Electronic and steric effects on the rate of the traceless Staudinger ligation

Article abstract of DOI:10.1039/b802336k

Interplay between electronic effects imparted by phosphinothiol substituents and steric effects imposed by amino-acid reactants affects the rate of the traceless Staudinger ligation of peptides in a predictable manner. This journal is The Royal Society of

Full text of DOI:10.1039/b802336k

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Electronic and steric effects on the rate of the traceless Staudinger
ligation†
Annie Tam,a Matthew B. Soellner‡a and Ronald T. Raines*a,b
Received 11th February 2008, Accepted 20th February 2008
First published as an Advance Article on the web 3rd March 2008
DOI: 10.1039/b802336k
Interplay between electronic effects imparted by phos-
phinothiol substituents and steric effects imposed by amino-
acid reactants affects the rate of the traceless Staudinger
ligation of peptides in a predictable manner.
Recently, we reported
a
new reagent, di(4-methoxy-
phenyl)phosphinomethanethiol (2), that is effective at mediating
ligations between encumbered residues.12 Results indicated that
electron-donating substituents on the aryl rings disfavor the
formation of a side product, thereby leading to an increased
yield (>80%) of the desired amide. Here, we report on electronic
and steric effects on the rate of different steps in the traceless
Staudinger ligation.
We surveyed the ligation rates mediated by a range of
phosphinothiol reagents (1–6) with varying degrees of electron-
donating character imparted by para substituents on their aryl
rings (Fig. 1). The phosphinothiols were synthesized via a route
analogous to that reported for similar phosphinothiols.13 Their
respective thioesters (7–18) were prepared by coupling the phos-
phinothiols to AcGlyOH or AcAlaOH to provide model reactants
of less and more sterically encumbered ligations, respectively.
Chemical synthesis enables the incorporation of nonnatural
functionality into proteins, harboring the potential to elucidate
function and enhance performance.1 The most common peptide
ligation strategy is “native chemical ligation”, but this method
is limited to ligations at junctions containing a cysteine residue.2
Efforts in our laboratory have been aimed at developing another
strategy for peptide ligation called the “traceless Staudinger
ligation”, which alleviates this limitation.
The Staudinger ligation joins a peptide having a C-terminal
phosphinothioester with a peptide having an N-terminal azide
through an intermediate iminophosphorane (Scheme 1). Notable
attributes of this ligation strategy include its ability to generate an
amide product without either residual atoms3 or racemization,4
and with a high reaction rate.5 (Diphenylphosphino)methanethiol
(1) is the most often used of known reagents for effecting the
traceless Staudinger ligation, and has mediated the orthogonal
assembly of a protein,6 site-specific immobilization of peptides
and proteins,7–9 and synthesis of glycopeptides.10,11
Fig. 1 Phosphinothiols, phosphinothioesters, and the rp values of their
aryl substituents.14
A detailed mechanism of the traceless Staudinger ligation is
depicted in Scheme 2. In this mechanism, phosphinothioester 19
and azide 20 react to form phosphazide 21 (Step 1), which gives
iminophosphorane 22 with concomitant release of N2(g) (Step 2).
Formation of the tetrahedral intermediate 23 follows (Step 3),
which collapses to amidophosphonium salt 24 (Step 4). Hydrolysis
of 24 yields the desired amide 25 and phosphine oxide 26 (Step 5).
A 13C NMR-based assay developed previously in our laboratory
was used to monitor the fate of azides enriched with 13C on their a-
carbon during the course of a traceless Staudinger ligation.5 This
assay enables the determination of the rate constants for both
the disappearance of the azide reactant and the appearance of the
amide product. Phosphinothioesters with various aryl substituents
(7–12) were reacted with 13C-labeled glycyl azide 27, and it was
Scheme 1 Putative mechanism of the traceless Staudinger ligation.
aDepartment of Chemistry, University of Wisconsin–Madison, Madison,
Wisconsin, 53706, USA
bDepartment of Biochemistry, University of Wisconsin–Madison, Madison,
Wisconsin, 53706, USA. E-mail: rtraines@wisc.edu
† Electronic supplementary information (ESI) available: Synthesis and
characterization of all new compounds. See DOI: 10.1039/b802336k
‡ Present address: Department of Chemistry, University of California,
Berkeley, California, 94720, USA.
