Reaction mechanism and kinetics of the traceless Staudinger ligation

Article abstract of DOI:10.1021/ja060484k

The traceless Staudinger ligation enables the formation of an amide bond between a phosphinothioester (or phosphinoester) and an azide without the incorporation of residual atoms. Here, the coupling of peptides by this reaction was characterized in detail

Full text of DOI:10.1021/ja060484k

Published on Web 06/20/2006
Reaction Mechanism and Kinetics of the Traceless Staudinger
Ligation
Matthew B. Soellner,†,| Bradley L. Nilsson,†,§ and Ronald T. Raines*,†,‡
Contribution from the Departments of Chemistry and Biochemistry, UniVersity of
Wisconsin-Madison, Madison, Wisconsin 53706
Received January 20, 2006; E-mail: raines@biochem.wisc.edu
Abstract: The traceless Staudinger ligation enables the formation of an amide bond between a
phosphinothioester (or phosphinoester) and an azide without the incorporation of residual atoms. Here,
the coupling of peptides by this reaction was characterized in detail. Experiments with [18O]H2O indicated
that the reaction mediated by (diphenylphosphino)methanethiol proceeded by SfN acyl transfer of the
iminophosphorane intermediate to form an amidophosphonium salt, rather than by an aza-Wittig reaction
and subsequent hydrolysis of the resulting thioimidate. A continuous 13C NMR-based assay revealed that
the rate-determining step in the Staudinger ligation of glycyl residues mediated by (diphenylphosphino)-
methanethiol was the formation of the initial phosphazide intermediate. Less efficacious coupling reagents
and reaction conditions led to the accumulation of an amine byproduct (which resulted from a Staudinger
reduction) or phosphonamide byproduct (which resulted from an aza-Wittig reaction). The Staudinger ligation
mediated by (diphenylphosphino)methanethiol proceeded with a second-order rate constant (7.7 × 10-3
M-1 s-1) and yield (95%) that was unchanged by the addition of exogenous nucleophiles. Ligations mediated
by phosphinoalcohols had lower rate constants or less chemoselectivity. Accordingly, (diphenylphosphino)-
methanethiol was judged to be the most efficacious known reagent for effecting the traceless Staudinger
ligation.
Introduction
ligation, a peptide with a C-terminal phosphinothioester is
coupled with a second peptide having an N-terminal azido acid
The advent of methodology for the chemoselective ligation
of peptide fragments has made proteins accessible targets for
total chemical synthesis.1 Already, many proteins have been
assembled from synthetic peptides using prior capture strategies.
“Native chemical ligation”, the coupling of a peptide containing
a C-terminal thioester with another peptide containing an
N-terminal cysteine residue, has been the most widely applied
of such strategies.2-4 “Expressed protein ligation” is an enabling
extension of native chemical ligation in which the C-terminal
thioester is accessed by using recombinant DNA technology.5
Although powerful, these methods are limited by their reliance
on cysteine, which is the second least common residue.6
to form a new peptide bond in a traceless manner, without
residual atoms.9,10 The reaction occurs without detectable
racemization.11 The Staudinger ligation has been used in the
orthogonal assembly of a fully functional enzyme12 and for the
site-specific immobilization of peptides and small molecules
on a surface.13,14 A variety of phosphines have been used to
apply the Staudinger ligation to other problems in chemical
biology,7 including glycopeptide synthesis,15 biomolecular label-
ing in vitro16 and in vivo,17,18 and drug delivery.19 Most of this
work has employed reagents that are engrammic, leaving a
phosphine oxide in the ligation product. This attribute has
negligible consequences for typical labeling experiments, in
which the creation of a stable linkage is of paramount
Emerging strategies for protein assembly avoid the need for
a cysteine residue at the ligation junction.1 The Staudinger
ligation is one such strategy.7,8 In one version of the Staudinger
(8) Bra¨se, S.; Gil, C.; Knepper, K.; Zimmermann, V. Angew. Chem., Int. Ed.
Engl. 2005, 44, 5188-5240.
(9) Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. Org. Lett. 2000, 2, 1939-
† Department of Chemistry.
1941.
(10) Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. Org. Lett. 2001, 3, 9-12.
(11) Soellner, M. B.; Nilsson, B. L.; Raines, R. T. J. Org. Chem. 2002, 67,
4993-4996.
‡ Department of Biochemistry.
§ Present address: Department of Chemistry, University of California,
Irvine, CA 92697.
(12) Nilsson, B. L.; Hondal, R. J.; Soellner, M. B.; Raines, R. T. J. Am. Chem.
Soc. 2003, 125, 5268-5269.
| Present address: Department of Chemistry, University of California,
Berkeley, CA 94720.
(13) Ko¨hn, M.; Wacker, R.; Peters, C.; Schro¨der, H.; Soule`re, L.; Breinbauer,
R.; Niemeyer, C. M.; Waldmann, H. Angew. Chem., Int. Ed. 2003, 42,
5830-5834.
(1) Nilsson, B. L.; Soellner, M. B.; Raines, R. T. Annu. ReV. Biophys. Biomol.
Struct. 2005, 34, 91-118.
(2) Dawson, P. E.; Kent, S. B. H. Annu. ReV. Biochem. 2000, 69, 923-960.
(3) Tam, J. P.; Xu, J.; Eom, K. D. Biopolymers 2001, 60, 194-205.
(4) Kent, S. J. Pept. Sci. 2003, 9, 574-593.
(14) Soellner, M. B.; Dickson, K. A.; Nilsson, B. L.; Raines, R. T. J. Am. Chem.
Soc. 2003, 125, 11790-11791.
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L. Org. Lett. 2004, 6, 4479-4482. (b) Bianchi, A.; Bernardi, A. Tetrahedron
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Tetrahedron: Asymmetry 2005, 16, 381-386. (d) Liu, L.; Hong, Z.-Y.;
Wong, C.-H. ChemBioChem 2006, 2006, 429-432.
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(6) McCaldon, P.; Argos, P. Proteins 1988, 4, 99-122.
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3116.
9
8820
J. AM. CHEM. SOC. 2006, 128, 8820-8828
10.1021/ja060484k CCC: $33.50 © 2006 American Chemical Society

Mechanism/Kinetics of the Traceless Staudinger Ligation
A R T I C L E S
Scheme 1. Putative Mechanisms for the Staudinger Ligation
importance. In synthetic applications, however, a residual
phosphine oxide is unacceptable.
Mediated by (Diphenylphosphino)methanethiol
Two distinct phosphinothiols have been used specifically for
the Staudinger ligation of peptides. The first, a phosphi-
nothiophenol, was able to provide dipeptides from phosphi-
nothioesters and azido acids, albeit in modest yield (e35%).9
The second was a phosphinomethanethiol that effected the
Staudinger ligation in high yield (>90%).10,11 These couplings
were performed in mixed THF/water or DMF/water solvents
with a stoichiometric ratio of reagents. To form an Xaa-Yaa
junction, however, either Xaa or Yaa (or both) must be a glycine
residue for the ligation to be efficient (B. L. Nilsson, M. B.
Soellner, and R. T. Raines, unpublished results).20
Herein, we report on a detailed analysis of the traceless
Staudinger ligation between a variety of phosphinothioesters
(or phosphinoesters) and azides. Our analyses rely on isotopic
labeling experiments and a continuous NMR-based assay for
the reaction. These analyses reveal much new and useful
information on the mechanism and kinetics of this versatile
synthetic reaction.
