Mechanistic investigation of the Staudinger ligation

Article abstract of DOI:10.1021/ja044461m

The Staudinger ligation of azides and phosphines has found widespread use in the field of chemical biology, but the mechanism of the transformation has not been characterized in detail. In this work, we undertook a mechanistic study of the Staudinger liga

Full text of DOI:10.1021/ja044461m

Published on Web 02/01/2005
Mechanistic Investigation of the Staudinger Ligation
Fiona L. Lin,† Helen M. Hoyt,† Herman van Halbeek,† Robert G. Bergman,† and
Carolyn R. Bertozzi*,†,‡,§,¶
Contribution from the Departments of Chemistry and Molecular and Cell Biology and
Howard Hughes Medical Institute, UniVersity of California, and Materials Sciences DiVision,
Lawrence Berkeley National Laboratory, Berkeley, California 94720
Received September 13, 2004; E-mail: crb@berkeley.edu
Abstract: The Staudinger ligation of azides and phosphines has found widespread use in the field of
chemical biology, but the mechanism of the transformation has not been characterized in detail. In this
work, we undertook a mechanistic study of the Staudinger ligation with a focus on factors that affect reaction
kinetics and on the identification of intermediates. The Staudinger ligation with alkyl azides was second-
order overall and proceeded more rapidly in polar, protic solvents. Hammett analyses demonstrated that
electron-donating substituents on the phosphine accelerate the overall reaction. The electronic and steric
properties of the ester had no significant impact on the overall rate but did affect product ratios. Finally, the
structure of an intermediate that accumulates under anhydrous conditions was identified. These findings
establish a platform for optimizing the Staudinger ligation for expanded use in biological applications.
Scheme 1
Introduction
Bioorthogonal chemical reactions, in which the coupling
partners react selectively without interference by biological
functionality, have become important tools in the field of
chemical biology.1 As an example, the Staudinger ligation of
azides and arylphosphines2,3 (Scheme 1) results in selective
formation of an amide bond within complex biological environs,
such as cells4 and living animals.5 This reaction has been
employed in a wide range of applications, including modification
of cell surfaces,2,4 protein engineering,6,7 specific labeling of
nucleic acids,8 proteomic studies,9,10 and as a general tool for
bioconjugation.11,12
for optimization of rates and yields. Herein, we report the first
mechanistic investigation of the Staudinger ligation, including
the determination of kinetic parameters and solvent effects,
Hammett analyses of substituent effects, and the structural
elucidation of a reaction intermediate.
While appreciation of the Staudinger ligation as a tool for
chemical biology continues to expand, little attention had been
directed to fundamental mechanistic studies that would allow
Results and Discussion
Investigation of the Kinetic Parameters of the Staudinger
Ligation by 31P NMR Spectroscopy. The Staudinger ligation
is a modified version of the classical Staudinger reaction13
between phosphines and azides. Mechanistic studies of the
classical reaction between triphenylphosphine (1, R ) Ph) and
phenyl azide (2, R′ ) Ph) suggest that the lone pair electrons
of 1 attack the terminal nitrogen atom of the azide (2) to yield
a linear phosphazide intermediate 3 (Scheme 2).14-18 This
intermediate can undergo intramolecular rearrangement via a
four-membered ring transition state to yield a second intermedi-
ate, azaylide 4 (also known as an iminophosphorane), with
concomitant loss of N2. In the presence of water, azaylide 4
undergoes hydrolysis to yield amine 5 and phosphine oxide 6.
† Department of Chemistry.
‡ Department of Molecular and Cell Biology.
§ Howard Hughes Medical Institute.
¶ Lawrence Berkeley National Laboratory.
(1) Hang, H. C.; Bertozzi, C. R. Acc. Chem. Res. 2001, 34, 727-736.
(2) Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007-2010.
(3) Kohn, M.; Breinbauer, R. Angew. Chem., Int. Ed. 2004, 43, 3106-3116.
(4) Saxon, E.; Luchansky, S. J.; Hang, H. C.; Yu, C.; Lee, S. C.; Bertozzi, C.
R. J. Am. Chem. Soc. 2002, 124, 14893-14902.
(5) Prescher, J. A.; Dube, D. H.; Bertozzi, C. R. Nature 2004, 430, 873-877.
(6) Nilsson, B. L.; Hondal, R. J.; Soellner, M. B.; Raines, R. T. J. Am. Chem.
Soc. 2003, 125, 5268-5269.
(7) Kiick, K. L.; Saxon, E.; Tirrell, D. A.; Bertozzi, C. R. Proc. Natl. Acad.
Sci. U.S.A. 2002, 99, 19-24.
(8) Wang, C. C.-Y.; Seo, T. S.; Li, Z.; Ruparel, H.; Ju, J. Bioconjugate Chem.
2003, 14, 697-701.
(9) Hang, H. C.; Yu, C.; Kato, D. L.; Bertozzi, C. R. Proc. Natl. Acad. Sci.
U.S.A. 2003, 100, 14846-14851.
(10) Vocadlo, D. J.; Hang, H. C.; Kim, E. J.; Hanover, J. A.; Bertozzi, C. R.
Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 9116-9121.
(11) Soellner, M. B.; Dickson, K. A.; Nilsson, B. L.; Raines, R. T. J. Am. Chem.
Soc. 2003, 125, 11790-11791.
(13) Staudinger, H.; Meyer, J. HelV. Chim. Acta 1919, 2, 635-646.
(14) Gololobov, Y. G.; Zhmurova, I. N.; Kasukhin, L. F. Tetrahedron 1981,
37, 437-472.
(15) Gololobov, Y. G.; Kasukhin, L. F. Tetrahedron 1992, 48, 1353-1406.
(16) Johnson, A. W. Ylides and Imines of Phosphorus; Wiley: New York, 1993.
(17) Leffler, J. E.; Tsuno, Y. J. Org. Chem. 1963, 28, 902-906.
(18) Leffler, J. E.; Temple, R. D. J. Am. Chem. Soc. 1967, 89, 5235-5246.
(12) Kohn, M.; Wacker, R.; Peters, C.; Schroder, H.; Soulere, L.; Breinbauer,
R.; Niemeyer, C. M.; Waldmann, H. Angew. Chem., Int. Ed. 2003, 42,
5830-5834.
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J. AM. CHEM. SOC. 2005, 127, 2686-2695
10.1021/ja044461m CCC: $30.25 © 2005 American Chemical Society

Mechanistic Investigation of the Staudinger Ligation
A R T I C L E S
Scheme 2
Scheme 3
Figure 1. Kinetic analysis of the Staudinger ligation of phosphine 7 and
benzyl azide (8). (A) Plot of ln kobs versus ln [8]0 to determine the second-
order rate constant under pseudo-first-order conditions, where 8 was used
in excess. Pseudo-first-order rate constants were measured at [7] ) 0.041
M and [H2O] ) 2.8 M in CD3CN at 20-21 °C. The second-order rate
constant is calculated to be (2.5 ( 0.2) × 10-3 M-1 s-1 from the y-intercept
of the least-squares fit, with an estimated 10% error. (B) Plot of 1/[8]0 versus
time to determine the second-order rate constant of the Staudinger ligation
between phosphine 7 and benzyl azide (8) under stoichiometric conditions
in CD3CN with 5% (v/v) H2O. The slopes of the linear fits represent the
second-order rate constants according to the kinetic model for a second-
order reaction. At each concentration of substrate, linear fits were obtained
Scheme 3 depicts a typical Staudinger ligation between
phosphine 7 and benzyl azide (8) to yield azaylide 10. We
presume that the early steps of the Staudinger ligation (steps 1
and 2, Scheme 3) are similar to those of the classical reaction.
The major difference between the Staudinger ligation and the
classical reaction is that azaylide 10 can undergo an intra-
molecular reaction (step 3) to produce a new intermediate, for
which we previously proposed structure 11.2 Finally, the
intermediate undergoes hydrolysis to afford amide-linked
product 12 (step 4).
with similar slopes of approximately 2 × 10-3 M-1 s-1
.
kinetics are observed, presumably due to the reversibility of
formation of the phosphazide intermediate (k-1, Scheme 2).18
We determined the kinetic parameters of the Staudinger
ligation of phosphine 7 and benzyl azide (8) (Scheme 3) using
31P NMR spectroscopy. When 7 (0.041 M) and 8 (0.41 M) were
combined in CD3CN with 5% water (v/v) (2.78 M), consumption
of 7 followed pseudo-first-order kinetics. Under these conditions,
the only species detectable by 31P NMR were the starting
material 7 and the ligation product 12. The pseudo-first-order
rate constants, kobs, for the consumption of 7 using different
concentrations of excess benzyl azide (0.41, 0.82, 1.24, and 1.65
M) were measured and used to determine the second-order rate
constant (k1, Scheme 3). By plotting ln kobs versus ln [8]0, the
overall second-order rate constant was determined to be (2.5 (
0.2) × 10-3 M-1 s-1 (Figure 1A). Consistent with that of the
mechanism of the classical Staudinger reaction, the rate-limiting
step of the Staudinger ligation is presumably the initial reaction
between 7 and 8 to yield phosphazide 9 (k1, Scheme 3). Since
no intermediates were detected and the observed kinetics were
in good agreement with a kinetic model for bimolecular
reactions, this first step is likely to be irreversible.