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Scheme 2 Putative mechanism of the traceless Staudinger ligation.
found that both of these rate constants correlate well with the
Hammett rp values of the substituents (Fig. 2, ᭺), which is
indicative of an electronic effect.15 In general, substituents with
greater electron-donating ability provide higher rates of reaction.
The trend was also evident in the more sterically encumbered
reaction between alanyl phosphinothioesters 13–18 and 13C-
labeled alanyl azide 28 (Fig. 2, ᭹). Interestingly, the rate constants
for azide consumption were similar for the Gly + Gly and
Ala + Ala ligations (Fig. 2A). This similarity indicates that the
intermolecular reaction rate of the phosphinothioester and azide
is influenced by electronic but not steric effects. On the other hand,
the rate constants for amide formation were markedly lower for
the ligation of the bulkier alanyl reactants (Fig. 2B). Apparently,
the rate of an intramolecular step is diminished by steric effects.
The values of q for amide formation are, however, highly negative,
indicating that this rate can benefit substantially from electron-
donating substituents.
Fig. 2 Hammett plots for the reaction of glycyl phosphinothioesters
7–12 with 13C-labeled glycyl azide 27 (᭺) and for the reaction of alanyl
phosphinothioesters 13–18 with 13C-labeled alanyl azide 28 (᭹) in DMF at
room temperature. Reactant concentrations: 0.146 M. Lines depict a linear
least-squares fit. (A) Second-order rate constants for azide consumption
(᭺: q = −0.62; ᭹: q = −0.55). (B) Second-order rate constants for amide
formation (᭺: q = −1.09; ᭹: q = −0.86).
Previous reports have assigned the rate-determining step in the
Staudinger ligation to be the association of starting phosphinoth-
ioester and azide to form phosphazide intermediate 21.16,5 This
assignment appears to be true for Gly + Gly ligations, as no
intermediates accumulate during the course of that reaction. In
an Ala + Ala ligation, however, an intermediate does accumulate
(Fig. 3), indicating that the rate of amide formation is indeed
influenced by the rate of an intramolecular step.
Fig. 3 13C NMR-based assay of an Ala + Ala traceless Staudinger ligation
in DMF at room temperature. Reactant concentrations: 0.146 M. Ninety
spectra were acquired over a 12-h time course.
To correlate the observed chemical shifts with discrete
intermediates along the reaction pathway for an Ala + Ala ligation,
mimics of the intermediate were synthesized and characterized
by 13C NMR spectroscopy.5 A mimic of iminophosphorane
29 in an Ala + Ala ligation was synthesized by the reaction of
1174 | Org. Biomol. Chem., 2008, 6, 1173–1175
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Table 1 Yields of amide and amine produced during Ala + Ala traceless
Staudinger ligations of 0.146 M reactants in DMF at room temperature
for 12 h
iminophosphorane nitrogen more susceptible to protonation by
trace amounts of water. That protonation would impair the desired
S→N acyl transfer, as well as make the adjacent phosphorus more
electrophilic and thereby expedite hydrolysis.
Phosphinothioester
rp
Azide
Amide yield
Amine yield
In conclusion, electronic and steric effects play multiple roles
in determining the rate and yield of traceless Staudinger ligations.
Most notably, electron-donating phosphinothiol substituents have
dichotomous consequences. They are advantageous in accelerat-
ing the intermolecular reaction, enhancing S→N acyl transfer
in the iminophosphorane, and discouraging undesirable P–O
bond formation in the ensuing tetrahedral intermediate. Electron-
donating substituents are deleterious in promoting the protona-
tion of the iminophosphorane nitrogen, which impairs S→N acyl
transfer in the iminophosphorane and leads to amine formation.
