a linkage at a natural Xaa-Cys bond is not always possible,
as cysteine comprises only 1.7% of the residues in globular
proteins.5 Installing an extra cysteine residue is often
undesirable. Cysteine is by far the most reactive residue
toward disulfide bonds, O2(g), and other electrophiles.6 In
addition, the sulfhydryl group of cysteine can suffer â-
elimination to form dehydroalanine, which can undergo
further reaction.7 Thus, the impact of native chemical ligation
would be even greater were it not limited to creating an Xaa-
Cys bond.
mechanism for this version of the “Staudinger ligation”11 is
shown in Scheme 2. The ligation begins by transthioesteri-
Scheme 2
Offer and Dawson have described a means to remove the
limitation inherent in native chemical ligation.8 In their
method, a peptide bond is formed from a thioester and an
o-mercaptobenzylamine. Though effective, this method is
engrammic, leaving o-mercaptobenzylamine in the ligation
product.
Here, we describe a method for peptide ligation that
eliminates the need for a cysteine residue and leaves no
residual atoms in the peptide product. Our method is inspired
by the Staudinger reaction.9 In the Staudinger reaction, a
phosphine is used to reduce an azide to an amine: PR3 +
N3R′ + H2O f OdPR3 + H2NR′ + N2(g). The intermediate
in the reaction is an iminophosphorane (R3P+--NR′), which
has a nucleophilic nitrogen. Vilarrasa and others have shown
that this nitrogen can attack an acyl donor in an intermo-
lecular or intramolecular reaction.10 The final product, after
hydrolysis of the amidophosphonium salt, is an amide. Saxon
and Bertozzi have shown that the acyl group can originate
from the phosphine itself and be transferred to the imino-
phosphorane nitrogen in an intramolecular reaction in water.11
Their product is an amide containing a phosphine oxide.
fication with the phosphinothiol. Coupling of the resulting
phosphinothioester with a peptide azide leads to the formation
of the reactive iminophosphorane. Attack of the iminophos-
phorane nitrogen on the thioester leads to an amidophos-
phonium salt. Hydrolysis of the amidophosphonium salt
produces the desired amide and a phosphine oxide. Signifi-
cantly, no atoms from the phosphinothiol remain in the amide
product.12
A critical aspect in effecting the Staudinger ligation of a
thioester and azide is selecting an appropriate phosphinothiol.
We chose an o-phosphinobenzenethiol (R2PC6H4-o-SH)
because it allows a six-membered ring to form in the
transition state for acyl transfer. Moreover, R2PC6H4-o-SH
does not allow for the formation of an episulfide and a stable
amidophosphine (R2PNR′C(O)R′′) by C-P bond cleavage
in the amidophosphonium salt, as would simple alkanethiols
such as R2PCH2CH2SH. Further, thiophenol itself is known
to effect the transthioesterification of thioesters during native
chemical ligation.13
We demonstrated the efficacy of the Staudinger ligation
by effecting the transformation shown in Scheme 3 (R )
Bn). In this transformation, the peptide AcPheGlyNHBn (5)
was synthesized from a phenylalanyl thioester (1) and a
glycyl azide (4) by the action of o-(diphenylphosphino)-
benzenethiol (2).14 Thioester 3 was prepared in quantitative
yield by the transthioesterification of thioester 1 with an
excess of phosphinobenzenethiol 2 in DMF containing
diisopropylethylamine (DIEA).15 Excess thiol was removed
by covalent immobilization to a Merrifield resin (chloro-
methylpolystyrene-divinylbenzene). Azide 4 (1 equiv) was
To apply the Staudinger reaction to peptide synthesis, we
use a phosphinothiol to unite a thioester and azide. A putative
(5) McCaldon, P.; Argos, P. Proteins 1988, 4, 99-122.
(6) (a) Schneider, C. H.; de Weck, A. L. Biochim. Biophys. Acta 1965,
168, 27-35. (b) Raines, R. T. Nat. Struct. Biol. 1997, 4, 424-427.
(7) Friedman, M. AdV. Exp. Med. Biol. 1999, 459, 145-159.
(8) Offer, J.; Dawson, P. E. Org. Lett. 2000, 2, 23-26.
(9) Staudinger, H.; Meyer, J. HelV. Chim. Acta 1919, 2, 635-646. For
reviews, see: (a) Gololobov, Yu. G.; Zhmurova, I. N.; Kasukhin, L. F.
Tetrahedron 1981, 37, 437-472. (b) Gololobov, Yu. G.; Kasukhin, L. F.
Tetrahedron 1992, 48, 1353-1406.
(10) For examples, see: (a) Bosch, I.; Romea, P.; Urpf, F.; Vilarrasa, J.
Tetrahedron Lett. 1993, 34, 4671-4674. (b) Bosch I.; Urpi F.; Vilarrasa J.
J. Chem. Soc., Chem. Commun. 1995, 91-92. (c) Shalev, D. E.; Chiacchiera,
S. M.; Radkowsky, A. E.; Kosower, E. M. J. Org. Chem. 1996, 61, 1689-
1701. (d) Bosch, I.; Gonzalez, A.; Urpi, F.; Vilarrasa, J. J. Org. Chem.
1996, 61, 5638-5643. (e) Tang, Z.; Pelletier, J. C. Tetrahedron Lett. 1998,
39, 4773-4776. (f) Ariza X.; Urpi, F.; Viladomat, C.; Vilarrasa J.
Tetrahedron Lett. 1998, 39, 9101-9102.
(11) Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007-2010.
(12) Amide formation by intramolecular acyl transfer from an N-acyl
imidazole to an iminophosphorane nitrogen is also traceless and effective
(Bertozzi, C. R. Presented at the 218th National Meeting of the American
Chemical Society, New Orleans, LA, August 1999; Paper ORGN 233).
(13) Dawson, P. E.; Churchill, M.; Ghadiri, M. R.; Kent, S. G. H. J.
Am. Chem. Soc. 1997, 119, 4325-4329.
(14) o-(Diphenylphosphino)benzenethiol (2) was prepared by reaction
of chlorodiphenylphosphine and ortholithiated thiophenol, as described by
Block, E.; Ofori-Okai, G.; Zubieta, J. J. Am. Chem. Soc. 1989, 111, 2327-
2329.
(15) Phosphines are remarkable catalysts of acyl transfer reactions
(Vedejs, E.; Diver, S. T. J. Am. Chem. Soc. 1993, 115, 3358-3359). Hence,
thioester 3 may result from the formation of an acylphosphonium salt
(Ph2P+(C6H4-o-SH)C(O)R), followed by intramolecular P- to S-acyl migra-
tion.
1940
Org. Lett., Vol. 2, No. 13, 2000