Kinetic studies of the classical Staudinger reaction have
shown that the process can be first- or second-order overall
depending on the exact nature of the reactants. For example,
the rate-limiting step in the reaction between triphenylphosphine
(1, Scheme 2, R ) Ph) and benzenesulfonyl azide (2, R′ )
PhSO2) is the unimolecular decomposition (k2, Scheme 2) of
the phosphazide complex.17 In contrast, the reaction of benzoyl
azide (2, R′ ) PhCO) and triphenylphosphine follows second-
order kinetics, indicating that the bimolecular step leading to
the formation of the phosphazide (k1, Scheme 2) is rate-
limiting.18 In reactions between substituted phenyl azides and
substituted triphenylphosphines, deviations from second-order
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J. AM. CHEM. SOC. VOL. 127, NO. 8, 2005 2687

A R T I C L E S
Lin et al.
Table 1. Pseudo-First-Order Rate Constants of the Staudinger Ligation of Phosphine 7 (0.044 M) and Benzyl Azide (0.83 M) in Different
Solventsa
1
1
1
kobs (10-3 M-1 s-
)
kobs (10-3 M-1 s-
)
kobs (10-3 M-1 s-
)
solvent
ꢀb
2.5% H2O (v/v) (1.39 M)
5% H2O (v/v) (2.78 M)
10% H2O (v/v) (5.56 M)
THF-d8
Acetone-d6
CD3OD
CD3CN
DMSO-d6
7.6
20.7
32.7
37.5
46.7
0.83 ( 0.02
1.3 ( 0.2
0.93 ( 0.03
1.2 ( 0.2
1.4 ( 0.3
1.44 ( 0.01
3.6 ( 0.7
2.4 ( 0.2
3.6 ( 0.5
2.6 ( 0.5
3.8 ( 0.1
1.9 ( 0.1
2.06 ( 0.04
1.9 ( 0.2
2.92 ( 0.08
a Reported rate constants represent the average of three runs under each condition (5% standard deviation. b From: Riddick, J. A.; Bunger, W. B.
Organic Solvents. In Techniques of Chemistry; Weissberger, A., Ed.; Wiley: New York, 1970; Vol. II.
To ensure that the Staudinger ligation behaves similarly under
stoichiometric conditions, the kinetics of the reaction using
equimolar amounts of both reactants and excess water were also
investigated. Under these conditions, the observed kinetics were
consistent with a second-order reaction characterized by a rate
constant of (1.9 ( 0.1) × 10-3 M-1 s-1 (Figure 1B). This rate
constant is in good agreement with that obtained under pseudo-
first-order conditions, indicating that the reaction behaves
similarly at different substrate concentrations.
Effects of Changes in Solvent Polarity on Staudinger
Ligation Rates. Using a series of organic solvents that are
miscible with water, we investigated the effect of changes in
solvent polarity on the rate of the Staudinger ligation. The results
are summarized in Table 1. In general, the reaction proceeded
faster in solvents with higher dielectric constants. These
observations indicate that the rate-limiting step involves a polar
transition state that can be stabilized by polar solvents.
Furthermore, the sensitivity of the observed rate constants to
different concentrations of water varied in different solvents.
For example, the rate constants observed in the presence of 1.39,
2.78, and 5.56 M water in acetone-d6 were similar, but in
DMSO-d6, the rate constants varied more significantly. It is also
interesting to note that the observed rate constants for reactions
performed in CD3OD are significantly higher than those in CD3-
CN, even though the two solvents have very similar dielectric
constants. These observations suggest additional contributions
of the solvent to transition state stabilization through hydrogen
bonding or other direct interactions.
Figure 2. Phosphines synthesized to investigate the effects of steric and
electronic modifications on the rate of the Staudinger ligation.
Effects of Phosphine Substituents on Staudinger Ligation
Rates. Three panels of phosphines were synthesized in order
to determine the effects of steric and electronic modifications
on the overall reaction (Figure 2). In series A, the steric bulk
of the ester was varied. Phosphines 13a-d were treated with
benzyl azide in CD3CN with 5% water (v/v), and the products
and pseudo-first-order rate constants were characterized (Table
2). The size of the leaving group did not have a significant effect
on the overall rate, indicating that ester cleavage is not involved
in the rate-limiting step. Interestingly, in the reactions of
phosphines with relatively small leaving groups (R ) Me, Et,
and i-Pr), only the Staudinger ligation product was observed.
However, when the leaving group was more sterically demand-
ing (R ) t-Bu), a significant amount of the phosphine oxide
derived from direct hydrolysis of the azaylide intermediate was
also observed (shown schematically in Table 2). This observa-
tion is consistent with a mechanism in which the rate-limiting
step is the bimolecular formation of the phosphazide (step 1,
Scheme 3), whereas the ligation:hydrolysis product ratio is
governed by the rate of intramolecular amide formation (step
Figure 3. Hammett analysis for substituents on the phenyl group directly
attached to the phosphorus center. Reactions were performed with phos-
phines 15a-f (0.020 M) and benzyl azide (8) (0.83 M) in 5% (v/v) H2O in
CD3CN at room temperature.
3) versus hydrolysis of the azaylide. Thus, better ligation yields
should be expected with relatively small ester leaving groups.
On the basis of the above results, one would not expect the
electronic properties of the ester leaving group to affect the
overall rate of the reaction. To test this hypothesis, we prepared
phosphines 14a-f (Figure 2, series B) functionalized with a
series of para-substituted phenol esters. The observed pseudo-
first-order rate constants were indeed very similar (Table 3).
Taken together, these results support a mechanism in which the
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2688 J. AM. CHEM. SOC. VOL. 127, NO. 8, 2005

Mechanistic Investigation of the Staudinger Ligation
A R T I C L E S
Table 2. Pseudo-First-Order Rate Constants and Product
Distribution for the Staudinger Ligation of Phosphines 13a-d and
Benzyl Azide in CD3CN with 5% (v/v) H2Oa
Scheme 4
M phosphine and 0.41 M azide in CD3CN with 5% water
(v/v)). The products and reaction rates were determined using
31P NMR spectroscopy. The reaction with phenyl azide (16a)
as a substrate proceeded rapidly to produce a stable intermediate
with a 31P resonance at ca. +13 ppm. This intermediate was
proposed to be azaylide 17a, consistent with 31P chemical shifts
reported for other azaylides.16 By contrast, no comparable
intermediate accumulated in the reaction with benzyl azide.
Conversion of azaylide 17a to the ligation product was rather
slow (>24 h at room temperature for complete conversion). This
is in sharp contrast to the reaction with benzyl azide, wherein
complete conversion of starting material (7) to the ligation
product occurred within 35 min under the same conditions.
Presumably, resonance stabilization of the phenyl-substituted
azaylide 17a (Scheme 4) reduces its reactivity relative to benzyl-
substituted azaylide 10 (Scheme 3).
1
phosphine
R
LP:HPb
kobs (10-3 M-1 s-
)
13a
13b
13c
13d
CH3
100:0
100:0
100:0
60:40
2.0 ( 0.1
1.9 ( 0.1
2.0 ( 0.2
1.8 ( 0.3
CH2CH3
CH(CH3)2
C(CH3)3
a Reported rate constants represent the average of three runs under each
condition (5% standard deviation. b LP ) ligation product; HP ) hydrolysis
product. Product ratios determined by 31P NMR peak integrations.
Table 3. Pseudo-First-Order Rate Constants of the Staudinger
Ligation of Phosphines 14a-f and Benzyl Azide in CD3CN with
5% (v/v) H2Oa
b
1
phosphine
X
σpara
kobs (10-3 M-1 s-
)
The reaction was repeated with p-methoxyphenyl azide (16b)
and p-nitrophenyl azide (16c). The Staudinger ligation with 16b
was complete within 8 h, but the reaction with 16c required
more than 48 h for complete conversion. In all cases, the
phosphine starting material was completely converted to the
azaylide intermediate in <5 min. Collectively, these observations
suggest that the rate-determining step in Staudinger ligation
reactions with aryl azides is the intramolecular amide bond-
forming step, rather than initial formation of the phosphazide
as observed with benzyl azide. Presumably, this change in the
rate-determining step is the consequence of enhanced azaylide
stability due to resonance stabilization.
14a
14b
14c
14d
14e
14f
OCH3
CH3
H
F
Cl
-0.27
-0.17
0
0.06
0.23
0.78
2.0 ( 0.2
2.0 ( 0.2
1.8 ( 0.2
1.9 ( 0
2.2 ( 0.1
1.9 ( 0.4
NO2
a Reported rate constants represent the average of three runs under each
condition (5% standard deviation. b Values are those given by Ritche, C.