Results and Discussion
Mechanism of the Staudinger Ligation. Our putative
mechanism for the Staudinger ligation is shown in Scheme 1.10
The nitrogen atom of an iminophosphorane such as 1 has
intrinsic nucleophilicity and can be acylated by a thioester
(Scheme 1, path A).21-28 This acylation likely proceeds via
tetrahedral intermediate 2 to form amidophosphonium salt 3.
Finally, the P-N bond of the amidophosphonium salt is
hydrolyzed to form amide 4 and phosphine oxide 5.
There is considerable literature precedent to indicate that the
iminophosphorane formed from a phosphinoester (as opposed
to a phosphinothioester) tends to undergo an aza-Wittig reac-
tion29 to produce a stable imidate (Scheme 1, path B).16f,p In
Scheme 1, the intramolecular aza-Wittig reaction of iminophos-
phorane 1 could also form a thioimidate, here 7. In contrast to
the stable imidate formed during Staudinger ligations mediated
by phosphinoalcohols,16f,p a thioimidate has not been observed
as a product of the reaction mediated by phosphinothiols.30,31
Nonetheless, the failure to observe thioimidate 7 does not
preclude its existence on the reaction pathway. Accordingly,
we did an experiment to search for evidence of the aza-Wittig
reaction of iminophosphorane 1.
In our proposed mechanism for the Staudinger ligation
mediated by a phosphinothiol (Scheme 1, path A),9,10 the oxygen
of phosphine oxide 5 is derived from water during the hydrolysis
of amidophosphonium salt 3. In the alternative aza-Wittig
mechanism (Scheme 1, path B), this oxygen originates from
the thioester. To distinguish between these two mechanisms,
we reacted phosphinothioester 11 (R ) AcNHCH2 in Scheme
1) and azide 12 (R′ ) CH2C(O)NHBn) in [18O]H2O and
examined the products with mass spectrometry. We found that
amide 4 contained exclusively 16O, and phosphine oxide 5
contained only 18O.32 These data demonstrate that an aza-Wittig
reaction does not occur to an appreciable extent in the traceless
Staudinger ligation to form a Gly-Gly junction using (diphen-
ylphosphino)methanethiol as the coupling reagent.
(16) For examples, see: (a) Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007-
2010. (b) Saxon, E.; Luchansky, S. J.; Hang, H. C.; Yu, C.; Lee, S. C.;
Bertozzi, C. R. J. Am. Chem. Soc. 2002, 124, 14893-14902. (c) Kiick, K.
L.; Saxon, E.; Tirrell, D. A.; Bertozzi, C. R. Proc. Natl. Acad. Sci. U.S.A.
2002, 99, 19-24. (d) Ovaa, H.; van Swieten, P. F.; Kessler, B. M.;
Leeuwenburgh, M. A.; Fiebiger, E.; van den Nieuwendijk, A. M. C. H.;
Galardy, P. J.; van der Marel, G. A.; Ploegh, H. L.; Overkleeft, H. S. Angew.
Chem., Int. Ed. 2003, 42, 3626-3629. (e) Wang, C. C.-Y.; Seo, T. S.; Li,
Z.; Ruparel, H.; Ju, J. Bioconjugate Chem. 2003, 14, 697-701. (f) Restituyo,
J. A.; Comstock, L. R.; Petersen, S. G.; Stringfellow, T.; Rajski, S. R.
Org. Lett. 2003, 5, 4357-4360. (g) 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. (h) Hang, H. C.; Yu, C.; Kato, D. L.; Bertozzi, C. R.
Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 14846-14851. (i) Luchansky, S.
J.; Argade, S.; Hayes, B. K.; Bertozzi, C. R. Biochemistry 2004, 43, 12358-
12366. (j) Hang, H. C.; Yu, C.; Pratt, M. R.; Bertozzi, C. R. J. Am. Chem.
Soc. 2004, 126, 6-7. (k) Hosoya, T.; Hiramatsu, T.; Ikemoto, T.; Nakanishi,
M.; Aoyama, H.; Hosoya, A.; Iwata, T.; Maruyama, K.; Endo, M.; Suzuki,
M. Org. Biomol. Chem. 2004, 2, 637-641. (l) Kho, Y.; Kim, S. C.; Jiang,
C.; Barma, D.; Kwon, S. W.; Cheng, J.; Jaunbergs, J.; Weinbaum, C.;
Tamanoi, F.; Falck, J.; Zhao, Y. Proc. Natl. Acad. Sci. U.S.A. 2004, 101,
12479-12484. (m) Tsao, M.-L.; Tian, F.; Schultz, P. G. ChemBioChem
2005, 6, 2147-2149. (n) Sprung, R.; Nandi, A.; Chen, Y.; Kim, S. C.;
Barma, D.; Falck, J. R.; Zhao, Y. J. Proteome Res. 2005, 4, 950-957. (o)
Comstock, L. R.; Rajski, S. R. Nucleic Acids Res. 2005, 33, 1644-1652.
(p) Rose, M. W.; Xu, J.; Kale, T. A.; O’Doherty, G.; Barany, G.; Distefano,
M. D. Peptide Sci. 2005, 80, 164-171.
(17) Prescher, J. A.; Dube, D. H.; Bertozzi, C. R. Nature 2004, 430, 873-877.
(18) Dube, D. H.; Prescher, J. A.; Quang, C. N.; Bertozzi, C. R. Proc. Natl.
Acad. Sci. U.S.A. 2006, 103, 4819-4824.
(19) Azoulay, M.; Tuffin, G.; Sallem, W.; Florent, J.-C. Bioorg. Med. Chem.
Lett. 2006, 16, 3147-3149.
(20) Yoon-Sik Lee and co-workers have reported that 14 different non-glycyl
couplings mediated by (diphenylphosphino)methanethiol (22) proceed in
“quantitative yield” (Kim, H.; Cho, J. K.; Aimoto, S.; Lee, Y.-S. Org. Lett.
2006, 8, 1149-1151). These results cannot be reproduced in our laboratory
and are inconsistent with reports from other laboratories (refs 15a and 44).
(21) Garc´ıa, J.; Urp´ı, F.; Vilarrasa, J. Tetrahedron Lett. 1984, 25, 4841-4844.
(22) Garc´ıa, J.; Vilarrasa, J.; Bordas, X.; Banaszek, A. Tetrahedron Lett. 1986,
27, 639-640.
(23) Urp´ı, F.; Vilarrasa, J. Tetrahedron Lett. 1986, 27, 4623-4624.
(24) Bosch, I.; Romea, P.; Urp´ı, F.; Vilarrasa, J. Tetrahedron Lett. 1993, 34,
4671-4674.
(29) Molina, P.; Vilaplana, M. J. Synthesis-Stuttgart 1994, 1197-1218.
(30) David, O.; Meester, W. J. N.; Bieraugel, H.; Schoemaker, H. E.; Hiemstra,
H.; van Maarseveen, J. H. Angew. Chem., Int. Ed. 2003, 42, 4373-4375.
(31) Grandjean, C.; Boutonnier, A.; Guerreiro, C.; Fournier, J. M.; Mulard, L.
