A proline-based phosphine template for Staudinger ligation

Article abstract of DOI:10.1021/ol3022484

A proline-based phosphine template enabling a rapid Staudinger ligation of azide-containing substrates under mild conditions is reported. This reaction has a second-order rate constant of 1.12 M^-1 s^-1. It is expected that the proline

Full text of DOI:10.1021/ol3022484

ORGANIC
LETTERS
2012
Vol. 14, No. 17
4694–4697
A Proline-Based Phosphine Template for
Staudinger Ligation
Chung-Min Park,† Wei Niu,‡ Chunrong Liu,† Tyler D. Biggs,† Jiantao Guo,‡ and
Ming Xian*,†
Department of Chemistry, Washington State University, Pullman, Washington 99164,
United States, and Department of Chemistry, University of Nebraska;Lincoln,
Lincoln, Nebraska 68588, United States
mxian@wsu.edu
Received August 14, 2012
ABSTRACT
A proline-based phosphine template enabling a rapid Staudinger ligation of azide-containing substrates under mild conditions is reported. This
reaction has a second-order rate constant of 1.12 MÀ1 sÀ1. It is expected that the proline-based Staudinger ligation strategy will be a useful method
for bioconjugation and proline based peptide coupling.
Bioorthogonal reactions, in which the coupling partners
react selectively without interference with other biological
functionalities, are important tools for chemical biology
research.1 The development of Staudinger ligation by
Bertozzi and co-workers is a seminal contribution to
bioorthogonal chemistry.1,2 Utilizing azides and phos-
phines asorthogonalspecies, Staudinger ligation promotes
a chemoselective amide bond formation under mild and
physiological conditions. It has been employed in labeling
a wide range of azide-containing biomolecules such as
glycans,2a DNA,3 lipids, and proteins.4 Typically, the
Staudinger ligation satisfies the criteria for bioorthogonal
reactions such as good reactivity, selectivity, biocompat-
ibility, etc. Currently the structural templates for Staudin-
ger ligation are limited. Almost all of the substrates used in
regular Staudinger ligation are triphenyl substituted phos-
phine derivatives. In the ‘traceless’ version of Staudinger
ligation developed by Raines et al., some alkyl-diphenyl-
substituted phosphine derivatives are also used.5 With
these substrates, the kinetics of the reaction are somewhat
slow (typical second-order rate constant of 0.002 MÀ1 sÀ1),
which mandates the use of high concentrations of phos-
phine reagents.6 Here we report a proline-based template
for the design of Staudinger ligation substrates. It allows
fast kinetics with azide substrates and provides an alter-
native way to construct peptide adducts.
† Washington State University.
‡ University of Nebraska;Lincoln.
The studies by Raines et al. revealed that a favorable
n f π* electrostatic interaction exists between the amide
oxygen and ester carbonyl in N-formylproline phenylesters
(1) (a) Sletten, E. M.; Bertozzi, C. R. Angew. Chem., Int. Ed. 2009, 48,
6974–6998. (b) Prescher, J. A.; Dube, D. H.; Bertozzi, C. R. Nature 2004,
430, 873–877. (c) Prescher, J. A.; Bertozzi, C. R. Nat. Chem. Biol. 2005, 1,
13–21. (d) Sletten, E. M.; Bertozzi, C. R. Acc. Chem. Res. 2011, 44, 666–
676. (e) van Berkel, S. S.; van Eldijk, M. B.; van Hest, J. C. M. Angew.
Chem., Int. Ed. 2011, 50, 8806–8827.
(2) (a) Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007–2010.
(b) Kohn, S. M.; Breinbauer, R. Angew. Chem., Int. Ed. 2004, 43,
3106–3116.
(3) (a) Franzini, R. M.; Kool, E. T. J. Am. Chem. Soc. 2009, 131,
16021–16023. (b) Comstock, L. R.; Rajski, S. R. J. Am. Chem. Soc. 2005,
127, 14136–14137.
(4) (a) Hang, H. C.; Geutjes, E. J.; Grotenbreg, G.; Pollington, A. M.;
Bijlmakers, M. J.; Ploegh, H. L. J. Am. Chem. Soc. 2007, 129, 2744–2745.
(b) Tsao, M. L.; Tian, F.; Schultz, P. G. ChemBioChem 2005, 6, 2147–
2149. (c) Yanagisawa, T.; Ishii, R.; Fukunaga, R.; Kobayashi, T.;
Sakamoto, K.; Yokoyama, S. Chem. Biol. 2008, 15, 1187–1197.
(5) (a) Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. Org. Lett. 2001,
3, 9–12. (b) McGrath, N. A.; Raines, R. T. Acc. Chem. Res. 2011, 44,
752–761.
(6) (a) Lin, F. L.; Hoyt, H. M.; van Halbeek, H.; Bergman, R. G.;
Bertozzi, C. R. J. Am. Chem. Soc. 2005, 127, 2686–2695. (b) Tam, A.;
Soellner, M. B.; Raines, R. T. J. Am. Chem. Soc. 2007, 129, 11421–
11430. (c) Ning, X.; Guo, J.; Wolfert, M. A.; Boons, G. J. Angew. Chem.,
Int. Ed. 2008, 47, 2253–2255. (d) Poloukhtine, A. A.; Mbua, N. E.;
Wolfert, M. A.; Boons, G. J.; Popik, V. V. J. Am. Chem. Soc. 2009, 131,
15769–15776. (e) Baskin, J. M.; Prescher, J. A.; Laughlin, S. T.; Agard,
N. J.; Chang, P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.; Bertozzi, C. R.
Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 16793–16797.
r
10.1021/ol3022484
Published on Web 08/27/2012
2012 American Chemical Society