D.; Sager, W. F. Prog. Phys. Org. Chem. 1964, 2, 323.
rate-limiting step occurs prior to intramolecular reaction of the
azaylide group with the ester functionality.
Unlike substituents on the ester leaving group, substituents
on a phenyl group directly attached to the phosphorus atom
(Figure 2, series C, 15a-f) had a pronounced effect on the
overall rate. Figure 3 shows the Hammett plot of log (kx/kH)
versus the σ+ values of the substituents. A linear fit (R2 ) 0.91)
with a small F value (-0.38) was obtained. The negative F value
suggests a buildup of positive charge in the rate-limiting
transition state, consistent with formation of the phosphazide
(step 1, Scheme 3) as the rate-determining step. From a practical
standpoint, these results suggest that the overall rate of the
Staudinger ligation can be accelerated by electron-donating
substituents on the phosphine. Indeed, in the future, we plan to
investigate the reactivity of triarylphosphines with electron-
donating substituents on each phenyl group.
Characterization of a Reaction Intermediate under An-
hydrous Conditions. We previously proposed the reaction
pathway shown in Scheme 3, involving compound 11 as a key
intermediate leading to the ligation product 12.2 In an earlier
study, we did not observe the accumulation of any intermediate
in reactions of phosphine 7 and benzyl azide in the presence of
excess water.4 However, we observed 31P resonances corre-
sponding to azaylide 10 (15 ppm) and a second intermediate
that accumulated under anhydrous conditions or in the presence
of substoichiometric amounts of water. On the basis of the 31
P
NMR spectrum of this second intermediate and those reported
for phosphetanes,16 we proposed the bridged bicyclic oxaza-
phosphetane structure 18 shown in Scheme 5.4 Addition of water
to the intermediate led to formation of ligation product 12. Since
our structural assignment was based on limited analytical data,
we undertook a more thorough characterization of the interme-
diate by NMR and X-ray analysis.
Effects of Azide Structure on Staudinger Ligation Rates.
We further probed the kinetic parameters of the Staudinger
ligation as a function of azide structure. Three aryl azides
(16a-c, Scheme 4) were prepared and treated with phosphine
7 using conditions previously applied with benzyl azide (0.041
The intermediate was isolated from the reaction of 7 and
benzyl azide in CH2Cl2 under rigorously anhydrous conditions
9
J. AM. CHEM. SOC. VOL. 127, NO. 8, 2005 2689

A R T I C L E S
Scheme 5
Lin et al.
(glovebox). The 1H NMR spectrum (Figure 4A) was not
consistent with that of the previously proposed structure 18.
The resonance corresponding to the methoxy protons (3.03 ppm)
was an apparent doublet with a relatively large J-coupling
constant of 11 Hz (Figure 4A, top). Broadband 31P decoupling
collapsed the doublet into a singlet (Figure 4A, bottom),
indicating coupling of the methoxy protons to the 31P nucleus.
In oxazaphosphetane 18, the methoxy protons should not exhibit
such a large coupling to the phosphorus nucleus.
1
Furthermore, the 2D H-13C HMBC spectrum (Figure 4B)
3
of the isolated intermediate did not show a JH-C cross-peak
between the methoxy protons (3.03 ppm) and the carbonyl
carbon (δ 165 ppm), arguing against 18 as the correct structure.
On the basis of these results, we propose a new structure for
the intermediate, namely, pentacoordinate phosphorane 19
(Figure 4C), which is consistent with the observed NMR data.
The connectivity of the intermediate was further confirmed by
X-ray crystallography (Figure 4C). In the solid state, the three
large phenyl substituents occupy the equatorial positions,
whereas the two more electronegative substituents occupy the
apical positions. The bond angles around the phosphorus center
are close to those predicted for the trigonal bipyramidal
geometry without significant distortion.
Addition of water to compound 19 resulted in the formation
of Staudinger ligation product 12 (Scheme 3). As shown in
Scheme 6, we propose a mechanism for this reaction that
involves the initial formation of phosphonium intermediate 20.
Presumably, this intermediate also forms during the Staudinger
ligation in aqueous media (Scheme 3) but is trapped by water
rather than methanol under these circumstances. In either case,
the oxygen atom of the phosphine oxide in the ligation product
should originate from water via the intermediacy of phos-
phorane 21.
To test this hypothesis, the isolated phosphorane intermediate
19 was hydrolyzed with 17O/18O-enriched water (76.7% 17O,
10.5% 16O, and 12.8% 18O). The 31P NMR spectra of the
hydrolysis products from either unlabeled (top) or labeled
(bottom) water are shown in Figure 5. A broad peak from 30
to 36 ppm was observed in the bottom spectrum, which we
attribute to a 31P nucleus coupled to 17O. The significant
broadening of the phosphorus resonance is a result of its fast
1
Figure 4. Characterization of intermediate 19. (A) (top) H spectrum of
intermediate 19 isolated from the reaction of phosphine 7 (0.40 M) and
benzyl azide (8) (0.44 M) in CH2Cl2 in the absence of water. The doublet
at 3.03 ppm corresponds to the methoxy group; (bottom) 1H{31P} spectrum
of 19. (B) 2D 1H-13C HMBC spectrum of intermediate 19. Note the absence
of the cross-peak between the methoxy protons (3.03 ppm) and carbonyl
carbon (165 ppm) that would have been expected for structure 18. (C)
Schematic and ORTEP diagrams illustrating the geometry and atom labeling
scheme for intermediate 19 (hydrogen atoms have been omitted for clarity).
Selected bond lengths (Å) and angles (deg): P-O(2) ) 1.687(1), P-N )
1.903(1), P-C(14) ) 1.817(2), P-C(15) ) 1.826(2), P-C(21) ) 1.823-
(2), O(2)-P-N ) 178.39(6), O(2)-P-C(14) ) 96.02(7), O(2)-P-C(15)
) 93.88(7), O(2)-P-C(21) ) 88.48(7), N-P-C(14) ) 84.40(7), N-P-
C(15) ) 87.27(7), N-P-C(21) ) 89.94(7), C(14)-P-C(15) ) 118.73-
(8), C(14)-P-C(21) ) 116.25(8), C(15)-P-C(21) ) 124.33(8).
spin-spin relaxation (short T2) induced by the quadrupolar 17
nucleus.19 Two additional closely spaced 31P resonances were
also observed, corresponding to 31P nuclei bound to either 16
O
O
or 18O. 16O and 18O are not NMR active; therefore, the 31P
resonances appear as singlets. The slight difference in chemical
shifts of the two 31P resonances is the result of an isotope
effect.20 Mass spectrometry analysis further confirmed the
(19) Boykin, D. W. 17O NMR Spectroscopy in Organic Chemistry; CRC Press:
New York, 1990.
(20) Quin, L. D.; Verkade, J. G. Phosphorus-31 NMR Spectral Properties in
Compound Characterization and Structural Analysis; VCH: New York,
1994.
presence of heavy oxygen isotopes in the Staudinger ligation
product (data not shown). These experiments provide direct
evidence for the mechanism of phosphorane hydrolysis shown
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Mechanistic Investigation of the Staudinger Ligation
A R T I C L E S
ligation shows some sensitivity to solvent polarity and is
expected to proceed most rapidly in polar, protic solvents, such
as water. This bodes well for most biological applications,
although reactions in lipophilic environments, such as mem-
branes, might be compromised. More generally, these results
provide a framework for tailoring the Staudinger ligation for
specific purposes.
Experimental Section
General. All chemical reagents were of analytical grade, obtained
from commercial suppliers (Sigma-Aldrich for chemicals, Cambridge
Isotope Laboratories for deuterated solvents, and Isotec for 17O/18O-
enriched water), and used without further purification. All Pd-catalyzed
cross-coupling reactions were performed under Ar using standard
Schlenk techniques. Reaction vessels were flame-dried prior to use.
Flash chromatography was performed using Merck 60 Å 230-400 mesh
silica gel. Analytical TLC was performed on Analtech Uniplate silica
gel plates and visualized by staining with ceric ammonium molybdate
or by absorbance of UV light at 254 nm. Acetonitrile (CH3CN),
deuterated acetonitrile (CD3CN), and triethylamine (TEA) were distilled
over calcium hydride, and methanol (MeOH) was dried over Mg°. All
solvents for Pd-catalyzed cross-coupling reactions and NMR studies
were degassed by three freeze-pump-thaw cycles and stored under
Ar. All organic extracts were dried over Na2SO4, filtered, and
concentrated under reduced pressure using a rotary evaporator.
1H NMR, 13C{1H} NMR, and 31P{1H} NMR spectra were obtained
with Bruker Avance AV-400 (9.4 T) or Bruker DRX-500 (11.7 T)
spectrometers equipped with a 5 mm z-gradient broadband probe. 19F
NMR spectra were obtained with a Bruker Avance 400 spectrometer
Figure 5. 31P{1H} spectra of the Staudinger ligation products obtained
from the hydrolysis of intermediate 19 using either H2O (top) or 17O/18O-
enriched H2O (bottom).
