 13C NMR
(S)-2-(t-Butoxycarbonylamino)-3-(4-(diethoxyphosphoryl)-
1H-1,2,3-triazol-1-yl)propanoic acid [Boc-4-
PO(OEt)2Tza-OH] (32).
(125 MHz, D2O):
d
173.9, 171.1, 139.9 (d,
JC,P = 223.7 Hz, CP), 131.5 (d, JC,P = 30.2 Hz, triazolyl),
52.2, 50.5, 21.5 (CH3). 31P NMR (120 MHz, D2O): d 2.68.
MS (ESI) m/z: 279 [M?H]?, 301 [M?Na]?. HRMS (ESI):
observed 279.0491 [C7H11N4O6P?H]? requires 279.0489.
[a]D = ?10.0° (c 1.0, H2O).
LiOH.H2O (5 mg, 0.1 mmol) was added to a stirred solu-
tion of 12a (51 mg, 0.13 mmol) in 3:1 MeOH/water
(4 mL) at 0°C. After 2 h, the reaction mixture was acidified
to pH 4 with saturated aqueous KHSO4, and then extracted
with DCM (3 9 40 mL). The extract was washed with
brine (10 mL), dried and evaporated, and the residue was
subjected to flash chromatography. Elution with 1:4
MeOH/DCM gave 32 as colourless oil (42 mg, 88%).
Rf = 0.10 (1:4 MeOH/DCM); 1H NMR (500 MHz,
CD3OD): d 8.39 (s, 1H, triazolyl), 5.01 (br dd, J = 17.5,
10.2 Hz, 1H, b-CH2a), 4.71 (m [apparent dd], 1H,
b-CH2b), 4.51 (m, 1H, a-CH), 4.17 (m, 4H, OCH2CH3),
Method B, from the carboxylic acid 27: A solution of 27
(0.20 g, 0.60 mmol) in 33% HBr in AcOH (5 mL,
20 mmol) was stirred for 48 h. The volatiles were evapo-
rated and the residue was subjected to Dowex anion-
exchange chromatography. Elution with water gave 30
(117 mg, 70%) as a white solid, [a]D = ?6.1° (c 1.0,
H2O), identical in every other respect with the material
described above.
1.37 (s, 9H, t-Bu), 1.32 (t, J = 7.1 Hz, 6H, OCH2CH3). 13
C
(S)-2-(t-Butoxycarbonylamino)-3-(5-(diethoxyphosphoryl)-
1H-1,2,3-triazol-1-yl)propanoic acid [Boc-5-
PO(OEt)2Tza-OH] (31).
NMR (125 MHz, CD3OD): d 175.2 (CO2), 158.2 (NCO2),
138.0 (d, JC,P = 242.2 Hz, CP), 134.2 (d, JC,P = 31.8 Hz,
triazolyl CH), 81.4 [OCCH3)3], 65.4 (d, JC,P = 3.4 Hz,
OCH2CH3a), 65.3 (d, JC,P = 3.3 Hz, OCH2CH3b), 57.5
(a), 54.0 (b), 29.5 (CH3)3, 17.4 (d, JC,P = 6.5 Hz,
OCH2CH3). MS (ESI) m/z: 415 [M?Na]?.
LiOH.H2O (5 mg, 0.1 mmol) was added to a stirred solu-
tion of 11a (51 mg, 0.13 mmol) in 3:1 MeOH/water
(4 mL) at 0°C. After 2 h, the reaction mixture was acidified
to pH 4 with sat. aq. KHSO4, and then extracted with DCM
(3 9 40 mL). The organic extract was washed with brine
(10 mL), dried and evaporated, and the residue was puri-
fied by flash chromatography. Elution with 1:9 MeOH/
DCM gave 26 as colourless oil (41 mg, 86%). Rf = 0.10
(1:9 MeOH/DCM); 1H NMR (500 MHz, CD3OD): d 8.09
(s, 1H, triazolyl), 5.15 (m, a-CH), 4.74 (m, 2H, b-CH2),
4.25 (m, 4H, OCH2CH3), 1.36 (t, J = 7.0 Hz, 6H,
OCH2CH3), 1.30 (s, 9H, t-Bu). 13C NMR (125 MHz,
CD3OD): 175.5 (CO2), 158.0 (NCO2), 142.3 (d,
A sample that had partially degraded was further puri-
fied by HPLC to compare with the reported spectra in
1
CDCl3. H NMR (600 MHz, CDCl3): d 8.28 (br s, 1H,
triazolyl), 6.4 (v br s, OH), 5.55 (br s, 1H, NH), 4.98 (m,
2H, b-CH2), 4.75 (br s, 1H, a-H), 4.20 (br m, 4H,
OCH2CH3), 1.43 (s, 9H, t-Bu), 1.34 (2 9 overlapping t at
1.34 and 1.33 [apparent q], J = 6.0 Hz, 6H, OCH2CH3).
