Synthesis of water-soluble phosphinophenol for traceless staudinger ligation

Article abstract of DOI:10.1055/s-0029-1219353

The traceless Staudinger ligation can be mediated in water without organic co-solvents if charged groups render the phenylphosphine reagent water soluble. Here the synthesis of a new water-soluble phosphine is presented based on a diphenylphosphino-phenol reagent. Staudinger ligation with this reagent and azido glycine amide showed conversion of 77%.

Full text of DOI:10.1055/s-0029-1219353

LETTER
787
Synthesis of Water-Soluble Phosphinophenol for Traceless Staudinger
Ligation
A
W
ater-Solub
a
le Phosphi
m
nophenol for Tracel
u
ess Stauding
e
er Ligationl H. Weisbrod, Andreas Marx*
Department of Chemistry and Konstanz Research School Chemical Biology, University of Konstanz, 78457 Konstanz, Germany
Fax +49(7531)885140; E-mail: andreas.marx@uni-konstanz.de
Received 6 December 2009
veloped by Bertozzi et al. consists of (diphenylphosphi-
no)phenol 3 (Figure 1, 1 nontraceless Staudinger ligation
phosphine for comparison).10 Both show comparable
reaction kinetics in ligation reactions with small mole-
Abstract: The traceless Staudinger ligation can be mediated in
water without organic co-solvents if charged groups render the phe-
nylphosphine reagent water soluble. Here the synthesis of a new
water-soluble phosphine is presented based on a diphenylphosphino-
phenol reagent. Staudinger ligation with this reagent and azido gly- cules.6 For labeling of biomolecules the water solubility
cine amide showed conversion of 77%.
of the phosphine reagent is critical because the ligation re-
action has to proceed in water. In case of the methanethiol
derivative 2 this problem has been extensively investigat-
ed by adding negatively or positively charged moieties to
the phenyl rings at different positions.8,11 It turned out
that N,N-dimethylbenzylamine-substituted phosphino-
methanethiols gave best yields in Staudinger ligation
reactions performed in water.11
Key words: amide, phenol, Staudinger ligation, conjugation, phos-
phine
The Staudinger ligation is a bioorthogonal conjugation
reaction which needs no catalyst to proceed.1 Thus it has
been employed for in vitro and in vivo labeling of several
biomolecules as carbohydrates, proteins, and nucleic ac-
ids.2 Developed by Bertozzi in 20003 the reaction is based
on the Staudinger reduction, wherein an azide is reduced
by a phosphine via an iminophosphorane intermediate.4 In
the ligation reaction an intramolecular acetylation of the
iminophosphorane takes place, and after hydrolysis an
amide bond is formed.5 In the traceless variant the
arylphosphine is part of the leaving group, and just a na-
tive amide bond between the ligation partners is formed
(Scheme 1).6 Thus the traceless Staudinger ligation is es-
pecially useful to ligate oligopeptides to form larger
polypeptides or peptide conjugates.7,8
Me
Me
N
O
O
Me
O
PPh2
PPh2
O
O
R
S
Ph2P
O
R
O
R
P
Me
N
O
NH
R
Me
1
2
3
4
Figure 1 Overview of phosphines for Staudinger ligations
There are mainly two types of traceless Staudinger phos-
phines, one is based on (diphenylphosphino)methanethiol Here, we target on the phosphinophenol 3 which has been
2 and has been developed by Raines et al.,9 the other de- used for peptide conjugation and glycoconjugation to pro-
teins so far.12 We reasoned that tertiary benzylic amines
should facilitate easy synthesis using inexpensive starting
O
O
materials. The modification should be placed at the un-
substituted phenyl rings of phosphinophenol 3 leading to
the structure of 4. Additionally, these positive charged
groups best mediated the Staudinger ligation in case of the
methanethiol derivatives.
R1
Ph
O
R2
N–
P+
Ph
O
R2
P
R1N3
+
Ph
– N2 (g)
Ph
iminophosphorane
Our synthesis starts with the protection of phenol with
ethyl vinyl ether (EVE) and catalytic amounts of pyridin-
ium p-toluenesulfonate (PPTS, Scheme 2).13 This protect-
ing group is also mediating directed metalation required
for subsequent directed ortho metalation (DOM). The
next step was the selective introduction of the phosphor
moiety in a DOM reaction with diethyl chlorophosphite.
The N,N-dimethylbenzylamine moiety was introduced by
halogen–lithium exchange using bromide 7 and subse-
quent addition to phosphonite 6. The yield was 66% and
the main side products were butyl-substituted phosphines.
Bromide 7 can be easily synthesized starting from p-bromo-
O
R1
N
R2
O
O
OH
H2O
P+
R1
P
+
N
R2
Ph
Ph
Ph
H
Ph
–O
Scheme 1 Putative mechanism of traceless Staudinger ligation
SYNLETT 2010, No. 5, pp 0787–0789
Advanced online publication: 25.01.2010
1
6.
3
.2
0
1
0
DOI: 10.1055/s-0029-1219353; Art ID: G40609ST
© Georg Thieme Verlag Stuttgart · New York