Scheme 6
1
equipped with a 5 mm z-gradient QNP probe. H and 13C chemical
shifts (δ) are reported in parts per million (ppm) referenced to TMS
(0 ppm) and were measured relative to the residual solvent peak. 31P
chemical shifts are referenced to 85% H3PO4 in D2O (0 ppm) and were
measured relative to trimethyl phosphate in C6D6 (3.0 ppm).
19F chemical shifts are referenced to CFCl3 (0 ppm). Coupling constants
(J) are reported in hertz (Hz). Due to 31P coupling, resonances for certain
carbon atoms in the phosphines listed below were observed as doublets.
1
One-dimensional 13C spectra with broadband H and 31P decoupling
were obtained for representative compounds to identify the carbon
resonances that are coupled to 31P. These spectra were recorded on a
Bruker Avance AV-500 spectrometer equipped with a 5 mm triple-
resonance, broadband, inverse probe with xyz gradients. JCP values were
measured from the 1D 13C{1H} spectra and are reported in hertz.
IR spectral data were obtained using a Perkin-Elmer 1600 FTIR
spectrometer. Low- and high-resolution fast atom bombardment (FAB)
and electrospray ionization (ESI) mass spectra were obtained at the
UCsBerkeley Mass Spectrometry Laboratory, and elemental analyses
were obtained at the UCsBerkeley Microanalytical Laboratory. Melting
points were determined using a Mel-Temp 3.0 melting point apparatus
and are uncorrected.
in Scheme 6, wherein the oxygen atom of the phosphine oxide
originates from water.
Conclusions
Synthetic Procedures and Spectral Data for Phosphines.21-23
Compounds 13a-d. The compounds with varied ester substituents
were prepared by Pd(II)-mediated coupling of diphenylphosphine with
the corresponding o-iodobenzoate esters, essentially as described by
Saxon et al.4
2-Diphenylphosphanyl Benzoic Acid Methyl Ester (13a). To a
solution of methyl 2-iodobenzoate (4.90 g, 18.7 mmol) dissolved in
20 mL of anhydrous, degassed CH3CN were added anhydrous TEA
(2.87 mL, 20.6 mmol), Pd(OAc)2 (42.0 mg, 0.187 mmol), and 1,3-
bisdiphenylphosphinopropane (dppp) (77.0 mg, 0.187 mmol). Diphen-
ylphosphine (5.0 mL, 18 mmol) was added to the mixture slowly via
syringe, upon which the solution turned deep red. The solution was
heated at reflux for 4 h, after which the reaction was complete, as
indicated by TLC analysis. The reaction mixture was cooled to 25 °C,
and excess solvent was removed in vacuo to yield a red solid. The
In summary, we have identified parameters that affect the
rate and yield of the Staudinger ligation and defined intermedi-
ates along the reaction pathway. Our results demonstrate that
the overall rate of the reaction is dependent on the electronic
properties of both the phosphine and the azide, making it
possible to tune the reactivity of the substrates for rate
enhancement. Moreover, the reaction tolerates a range of ester
leaving groups, although significant addition of steric bulk can
direct the reaction toward azaylide hydrolysis products at the
expense of ligation products. Still, several alkyl and aryl alcohols
function as efficient leaving groups, and these might be
engineered for specific biological applications. For example,
the leaving groups could be designed with chromogenic or
fluorogenic properties or used as prodrugs. The Staudinger
9
J. AM. CHEM. SOC. VOL. 127, NO. 8, 2005 2691

A R T I C L E S
Lin et al.
crude product was purified by flash chromatography (1:9 EtOAc/
hexanes) under N2 to yield a white solid. This solid was further purified
by recrystallization from MeOH to yield 4.6 g (76%) of a white
crystalline solid: mp 99-100 °C (lit.4 96 °C); 1H NMR (500
MHz, CDCl3) δ 3.75 (s, 3), 6.92-6.98 (m, 1), 7.26-7.84 (m, 12),
8.03-8.07 (m, 1); 13C NMR (125 MHz, CDCl3) δ 52.32, 128.57,
128.73, 128.79, 129.00, 130.96, 132.23, 134.06, 134.22, 134.59,
167.46; 31P NMR (162 MHz, CDCl3) δ -4.5. FAB-HRMS calcd
for C20H17O2P: m/z 321.1044, found 321.1127. Anal. Calcd for
C20H17O2P: C, 74.99; H, 5.38. Found: C, 74.98; H, 5.38.
(s, 3), 6.78-6.87 (m, 4), 6.98-7.00 (m, 1), 7.28-7.37 (m, 10), 7.43-
7.52 (m, 2), 8.24-8.27 (m, 1); 13C NMR (125 MHz, CDCl3) δ 55.81,
114.56, 122.62, 128.52, 128.74, 128.80, 128.96, 311.50, 132.65, 133.96,
134.21, 134.61, 137.90, 141.51, 144.26, 157.45, 165.86; 31P NMR (162
MHz, CDCl3) δ -4.3; IR (thin film, cm-1) ν 2919, 2857, 1728, 1499,
1238, 1189, 1040. FAB-LRMS calcd for C26H21O3P: m/z 412.4, found
[M + H]+ 413.4. Anal. Calcd for C26H21O3P: C, 75.72; H, 5.13.
Found: C, 75.80; H, 4.74.
2-Diphenylphosphanyl Benzoic Acid 4-Tolyl Ester (14b). The
procedure was similar to that described for 14a. The reaction was
performed with 430 mg (1.40 mmol) of 2-diphenylphosphanyl benzoic
acid and 152 mg (1.40 mmol) of p-cresol to afford 500 mg (91%) of
a white crystalline solid: mp 140-141 °C; 1H NMR (500 MHz, CDCl3)
δ 2.32 (s, 3), 6.81-6.83 (m, 2), 6.98-7.00 (m, 1), 7.10-7.12 (m, 2),
7.28-7.37 (m, 11), 7.42-7.49 (m, 2), 8.25-8.27 (m, 1); 13C NMR
(125 MHz, CDCl3) δ 20.80, 121.25, 128.24, 128.47 (d, JCP ) 7.3),
128.67, 129.74, 131.20 (d, JCP ) 2.9), 132.31, 133.78 (d, JCP ) 18.3),
134.02 (d, JCP ) 20.5), 134.33, 135.30, 137.70 (d, JCP ) 11.0), 141.15
(d, JCP ) 27.1), 148.33, 165.40 (d, JCP ) 2.9); 31P NMR (162 MHz,
CDCl3) δ -4.3; IR (thin film, cm-1) ν 3062, 1722, 1499, 1431, 1239,
1189, 1083, 1033. FAB-LRMS calcd for C26H21O2P: m/z 396, found
[M + H]+ 397. Anal. Calcd for C26H21O2P: C, 78.78; H, 5.34. Found:
C, 78.39; H, 5.36.
2-Diphenylphosphanyl Benzoic Acid Ethyl Ester (13b). The
procedure was similar to that described for 13a. The reaction was
performed with 1.95 g (7.06 mmol) of 2-iodobenzoic acid ethyl ester
and 1.3 mL (7.0 mmol) of diphenylphosphine, and 0.80 g (34%) of a
white crystalline solid was obtained after recrystallization from
1
MeOH: mp 114 °C; H NMR (500 MHz, CDCl3) δ 1.20 (t, 3, J )
7.0), 4.22 (q, 2, J ) 7.0), 6.92-6.94 (m, 1), 7.26-7.42 (m, 12), 8.05-
8.07 (m, 1); 13C NMR (125 MHz, CDCl3) δ 14.02, 61.20, 128.21,
128.41, 128.46, 130.59, 131.81, 133.80, 133.96, 134.29, 134.61, 137.75,
137.83, 166.87; 31P NMR (162 MHz, CDCl3) δ -4.7; IR (thin film,
cm-1) ν 3056, 2796, 1709, 1430, 1269, 1102. FAB-LRMS calcd for
C21H19O2P: m/z 334.1, found [M + H]+ 335.1. Anal. Calcd for
C21H19O2P: C, 75.44; H, 5.35. Found: C, 75.63; H, 5.75.