13C NMR (150 MHz, CDCl3): d 170.5 (br CO2), 155.4
(NCO), 136.4 (br d, JC,P = 231.6 Hz, CP), 132.5 (br s,
triazolyl CH), 80.7 [OCCH3)3], 63.85 (br s, OCH2CH3a),
63.76 (br s, OCH2CH3b), 53.7 (br, a), 51.2 (b), 28.2
123

Stable triazolylphosphonate analogues of phosphohistidine
869
1
(CH3)3, 16.1 (br s, OCH2CH3); [a]D = ?40.5° (c 0.7,
CHCl3). The NMR data were similar to those reported (Kee
et al. 2010). See Table 3 for enantiopurity.
NH3 ? OHs), 1,716 (C=O); H NMR (600 MHz, D2O) d:
8.12 (s, 1H, triazolyl), 4.98–5.00 (m, 2H, b-H), 4.47 (dd,
J = 5.4, 4.8 Hz, 1H, a-H). 13C NMR (125 MHz, D2O): d
170.7 (CO), 143.7 (d, JC,P = 217.6 Hz, CP), 130.2 (d,
JC,P = 31.7 Hz, triazolyl CH), 54.5 (a), 50.0 (b). 31P NMR
(120 MHz): d 1.95; MS (ESI) m/z: 237 [M?H]? (where
M = free base); HRMS (ESI): observed, 237.0385,
[C5H10N4O5P]? requires 237.0383. [a]D = -25.0° (c 0.5,
H2O).
(S)-1-carboxy-2-(5-phosphono-1H-1,2,3-triazol-1-
yl)ethanaminium bromide [H2N-5-PO(OH)2Tza-OH.HBr]
(33)
Method A, from the carboxylic acid 31: A solution of 31
(0.090 g, 0.23 mmol) in 33% HBr in AcOH (2 mL,
8 mmol) was stirred for 48 h. The volatiles were evapo-
rated and the residue was subjected to Dowex anion-
exchange chromatography. Elution with water gave 33 as a
white solid (51 mg, 70%), identical in every respect except
optical rotation with the material described below.
(S)-Benzyl 2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-
3-hydroxypropanoate [Fmoc-Ser-OBn] (36)
Benzyl bromide (1.40 mL, 11.8 mmol) was added to a
solution of Fmoc-L-serine monohydrate (2.00 g,
5.79 mmol) and DIPEA (1.00 mL, 11.6 mmol) in DMF
(20 mL) at 0°C. The solution was allowed to warm to room
temperature, stirred for 18 h, diluted with sat. aq. NH4Cl
(30 mL), and extracted with EtOAc (3 9 50 mL). The
organic extract was washed with brine (50 mL), dried and
evaporated, and the residue was subjected to flash chro-
matography. Elution with 2:3 EtOAc/hexanes gave 36 as a
Method B, from the benzyl ester 11e: A solution of 11e
(0.100 g, 0.207 mmol) in 33% HBr in AcOH (2 mL,
8 mmol) was stirred for 48 h. The volatiles were evapo-
rated and the residue was subjected to Dowex anion-
exchange chromatography. Elution with water gave 33 as a
white solid (43 mg, 65%), mp 138–140°C. IR (KBr disk)
cm-1: 3,200–3,500 (br, NH3 ? OHs), 1,600–1,750 (br,
1
white solid (2.18 g, 90%). H NMR (500 MHz, CDCl3): d
1
obscures C=O); H NMR (600 MHz, D2O) d: 7.86 (s, 1H,
7.81 (d, J = 7.5 Hz, 2H, Ar), 7.70 (br d, J = 5.5 Hz, 2H,
Ar), 7.46 (apparent t, J = 7.5 Hz, 4H, Ar), 6.47 (br d,
J = 5.0 Hz, 1H, NH), 5.27 (s, 2H, OCH2Ph), 4.66 (m, 1H,
a-H), 4.53 (m [apparent t], 1H, NCO2CH2a), 4.43 (m
[apparent t], 1H, NCO2CH2b), 4.66 (t, J = 7.0 Hz, 1H,
H90), 4.12 (m [apparent br d], 1H, b-Ha), 3.99 (m [apparent
br d], 1H, b-Hb). 13C NMR (125 MHz, CD3OD): d 170.4
(CO2), 156.3 (NCO2), 143.5 (Ar), 143.3 (Ar), 140.9 (Ar),
134.8 (Ar), 128.2 (Ar), 128.0 (Ar), 127.7 (Ar), 127.3 (Ar),
126.7 (Ar), 119.6 (Ar), 67.0, 66.9, 62.4, 56.0, 46.6. MS
(ESI) m/z: 440 [M?Na]?. [a]D = ?0.7° (c 1.0, DCM) [lit.