788
S. H. Weisbrod, A. Marx
LETTER
benzyl bromide and is also commercially available. The stable towards oxidation at least for one week in solution.
final deprotection of the phenol was performed in 1 M Although the triphenylphosphine core is highly hydro-
aqueous HCl since catalytic amounts of PPTS were not phobic, the two introduced tertiary amines increase the
sufficient presumably due to the presence of the tertiary water solubility of the phosphine sufficiently and thus, re-
amines. The final phosphinophenol 9 is water soluble and actions in water without organic co-solvents are possible.
stable to air at least for one week in solution as checked by The conversion of the Staudinger ligation reaction is com-
31P NMR.
parable to the water-soluble methanethiol derivatives,11
which seem to be more susceptible to oxidation since only
two phenyl rings stabilize the phosphine.
OH
O
O
O
PPTS
OEt
P
O
O
1. n-BuLi
2. ClP(OEt)2
EtO
Diethyl Arylphosphonite 6
80%
56%
5
6
To a solution of EVE-protected phenol (2.99 g, 18.0 mmol) in an-
hyd THF n-BuLi (1.6 M in hexane, 12.4 mL, 19.9 mmol) was added
dropwise at 0 °C, stirred for 1.5 h hours at 0 °C and further 1.5 h at
r.t. The dark solution was added to a cooled (–78 °C) solution of
(EtO)2ClP in THF (10 mL) and stirred 16 h with warming to r.t. Af-
ter evaporation of THF the residue was distilled in vacuo (1 mbar,
104 °C) and isolated as colorless liquid (2.9 g, 56%). 1H NMR (400
MHz, CDCl3): d = 7.63 (1 H, m), 7.31 (1 H, m), 7.07 (dd, J = 8.0,
4.1 Hz, 1 H), 7.01 (t, J = 7.4 Hz, 1 H), 5.46 (q, J = 5.3 Hz, 1 H), 3.92
(m, 2 H), 3.80 (m, 3 H), 3.56 (m, 1 H), 1.51 (d, J = 5.4 Hz, 3 H),
1.25 (dd, J = 12.9, 6.9 Hz, 7 H), 1.18 (t, J = 7.1 Hz, 3 H). 13C NMR
(100 MHz, CDCl3): d = 159.5 (d), 131.5, 130.6 (d), 130.1 (d), 121.7,
115.3, 99.5, 63.0 (d), 62.8 (d), 60.8, 20.1, 17.4, 17.3, 15.4. 31P NMR
(162 MHz, CDCl3): d = 150.3.
Me
Me2NH
76%
n-BuLi, then 6
Br
N
66%
Me
Br
Me
Br
7
Me
N
N
Me
Me
OH
aq HCl
O
O
48%
P
P
Phosphine 8
To a solution of compound 7 (3.43 g, 16 mmol) in anhyd THF (20
mL), n-BuLi (1.6 M in hexane, 10 mL, 16 mmol) was added drop-
wise at –78 °C and stirred for 1 h. Diethyl arylphosphonite 6 (2.15
g, 7.5 mmol) in THF (10 mL) was added dropwise and stirred for 20
h with warming to r.t. and quenched with distilled H2O (10 mL). Af-
ter phase separation the water layer was extracted with CH2Cl2
(2 × 10 mL), and the combined organic phases were dried (MgSO4)
and concentrated in vacuo. The residue was purified by flash col-
umn chromatography (EtOAc, then 2% MeOH in CH2Cl2 contain-
9
8
N
N
Me
Me
Me
Me
Scheme 2 Synthesis of water-soluble phosphinophenol
Next, the Staudinger ligation with this new water-soluble
phosphinophenol was investigated. Therefore compound
9 has been acetylated in pyridine using acetic anhydride
yielding phosphine 10 which should be capable to transfer
ing 1% Et3N) to afford triarylphosphine 8 (2.29 g, 66%) as slightly
1
the acyl group in a Staudinger ligation reaction. Water- yellow oil. H NMR (400 MHz, CDCl3): d = 7.25–7.17 (m, 9 H),
7.04 (dd, J = 7.4, 4.4 Hz, 1 H), 6.80 (t, J = 7.4 Hz, 1 H), 6.63 (m, 1
soluble azidoglycine amide has been chosen as ligation
partner. To increase the water solubility of acetylated
phosphinophenol 10 the TFA salt was used which is hy-
H), 5.25 (q, J = 5.3 Hz, 1 H), 3.42–3.31 (m, 1 H), 3.36 (s, 4 H), 3.23
(m, 1 H), 2.18 (s, 12 H), 1.14 (d, J = 5.4 Hz, 3 H), 0.98 (t, J = 7.1
Hz, 3 H). 13C NMR (100 MHz, CDCl3): d = 158.4 (d), 134.3, 134.1,
groscopic and well soluble in water (> 0.1 M). The reac-
134.0, 133.8, 133.3 (d), 130.0, 129.2 (d), 129.1 (d), 124.4 (d) 121.7,
tion proceeded slowly as monitored by 1H NMR, but after
30 hours conversion stopped, and the product was formed
in 77% yield (Scheme 3).
114.5, 98.8, 64.1, 59.6, 45.3, 19.3, 15.2. 31P NMR (162 MHz,
CDCl3): d = –17.2. ESI-HRMS: m/z calcd for C28H38N2O2P [M +
H]+: 465.2665; found: 465.2664.
In summary, here we present a five-step synthesis of a
water-soluble, unsymmetrically substituted triphe-
nylphosphine-based reagent for traceless Staudinger liga-
Phosphinophenol 9
Phosphine 8 (1.10 g, 2.37 mmol) was dissolved in aq HCl (1 M, 30
mL) and stirred for 16 h at r.t. Water was evaporated and the residue
tion in water at neutral pH. The employed phosphine 9 is chromatographed with RP-MPLC (Büchi C18 column, linear gradi-
ent H2O–MeCN, 0.1% TFA). The free amino compound was isolat-
Me
ed by extraction of sat. NaHCO3 solution (50 mL) with CH2Cl2
(3 × 30 mL) and yielded 0.45 g, 48%. 1H NMR (400 MHz, CDCl3):
N
Me
d = 7.28–7.20 (m, 8 H), 7.16 (m, 1 H), 6.76–6.67 (m, 3 H), 3.42 (s,
O
4 H), 2.21 (s, 12 H). 13C NMR (100 MHz, CDCl3): d = 159.6 (d),
135.7, 135.6, 134.1, 133.9, 133.7, 132.0 (d), 130.4, 129.3 (d), 129.2
O
(d), 123.2 (d) 119.8, 115.3, 98.8, 63.9, 45.9. 31P NMR (162 MHz,
P
Me
CDCl3): d = –20.0. ESI-HRMS: m/z calcd for C24H30N2OP [M +
O
10
O
N
H
H]+: 393.2090; found: 393.2084.
Me
N
N3
NH2
NH2
D2O, 77%
11
O
Acetylphosphinophenol 10
To a solution of phosphine 9 (50 mg, 0.12 mmol) in anhyd pyridine
(1 mL) Ac2O (14 mL, 0.15 mmol) was added and stirred for 2 h. Af-
Scheme 3 Investigated traceless Staudinger ligation in water
Synlett 2010, No. 5, 787–789 © Thieme Stuttgart · New York