2-Diphenylphosphanyl Benzoic Acid Isopropyl Ester (13c). The
procedure was similar to that described for 13a. The reaction was
performed with 1.34 g (4.62 mmol) of 2-iodobenzoic acid isopropyl
ester and 1.3 mL (5.1 mmol) of diphenylphosphine to give 0.60 g (37%)
of a white crystalline solid after recrystallization from MeOH: mp 98-
100 °C; 1H NMR (400 MHz, CDCl3) δ 1.19 (d, 6, J ) 6.4), 5.12 (app
septet, 1, J ) 6.4), 6.92-6.95 (m, 1), 7.28-7.88 (m, 12), 8.04-8.07
(m, 1); 13C NMR (125 MHz, CDCl3) δ 21.55, 68.75, 127.98, 128.25
(d, JCP ) 7.2), 130.32, 131.49, 133.77 (d, JCP ) 20.6), 134.08, 135.30
(d, JCP ) 11.2), 138.04 (d, JCP ) 11.5), 139.70 (d, JCP ) 26.4), 166.28
(d, JCP ) 2.2); 31P NMR (162 MHz, CDCl3) δ -4.9; IR (thin film,
cm-1) ν 3062, 2981, 1703, 1263, 1102. FAB-LRMS calcd for
C22H21O2P: m/z 348, found [M + H]+ 349. Anal. Calcd for
C22H21O2P: C, 75.85; H, 6.08. Found: C, 75.94; H, 6.12.
2-Diphenylphosphanyl Benzoic Acid tert-Butyl Ester (13d). The
procedure was similar to that described for 13a. The reaction was
performed with 4.2 g (14 mmol) of 2-iodobenzoic acid tert-butyl ester
and 3.8 mL (15 mmol) of diphenylphosphine to give 3.0 g (60%) of a
white crystalline solid after recrystallization from MeOH: mp 127-
128 °C; 1H NMR (400 MHz, CDCl3) δ 1.17 (s, 9), 6.89-6.92 (m, 1),
7.29-7.53 (m, 12), 7.95-7.98 (m, 1); 13C NMR (125 MHz, CDCl3) δ
27.82, 82.12, 128.21, 128.37, 128.42, 128.64, 130.29, 131.31, 133.88,
134.04, 166.51; 31P NMR (162 MHz, CDCl3) δ -4.4; IR (thin film,
cm-1) ν 3062, 2981, 1703, 1598, 1437, 1294. FAB-LRMS calcd for
C23H23O2P: m/z 362, found [M + H]+ 363. Anal. Calcd for
C23H23O2P: C, 76.23; H, 6.40. Found: C, 76.22; H, 6.40.
2-Diphenylphosphanyl Benzoic Acid Phenyl Ester (14c). The
procedure was similar to that described for 14a. The reaction was
performed with 220 mg (0.71 mmol) of 2-diphenylphosphanyl benzoic
acid and 67 mg (0.71 mmol) of phenol to afford 220 mg (77%) of pale
yellow crystals: mp 126-127 °C; 1H NMR (500 MHz, CDCl3) δ 6.94-
7.00 (m, 2), 7.00-7.01 (m, 1), 7.17-7.20 (m, 1), 7.26-7.35 (m, 13),
7.36-7.49 (m, 2), 8.25-8.28 (m, 1); 13C NMR (125 MHz, CDCl3) δ
121.59, 125.72, 128.25, 128.45, 128.51, 128.69, 129.24, 131.24, 132.41,
133.48, 133.92, 134.08, 134.37, 137.60, 137.68, 141.16, 141.38, 150.52,
165.18; 31P NMR (162 MHz, CDCl3) δ -4.3; IR (thin film, cm-1) ν
1728, 1586, 1238, 1189, 1090, 1040. FAB-LRMS calcd for
C25H19O2P: m/z 382, found [M + H]+ 383. Anal. Calcd for
C25H19O2P: C, 78.52; H, 5.01. Found: C, 78.37; H, 4.84.
2-Diphenylphosphanyl Benzoic Acid 4-Fluorophenyl Ester (14d).
The procedure was similar to that described for 14a. The reaction was
performed with 362 mg (1.18 mmol) of 2-diphenylphosphanyl benzoic
acid and 132 mg (1.18 mmol) of p-fluorophenol to afford 340 mg (71%)
1
of white crystals: mp 109-110 °C; H NMR (500 MHz, CDCl3) δ
6.88-6.92 (m, 2), 6.97-7.02 (m, 3), 7.28-7.36 (m, 10), 7.44-7.50
(m, 2), 8.23-8.24 (m, 1); 13C NMR (125 MHz, CDCl3) δ 115.77,
115.96, 122.94, 123.01, 128.30, 128.53 (d, JCP ) 7.7), 128.77, 131.24
(d, JCP ) 2.2), 132.52 (d, JCP ) 17.6), 134.00 (d, JCP ) 20.5), 134.40,
137.52 (d, JCP ) 11.0), 141.25 (d, JCP ) 27.8), 146.25, 159.07, 161.50,
165.34 (d, JCP ) 2.2); 31P NMR (162 MHz, CDCl3) δ -4.2; 19F NMR
(376 MHz, CDCl3) δ -116.20 (m); IR (thin film, cm-1) ν 3056, 2956,
1735, 1499, 1430, 1244, 1182, 1090, 1040. FAB-LRMS calcd for
C25H18FO2P: m/z 400, found [M + H]+ 401. Anal. Calcd for C25H18-
FO2P: C, 75.00; H, 4.53. Found: C, 75.13; H, 4.35.
Synthesis and Spectral Data for Phosphines 14a-f. Compounds
14a-f were prepared by dicyclohexyl carbodiimide (DCC)-mediated
coupling of diphenylphosphanyl benzoic acid with the corresponding
phenol derivatives.
2-Diphenylphosphanyl Benzoic Acid 4-Chlorophenyl Ester (14e).
The procedure was similar to that described for 14a. The reaction was
performed with 350 mg (1.15 mmol) of 2-diphenylphosphanyl benzoic
acid and 147 mg (1.15 mmol) of p-chlorophenol to afford 210 mg (43%)
2-Diphenylphosphanyl Benzoic acid 4-Methoxyphenyl Ester
(14a). 2-Diphenylphosphanyl benzoic acid (367 mg, 1.20 mmol) was
dissolved in 50 mL of anhydrous CH2Cl2 under Ar. DCC (247 mg,
1.20 mmol) and DMAP (7.32 mg, 0.060 mmol) were added, and the
reaction mixture was stirred at room temperature for 30 min. 4-Meth-
oxyphenol (148 mg, 1.20 mmol) was added, and the resulting mixture
was stirred at room temperature overnight. The reaction mixture was
then concentrated to yield a slightly yellow solid. Ice-cold acetone was
added to dissolve the crude product, and the insoluble urea byproduct
was removed by filtration. The filtrate was concentrated, the residue
purified by silica gel chromatography (1:9 EtOAc/hexanes), and the
product recrystallized from MeOH to yield 200 mg (39%) of a white
crystalline solid: mp 169-170 °C; 1H NMR (500 MHz, CDCl3) δ 3.78
1
of a white crystalline solid: mp 153-154 °C; H NMR (500 MHz,
CDCl3) δ 6.87-6.88 (m, 2), 6.98-7.02 (m, 1), 7.29-7.34 (m, 12),
7.44-7.50 (m, 2), 8.23-8.26 (m, 1); 13C NMR (125 MHz, CDCl3) δ
122.97, 128.30, 128.52, 128.79, 129.31, 131.12, 131.30, 132.63, 133.02,
133.92, 134.08, 134.41, 137.40, 137.49, 141.28, 141.50, 148.93, 164.98;
31P NMR (162 MHz, CDCl3) δ -4.1; IR (thin film, cm-1) ν 3062,
2957, 2864, 1735, 1480, 1431, 1239, 1195, 1083, 1033. FAB-LRMS
calcd for C25H18ClO2P: m/z 416, found [M + H]+ 417. Anal. Calcd
for C25H18ClO2P: C, 72.03; H, 4.35. Found: C, 72.00; H, 4.35.
2-Diphenylphosphanyl Benzoic Acid 4-Nitrophenyl Ester (14f).
The procedure was similar to that described for 14a. The reaction was
9
2692 J. AM. CHEM. SOC. VOL. 127, NO. 8, 2005

Mechanistic Investigation of the Staudinger Ligation
A R T I C L E S
performed with 500 mg (1.63 mmol) of 2-diphenylphosphanyl benzoic
acid and 227 mg (1.63 mmol) of p-nitrophenol to afford 400 mg (60%)
of a pale yellow solid: mp 111-113 °C; 1H NMR (400 MHz, CDCl3)
δ 7.00-7.03 (m, 1), 7.08-7.13 (m, 2), 7.29-7.38 (m, 10), 7.47-7.51
(m, 2), 8.15-8.26 (m, 3); 13C NMR (125 MHz, CDCl3) δ 122.42,
125.01, 128.39, 128.63, 128.94 (d, JCP ) 7.3), 131.44 (d, JCP ) 2.2),
132.52 (d, JCP ) 17.6), 132.99, 134.01, 134.53 (d, JCP ) 20.5), 137.20
(d, JCP ) 10.2), 141.76 (d, JCP ) 27.8), 145.30, 155.25, 164.28 (d, JCP
) 2.9); 31P NMR (162 MHz, CDCl3) δ -3.7. FAB-LRMS calcd for
C25H18NO4P: m/z 427, found [M + H]+ 428. Anal. Calcd for C25H18-
NO4P: C, 70.26; H, 4.25; N, 3.28. Found: C, 69.90; H, 4.06; N, 3.24.