triazolyl), 5.11 (dd, J = 15.0, 3.6 Hz, 1H, b-Ha) 4.98 (dd,
J = 15.6, 7.2 Hz, 1H, b-Hb), 4.27 (dd, J = 7.8, 3.6 Hz,
1H, a-H); 13C NMR (125 MHz, D2O): d 169.6 (CO), 139.9
(d,
JC,P = 18.4 Hz,
triazolyl
CH),
135.6
(d,
JC,P = 191.8 Hz, CP), 53.2 (a), 49.0 (b); 31P NMR
(120 MHz): d –3.14; MS (ESI) m/z: 237 [M?H]?, 259
[M?Na]?, 281 [M?2 Na]? (where M = free base);
HRMS (ESI): observed, 259.0201, [C5H10N4O5P?Na]?
requires 259.0203; [a]D = -7.7° (c 0.5, H2O).
1
(S)-1-Carboxy-2-(4-phosphono-1H-1,2,3-triazol-1-
yl)ethanaminium bromide [H2N-4-PO(OH)2Tza-OH.HBr]
(34).
(Huang et al. 2004) ?0.6° (c 1.0, DCM)]. The H and 13C
NMR data are similar to those reported (Huang et al. 2004).
Method A, from the carboxylic acid 32: A solution of 32
(0.090 g, 0.23 mmol) in 33% HBr in AcOH (2 mL,
8 mmol) was stirred for 48 h. The volatiles were evapo-
rated and the residue was subjected to Dowex anion-
exchange chromatography. Elution with water 34 as a
white solid (52 mg, 72%), identical in every respect, except
optical activity, with the material described below.
Results and discussion
Our synthetic endeavours began with the thermal Huisgen
cycloaddition (Dondoni et al. 2004) of the known phos-
phonate 10 (Vuilhorgne et al. 2003) and serine-derived
azide 9a (Boger et al. 1994). Gratifyingly, this reaction
gave both of the desired triazole regioisomers 11a and 12a
(Scheme 1; Table 1), which were easily separable by col-
umn chromatography. The constitution of the adducts was
established by an HMBC experiment; specifically, a three-
bond correlation between the triazolyl proton and the
b-carbon identified the 1,4-substituted triazole 12a.
Method B, from the benzyl ester 12e: Asolution of 12e
(0.100 g, 0.207 mmol) in 33% HBr in AcOH (2 mL,
8 mmol) was stirred at room temperature for 48 h. The
volatiles were evaporated and the residue was subjected to
Dowex anion-exchange chromatography. Elution with
water gave 34 as a white solid (44 mg, 67%), mp
133–135°C. IR (KBr disk) cm-1: 3,300–3,500 (br,
As expected, the Cu(I)-catalysed ‘click’ reaction (Ros-
tovtsev et al. 2002) afforded the 1,4-disubstituted isomer
12a exclusively, in excellent yield, whereas Ru(II)
123