A. J. Org. Chem. 2005, 70, 7123-7132.
(25) Bosch, I.; Urp´ı, F.; Vilarrasa, J. Chem. Commun. 1995, 91-92.
(26) Bosch, I.; Gonzalez, A.; Urp´ı, F.; Vilarrasa, J. J. Org. Chem. 1996, 61,
5638-5643.
(27) Ariza, X.; Urp´ı, F.; Viladomat, C.; Vilarrasa, J. Tetrahedron Lett. 1998,
39, 9101-9102.
(32) Amide 4, MS (ESI) m/z 286.1149 (MNa+ [C13H17N3O3Na+] ) 286.1162);
phosphine oxide 5, MS (ESI) m/z 273.0366 (MNa+ [C13H1318OPSNa+] )
273.0359).
(28) Ariza, X.; Pineda, O.; Urp´ı, F.; Vilarrasa, J. Tetrahedron Lett. 2001, 42,
4995-4999.
9
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A R T I C L E S
Soellner et al.
Figure 1. 13C NMR-based assay of a traceless Staudinger ligation. The reaction was performed at room temperature with reagent concentrations of 0.175
M. 90 spectra were acquired over a 12-h time course, scaled to enable the calculation of the second-order rate constant and reaction yield. Peak integrations
were calibrated with a standard curve generated with purified 13C-labeled species.
Scheme 2. 13C Chemical Shift of Mimics of Reaction Intermediates
NMR-Based Assay To Monitor the Staudinger Ligation.
We next sought to obtain detailed mechanistic and kinetic
information about the Staudinger ligation. To do so, we needed
an appropriate assay. NMR spectroscopy offers the ability to
observe a chemical reaction continuously and without its
perturbation. The resulting data can be used to identify reaction
intermediates, as well as obtain quantitative data on the rate of
their appearance and disappearance. Perhaps most importantly,
NMR spectroscopy can reveal a product-distribution profile that
is unperturbed by the vagaries of chemical purification. Finally,
an assay based on NMR spectroscopy is facile, allowing many
coupling reagents and reaction conditions to be surveyed rapidly.
in both the presence and absence of water (Scheme 2).
Iminophosphorane 18 was found to have a chemical shift of
49.54 ppm, which indicated that the signal at 49.20 ppm in
Figure 1 was due to iminophosphorane 13 in the Staudinger
ligation. This model reaction also provided information for the
chemical shift of the amine 16 byproduct, observed at 44.45
ppm. During the Staudinger ligation, the chemical shift of amine
16 changed from 44.58 to 44.05 ppm (Figure 1), which is likely
due to a decrease in solution pH and consequent amine
protonation during the course of the reaction.
To obtain chemical shift information on the amidophospho-
nium salt intermediate, the reaction of 11 and 12 was monitored
in anhydrous DMF. Although the reaction in the absence of
water did provide amide 15 (thereby suggesting that amidophos-
phonium salt 3 in Scheme 1 can fragment to form amide 4 and
a thiaphosphiranium salt), it also led to a marked accumulation
of a species with a chemical shift of 45.08 ppm, which was
likely amidophosphonium salt 14. Likewise, Staudinger ligation
mediated by (diphenylphosphino)ethanethiol, which has an
additional methylene group between its phosphorus and sulfur
atoms, implicated the chemical shift at 45.08 in Figure 1 as
arising from amidophosphonium salt 14 (vida infra). The
compound conferring the signal at 43.69 ppm was isolated from
We developed the means to monitor the Staudinger ligation
by 13C NMR spectroscopy using an organic azide enriched with
13C at its R-carbon (Figure 1). Detection by 13C NMR
spectroscopy was chosen after attempts to follow the reaction
by 15N NMR spectroscopy with a uniformly 15N-labeled azide
were unsuccessful due to low sensitivity. 31P NMR spectroscopy,
which was also considered, reports not on the desired product
(amide 4) but on a side product (phosphine oxide 5), which
can form disulfides that further complicate analyses. DMF was
chosen as the solvent, as this solvent had been shown previously
to be conducive to the Staudinger ligation.12,14 In addition, DMF
has a high boiling point (153 °C), which allows for monitoring
overnight reactions with no significant change in solute con-
centrations due to solvent evaporation.
To correlate the observed chemical shifts with discrete
intermediates along the reaction pathway, mimics of the
intermediates were synthesized and characterized further by 13
C
NMR spectroscopy. To obtain a mimic for the iminophospho-
rane intermediate, the reaction of diphenylethylphosphine and
13C-labeled azide 12 was monitored by 13C NMR spectroscopy,
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8822 J. AM. CHEM. SOC. VOL. 128, NO. 27, 2006

Mechanism/Kinetics of the Traceless Staudinger Ligation
A R T I C L E S
Scheme 3. Staudinger Ligation in the Presence of Amino Acids
Figure 2. Rate of amide 15 formation during an intramolecular (b) and
intermolecular (O) Staudinger ligation. The intramolecular reaction was
between phosphinothioester 11 and azide 12. The intermolecular reaction
was between (diphenylethyl)phosphine, azide 12, and thioester 21 (Scheme
4). Reactions were performed at room temperature with reagent concentra-
tions of 0.175 M and were monitored by 13C NMR spectroscopy as in Figure
1. The initial rate constants for amide formation were (7.7 ( 0.3) × 10-3
M-1 s-1 (intramolecular) and (6.5 ( 0.1) × 10-4 M-2 s-1 (intermolecular).
Scheme 4. Intermolecular Staudinger Ligation
the completed reaction and found to be a previously unknown
byproduct, phosphonamide 17. Finally, the chemical shift of
isolated amide 15 was observed to be 42.52 ppm.
13C NMR spectroscopy enabled us to observe each putative
intermediate in the traceless Staudinger ligation of phosphi-
nothioester 11 and azide 12 (Figure 1). Conveniently, the 13C
chemical shifts of the relevant species (azide f iminophos-
phorane f amidophosphonium salt f amide) decrease mono-
tonically as the reaction progresses. Significantly, the Staudinger
ligation of phosphinothioester 11 and azide 12 proceeds without
the significant accumulation of any intermediate.
Mechanism for Phosphonamide Byproduct Formation. To
reveal the source of the phosphonamide 17 byproduct, phos-
phinothioester 11 and azide 12 were reacted in the presence of
[18O]H2O. The phosphonamide 17 byproduct produced during
this reaction was isolated and found to contain exclusively 16O.33
This result indicates that phosphonamide byproduct forms via
an aza-Wittig reaction of the iminophosphorane (Scheme 1), in
which oxazaphosphetane 6 is transformed into phosphonamide
10 by a mechanism that is (as yet) unknown. Discerning the
fate of the sulfur atom during the 6f10 conversion would likely
illuminate this mechanism.