(Figure 1).7 Such type of effects contribute to the endo-
genous preference for the trans-peptide bonds. These
results led us to consider the structures of proline-based
iminophosphoranes such as 2 (Figure 1), which could be
generated through the Staudinger reaction of azides. We
anticipated that a similar n f π* interaction might occur
and fix the iminophosphorane conformation as shown in
Figure 1. This could lead to a favorable ligation process. In
addition, the electron-rich phosphorus atom adjacent to the
proline nitrogen could facilitate the formation of the azay-
lide intermediate 2. Finally the reactive species 2 having a
pyrrolidine enamine-like skeleton8 could also enhance the
desired intramolecular acyl transfer to complete the ligation
process. Given these factors, it may provide a new Staudinger
ligation template with improved kinetics.
were prepared and tested (Table 1). As expected, the
desired ligation product 7 was obtained in excellent yields
with these substrates. These ligations proved to be fast, as
all reactions completed within 30 min. The change of
organic solvents (THF, DMF, and acetonitrile) had little
effect on the reaction yields. It should also be noted that
these proline-based substrates (3aÀ3d) are quite stable
toward air and moisture and can be easily handled in the
laboratory.
Table 1. Staudinger Ligation of 3bÀ3d with Benzyl Azide 4a
entry
substrate
solvent
yield (%)b
1
2
3
4
5
6
7
8
9
3b
3c
3d
3b
3c
3d
3b
3c
3d
DMF/buffer (3:1)
96
95
84
90
92
84
96
94
82
DMF/buffer (3:1)
DMF/buffer (3:1)
THF/buffer (3:1)
Figure 1. Design of proline-based template.
THF/buffer (3:1)
THF/buffer (3:1)
MeCN/THF/buffer (1.5:1.5:1)
MeCN/THF/buffer (1.5:1.5:1)
MeCN/THF/buffer (1.5:1.5:1)
To test this idea, we prepared a proline-based diphenyl-
phosphine derivative 3a (Scheme 1; see Supporting Infor-
mation for preparation). When 3a was treated with a
model azide 4 in a mixture solvent system (organic sol-
vent/pH 7.4 phosphate buffer = 3/1) at rt, the aza-ylide
intermediate 5 should be formed. Interestingly this inter-
mediate did not proceed to form the desired ligation product
7 at rt. Instead, we isolated the protonated azaylide inter-
mediate in 55% yield (Supporting Information). We were
able to promote the ligation process by increasing reaction
temperature to 50 °C. Under such conditions, the ligation
product 7 was obtained in moderate yields. Several different
solvents (THF, DMF, a mixture of MeCN/THF) were
tested, and the yields were consistent in the 30À40% range.
a Reaction conditions: organic solvent with pH 7.4 phosphate buffer
(20 mM with 0.15 M NaCl) at rt for 30 min. b Isolated yield.
It is known that pH values could affect the efficiency of
the Staudinger ligation.6b We then tested the reactivity of
3bÀ3d under various pH’s. As shown in Figure S1
(Supporting Information), the yields of the ligation pro-
duct 7 did not change much in pH 5.5À8.5. These results
suggested that the azaylide intermediates were very reac-
tive toward the ester function groups in 3bÀ3d. We noticed
that under basic conditions (pH 8.5) the yield from 3d
dropped slightly. This was possibly due to partial hydro-
lysis of the thioester group in 3d under such conditions.
Among these phosphine substrates, we expected 3b to be
the most promising for Staudinger ligation, given the
better stability of the ester functional group compared to
thioester groups. We then measured the kinetics of the
reaction between 3b and azide 4 using a 31P NMR method
as described before.6a,9 As such, 3b (0.031 M) and 4 (0.31
M) were combined in CD3CN with 5% H2O (v/v) with
triphenylphophine oxide as the internal standard. Under
these conditions, we only observed two species by 31P
NMR, i.e. the starting material 3b (δ = 54.5 ppm) and
the ligation product 7 (δ = 35.2 ppm). As shown in Figure
2A, the reaction completed within 30 min at rt. The
pseudo-first-order rate constants kobs for the consumption
of 3b using different concentrations of excess azide 4 (0.31,
Scheme 1. Staudinger Ligation of 3a with Benzyl Azide 4
To improve the ligation process with the proline-based
template, we decided to introduce better leaving groups in
the ester moiety. Three different substrates 3b, 3c, and 3d
(7) (a) Hodges, J. A.; Raines, R. T. Org. Lett. 2006, 8, 4695–4697.
(b) Bretscher, L. E.; Jenkins, C. L.; Taylor, K. M.; DeRider, M. L.;
Raines, R. T. J. Am. Chem. Soc. 2001, 123, 777–778. (c) DeRider, M. L.;
Wilkens, S. J.; Waddell, M. J.; Bretscher, L. E.; Weinhold, F.; Raines,
R. T.; Markley, J. L. J. Am. Chem. Soc. 2002, 124, 2497–2505.
(d) Hinderaker, M. P.; Raines, R. T. Protein Sci. 2003, 12, 1188–1194.
(8) Mayr, H.; Kempf, B.; Ofial, A. R. Acc. Chem. Res. 2003, 36,
66–77.
(9) Quin, L. D.; Verkade, J. G. Phosphorus-31 NMR Spectral Proper-
ties in Compound Characterization and Structural Analysis; VCH: New
York, 1994.
Org. Lett., Vol. 14, No. 17, 2012
4695