870
S. Mukai et al.
O
P
O
O
O
Br
H3N
H
H
OEt
OEt
BocN
BocN
O
O
O
OMe
a
N
H
N
H
H
H
H
RN
RN
RN
'
OR'
10
conditions
OR'
OR
N
N
N
N
N
b
N
N
N
N
+
N
N
O
O
O
N3
N
N
N
N
P
P
P
P
EtO
EtO
HO
O
OEt
OEt
OH
P
EtO
EtO
11a
13
15
EtO
OEt
O
−
−
−
9a
f
11a
f
12a f
O
O
O
Br
H3N
H
H
BocN
BocN
Scheme 1 Carboxyl-protecting groups in azidoalanine derivatives
significantly affect the regioselectivity of dipolar cycloadditions to
give the corresponding triazolylalanine phosphonates. See Table 1 for
the nature of the R/R0 groups, conditions and yields
OMe
a
N
H
N
H
N
N
N
N
N
b
N
N
N
P
N
P
EtO
EtO
EtO
HO
HO
P
EtO
O
O
O
12a
14
16
Table 1 Dipolar cycloadditions of 9a–f and 10 under various
conditions
c
O
O
H
Azide
R
R0
Product
Yield
H2N
N
OMe
AcN
H
OMe
D
CuI
RuII
O
N
e
N
N
N
N
9a
9b
9c
9d
9e
9f
Boc
Ac
Me
Me
All
Bn
Bn
Bn
11a
12a
11b
12b
11c
12c
11d
12d
11e
12e
11f
21
58
26
60
41
44
43
45
41
42
39
41
–
56
21
57
19
73
10
81
–
N
EtO
P
95
–
EtO
O
P
EtO
O
EtO
17
19
91
–
Ac
d
84
–
O
H
AcN
Ac
OMe
87
–
N
N
N
Boc
Fmoc
80
–
88
–
EtO
P
EtO
O
78
–
18
12f
85
Scheme 2 Reagents, conditions and yields: a MeNH2, MeOH/H2O
(13, 85%; 14 90%); b 33% HBr/AcOH (15, 71%; 16, 73%); c TFA,
DCM (80%); d Ac2O or AcCl, NEt3, DCM (0%); e HCTU, i-Pr2NEt,
DCM (0%)
Conditions: D: PhMe, 110°C; CuI: Cu2SO4, sodium ascorbate, 1:1 t-
BuOH-H2O; RuII: Cp*RuCl(PPh3)2, PhMe, 60°C
catalysis (Zhang et al. 2005) allowed a substantial
improvement in the yield of the 1,3-isomer 11a (Table 1).
Amidation (Lesma et al. 2007) of the methyl esters 11a
and 12a proceeded smoothly, providing the N-methyla-
mides 13 and 14, respectively, in excellent yields
(Scheme 2). Protonolysis then provided the first target
haptens, ready for bioconjugation through the amino group,
as the hydrobromides 15 and 16.
hydrazoic acid (Scheme 3). The azide-alkyne cycloaddi-
tions of 9b under thermal, Cu(I)- or Ru(II)-catalysed con-
ditions were very similar to those for the analogues
carbamate 9a (Scheme 1; Table 1).
Saponification of the resultant triazoylalanines 11b and
12b proved problematic (Scheme 4). The reaction of 12b
with LiOH at room temperature gave only N-acetyldehy-
droalanine (28). At lower temperature, 28 was not
observed, but the yield of the desired carboxylic acid 27
was poor. This, coupled with the earlier observation of 28,
the product of an E1cb reaction, indicated the likely for-
mation of an intermediate enolate and hence at least partial
racemisation of 12b/27 under the reaction conditions.
Indeed, when the saponification of 12b was repeated with
the weaker base K2CO3, at room temperature, the desired
carboxylic acid 27 was isolated in improved, although still
modest, yield, and the specific rotation of this material was
greater than that of the LiOH-derived product (Table 2). At
In contrast, although efficient and selective removal of
the Boc group of 12a was achieved, all efforts to acetylate
the resulting amine 17 to give 18 were unsuccessful. An
attempted coupling with N-acetylserine to give 19 also
failed. It is unclear at this stage what prevents these
seemingly simple transformations. Thus, to access haptens
7 and 8 (Fig. 1) it was necessary to acetylate at an earlier
point.