Chemoselectivity of the Staudinger Ligation. To examine
its chemoselectivity, the Staudinger ligation of equimolar
amounts of phosphinothioester 11 and azide 12 in DMF/D2O
(6:1) was examined by 13C NMR spectroscopy in the presence
of compounds containing functional groups present in pro-
teinogenic amino acids (Scheme 3). No significant changes in
the spectra were observed upon addition of stoichiometric
amounts of GlyNHBn (19, which is unlabeled 16) or AspNHBn
(20). These data assert further to the chemoselectivity of the
reaction with (diphenylphosphino)methanethiol as a coupling
reagent.14
Rate of Glycyl Couplings Using (Diphenylphosphino)-
methanethiol. The reaction of phosphinothioester 11 and azide
12 has a half-life of t1/2 ) 7 min with reactant concentrations
of 0.175 M (Figure 1). These data were used to calculate a
second-order rate constant for the appearance of the amide 15
product of k2 ) (7.7 ( 0.3) × 10-3 M-1 s-1. This value is
similar to that reported previously for the Staudinger reduction
of ethyl(2-azido)acetate by triphenylphosphine in benzene (k2
) 0.01 M-1 s-1).34 A recent analysis by Bertozzi and co-workers
of a nontraceless Staudinger ligation with an oxygen ester
revealed a similar rate constant.35 As with the Staudinger
reduction,34 the lack of intermediate accumulation (Figure 1)
indicates that the encounter of the phosphine and azide is the
rate-determining step in the traceless Staudinger ligation.
To quantify the effect of tethering the phosphine functionality
to the thioester functionality, we used our NMR-based assay to
examine an intermolecular version of the traceless Staudinger
ligation (Scheme 4). This reaction proceeded in low yield
relative to their intramolecular counterparts.25 Based on the
derived rate constants (Figure 2), the effective concentration of
(34) Leffler, J. E.; Temple, R. D. J. Am. Chem. Soc. 1967, 89, 5235-46.
(35) Lin, F. L.; Hoyt, H. M.; van Halbeek, H.; Bergman, R. G.; Bertozzi, C. R.
J. Am. Chem. Soc. 2005, 127, 2686-2695.
(33) MS (ESI) m/z 388.1251 (MNa+ [C2013CH21N2O2PNa+] ) 388.1266).
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A R T I C L E S
Soellner et al.
Table 1. Effect of Solvent Polarity on the Rate of the Staudinger
Ligation
as determined herein by our NMR-based assay, was 38%. This
yield is similar to the 35% yield determined previously by
product isolation.9 No buildup of intermediates was observed
during the course of the reaction. After 12 h, the reaction
consisted solely of the amide 15 product and amine 16
byproduct. The rate constant for the reaction of AcGlySC6H4-
o-PPh2 and azide 12 was found to be k2 ) (1.04 ( 0.05) ×
10-3 M-1 s-1, which is 14% of that for the same reaction
mediated by phosphinothiol 22. Using phosphinothiophenol 23
as the coupling reagent has another consequence, an increase
in the production of the amine 16 byproduct, presumably
because hydrolysis of the iminophosphorane intermediate is able
to compete more successfully with SfN acyl transfer.
1
solvent
SPP
k
2 (10-3 M-1 s-
)
DMF
CH3CN
THF
0.954
0.895
0.838
7.7
4.7
1.3
the nitrogen nucleophile in iminophosphorane 1 is estimated to
be EC ) (7.7 × 10-3 M-1 s-1)/(6.5 × 10-4 M-2 s-1) ) 12 M.
Effect of Solvent Polarity on Reaction Rate. The Staudinger
ligation of phosphinothioester 11 and azide 12 was assayed in
three solvents of different polarities. The observed rate constants
are listed in Table 1. In general, the reaction rate was higher in
more polar solvents, as denoted by the solvent polarity-
polarizability scale (SPP).36 These data indicate that the rate-
determining step involves a polar transition state that is stabilized
by polar solvents. This transition state is likely that for the
formation of the initial R3sP+sNdNsNsR′ phosphazide
intermediate.37,38
Comparison of Coupling Reagents in the Staudinger
Ligation. Several reagents other than phosphinomethanethiol
22 have been used in the traceless Staudinger ligation, with
varied success.39-41 These compounds include phosphinothio-
phenol 23,9 phosphinomethanol 24,39 phosphinoethanethiol 25,41
and phosphinophenol 2639 (Figures S1-S4 in the Supporting
Information). We used our NMR-based assay to compare the
efficacy of reagents 22-26 in mediating the coupling of
acetylglycine and azide 12 in DMF/D2O (6:1). The results are
listed in Table 2.
The next coupling reagent examined was (diphenylphosphi-
no)methanol (24). This reagent was described by Bertozzi and
co-workers and differs from (diphenylphosphino)methanethiol
(22) only in its bridging oxygen,39 enabling a direct comparison
of an ester and thioester reagent in mediating a traceless
Staudinger ligation. The rate constant for the formation of amide
15 was k2 ) (1.2 ( 0.1) × 10-4 M-1 s-1, which is merely 2%
that with (diphenylphosphino)methanethiol 22. Moreover, the
reaction of phosphinoester 27 and azide 12 resulted in a low
(11%) yield of the amide 15 product (Table 2). Bertozzi and
co-workers reported that the Staudinger ligation mediated by
(diphenylphosphino)methanol yielded only the amine byprod-
uct.39 In contrast, we observed nearly exclusive formation of
aza-Wittig byproducts with our NMR-based assay. Mass
spectrometric analysis indicates that the aza-Wittig adduct is
likely to be oxazaphosphetane 29 or imidate 30 (Scheme 5),
which have identical mass.42 The presence of a cross-peak (13C-
31P 3J ) 20.2 Hz) in the 13C-31P two-dimensional COSY NMR
spectrum provides additional support for the presence of
oxazaphosphetane 29 (data not shown). Attempted purification
of this adduct led to its decomposition.
The first traceless Staudinger ligation used phosphinothio-
phenol 23 as the coupling reagent.9 The yield of this reaction,
Table 2. Effect of Coupling Reagent on the Rate and Product Distribution of the Staudinger Ligation
9
8824 J. AM. CHEM. SOC. VOL. 128, NO. 27, 2006

Mechanism/Kinetics of the Traceless Staudinger Ligation
A R T I C L E S
Scheme 5. Staudinger Ligation with (Diphenylphosphino)methanol
(24)
Scheme 7. Staudinger Ligation with (o-Diphenylphosphino)phenol
(26)
thiol [alkyl (22 and 25) versus aryl (23)]. The larger ring size
of the tetrahedral intermediate derived from 23 and 25 also
appears to change the rate-determining step for the formation
of amide 15. The lower reaction rate and accumulation of the
amine 16 byproduct are consistent with the formation of
tetrahedral intermediate 2 (rather than the initial phosphazide
intermediate) limiting the rate of the Staudinger ligation
mediated by 23 and 25.
Scheme 6. Staudinger Ligation with
(Diphenylphosphino)ethanethiol (25)
Finally, we examined (o-diphenylphosphino)phenol (26). This
coupling reagent was first described by Bertozzi and co-workers
and later used by Liskamp and co-workers in the synthesis of
tetrapeptides with non-glycyl residues.39,40 Bertozzi and co-
workers obtained a yield of >95% using this reagent for acyl
transfers to an azido nucleotide.39 Likewise, our yield for the
reaction of phosphinoester 32 and azide 12 was nearly quantita-
tive (99%) with no observable formation of the amine 16
byproduct or accumulation of reaction intermediates (Table 2).
The rate constant for the formation of amide 15 was k2 ) (7.43
( 0.03) × 10-3 M-1 s-1, which is indistinguishable from that
with (diphenylphosphino)methanethiol (22)[k2 ) (7.7 ( 0.3)
× 10-3 M-1 s-1].