0.62, and 1.24M) were measured and usedtodetermine the
second-order rate constant. By plotting ln kobs versus ln
[4]0, the overall second-order rate constant was determined
to be 1.12 ( 0.03 MÀ1 sÀ1 (Figure 2B). This was approxi-
mately 500 times faster than the triphenylphosphine-based
Staudinger ligation (including the corresponding phenyl
ester substrate6a).
Table 2. Staudinger Ligation of 3b with Azide-Containing
Substrates
Figure 2. Kinetic analysis of the Staudinger ligation of 3b
and azide 4. (A) 31P NMR spectra recorded at 20 °C with 3b
(0.031 M) and 4 (0.31 M) in CD3CN containing 5% H2O. The
spectra were recorded every 3 min for 30 min. (B) Plot of ln kobs
versus ln [4]0 to determine the second-order rate constant, where
4 was used in excess. The second-order rate constant is calcu-
substrate system. To prove this hypothesis, a trans-hydro-
xyproline-phosphine derivative 10 was prepared. The
compound has excellent stability under a normal environ-
ment. Control experiments demonstrated that 10 did not
react with free thiols (like cysteine), disulfides, or amines
lated to be 1.12 ( 0.03 MÀ1 sÀ1
.
To further demonstrate the generality of using phos-
phine 3b in Staudinger ligation, a series of azide-containing
substrates (8aÀ8i) were prepared and tested. All reactions
were carried out with a 1:1 ratio of the phosphine and azides.
As shown in Table 2, the reaction worked smoothly for all the
cases and the ligation products were obtained in high yields.
Steric hindrance did not seem to affect the reaction much, as
the structural changes adjacent to the azide group had little
effect on the reaction yields. It should be noted that substrates
with unprotected functional groups such as ÀCO2H and
ÀNH2 (entries 7 and 8) also showed good reactivity.
Table 3. Staudinger Ligation of Phosphine 10 with
Azide-Containing Substrates
entry
1
N3ÀR
product
yield (%)
91
N3Bn
Ph2OP-Hyp[Gly(Ge)]
-NHBn 11a
4
2
3
N3-Leu-NHBn
Ph2OP-Hyp[Gly(Ge)]
-Leu-NHBn 11b
86
83
We envisioned that the proline moiety on phosphine
substrates could allow us to easily introduce chemical
handles or report molecules for bioconjugation. This
change should not affect the ligation efficacy of this
8b
N3-Leu-Phe-OMe
Ph2OP-Hyp[Gly(Ge)]
-Leu-Phe-OMe 11c
8d
4696
Org. Lett., Vol. 14, No. 17, 2012