Synthesis and Spectral Data for Phosphines 15a-f. The unsym-
metrical phosphines were synthesized using a procedure similar to that
described in refs 22 and 23. Briefly, methyl 2-iodobenzoate and
phenylphosphine were coupled using Pd(OAc)2. The diarylphosphine
was then coupled in a manner similar to that of the appropriate aryl
iodide to afford the triarylphosphine product.
3056, 2950, 1716, 1269, 1102, 1058. ESIMS calcd for C20H17O3P: m/z
336.1, found [M + H]+ 337.1. Anal. Calcd for C20H17O3P: C, 71.42;
H, 5.09. Found: C, 71.11; H, 5.15.
2-[(4-Methoxyphenyl)phenylphosphanyl] Benzoic Acid Methyl
Ester (15c). The procedure was similar to that described for 15a. The
reaction was performed with 1.31 g (5 mmol) of methyl 2-iodobenzoate,
0.55 mL (5 mmol) of phenylphosphine, and 1.17 g (5 mmol) of
4-iodoanisole to afford 105 mg (6%) of an off-white solid (mp 89-90
°C) after two rounds of flash chromatography (1:9 EtOAc/hexanes)
under N2: 1H NMR (400 MHz, CDCl3) δ 3.75 (s, 3), 3.82 (s, 3), 6.88-
6.90 (m, 2), 6.95-6.98 (m, 1), 7.23-7.40 (m, 9), 8.03-8.06 (m, 1);
13C NMR (125 MHz, CDCl3) δ 52.27, 55.38, 114.43, 114.50, 128.39,
128.64, 128.69, 128.74, 132.14, 133.62, 133.78, 134.39, 161.51; 31P
NMR (162 MHz, CDCl3) δ -5.9; IR (thin film, cm-1) ν 2950, 2832,
1716, 1585, 1499, 1430, 1288, 1244. 1176. ESIMS calcd for
C21H19O3P: m/z 350.1, found [M + H]+ 351.1. Anal. Calcd for
C21H19O3P: C, 71.99; H, 5.47. Found: C, 71.76; H, 5.49.
2-[(4-N,N-(Dimethylamino)phenyl)phenylphosphanyl] Benzoic
Acid Methyl Ester (15a). To a solution of methyl 2-iodobenzoate
(4.0 g, 15 mmol) dissolved in 100 mL of anhydrous and degassed
CH3CN were added anhydrous TEA (23 mL, 15 mmol), Pd(OAc)2
(110 mg, 0.5 mmol), and 1,3-bisdiphenylphosphinopropane (dppp)
(206 mg, 0.5 mmol). Phenylphosphine (1.7 mL, 15 mmol) was added
to the above mixture over a 5 min period via syringe, during which
time the solution turned deep red. The reaction mixture was heated at
reflux, and reaction progress was monitored by 31P NMR. A new 31P
resonance at about -48 ppm was observed after 30 min, consistent
with formation of a secondary phosphine.20 The reaction mixture was
heated for 10 h, at which point most of the phenylphosphine was
consumed (∼70%), as determined by 31P NMR analysis. Complete
conversion to the diaryl phosphine could not be achieved even after
prolonged heating (>24 h). The diaryl phosphine was carried on to
the next step without purification. The reaction mixture was cooled to
25 °C, and a second equivalent of TEA (0.77 mL, 0.55 mmol) and
N,N-(dimethylamino)-4-iodobenzene (3.8 g, 15 mmol) was added. The
resulting mixture was heated at reflux for 14 h, after which TLC and
31P NMR analysis indicated complete consumption of the diaryl
phosphine. The crude mixture was concentrated, and the product was
purified by silica gel chromatography (1:9 EtOAc/hexanes) and
recrystallization from MeOH to give 1.0 g (18%) of a yellow crystalline
2-[(4-Methylphenyl)phenylphosphanyl] Benzoic Acid Methyl
Ester (15d). The procedure was similar to that described for 15a. The
reaction was performed with 1.3 g (5 mmol) of methyl 2-iodobenzoate,
0.55 mL (5 mmol) of phenylphosphine, and 1.2 g (5 mmol) of
4-iodotoluene to afford 124 mg (7%) of a bright yellow oil after
purification by silica gel chromatography (1:9 EtOAc/hexanes):
1H NMR (500 MHz, CDCl3) δ 2.36 (s, 3), 3.75 (s, 3), 6.94-6.97 (m,
1), 7.15-7.21 (m, 4), 7.28-7.40 (m, 7), 8.04-8.06 (m, 1); 13C NMR
(125 MHz, CDCl3) δ 21.30, 51.98, 128.04, 128.36, 128.41, 128.46,
129.29, 129.35, 130.63, 131.84, 133.59, 133.75, 133.90, 134.07, 134.18,
138.66, 154.01; 31P NMR (162 MHz, CDCl3) δ -5.9. ESIMS calcd
for C21H20O2P [M + H]+: m/z 334.1, found [M + H]+ 335.1. FAB-
HRMS calcd for C21H20O2P [M + H]+: m/z 335.1201, found 335.1205.
2-[(4-Bromophenyl)phenylphosphanyl] Benzoic Acid Methyl
Ester (15e). The procedure was similar to that described for 15a. The
reaction was performed with 2.6 g (10 mmol) of methyl 2-iodobenzoate,
1.1 mL (10 mmol) of phenylphosphine, and 2.8 g (10 mmol) of
1-bromo-4-iodobenzene to afford 457 mg (6%) of an off-white solid:
1
mp 113-114 °C; H NMR (500 MHz, CDCl3) δ 3.82 (s, 3), 6.92-
6.94 (m, 1), 7.27-7.39 (m, 2), 7.40-7.50 (m, 7), 8.13-8.16 (m, 3);
13C NMR (125 MHz, CDCl3) δ 52.05, 123.23, 128.40, 128.58 (d, JCP
) 7.3), 128.86, 130.76 (d, JCP ) 2.2), 131.59 (d, JCP ) 7.3), 132.05,
133.87, 134.16 (d, JCP ) 21.2), 135.30 (d, JCP ) 21.2), 137.28 (d, JCP
1
solid: mp 113-114 °C; H NMR (500 MHz, CDCl3) δ 2.97 (s, 6),
) 7.3), 137.34 (d, JCP ) 6.6), 139.88 (d, JCP ) 26.3), 167.05 (d, JCP
)
3.73 (s, 3), 6.70 (d, 2), 7.01-7.03 (m, 1), 7.18-7.39 (m, 9), 8.01-
8.03 (m, 1); 13C NMR (125 MHz, CDCl3) δ 40.16, 51.81, 111.13,
112.37, 127.77, 128.04, 128.21 (d, JCP ) 6.6), 130.49 (d, JCP ) 2.2),
131.46, 131.64, 133.22 (d, JCP ) 19.8), 133.29, 134.03, 134.16 (d, JCP
) 18.3), 135.66 (d, JCP ) 22.7), 139.17 (d, JCP ) 8.8), 141.62 (d, JCP
) 27.1), 167.39 (d, JCP ) 2.9); 31P NMR (202 MHz, CDCl3) δ -7.0;
IR (thin film, cm-1) ν 3056, 2950, 1716, 1269, 1102, 1058. ESIMS
calcd for C22H22NO2P: m/z 363.1, found [M + H]+ 364.1. Anal. Calcd
for C22H22NO2P: C, 72.71; H, 6.10; N, 3.85. Found: C, 72.64; H, 5.93;
N, 3.87.
2-[(4-Hydroxyphenyl)phenylphosphanyl] Benzoic Acid Methyl
Ester (15b). The procedure was similar to that described for 15a. The
reaction was performed with 2.6 g (10 mmol) of methyl 2-iodobenzoate,
1.1 mL (10 mmol) of phenylphosphine, and 2.8 g (10 mmol) of
4-iodophenol to afford 457 mg (6%) of pale yellow crystals: mp 113-
114 °C; 1H NMR (500 MHz, CDCl3) δ 3.73 (s, 3), 6.78 (d, 2), 6.93-
6.96 (m, 1), 7.15-7.18 (m, 2), 7.25-7.39 (m, 7), 8.02-8.05 (m, 1);
13C NMR (100 MHz, CDCl3) δ 52.09, 115.75, 115.83, 123.10, 128.42,
128.49, 130.71, 131.94, 133.45, 133.65, 134.12, 134.28, 135.80, 136.02,
156.48; 31P NMR (162 MHz, CDCl3) δ -5.9; IR (thin film, cm-1) ν
2.2); 31P NMR (162 MHz, CDCl3) δ -4.3; IR (thin film, cm-1) ν 3056,
2950, 1716, 1269, 1102, 1058. ESIMS calcd for C20H16BrO2P: m/z
399, found [M + H]+ 400. Anal. Calcd for C20H16BrO2P: C, 60.17;
H, 4.04. Found: C, 60.12; H, 3.99.