The inability of phosphinoalcohol 24 to mediate the Staudinger
ligation indicates that the nature of the leaving group plays an
important role in the partitioning of tetrahedral intermediate 2
toward a Staudinger ligation (Scheme 1, path A) or aza-Wittig
reaction (Scheme 1, path B). With a poor leaving group, such
as an alkoxide, the oxyanion of the tetrahedral intermediate is
long-lived enough to react with the oxophilic phosphorus to
form an oxazaphosphetane (e.g., 29 in Scheme 5). In contrast,
a good leaving group, such as a thiolate, is displaced quickly
from the tetrahedral intermediate, leading to the formation of
an amidophosphonium salt. Thus, these results highlight a key
advantage of using a phosphinothiol (22) rather than a phos-
phinoalcohol (24) to mediate the traceless Staudinger ligation.
As phosphinophenol 26 was as efficacious as (diphenylphos-
phino)methanethiol (22) in mediating a traceless Staudinger
ligation, its reactivity was investigated further. The presence
of a stoichiometric amount of GlyNHBn (19) during the reaction
of azide 12 and phosphinoester 32 decreased the yield of amide
15 from 97% to 64% (Scheme 7). This decrease in yield with
phosphinoester 32 is in contrast to the reaction with phosphi-
nothioester 11, which produced no less amide 15 in the presence
of a stoichiometric amount of GlyNHBn (Scheme 3). Presum-
ably, the diminished yield with phosphinoester 32 was due to
the greater electrophilicity of the aryl ester of 32 than the alkyl
thioester of 11. This result is consistent with the findings of
Liskamp and co-workers, who reported that both N-terminal
and lysyl ꢀ-amino groups can undergo nonspecific reaction with
esters derived from phosphinophenol 26.40 Thus, phosphino-
phenol 26 can be an exceptional reagent for mediating the
traceless Staudinger ligation, but its intrinsic lack of chemose-
lectivity constrains its use.
Recently, Viola and co-workers examined the Staudinger
ligation mediated by (diphenylphosphino)ethanethiol (25).41 This
reaction was reported to proceed in quantitative yield, as
monitored by thin-layer chromatography. With our NMR-based
assay, however, we found that the reaction of phosphinothioester
31 and azide 12 in DMF/D2O (6:1) yields mostly the amine 16
byproduct (Table 2; Scheme 6). The rate constant for the
formation of amide 15 was k2 ) (6.5 ( 0.1) × 10-4 M-1 s-1
,
which is only 8% that with (diphenylphosphino)methanethiol
(22). The low rate and high level of amine are likely due to the
increased size of the ring that is formed during the nucleophilic
attack of the iminophosphorane nitrogen on the thioester (e.g.,
to produce tetrahedral intermediate 2 in Scheme 1). Indeed, a
comparison of the reaction rates and product distributions of
the ligations mediated by 22, 23, and 25 indicates that ring size
[5 atoms (22) being better than 6 atoms (23 and 25)] is a more
significant determinant of the efficacy of a phosphinothiol in
mediating the traceless Staudinger ligation than is the type of
(36) Catalan, J.; Lopez, V.; Perez, P.; Martinvillamil, R.; Rodriguez, J. G. Liebigs
Ann. 1995, 241-252.
(37) Gololobov, Y. G.; Zhmurova, I. N.; Kasukhin, L. F. Tetrahedron 1981,
37, 437-472.
(38) Gololobov, Y. G.; Kasukhin, L. F. Tetrahedron 1992, 48, 1353-1406.
(39) Saxon, E.; Armstrong, J. I.; Bertozzi, C. R. Org. Lett. 2000, 2, 2141-
2143.
(40) Merkx, R.; Rijkers, D. T. S.; Kemmink, J.; Liskamp, R. M. J. Tetrahedron
Lett. 2003, 44, 4515-4518.
(41) Han, S.; Viola, R. E. Protein Pept. Lett. 2004, 11, 107-114.
(42) MS (ESI) m/z 388.1251 (MNa+ [C2013CH21N2O2PNa+] ) 388.1266).
9
J. AM. CHEM. SOC. VOL. 128, NO. 27, 2006 8825

A R T I C L E S
Soellner et al.
Scheme 8. Staudinger Ligation of Non-Glycyl Residues
the reaction of D-alanyl phosphinothioester 39 (which is the
enantiomer of 34) and azide 35 was examined with our NMR-
based assay. The yield of amide from this reaction was
indistinguishable from that for L-alanyl phosphinothioester 34,
indicating that amino acid stereochemistry does not play a role
in the diminished yields of non-glycyl couplings.
The ratio of amide 36 product to phosphonamide 38 byprod-
uct did not rely on the presence of water. In anhydrous DMF,
the reaction of alanyl phosphinothioester 34 and azide 35 results
in 36% amide 36 product and 64% phosphonamide 38 byprod-
uct. This nearly 1:2 ratio is similar to that observed in DMF/
D2O (6:1) (Scheme 8). This finding is consistent with the
putative mechanism in Scheme 1, as the hydrolysis of the
iminophosphorane intermediate occurs prior to the partitioning
toward path A (amide product) or path B (phosphonamide
byproduct).
Finally, increasing the temperature of the reaction of alanyl
phosphinothioester 34 and alanyl azide 35 increased the ratio
of amide 36 product to phosphonamide 38 byproduct. Still, at
the highest temperature tested (50 °C), the yield of amide 36
remained moderate (48%). This yield for a non-glycyl coupling
with (diphenylphosphino)methanethiol (22) is comparable to that
reported previously with phosphinophenol 26.40
Table 3. Yield of Amide from the Staudinger Ligation of Glycyl
and Non-Glycyl Residues
Conclusions
We have elucidated the mechanism for the traceless Staudinger
ligation mediated by phosphinothiols and phosphinoalcohols.
By developing and using a sensitive and continuous assay based
on 13C NMR spectroscopy, we were able to probe for reaction
intermediates and determine reaction rate constants. In addition,
we were able to compare reagents for their efficacy in mediating
the Staudinger ligation. Based on its high rate constant and
chemoselectivity, (diphenylphosphino)methanethiol (22)10 was
judged to be the most efficacious of known coupling reagents.
Efforts are underway in our laboratory to use the extant
understanding of the mechanism and kinetics of the Staudinger
ligation to develop new reagents and reaction conditions for
the high-yielding, traceless coupling of non-glycyl residues and
other chemical transformations.
Non-Glycyl Couplings. High (>90%) yields in the Staudinger
ligation mediated by (diphenylphosphino)methanethiol (22) are
obtained routinely if a glycine residue is at the N- or C-terminus
of the ligation junction.10,11 Couplings involving two non-glycyl
residues provide low (20-50%) yields.20 The origin of these
low yields is not readily apparent upon consideration of the
putative reaction mechanism (Scheme 1). Accordingly, we used
our NMR-based assay to examine reactions involving a non-
glycyl phosphinothioester and non-glycyl azide.