(Supporting Information). Its reactions with azide sub-
strates went smoothly, and the desired ligation products
were obtained in good yields (Table 3).
The diphenylphosphinyl moiety on the ligation products
could be considered the protection group of proline and
should be readily removed under acidic conditions.10 We then
tested a ‘one-pot’ ligationÀdeprotection sequence (Scheme 2).
Indeed the ligation followed by acid-mediated depro-
tection provided diphenylphosphine oxide-free pro-
ducts in good yields. Given the efficiency of this
protocol, it could see some applications in proline-
based peptide coupling.
Scheme 2. ‘One-Pot’ LigationÀDeprotection Sequence
Figure 3. ESI-MS analysis of the reaction product from the
reaction of azide-containing Z-domian protein with phos-
phine 3b. Top spectrum: unreacted protein, calculated mass:
7822 Da (without N-terminal methionine); observed mass:
7822 Da. Bottom spectrum: reacted protein; calculated mass:
8093 Da (without N-terminal methionine); observed mass:
8093 Da.
Finally we validated this strategy in bioconjugation
using an azide-containing protein (Figure 3). A noncanonical
amino acid, 4-azido-L-phenylalanine, was genetically incorpo-
rated at position 7 of the Z-domain protein11 in Escherichia
coli by means of an orthogonal amber suppressor tRNA/
aminoacyl-tRNA synthetase pair.12 The azide-containing
Z-domain protein was partially purified by Ni-NTA affinity
chromatography and characterized by mass spectrometry
(Figure 3A). The azide-containing Z-domain protein was
subsequently reacted with phosphine 3b in a mixed solvent
system (DMF/pH 7.4 phosphate buffer). After shaking
gently for 4 h at room temperature, the reaction mixture
was directly analyzed by LC-ESI. The major peak of the
observed spectrum (Figure 3B) matched the expected con-
jugation product. The reaction also proceeded in high yield,
and only a trace amount of the azide-containing Z-domain
protein was detected after the reaction.
In summary, we report in this study a proline-based
phosphine template which can be used to promote a fast
Staudinger ligation. The proline functional group of the
substrates provides an opportunity toreadily introduce the
reporting molecules. We expect this strategy will be useful
for bioconjugation and proline-based peptide coupling.
Acknowledgment. This work is supported by a CA-
REER award from the NSF (0844931) to M.X and the
New Faculty Startup Fund to J.G. from the Chemistry
Department of University of Nebraska-Lincoln.
Supporting Information Available. Synthetic proce-
dures, spectroscopic data, and experimental procedures.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(10) Ramage, R.; Hopton, D.; Parrott, M. J.; Kenner, G. W.; Moore,
G. A. J. Chem. Soc., Perkin Trans. 1 1984, 1357–1370.
(11) Wang, L.; Zhang, Z.; Brock, A.; Schultz, P. G. Proc. Natl. Acad.
Sci. U.S.A. 2003, 100, 56–61.
(12) Young, T. S.; Ahmad, I.; Yin, J. A.; Schultz, P. G. J. Mol. Biol.
2010, 395, 361–374.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 17, 2012
4697