To this end, the known azide 9b (Davoli et al. 1995) was
prepared in 60% yield from methyl N-acetylserinate (20)
(Maruyama et al. 1992) using a Mitsunobu reaction with
123

Stable triazolylphosphonate analogues of phosphohistidine
871
O
O
Table 2 Deprotection of N-acetyltriazolylalaninephosphonic acid
derivatives (Scheme 4)
H
H
AcN
AcN
OMe
OR
a
Compound
Conditions
Yield
(%)
Specific
rotation
Solvent
(c 1.0)
HO
N3
20
9b, R = Me
9c, R = All
9d, R = Bn
27
27
27
27
29
30
30
LiOH, 0°C
LiOH, RT
K2CO3, 0°C
K2CO3, RT
from 11d
from 27
31
0
?5.1°
–
MeOH
–
c
O
O
H
O
0
–
–
H
H3N
HO
AcN
AcN
47
69
70
67
?8.8°
-23.8°
?6.1°
?10.0°
MeOH
H2O
H2O
H2O
O
OR
OR
b
+
HO
22 R = All
23 R = Bn
AcO
21
24, R = All
25, R = Bn
from 12d
Scheme 3 Reagents, conditions and yields: a PPh3, HN3, DIAD,
THF, PhMe (9b, 60%); b (1) Ac2O, AcOH; (2) AllBr or BnBr, i-
Pr2NEt, DMF (22, 21%; 24, 45%; 23, 31%; 25, 45% [all over two
steps]); c PPh3, HN3, DEAD (9c) or DIAD (9d), THF, PhMe (9c,
61%; 9d, 61%)
followed by selective alkylation gave the allyl and benzyl
esters, 22 and 23, respectively, after separation from the
N,O-diacetyl derivatives, 24 and 25. Mitsunobu reactions
of 22 and 23 with hydrazoic acid provided the desired
azides.
O
O
O
H
H
H
AcN
AcN
AcN
OMe
OH
OH
N
N
a
N
N
The thermal Huisgen cycloadditions of 9c and 9d with
alkyne 10 produced more of the 1,5-regiosiomers 11c and
11d relative to the reaction of the corresponding methyl
ester 9b (Scheme 1; Table 1). This innate tendency had
little effect on the Cu(I)-catalysed reactions, with only the
1,4-disubstituted triazoles 12c and 12d observed in both
cases. Conversely, relative to the methyl ester 9b, the allyl-
protecting group in 9c improved the regioselectivity of the
Ru(II)-catalysed reaction, giving the 1,5-isomer 11c as the
predominant product. The reaction of the still bulkier
benzyl ester 9d was completely regioselective, with only
the desired 1,5-triazole 11d observed, and isolated in
excellent yield. At this stage the enhanced regioselectivity
is attributed to steric effects; however, chelation of the
catalyst by the pendant p-systems cannot be ruled out.
Attempts to remove the allyl-protecting groups in 11c/
12c under standard conditions [Pd(PPh3)4, morpholine,
THF] (Schmittberger and Cotte 1998) returned only start-
ing material. The failure of this normally reliable reaction
could be due to the formation of a stable Pd complex with
the abundant coordinating groups in the substrate. Alter-
native deprotection conditions were not pursued as the
chemistry of the benzyl esters proved more fruitful. Thus,
protonolysis of 11d and 12d gave the desired haptens 29
and 30 (the protonated forms of 7 and 8), ready for bio-
conjugation through the carboxyl terminus (Scheme 4).
The specific rotation of the hapten 30 derived in one step
from 12d was slightly larger than that of the material
prepared in two steps from the analogous methyl ester 12b
(10.0° vs. 6.1°), indicating that partial racemisation had
indeed occurred in the saponification step.
N
N
+
28
P
P
O
EtO
O
EtO
EtO
EtO
11b (1,5)
12b (1,4)
26 (1,5)
27 (1,4)
b
O
H
AcN
O
H
AcN
OBn
OH
N
N
b
N
N
N
N
P
P
O
O
EtO
EtO
HO
HO
29 (1,5)
30 (1,4)
11d (1,5)
12d (1,4)
Scheme 4 Reagents, conditions and yields: a base, MeOH, H2O,
then H3O?; b 33% HBr/AcOH. See Table 2 for further details
0°C, 12b failed to react with K2CO3. The saponification of
the regioisomer 11b was also complicated by competing
elimination, and in this case the desired product 26 had
almost identical chromatographic mobility to the N-acety-
ldehydroalanine (28) and could not be isolated.
Protonolysis of the 1,4-triazolylphosphonate 27 derived
from 12b gave the target hapten 30 (the protonated form of
7, Fig. 1) in 70% yield (Scheme 4), but the poor yields of
the saponifications, coupled with the likelihood that partial
racemisation had occurred, even with K2CO3 (which, as
described below, was ultimately shown to be case), meant
an alternative carboxyl-protecting group strategy had to be
devised.
Allyl and benzyl esters were considered simultaneously.
The azidoalanine derivatives 9c and 9d were prepared in
three steps from L-serine (21) (Scheme 3). Acetylation
With the two desired haptens in hand, we then targeted
the free phosphonoamino acids 33 and 34 (Scheme 5).