Overall, electron-donating substituents increase the ligation rate
of sterically encumbered reactants (Fig. 2), but decrease the yield
of amide product (Table 1). The optimal choice of phosphinothiol
for a particular ligation (especially between non-glycyl reactants)
requires consideration of these electronic and steric effects.17
16
17
18
−0.27
−0.40
−0.83
28
28
28
51%
45%
36%
49%
55%
59%
diphenylethylphosphine and 13C-labeled alanyl azide 28, both
in the absence of water (to generate iminophosphorane 32) and
in the presence of water (to generate amine 33; Scheme 3). The
chemical shifts of iminophosphorane 32 and amine 33 were
obtained by 13C NMR spectroscopy.
Notes and references
1 B. L. Nilsson, M. B. Soellner and R. T. Raines, Annu. Rev. Biophys.
Biomol. Struct., 2005, 34, 91–118.
2 P. E. Dawson and S. B. H. Kent, Annu. Rev. Biochem., 2000, 69, 923–
960.
Scheme 3 13C Chemical shifts of reaction-intermediate mimics.
3 B. L. Nilsson, L. L. Kiessling and R. T. Raines, Org. Lett., 2001, 3,
Iminophosphorane 32 was found to have a chemical shift
of 54.66 ppm, which supports the assignment of the signal at
54.64 ppm in Fig. 3 to iminophosphorane 29. Thus, the inter-
mediate that accumulates in ligations between bulkier reactants
is the iminophosphorane, and Step 3 partially limits the rate
of amide formation. Presumably, the steric effects hinder S→N
acyl transfer. Analogous steric effects have been observed in
non-traceless Staudinger ligations.16 In the traceless Staudinger
ligation, these steric effects can be mitigated by electron-donating
substituents (Fig. 2B).
9–12.
4 M. B. Soellner, B. L. Nilsson and R. T. Raines, J. Org. Chem., 2002, 67,
4993–4996.
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Electron-donating substituents are also known to benefit Step 4
of the reaction pathway (Scheme 2). Previously, we demonstrated
that increasing electron density of the phosphorus increases
the yield at bulkier ligation sites by discouraging tetrahedral
intermediate 23 from forming a P–O bond and hence an
oxazaphosphetane.12 Thus, electron-rich phosphinothiols aid in
both Step 3 and Step 4 of the traceless Staudinger ligation.
Electron-donating substituents do, however, have a detrimental
effect. Hydrolysis of 32 gave a signal at 51.02 ppm for amine 33,
which is similar to the 51.27 ppm signal in Fig. 3. Thus, an amine
is the major byproduct of more sterically encumbered ligations.
Furthermore, amine was formed to a greater extent when azide 28
was reacted with the more electron-donating phosphinothioesters
(Table 1). We suspect that increased electron density renders the
9 C. Gauchet, G. R. Labadie and C. D. Poulter, J. Am. Chem. Soc., 2006,
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10 Y. He, R. J. Hinklin, J. Y. Chang and L. L. Kiessling, Org. Lett., 2004,
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11 L. Liu, Z. Y. Hong and C. H. Wong, ChemBioChem, 2006, 7, 429–432.
12 M. B. Soellner, A. Tam and R. T. Raines, J. Org. Chem., 2006, 71,
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14 C. Hansch, A. Leo and R. W. Taft, Chem. Rev., 1991, 91, 165–195.
15 L. P. Hammett, Chem. Rev., 1935, 17, 125–136.
16 F. L. Lin, H. M. Hoyt, H. Van Halbeek, R. G. Bergman and C. R.
Bertozzi, J. Am. Chem. Soc., 2005, 127, 2686–2695.
17 We thank Dr E. L. Myers for contributive discussions. This work was
supported by grant GM044783 (NIH). M.B.S. was supported by an
ACS Division of Organic Chemistry Fellowship, sponsored by Abbott
Laboratories.
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 1173–1175 | 1175
©