2-[(4-Nitrophenyl)phenylphosphanyl] Benzoic Acid Methyl Ester
(15f). The procedure was similar to that described for 15a. The reaction
was performed with 1.3 g (5 mmol) of methyl 2-iodobenzoate, 0.55
mL (5 mmol) of phenylphosphine, and 1.2 g (5 mmol) of 1-iodo-4-
nitrobenzene to afford 124 mg (7%) of a bright yellow oil after
purification by silica gel chromatography (1:9 EtOAc/hexanes):
1H NMR (400 MHz, CDCl3) δ 3.82 (s, 3), 6.92-6.94 (m, 1), 7.27-
7.39 (m, 2), 7.40-7.50 (m, 7), 8.13-8.16 (m, 3); 13C NMR (125 MHz,
CDCl3) δ 52.31, 123.08, 128.85, 128.91, 129.49, 130.94, 132.45,
133.72, 133.88, 134.28, 134.38, 134.55, 136.13, 136.22, 147.74, 148.22,
148.35, 166.93; 31P NMR (162 MHz, CDCl3) δ -4.3; IR (thin film,
cm-1) ν 2919, 2845, 1709, 1518, 1338, 1270, 1108. FAB-HRMS calcd
for C20H17NO4P [M + H]+: m/z 366.0895, found 366.0893.
Characterization of Staudinger Ligation Products from the
Reactions of Phosphines 13a-d, 14a-f, and 15a-f and Benzyl
Azide. The Staudinger ligation products formed in the reactions of
benzyl azide with phosphines13a-d, 14a-f, or 15a-f were isolated
from the reaction mixtures used in the kinetics experiments. During
the course of these reactions, no background oxidation of the phosphines
was observed.
(21) Quin, L. D. A Guide to Organophosphorus Chemistry; Wiley: New York,
2000.
(22) Brauer, D. J.; Hingst, M.; Kottsieper, K. W.; Liek, C.; Nickel, T.; Tepper,
M.; Stelzer, O.; Sheldrick, W. S. J. Organomet. Chem. 2002, 645, 14-26.
(23) Herd, O. J.; Hessler, A.; Hingst, M.; Tepper, M.; Stelzer, O. J. Organomet.
Chem. 1996, 522, 69-76.
9
J. AM. CHEM. SOC. VOL. 127, NO. 8, 2005 2693

A R T I C L E S
Lin et al.
N-Benzyl-2-(diphenylphosphinoyl)benzamide.4 This compound is
the product of the Staudinger ligation of benzyl azide with phosphines
13a-d and 14a-f. The product was purified by silica gel chromatog-
raphy (5:95 MeOH/CH2Cl2) and recrystallized from MeOH to yield a
127.06, 127.94, 128.45, 128.62, 128.72, 129.12, 129.45, 129.55, 129.74,
131.56, 131.64, 131.70, 131.81, 132.23, 132.55, 133.32, 137.59, 142.94,
167.30; 31P NMR (162 MHz, CDCl3) δ 35.5. FAB-LRMS calcd for
C27H24NO2P: m/z 425, found [M + H]+ 426. Anal. Calcd for C27H24-
NO2P: C, 76.22; H, 5.69; N, 3.29. Found: C, 75.91; H, 5.84; N, 3.55.
N-Benzyl-2-[(4-bromophenyl)phenylphosphinoyl]benzamide. This
compound is the product of the Staudinger ligation of phosphine 15e
and benzyl azide. The product was purified by silica gel chromatography
(5:95 MeOH/CH2Cl2) and recrystallized from MeOH to yield a white
1
white solid: mp 195-196 °C; H NMR (500 MHz, CDCl3) δ 4.08-
4.09 (d, 2, J ) 5.5), 7.05-7.09 (dq, 1, J ) 0.9, 7.7), 7.21-7.28
(m, 5), 7.35-7.39 (m, 1), 7.46-7.50 (m, 4), 7.55-7.67 (m, 7), 8.02-
8.04 (dq, 1, J ) 1.0, 4.0), 9.05-9.08 (br s, 1); 13C NMR (125 MHz,
CDCl3) δ 44.00, 127.09, 127.92, 128.48, 128.66, 128.75, 128.93,
129.72, 129.75, 129.84, 130.59, 131.44, 131.61, 131.69, 131.75, 132.29,
132.32, 132.61, 133.34, 137.57, 140.98, 141.04, 167.21, 167.24; 31P
NMR (162 MHz, CDCl3) δ 34.9; IR (thin film, cm-1) ν 3050, 1660,
1579, 1480, 1437, 1300, 1172. EI-LRMS calcd for C26H22NO2P: m/z
411, found (M+) 411.
1
solid: mp 206-207 °C; H NMR (500 MHz, CDCl3) δ 4.06-4.18
(m, 2), 7.06-7.11 (m, 1), 7.21-7.67 (m, 16), 8.02-8.06 (m, 1), 8.91-
8.98 (m, 1); 13C NMR (125 MHz, CDCl3) δ 44.06, 127.19, 127.58,
128.00, 128.31, 128.51, 128.77, 128.87, 129.11, 130.10, 131.59, 131.67,
131.74, 131.82, 131.98, 132.08, 132.53, 132.86, 132.99, 133.08, 133.23,
133.33, 137.45, 140.97, 167.08; 31P NMR (162 MHz, CDCl3) δ 34.7.
FAB-LRMS calcd for C26H21BrNO2P: m/z 490, found (M+) 490. Anal.
Calcd for C26H21BrNO2P: C, 63.69; H, 4.32; N, 2.86. Found: C, 63.30;
H, 4.45; N, 2.86.
N-Benzyl-2-[(4-nitrophenyl)phenylphosphinoyl]benzamide. This
compound is the product of the Staudinger ligation of phosphine 15f
and benzyl azide. The product was purified by silica gel chromatography
(5:95 MeOH/CH2Cl2) and recrystallized from MeOH to yield a pale
yellow solid: mp 142-144 °C; 1H NMR (500 MHz, CDCl3) δ 4.04-
4.19 (m, 2), 7.14-7.26 (m, 5), 7.36-7.90 (m, 8), 8.00-8.02 (m, 1),
8.24-8.26 (m, 2), 8.54-8.57 (m, 1); 13C NMR (125 MHz, CDCl3) δ
44.07, 123.38, 123.48, 127.36, 128.11, 129.03, 130.09, 130.19, 131.71,
131.79, 132.60, 132.68, 132.87, 133.31, 137.25, 158.23; 31P NMR (162
MHz, CDCl3) δ 32.9. FAB-HRMS calcd for C26H22N2O4P [M + H]+:
m/z 457.1317, found 457.1320.
Isolation and Characterization of Intermediate 19. All compounds
were manipulated in a glovebox under an inert atmosphere (N2).
Glassware was dried overnight at 150 °C or flame-dried under vacuum
immediately prior to use. NMR tubes were sealed by attaching the tube
directly to a Kontes high-vacuum stopcock via a Cajon Ultra-Torr
reducing union and flame-sealing on a vacuum line.
Anhydrous CH2Cl2, pentane, and diethyl ether were obtained by
passage through a column of activated alumina (type A2, size 12 ×
32, Purify Co.) under N2 pressure, sparged with N2, and stored over 3
Å molecular sieves in the glovebox under N2. CD2Cl2 was dried over
calcium hydride, degassed by three freeze-pump-thaw cycles, vacuum
transferred to a glass bomb, and stored over 3 Å molecular sieves under
an atmosphere of N2. Benzyl azide was additionally dried over 3 Å
sieves in a vial in the glovebox for 4 days prior to use.
A solution of phosphine 13a (0.637 g, 1.99 mmol) and benzyl azide
(0.295 g, 2.22 mmol) in CH2Cl2 (5.0 mL) was stirred at room
temperature in the glovebox for 3 days. Upon mixing, vigorous bubbling
was observed in the pale yellow solution. The volatile materials were
removed under vacuum to yield a brown oil. Upon adding pentane
(10 mL) to this oil, a white solid precipitated from the brown solution.
The solid was dried under vacuum then washed twice with toluene
(2 × 10 mL). The resulting off-white powder (mp 141-143 °C) was
then dried under vacuum to yield compound 19 (0.662 g, 78%), which
was analytically pure. A small amount of compound 19 (∼20 mg) was
crystallized at -30 °C from diethyl ether (∼1.5 mL) to yield colorless
bladelike crystals in the space group P21/n: 1H NMR (500 MHz, CD2-
Cl2) δ 3.02-3.05 (d, 3, J ) 11.0), 3.70-3.71 (d, 2, J ) 4.0), 6.63-
6.65 (d, 2, J ) 8.0), 6.90-7.00 (m, 3), 7.22-7.50 (m, 6), 7.63-7.79
(m, 5), 7.92-7.98 (m, 1), 7.98-8.00 (m, 1), 8.33-8.35 (m, 1);
13C NMR (125 MHz, CD2Cl2) δ 46.00, 54.17 (d, JCP ) 8.2), 125.62
(d, JCP ) 13.0), 125.83, 127.57, 128.21, 128.74 (d, JCP ) 16.8), 129.73
(d, JCP ) 159.3), 130.29 (d, JCP ) 3.4), 130.56 (d, JCP ) 11.0), 131.81
(d, JCP ) 15.4), 134.31 (d, JCP ) 3.4), 135.11 (d, JCP ) 170.8), 135.89
(d, JCP ) 11.5), 139.84, 143.28 (d, JCP ) 11.5), 165.10 (d, JCP ) 8.2);
31P NMR (162 MHz, CD2Cl2) δ -56.0. ESI-LRMS calcd for C27H24-
NO2P: m/z 425, found [M + H]+ 426. Anal. Calcd for C27H24NO2P:
C, 76.22; H, 5.69; N, 3.29. Found: C, 76.11; H, 5.69; N, 3.44.