123

872
S. Mukai et al.
O
O
(Scheme 5). The NMR spectra of 31 and 32 matched those
1
reported except that in our case, most signals in the H
H
H
BocN
BocN
OMe
OBn
NMR spectra, and several signals in the 13C NMR spectra,
in CDCl3 solution, were significantly broadened in com-
parison with the published spectra, presumably due to some
dynamic phenomenon. In contrast, the NMR spectra in
CD3OD exhibited sharp signals.
N
N
N
N
N
N
P
P
O
O
EtO
EtO
EtO
EtO
11a (1,5)
12a (1,4)
11e (1,5)
12e (1,4)
The specific rotations of both regioisomeric carboxylic
acids were significantly lower in magnitude than those
reported (Kee et al. 2010) (Table 3), suggesting that partial
racemisation had again occurred during the saponification
step. Enantioselective chromatography showed this to be
the case; the enantiomeric excesses calculated from the
HPLC peak integrals are close to those determined from
the specific rotations. Although protonolysis afforded the
‘free’ phosphonoamino acids 33 and 34, it was necessary to
revise the protecting group strategy again to avoid the
complication of partial racemisation.
a
b
O
H
O
Br
H3N
BocN
OH
OH
N
N
N
N
b
N
N
P
P
O
O
EtO
HO
EtO
HO
31 (1,5)
32 (1,4)
33 (1,5)
34 (1,4)
Scheme 5 Reagents and conditions: a LiOH, MeOH, H2O, then
H3O?; b 33% HBr, AcOH (see Table 3 for yields)
The azide-alkyne cycloadditions of benzyl N-t-butox-
ycarbonylazidoalanine 9e (Kogan and Rawson 1992)
reflected those of the corresponding acetyl derivative 9d
(Scheme 1; Table 1), indicating that, while the ester group
significantly influences the outcome of these reaction, they
are largely unaffected by the N-protecting group. The
Cu(I)-catalysed cycloaddition of the benzyl ester 9e under
aqueous conditions was higher yielding than the analogous
reaction of the free carboxylic acid in DMF reported by
Muir and co-workers (Kee et al. 2010). It is more difficult
to compare the Ru(II)-catalysed reaction of 9e in this work
with that reported by Muir, as they immediately removed
the benzyl-protecting group, providing acid 31 in 68%
yield over two steps. Nevertheless, the desired 1,5-triazole
11e was formed exclusively in excellent yield.
Although not particularly useful for synthesis or antibody
generation, the free amino acids might possess interesting
biological activity, such as histidine kinase inhibition.
While extremely polar and almost certainly not able to
passively cross cell membranes, it is possible that they may
be substrates for an amino acid transporter.
Attempts to effect global deprotection of the methyl
ester 11a with aqueous hydrochloric or hydrobromic acid
were unsuccessful, so a two-step saponification-proto-
nolysis was investigated. Although partial racemisation of
the acetamide 11b was observed during saponification, we
were optimistic that racemisation could be avoided in this
case due to the decreased acidity of the a-protons in t-butyl
carbamates compared with analogous N-acylamino esters.
Careful saponification of 11a and 12a gave the carboxylic
acids 31 and 32 reported by Muir (Kee et al. 2010)
Global deprotection of 11e and 12e by protonolysis
revealed the free amino acid hydrobromides 33 and 34 as
Table 3 Deprotections to give the free phosphonoamino acids (Scheme 6)
Compound
Yield (%)
[a]aD
ee
Muirb
This work
[a]D
HPLCe
31
86
88
70
65
72
67
?1.8° (1.0)
?60° (1.0)
–
–
–
–
?1.2° (0.5)
?40.5° (0.7)
-5.6° (0.8)
-7.7° (0.5)
-18.8° (0.5)
-25.0° (0.5)
67%c
68%c
72%d
–
70%
32
74%
33 (from 31)
33 (from 11e)
34 (from 32)
34 (from 12e)
a
–
–
–
–
75%d
–
[a]D values were recorded at 21°C (Muir) and ambient temperature (this work). Concentrations in g 100 mL-1 in CHCl3 (31, 32) or H2O (33,
34) are given in brackets
b
(Kee et al. 2010)
c
Determined by comparison with the specific rotations reported by Muir and co-workers (Kee et al. 2010)
d
Determined by comparison with the specific rotation of the same material synthesised by protonolysis of the benzyl esters 11e/12e
e
Determined from the relative integrals of the peaks corresponding to the enantiomers by enantioselective HPLC
123

Stable triazolylphosphonate analogues of phosphohistidine
873
O
O
O
The haptens described herein should prove valuable in
the ongoing search for generic phosphohistidine antibodies
and for otherwise exploring the biochemistry of histidine-
phosphorylated proteins, the corresponding kinases, and
associated cell-signalling pathways.