Experimental Procedures
As a model coupling for non-glycyl residues, alanyl phos-
phinothioester 34 and alanyl azide 35 were allowed to react in
DMF/D2O (6:1). At room temperature, the reaction resulted in
27% amide 36 product, 19% amine 37 byproduct, and 54%
phosphonamide 38 byproduct (Scheme 8). Moreover, the rate
constant for amide formation was found to be k2 ) (3.7 ( 0.3)
× 10-3 M-1 s-1, which is half that for the analogous all-glycyl
coupling. When the reaction was performed in the presence of
[18O]H2O, the phosphonamide 38 byproduct contained exclu-
sively 16O,42 indicating once again that this byproduct results
from an aza-Wittig reaction. A comparison of the quantity of
amide produced during the four possible glycyl and alanyl
couplings (Table 3) indicates that steric encumbrance during
the reaction of alanyl phosphinothioester 34 and alanyl azide
35 (27% yield) is significantly more severe than that during a
glycyl coupling (93-96% yield). This strain could arise in
tetrahedral intermediate 2, in which the bulky R and R′ groups
of a non-glycyl coupling are brought into close proximity.
To acertain whether the stereochemistry at the R-carbon plays
a role in the decreased amide yield of a non-glycyl coupling,
General. Reactions were monitored by thin-layer chromatography
with visualization by UV light or staining with ninhydrin or I2.
Compound purification was carried out with an Argonaut Flashmaster
Solo automated chromatography system. Silica gel used in flash
chromatography had a 230-400 mesh and 60 Å pore size. Reagent
chemicals were obtained from commercial suppliers, and reagent grade
solvents were used without further purification. NMR spectra were
obtained with a 500 or 400 MHz spectrometer at the National Magnetic
Resonance Facility at Madison (NMRFAM) or the University of
Wisconsin Nuclear Magnetic Resonance Facility, respectively. Carbon-
13 and phosphorus-31 spectra were both proton-decoupled, and
phosphorus-31 spectra were referenced against an external standard of
deuterated phosphoric acid (0 ppm). Mass spectra were obtained with
electrospray ionization (ESI) techniques.
General Procedures for Kinetics Experiments Using 13C NMR
Spectroscopy. A phosphinothioester or phosphinoester (0.105 mmol
in 400 µL of DMF) was mixed with a 13C-labeled azide (0.105 mmol
in 300 µL of DMF/D2O (2:1)) in a vial. After mixing, the solution was
transferred to an NMR tube. (Due to the evolution of N2(g), NMR tubes
were not capped during experiments.) The NMR tube containing the
reaction mixture was then inserted into an NMR spectrometer that had
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8826 J. AM. CHEM. SOC. VOL. 128, NO. 27, 2006

Mechanism/Kinetics of the Traceless Staudinger Ligation
A R T I C L E S
been prelocked on a sample containing 13C-labeled azide in 700 µL of
DMF/D2O (6:1). After an initial delay of 45 s, the acquisition of NMR
spectra was initiated. 90 spectra were acquired over a scaled time course
of 16 h (15 spectra during the first 15 min, 15 spectra during the next
30 min, 15 spectra during the next 60 min, 15 spectra during the next
120 min, 15 spectra during the next 240 min, and 15 spectra during
the final 480 min). Each time point was designed to consume 44 s,
with the remaining time being a preacquisition delay before the next
scan. An appropriate flip angle (30° pulse) and relaxation delay (10 s)
were chosen to obtain fully quantitative spectra at each time point and
for each intermediate. In addition, the decoupler was turned on solely
during the acquisition to prevent any NOE buildups. To confirm that
the NMR-based assay provided quantitative results, a standard curve
was made for each starting material and available intermediate and
shown to correlate well with the spectral integration during a reaction.
General Procedures for Staudinger Ligations. Unless noted
otherwise, Staudinger ligations were performed at room temperature
with equimolar amounts of phosphinothioester (or phosphinoester) and
azide (0.105 mmol) in DMF/D2O (6:1; 600 µL).
The spectral data for the isolated and synthesized compounds were
indistinguishable.
Spectral Data. H NMR (CD3OD, 400 MHz) δ 7.91-7.86 (m, 5
1
H), 7.55-7.50 (m, 5H), 7.34-7.26 (m, 5H), 4.42 (s, 2H), 3.94 (s, 2H)
ppm; 13C NMR (CD3OD, 125 MHz) δ 168.63, 138.19, 134.06, 132.75,
131.63, 131.60, 131.42, 131.32, 128.26, 128.18, 128.12, 127.22, 126.94,
51.71, 42.69 ppm; 31P NMR (CD3OD, 161 MHz) δ 24.55 ppm; MS
(ESI) m/z 388.1251 (MNa+ [C2013CH21N2O2PNa+] ) 388.1266).
GlyNHBn (19) and AspNHBn (20). Amines 19 (which is unlabeled
16)43 and 2044 were prepared according to procedures reported
previously. Spectral Data. Spectral data were as reported previously.43,44
AcGlySCH2C(O)NHMe (21). N-Acetylglycine (4.0 g, 34.2 mmol)
was dissolved in anhydrous DMF (100 mL). DCC (7.06 g, 34.2 mmol)
was then added. Once precipitate (DCU) was observed, N-methyl
mercaptoacetamide was added (3.6 g, 34.2 mmol). The reaction mixture
was allowed to stir under Ar(g) for 18 h. The precipitate was removed
by filtration, and the filtrate was concentrated under reduced pressure
to give a white solid. The solid was dissolved in ethyl acetate and
purified by flash chromatography (silica gel, ethyl acetate/hexanes).
Thioester 21 was isolated as an off-white solid in 91% yield. Spectral
AcGlySCH2PPh2 (11). N-Acetylglycine (1.90 g, 16.2 mmol) was
dissolved in anhydrous DMF (75 mL). HOBt (2.48 g, 16.2 mmol) was
added to the resulting solution, followed by DCC (3.34 g, 16.2 mmol).
Once precipitate (DCU) was observed, phosphinothiol 26 was added
(3.77 g, 16.2 mmol). The reaction mixture was allowed to stir under
Ar(g) for 3 h. The precipitate was removed by filtration, and the filtrate
was concentrated under reduced pressure to give a white solid. The
solid was dissolved in ethyl acetate and purified by flash chromatog-
raphy (silica gel, ethyl acetate). Phosphinothioester 11 was isolated in
96% yield. Spectral Data. Spectral data were as reported previously.10
[2-13C]-2-Azido-N-benzyl-acetamide (12). Azide 12 was synthe-
sized from 2-13C bromoacetic bromide via methods reported previously10
and isolated in 98% yield. Spectral Data. 1H NMR (CDCl3, 400 MHz)
δ 7.39-7.27 (m, 5H), 6.71 (bs, 1H), 4.47 (d, J ) 5.7 Hz, 2H), 4.00 (s,
2H) ppm; 13C NMR (CDCl3, 100 MHz) δ 166.66, 137.39, 128.43,
1
Data. H NMR (CDCl3, 400 MHz) δ 6.09 (bs, 1H), 4.26 (bs, 1H),
3.59 (s, 2H), 2.82 (s, 2H), 2.18 (s, 3H), 2.10 (s, 3H) ppm; 13C NMR
(CDCl3/CD3OD (1:1), 125 MHz) δ 196.99, 172.23, 169.07, 31.43,
31.40, 26.04, 25.90, 21.65 ppm; MS (ESI) m/z 227.0457 (MNa+
[C7H12N2O3SNa+] ) 227.0461).