LETTER
A Water-Soluble Phosphinophenol for Traceless Staudinger Ligation
789
ter evaporation of solvents the residue was extracted with CH2Cl2
(3 × 10 mL) from sat. NaHCO3 solution (20 mL) and further puri-
fied by RP-MPLC (Büchi C18 column, linear gradient H2O–MeCN,
C. A.; Chaikof, E. L. Bioconjugate Chem. 2007, 18, 1713.
(e) Verdoes, M.; Florea, B. I.; Hillaert, U.; Willems, L. I.;
van der Linden, W. A.; Sae-Heng, M.; Filippov, D. V.;
Kisselev, A. F.; van der Marel, G. A.; Overkleeft, H. S.
ChemBioChem 2008, 9, 1735. (f) Yanagisawa, T.; Ishii, R.;
Fukunaga, R.; Kobayashi, T.; Sakamoto, K.; Yokoyama, S.
Chem. Biol. 2008, 15, 1187.
1
0.1% TFA) to yield 36 mg, 66%. H NMR (400 MHz, D2O): d =
7.42–7.30 (m, 9 H), 7.14 (m, 1 H), 7.07 (m, 1 H), 6.88 (m, 1 H), 4.32
(s, 4 H), 2.78 (s, 12 H), 1.94 (s, 3 H). 31P NMR (162 MHz, D2O):
d = –18.8. ESI-HRMS: m/z calcd for C26H32N2O2P [M + H]+:
435.2196; found: 435.2190.
(3) Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007.
(4) Staudinger, H.; Meyer, J. Helv. Chim. Acta 1919, 2, 635.
(5) Lin, F. L.; Hoyt, H. M.; Van Halbeek, H.; Bergman, R. G.;
Bertozzi, C. R. J. Am. Chem. Soc. 2005, 127, 2686.
(6) Soellner, M. B.; Nilsson, B. L.; Raines, R. T. J. Am. Chem.
Soc. 2006, 128, 8820.
Staudinger Ligation
TFA salt of phosphine 10 (60 mmol) and azidoglycine amide (78
mmol) were combined in D2O (0.8 mL) and the reaction monitored
by 1H NMR. Conversion has been determined by peak integration.
(7) (a) Kleineweischede, R.; Hackenberger, C. P. Angew. Chem.
Int. Ed. 2008, 47, 5984. (b) Liu, L.; Hong, Z. Y.; Wong,
C. H. ChemBioChem 2006, 7, 429.
Acknowledgment
(8) Tam, A.; Soellner, M. B.; Raines, R. T. J. Am. Chem. Soc.
2007, 129, 11421.
(9) Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. Org. Lett.
2001, 3, 9.
The authors would like to thank DFG (SPP 1243) for financial sup-
port.
(10) Saxon, E.; Armstrong, J. I.; Bertozzi, C. R. Org. Lett. 2000,
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Synlett 2010, No. 5, 787–789 © Thieme Stuttgart · New York

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