H
H
H
FmocN
FmocN
FmocN
a
b
OH
OBn
OBn
HO
HO
N3
35
36
9f
Scheme 6 Reagents and conditions: a BnBr, i-Pr2NEt, DMF (90%);
b PPh3, HN3, DEAD, THF, PhMe (65%)
Acknowledgments SM was the recipient of a UWA full PhD
scholarship.
slightly hygroscopic solids, which were purified by ion
exchange chromatography (Scheme 5). The specific rota-
tions of the free amino acids 33 and 34 prepared in this way
were consistent with those expected for enantiopure
material, based on the specific rotations of 33 and 34
prepared from partially racemised carboxylic acids 31 and
32 (Table 3).
Conflict of interest The authors declare that they have no conflict
of interest.
References
Attwood PV, Piggott MJ, Zu XL, Besant PG (2007) Focus on
phosphohistidine. Amino Acids 32:145–156
Finally, to provide access to derivatives suitable for use
in solid-phase peptide synthesis, we targeted the Fmoc-
protected triazolylalaninephosphonates 11f and 12f
(Scheme 1). Fmoc-Ser-OBn (36), prepared by esterifica-
tion of commercially available Fmoc-serine (35) with
benzyl bromide, underwent a Mitsunobu reaction with
hydrazoic acid to provide the novel azide 9f (Scheme 6).
Thermal cycloaddition of this compound with alkyne 10
gave a mixture of the regioisomeric triazoles 11f and 12f
(Scheme 1; Table 1) and Cu(I)-catalysis provided the
1,4-regioisomer 12f exclusively. Webb and co-workers
have recently prepared the corresponding carboxylic acid
by Cu(I)-catalysed cycloaddition of Fmoc-azidoalanine
(McAllister et al. 2011). Ru(II)-catalysis is not compatible
with a free carboxylic acid (Kee et al. 2010); the use of the
benzyl ester-protecting group in the present work allows
Ru(II)-catalysis, providing the 1,5-regioisomer 11f exclu-
sively, and in good yield (Table 1).
Besant PG, Attwood PV (2005) Mammalian histidine kinases.
Biochim Biophys Acta, Proteins Proteomics 1754:281–290
Besant PG, Attwood PV (2009) Detection and analysis of protein
histidine phosphorylation. Mol Cell Biochem 329:93–106
Besant PG, Tan E, Attwood PV (2003) Mammalian protein histidine
kinases. Int J Biochem Cell Biol 35:297–309
Boger DL, Honda T, Menezes RF, Colletti SL, Dang Q, Yang W
(1994) Total syntheses of (?)-P-3a, epi-(-)-P-3a, and (-)-
desacetamido P-3a. J Am Chem Soc 116:82–92
Davoli P, Forni A, Moretti I, Prati F (1995) Stereochemistry of
nucleophilic ring-opening reactions of optically active N-acetyl-2-
methoxycarbonylaziridine. Tetrahedron Asymmetry 6:2011–2016
Dondoni A, Giovannini PP, Massi A (2004) Assembling heterocycle-
tethered C-glycosyl and a-amino acid residues via 1,3-dipolar
cycloaddition reactions. Org Lett 6:2929–2932
Huang Y, Dey S, Zhang X, Soennichsen F, Garner P (2004) The
a-helical peptide nucleic acid concept: merger of peptide
secondary structure and codified nucleic acid recognition.
J Am Chem Soc 126:4626–4640
Janiak-Spens F, West AH (2004) Histidine kinases. Handbook of Cell
Signaling 1:563–566
Kee J-M, Villani B, Carpenter LR, Muir TW (2010) Development
of stable phosphohistidine analogues.
132:14327–14329
Kogan TP, Rawson TE (1992) The synthesis of chiral 3-oxo-6-
[(phenylmethoxy)carbonyl]-2-piperazineacetic acid esters
J Am Chem Soc
Conclusion
designed for the presentation of an aspartic acid side chain. A
subsequent novel Friedel Crafts reaction. Tetrahedron Lett
33:7089–7092
The synthesis of several stable triazolylphosphonate ana-
logues of N1- and N3-phosphohistidine has been achieved.