(Diphenylphosphino)methanethiol (22), (o-Diphenylphosphino)-
benzenethiol (23), (diphenylphosphino)methanol (24), (Diphen-
ylphosphino)ethanethiol (25), and (o-Diphenylphosphino)phenol
(26). Compounds 22,11 23,9 24,39 25,41 and 2639 were prepared according
to reports published previously. Spectral Data. Spectral data were as
reported previously.9,11,39,41
AcGlyOCH2PPh2 (27). N-Acetylglycine (1.12 g, 10.0 mmol) was
dissolved in anhydrous CH2Cl2 (30 mL). DMAP (0.12 g, 1.0 mmol)
was added to the resulting solution, followed by DCC (2.06 g, 10.0
mmol). Once precipitate (DCU) was observed, (diphenylphosphino)-
methanol (24; 2.16 g, 10.0 mmol) was added, and the reaction mixture
was allowed to stir under Ar(g) for 18 h. The precipitate was removed
by filtration, and the filtrate was concentrated under reduced pressure
to give a clear oil. The oil was dissolved in ethyl acetate and purified
by flash chromatography (silica gel, ethyl acetate/hexanes). Phosphi-
noester 27 was isolated as a colorless oil in 56% yield. Spectral Data.
1H NMR (CDCl3, 400 MHz) δ 7.47-7.42 (m, 4H), 7.39-7.37 (m,
6H), 5.89 (bt, 1H), 4.92 (d, J ) 6.2 Hz, 2H), 3.98 (d, J ) 5.0 Hz, 2H),
2.00 (s, 3H) ppm; 13C NMR (CDCl3, 125 MHz) δ 185.35, 169.59,
132.65, 132.50, 128.85, 128.27, 128.22, 48.13, 33.27, 25.17, 24.47 ppm;
31P NMR (CDCl3, 161 MHz) δ -20.02 ppm; MS (ESI) m/z 338.0920
(MNa+ [C17H18NO3PNa+] ) 338.0916).
127.45, 127.35, 52.06, 43.08 ppm; MS (ESI) m/z 214.0789 (MNa+ [C813
CH10N4ONa+] ) 214.0780).
-
AcGly[13CR]GlyNHBn (15). Amide 15 was synthesized by using
the general procedures for Staudinger ligation (vide supra). Spectral
Data. Spectral data were the same as reported previously.10
[13CR]GlyNHBn (16). Azide 12 (951 mg, 5.0 mmol) was dissolved
in anhydrous THF (30 mL) in a flame-dried flask under Ar(g). Ethyl
diphenyl phosphine (1.29 g, 6.0 mmol) was added to the resulting
solution, which was then allowed to stir under Ar(g) for 8 h. Water
(3.3 mL, to 10% v/v) was added, and the reaction mixture was stirred
for an additional 1 h. The solvent was then removed under reduced
pressure, and the resulting oil was purified by flash chromatography
(silica gel, 3% v/v MeOH in CH2Cl2). Amine 16 was isolated in 97%
yield as a clear oil. Spectral Data. Spectral data were the same as
reported previously.43
AcGlySCH2CH2PPh2 (31). N-Acetylglycine (475 mg, 4.06 mmol)
was dissolved in anhydrous DMF (20 mL). HOBt (549 mg, 4.06 mmol)
was added to the resulting solution, followed by DCC (838 mg, 4.06
mmol). Once precipitate (DCU) was observed, (diphenylphosphino)-
ethanethiol (25; 1.0 g, 4.06 mmol) was added. The reaction mixture
was allowed to stir under Ar(g) for 3 h. The precipitate was removed
by filtration, and the filtrate was concentrated under reduced pressure
to give a clear oil. The oil was dissolved in ethyl acetate and purified
by flash chromatography (silica gel, 70% v/v ethyl acetate in hexanes).
Phosphinoester 31 was isolated as a white solid in 92% yield. Spectral
Ph2P(O)[13CR]GlyNHBn (17). Phosphonamide 17 was isolated from
a Staudinger ligation of phosphinothioester 11 and azide 12, performed
as described above. The solvent of the reaction was removed under
reduced pressure, and the resulting oil was washed with CH2Cl2. The
suspension was filtered, and phosphonamide 17 was isolated in 1%
yield.
Phosphonamide 17 was also synthesized directly by the reaction of
diphenylphosphinic chloride and GlyNHBn (16). Diphenylphosphinic
chloride (1.0 g, 4.2 mmol) was dissolved in anhydrous THF (20 mL).
DMAP (60 mg, 0.5 mmol) was added to the resulting solution, followed
by GlyNHBn (700 mg, 4.2 mmol). The reaction mixture was allowed
to stir under Ar(g) for 8 h. The solvent was then removed under reduced
pressure, and the resulting oil was purified by flash chromatography
(silica gel, 3% v/v MeOH in CH2Cl2). Phosphonamide 17 was isolated
in 81% yield.
1
Data. H NMR (CDCl3, 400 MHz) δ 7.47-7.43 (m, 4H), 7.37-7.36
(m, 6H), 6.06 (bs, 1H), 4.20 (d, J ) 5.6 Hz, 2H), 3.00 (m, 2H), 2.46
(m, 2H), 2.07 (s, 3H) ppm; 13C NMR (CDCl3, 125 MHz) δ 196.78,
170.65, 137.23, 137.14, 132.68, 132.52, 128.81, 128.53, 128.47, 126.33,
125.53, 49.15, 28.27 (d, J ) 15.4 Hz), 25.60 (d, J ) 23.1 Hz), 22.81
ppm; 31P NMR (CDCl3, 161 MHz) δ -19.88 ppm; MS (ESI) m/z
368.0852 (MNa+ [C18H20NO2PSNa+] ) 368.0845).
(43) Balboni, G.; Guerrini, R.; Salvadori, S.; Bianchi, C.; Rizzi, D.; Bryant, S.
(44) van Leeuwen, S. H.; Quaedflieg, P.; Broxterman, Q. B.; Liskamp, R. M.
D.; Lazarus, L. H. J. Med. Chem. 2002, 45, 713-720.
J. Tetrahedron Lett. 2002, 43, 9203-9207.
9
J. AM. CHEM. SOC. VOL. 128, NO. 27, 2006 8827

A R T I C L E S
Soellner et al.
AcGlyOC6H4-o-PPh2 (32). N-Acetylglycine (562 mg, 4.8 mmol)
was dissolved in anhydrous CH2Cl2 (50 mL). DMAP (59 mg, 0.5 mmol)
was added to the resulting solution, followed by DCC (990 mg, 4.8
mmol). Once precipitate (DCU) was observed, (o-diphenylphosphino)-
phenol (26; 2.16 g, 10.0 mmol) was added, and the reaction mixture
was allowed to stir under Ar(g) for 4 h. The precipitate was removed
by filtration, and the filtrate was concentrated under reduced pressure
to give a clear oil. The oil was dissolved in ethyl acetate and purified
by flash chromatography (silica gel, ethyl acetate/hexanes (1:1)).