N-Benzyl-2-[(4-N,N-(dimethylamino)phenyl)phenylphosphinoyl]-
benzamide. This compound is the product of the Staudinger ligation
of phosphine 15a and benzyl azide. The product was purified by silica
gel chromatography (5:95 MeOH/CH2Cl2) and recrystallized from
1
MeOH to yield a white solid: mp 183-184 °C; H NMR (500 MHz,
CDCl3) δ 3.02 (s, 6), 4.06-4.10 (dd, 1, J ) 12.7, 5.3), 4.17-4.21 (dd,
1, J ) 12.7, 5.3), 6.70-6.72 (d, 2, J ) 11.0), 7.06-7.10 (m, 1), 7.17-
7.64 (m, 14), 8.02-8.04 (dd, 1, J ) 7.6, 2.2), 9.37-9.48 (m, 1); 13C
NMR (125 MHz, CDCl3) δ 39.97, 44.06, 111.40, 111.51, 127.00,
127.92, 128.42, 128.49, 128.59, 129.60, 130.07, 130.84, 131.44, 131.52,
131.71, 131.77, 131.89, 132.22, 132.56, 133.07, 133.16, 133.26, 133.36,
137.76, 140.81, 140.87, 152.46, 167.55, 167.58; 31P NMR (162 MHz,
CDCl3) δ 35.9. FAB-LRMS calcd for C28H27N2O2P: m/z 454, found
[M + H]+ 455. Anal. Calcd for C28H27N2O2P: C, 73.99; H, 5.99; N,
6.16. Found: C, 73.74; H, 6.09; N, 6.15.
N-Benzyl-2-[(4-hydroxy)phenylphosphinoyl]benzamide. This com-
pound is the product of the Staudinger ligation of phosphine 15b and
benzyl azide. The product was purified by silica gel chromatography
(5:95 MeOH/CH2Cl2) and recrystallized from MeOH to yield a white
1
solid: mp 194-195 °C; H NMR (500 MHz, CDCl3) δ 3.96-4.00
(dd, 1, J ) 15.0, 4.0), 4.35-4.40 (dd, 1, J ) 15.0, 6.0), 6.79-6.82
(m, 2), 7.04-7.09 (m, 1), 7.23-7.26 (m, 4), 7.37-7.70 (m, 10), 8.00-
8.02 (m, 1), 9.36-9.37 (br s, 1); 13C NMR (125 MHz, CDCl3) δ 44.41,
116.30, 116.44, 120.04, 127.27, 127.89, 128.53, 128.65, 128.77, 129.75,
130.18, 130.30, 130.72, 131.38, 132.07, 132.17, 132.37, 132.65, 133.06,
133.18, 133.39, 133.51, 137.00, 140.09, 161.49, 168.81; 31P NMR
(162 MHz, CDCl3) δ 36.1. FAB-LRMS calcd for C26H22NO3P: m/z
427, found [M + H]+ 428. Anal. Calcd for C26H22NO3P: C, 73.06; H,
5.19; N, 3.28. Found: C, 72.73; H, 5.19; N, 3.59.
N-Benzyl-2-[(4-methoxyphenyl)phenylphosphinoyl]benzamide. This
compound is the product of the Staudinger ligation of phosphine 15c
and benzyl azide. The product was purified by silica gel chromatography
(5:95 MeOH/CH2Cl2) and recrystallized from MeOH to yield a white
1
solid: mp 209-210 °C; H NMR (500 MHz, CDCl3) δ 3.86 (s, 3)
4.10-4.15 (m, 2), 6.96-6.98 (m, 2), 7.05-7.09 (m, 1), 7.22-7.28 (m,
5), 7.41-7.67 (m, 9), 8.02-8.04 (m, 1), 9.18-9.21 (m, 1); 13C NMR
(125 MHz, CDCl3) δ 44.06, 55.32, 114.28, 114.39, 122.14, 127.06,
127.95, 128.45, 128.62, 128.71, 130.09, 131.53, 131.61, 131.82, 131.87,
132.20, 133.52, 133.61, 137.61, 162.74, 167.90; 31P NMR (202 MHz,
CDCl3) δ 34.1. FAB-LRMS calcd for C27H24NO3P: m/z 441, found
(M+) 441. Anal. Calcd for C27H24NO3P: C, 73.46; H, 5.48; N, 3.17.
Found: C, 73.28; H, 5.52; N, 3.14.
N-Benzyl-2-[(4-methylphenyl)phenylphosphinoyl]benzamide. This
compound is the product of the Staudinger ligation of phosphine 15d
and benzyl azide. The product was purified by silica gel chromatography
(5:95 MeOH/CH2Cl2) and recrystallized from MeOH to yield a white
1
solid: mp 226-227 °C; H NMR (500 MHz, CDCl3) δ 2.42 (s, 3),
4.09-4.11 (m, 2), 7.06-7.11 (m, 1), 7.21-7.67 (m, 16), 8.02-8.06
(m, 1), 8.91-9.00 (m, 1); 13C NMR (125 MHz, CDCl3) δ 21.66, 44.02,
9
2694 J. AM. CHEM. SOC. VOL. 127, NO. 8, 2005

Mechanistic Investigation of the Staudinger Ligation
A R T I C L E S
General Procedures for Kinetics Experiments using 31P NMR
Spectroscopy.20 Stock solutions of phosphine, azide, and the internal
standard (triphenylphosphine oxide) were prepared as follows. The
phosphine was placed in a 1.00 mL volumetric tube, and anhydrous,
degassed CD3CN was added to yield a total volume of 1.00 mL and
final phosphine concentrations between 0.064 and 0.26 M. Gentle
heating in a water bath (∼45 °C) was required to dissolve the phosphine
completely. The phosphine stock solution was transferred to a flame-
dried screw-capped vial, purged with Ar for 5 min, and stored at
-20 °C until use. Stock solutions of the azide (4.95 M) and
triphenylphosphine oxide (0.31 M) were prepared and stored in a similar
manner.
In a typical experiment, 100 µL each of the phosphine and phosphine
oxide stock solutions was transferred to a 5 mm NMR tube using a
pipetman. Distilled water (30 µL, 1.7 mmol) and 270 µL of CD3CN
were added to give a final volume of 500 µL. An aliquot of the azide
stock solution (100 µL) was added immediately before the start of
kinetic runs. Volumes of water, phosphine, or azide used were adjusted
accordingly in experiments where the concentrations of these reagents
were varied.
a neat methanol sample. 31P NMR spectra were obtained using a 90°
pulse (6.1 µs) with a 60 s delay between scans to allow full relaxation
(typical T1 values for the various 31P species were measured to be <10
s). One scan was acquired for each time point. The first scan was
typically taken within 3 min after addition of the azide. All kinetics
experiments were performed in triplicate.
31P NMR spectra were processed using the Bruker XWinNMR
software v3.5 with a line broadening of 2 Hz. Peak areas were integrated
automatically and normalized to the triphenylphosphine oxide reso-
nance. Data were plotted and analyzed using Microsoft Excel X.
Acknowledgment. We thank Dr. Fred Hollander for solving
the crystal structure of intermediate 19, and Dr. Ulla Andersen
for mass spectrometry analyses. This work was supported by
grants to C.R.B. from the National Institutes of Health
(GM58867), and the Director, Office of Science, Office of Basic
Energy Sciences, Division of Materials Science and Engineering
and the Office of Energy Biosciences of the U.S. Department
of Energy under Contract DE-AC03-76SF00098, and a grant
to R.G.B. from the NSF (CHE_0345488).
NMR experiments designed to investigate the kinetic order of the
Staudinger ligation with respect to phosphine, benzyl azide, and water
were performed with either a Bruker DRX-500 spectrometer equipped
with a 5 mm z-gradient broadband probe or a Bruker AV-400
spectrometer equipped with a 5 mm z-gradient broadband probe at
ambient temperature (20 ( 0.5 °C), as verified by ∆δ(CH3-δOH) for
Supporting Information Available: CIF data for compound
19. This material is available free of charge via the Internet at
http://pubs.acs.org.
JA044461M
9
J. AM. CHEM. SOC. VOL. 127, NO. 8, 2005 2695