In addition to the ‘free’ amino acids, derivatives suitable
for bioconjugation through amide linkages with lysine or
aspartate/glutamate residues of carrier proteins were effi-
ciently prepared.
Lecercle D, Sawicki M, Taran
F (2006) Phosphine-catalyzed
a-P-addition on activated alkynes: a new route to P-C-P
backbones. Org Lett 8:4283–4285
Lesma G, Meschini E, Recca T, Sacchetti A, Silvani A (2007)
Synthesis of tetrahydroisoquinoline-based pseudopeptides and
their characterization as suitable reverse turn mimetics. Tetra-
hedron 63:5567–5578
Maruyama K, Hashimoto M, Tamiaki H (1992) Intramolecular
photoreaction of synthetic oligopeptide-linked anthraquinone
molecules. J Org Chem 57:6143–6150
McAllister TE, Nix MG, Webb ME (2011) Fmoc-chemistry of a stable
phosphohistidine analogue. Chem Commun 47:1297–1299
Meldal M, Tornoe CW (2008) Cu-catalyzed azide-alkyne cycloaddi-
tion. Chem Rev 108:2952–3015
A survey of the azide-alkyne cycloadditions of various
L-azidoalanine derivatives with diethyl ethynylphosphonate
has revealed that the amino-protecting group has little impact
on the regioselectivity of the reactions. In contrast, bulkier
carboxyl-protecting groups increase the relative proportion
of the 1,5-disubstituted triazoles under thermal conditions,
and with Ru(II) catalysis, allow exclusive and high-yielding
formation of these products. Irrespective of the protecting
groups, Cu(I) catalysis is regiospecific, providing the
1,4-disubstituted triazoles cleanly and in excellent yields.
Nakata T, Nakatani M, Takahashi M, Okai J, Kawaoka Y, Kouge K,
Okai H (1996) Peptide synthesis in aqueous solution. V. Properties
and reactivities of (p-hydroxyphenyl)benzylmethylsulfonium salts
123

874
S. Mukai et al.
for direct benzyl esterification of N-acylpeptides. Bull Chem Soc
Jpn 69:1099–1106
Pirrung MC, James KD, Rana VS (2000) Thiophosphorylation of
histidine. J Org Chem 65:8448–8453
Tan E, Besant PG, Zu XL, Turck CW, Bogoyevitch MA, Lim SG,
Attwood PV, Yeoh GC (2004) Histone H4 histidine kinase
displays the expression pattern of a liver oncodevelopmental
marker. Carcinogenesis 25:2083–2088
Rostovtsev VV, Green LG, Fokin VV, Sharpless KB (2002) A
stepwise Huisgen cycloaddition process: copper(I)-catalyzed
regioselective ‘‘ligation’’ of azides and terminal alkynes. Angew
Chem Int Ed 41:2596–2599
Schenkels C, Erni B, Reymond J-L (1999) Phosphofurylalanine, a
stable analog of phosphohistidine. Bioorg Med Chem Lett
9:1443–1446
Schmittberger T, Cotte A (1998) Synthesis of characteristic lipopep-
tides of lipid modified proteins employing the allyl ester as
protecting group. Chem Commun 937–938
Steeg PS, Palmieri D, Ouatas T, Salerno M (2003) Histidine kinases
and histidine phosphorylated proteins in mammalian cell biol-
ogy, signal transduction and cancer. Cancer Lett 190:1–12
Stock AM, Robinson VL, Goudreau PN (2000) Two-component
signal transduction. Annu Rev Biochem 69:183–215
Tummatorn J, Albiniak PA, Dudley GB (2007) Synthesis of benzyl
esters using 2-benzyloxy-1-methylpyridinium triflate. J Org
Chem 72:8962–8964
Vuilhorgne M, Malpart J, Mutti S, Mignani S (2003) Preparative
route to 2-ethoxycarbonylimidazole-4-phosphonate and diethyl
imidazole-2,4-dicarboxylate. J Het Chem 40:159–162
Wu P, Fokin VV (2007) Catalytic azide-alkyne cycloaddition:
reactivity and applications. Aldrichimica Acta 40:7–17
Yeager AR, Finney NS (2004) The first direct evaluation of the two-
active site mechanism for chitin synthase. J Org Chem 69:613–618
Zhang L, Chen X, Xue P, Sun HHY, Williams ID, Sharpless KB,
Fokin VV, Jia G (2005) Ruthenium-catalyzed cycloaddition of
alkynes and organic azides. J Am Chem Soc 127:15998–15999
123