Phosphinoester 32 was isolated as a white solid in 36% yield. Spectral
Data. 1H NMR (CDCl3, 400 MHz) δ 7.42-7.28 (m, 12H), 7.19-7.17
(m, 2H), 6.87 (bt, 1H), 4.00 (d, J ) 5.0 Hz, 2H), 1.98 (s, 3H) ppm;
13C NMR (CDCl3, 125 MHz) δ 169.98, 168.03, 135.24, 135.14, 133.99,
133.82, 130.12, 129.18, 128.73, 128.67, 126.63, 122.37, 41.27, 22.88
ppm; 31P NMR (CDCl3, 161 MHz) δ -19.07 ppm; MS (ESI) m/z
400.1071 (MNa+ [C22H20NO3PNa+] ) 400.1073).
v/v ethyl acetate in hexanes and purified by flash chromatography (silica
gel, 35% v/v ethyl acetate in hexanes). Azide 35 was isolated in 90%
yield as an off-white solid. Spectral Data. 1H NMR (CDCl3, 400 MHz)
δ 7.37-7.28 (m, 5 H), 6.67 (bs, 1H), 4.46 (s, 2H), 4.13 (dt, J ) 144.7,
6.9 Hz, 1H), 1.60 (d, J ) 2.3 Hz, 3H) ppm; 13C NMR (CDCl3, 125
MHz) δ 128.78, 127.74, 59.29, 43.52 ppm; MS (ESI) m/z 228.0935
(MNa+ [C913CH12N4ONa+] ) 228.0937).
AcAlaAlaNHBn (36). Amide 36 was prepared by using the general
1
procedures for Staudinger ligations (vide supra). Spectral Data. H
NMR (300 MHz, CDCl3/CD3OD (1:1)) δ 7.32-7.35 (m, 5 H), 4.51-
4.22 (m, 4H), 1.98 (1.87) (s, 3H), 1.41 (d, J ) 7.5 Hz, 3H), 1.34 (d, J
) 6.9 Hz, 3H) ppm; 13C NMR (75 MHz, CDCl3/CD3OD (1:1), numbers
in parentheses indicate doubling due to rotational isomerism) δ 173.27
(172.78), 172.67 (172.54), 171.44, 137.59 (137.51), 127.39 (127.49),
126.28 (126.36), 126.02 (126.17), 48.92 (48.70), 48.59 (48.34), 41.99
(42.06), 20.50 (20.76), 15.99 (16.23), 15.45 ppm; MS (ESI) m/z
315.1511 (MNa+ [C1413CH21N3O3Na+] ) 315.1509).
Ph2P(O)[13CR]AlaNHBn (38). Phosphonamide 38 was isolated from
a Staudinger ligation of phosphinothioester 34 and azide 35, performed
as described above. The solvent of the reaction was removed under
reduced pressure, and the resulting oil was washed with CH2Cl2. The
suspension was then filtered, and phosphonamide 38 was isolated in
54% yield. Spectral Data. 1H NMR (CD3OD, 400 MHz) δ 7.92-7.86
(m, 5H), 7.58-7.48 (m, 5H), 7.35-7.26 (m, 5H), 4.41 (s, 2H), 4.00
(q, J ) 7.1 Hz, 1H), 1.47 (d, J ) 7.1 Hz, 3H) ppm; 13C NMR (CD3-
OD, 125 MHz) δ 171.58, 138.26, 134.07, 132.76, 131.63, 131.60,
131.42, 131.32, 128.26, 128.18, 128.13, 127.12, 126.92, 58.23, 42.64,
15.97 ppm; 31P NMR (CD3OD, 161 MHz) δ 24.63 ppm; MS (ESI)
m/z 402.1432 (MNa+ [C2113CH23N2O2PNa+] ) 402.1423).
AcGlyGlyNHBn (33). Amide 33 (which is unlabeled 15) was
synthesized using the general procedures for Staudinger ligations (vide
supra). Spectral Data. Spectral data were the same as that reported
previously.10
AcAlaSCH2PPh2 (34). N-Acetylalanine (557 mg, 4.25 mmol) was
dissolved in anhydrous DMF (20 mL). HOBt (527 mg, 3.90 mmol)
was added to the resulting solution, followed by DCC (805 mg, 3.90
mmol). Once precipitate (DCU) was observed, (diphenylphosphino)-
methanethiol (22; 900 mg, 3.87 mmol) was added, and the reaction
mixture was allowed to stir under Ar(g) for 4 h. The precipitate was
removed by filtration, and the filtrate was concentrated under reduced
pressure to give a clear oil. The oil was dissolved in ethyl acetate and
purified by flash chromatography (silica gel, 70% v/v ethyl acetate in
hexanes). Phosphinothioester 34 was isolated as a white solid in 83%
yield.
Acknowledgment. We are grateful to Drs. L. L. Kiessling,
H. J. Reich, and J. A. Hodges and to A. Tam and L. D. Lavis
for contributive discussions, and to Drs. Milo Westler and C.
G. Fry for technical advice during the development of our NMR-
based assay. This work was supported by Grant GM44783
(NIH). M.B.S. was supported by an ACS Division of Organic
Chemistry Fellowship, sponsored by Abbott Laboratories.
B.L.N. was supported by the Abbott Laboratories Fellowship
in Synthetic Organic Chemistry (University of Wisconsin-
Madison). NMRFAM was supported by Grant P41RR02301
(NIH); the University of Wisconsin Nuclear Magnetic Reso-
nance Facility was supported by Grants CHE-9208463 (NSF)
and RR08389 (NIH).
This procedure was repeated with Ac-D-AlaOH to give Ac-D-
AlaSCH2PPh2 (39) as a white solid in 92% yield. The spectral data for
AcAlaSCH2PPh2 and Ac-D-AlaSCH2PPh2 were indistinguishable.
1
Spectral Data. H NMR (CDCl3, 400 MHz) δ 7.44-7.37 (m, 10
H), 5.89 (bt, 1H), 4.71 (bt, 1H), 3.53 (s, 2H), 2.02 (s, 3H), 1.64 (m,
1H), 1.32 (d, J ) 5.8 Hz, 3H) ppm; 13C NMR (CDCl3, 125 MHz) δ
199.85, 169.70, 136.51, 132.74, 132.59, 132.57, 129.10, 128.51, 128.46,
54.83, 25.29 (d, J ) 24.3 Hz), 22.99, 18.66 ppm; 31P NMR (CDCl3,
161 MHz) δ -17.97 ppm; MS (ESI) m/z 368.0853 (MNa+ [C18H20-
NO2PSNa+] ) 368.0845).
[2-13C]-(2S)-2-Azido-N-benzyl-1-propionamide (35). [2-13C]-(2S)-
2-Azido-1-propionic acid was synthesized from [13CR]-(2S)-alanine
according to the procedure of Lundquist and Pelletier.45 [2-13C]-(2S)-
2-Azido-1-propionic acid (280 mg, 2.8 mmol) was dissolved in
anhydrous DMF (15 mL). Hydroxybenzotriazole (397 mg, 2.9 mmol)
was then added, followed by DCC (607 mg, 2.9 mmol). Once precipitate
(DCU) was observed, benzylamine (0.370 mL, 3.4 mmol) was added,
and the reaction mixture was allowed to stir under Ar(g) for 3 h. The
precipitate was removed by filtration, and the filtrate was removed under
reduced pressure to yield a yellow oil. This oil was dissolved in 35%
Supporting Information Available: Four figures such as
Figure 1 depicting the results of 13C NMR-based assays of the
reaction of azide 12 with phosphinothiophenol 23, phosphi-
noalcohol 24, phosphinothiol 25, and phosphinophenol 26. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(45) Lundquist, J. T.; Pelletier, J. C. Org. Lett. 2001, 3, 781-783.
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