Staudinger ligation of α-azido acids retains stereochemistry

Article abstract of DOI:10.1021/jo025631l

The Staudinger ligation of peptides with a C-terminal phosphinothioester and N-terminal azide is an emerging method in protein chemistry. Here, the first Staudinger ligations of nonglycyl azides are reported and shown to proceed both in nearly quantitative yield and with no detectable effect on the stereochemistry at the α-carbon of the azide. These results demonstrate further the potential of the Staudinger ligation as a general method for the total synthesis of proteins from peptide fragments.

Full text of DOI:10.1021/jo025631l

SCHEME 1
Sta u d in ger Liga tion of r-Azid o Acid s
Reta in s Ster eoch em istr y
Matthew B. Soellner,† Bradley L. Nilsson,† and
Ronald T. Raines*,†,‡
Departments of Chemistry and Biochemistry, University of
WisconsinsMadison, Madison, Wisconsin 53706
raines@biochem.wisc.edu
Received February 19, 2002
Abstr a ct: The Staudinger ligation of peptides with a
C-terminal phosphinothioester and N-terminal azide is an
emerging method in protein chemistry. Here, the first
Staudinger ligations of nonglycyl azides are reported and
shown to proceed both in nearly quantitative yield and with
no detectable effect on the stereochemistry at the R-carbon
of the azide. These results demonstrate further the potential
of the Staudinger ligation as a general method for the total
synthesis of proteins from peptide fragments.
The chemoselective ligation of peptides can be used to
effect the total chemical synthesis of proteins.1 The most
common ligation method, native chemical ligation, relies
on the presence of a cysteine residue at the N-terminus
of each ligation junction.2,3 We have identified the
“Staudinger ligation” as a peptide ligation method that
has the potential to be universalsindependent of the
presence of any particular side chain.4 This method is
based on the Staudinger reaction, wherein a phosphine
reduces an azide via a stable iminophosphorane inter-
mediate.5 Acylation of this iminophosphorane yields an
amide.6,7
thioester (2) reacts with another peptide fragment having
an N-terminal azide (3). The resulting iminophosphorane
(4) leads, after an S- to N-acyl shift, to an amidophos-
phonium salt (5). The P-N bond of the amidophospho-
nium salt is hydrolyzed readily to produce the amide
product (6) and a phosphine oxide (7). Importantly, no
residual atoms remain in the amide product.4,6b Previ-
ously, we showed that the ligation of glycyl and phenyl-
alanyl thioesters of phosphinothiol 1 with glycyl azides
proceeds in high yield.4b
In our version of the Staudinger ligation (Scheme 1),4
a peptide fragment having a C-terminal phosphino-
All natural R-amino acids except glycine have a ste-
reogenic center at their R-carbon.8 To be an effective tool
for the total chemical synthesis of proteins, a peptide
ligation reaction must proceed without epimerization.
* To whom correspondence should be addressed at the Department
of Biochemistry, University of WisconsinsMadison, 433 Babcock Dr.,
Madison, WI 53706-1544. Telephone: (608) 262-3453. Fax: (608) 262-
3453.
† Department of Chemistry.
(7) For examples in which the acyl donor is not attached to the
phosphine, see: (a) Garcia, J .; Urpı´, F.; Vilarrasa, J . Tetrahedron Lett.
1984, 25, 4841-4844. (b) Garcia, J .; Vilarrasa, J . Tetrahedron Lett.
1986, 27, 639-640. (c) Urp´ı, F.; Vilarrasa, J . Tetrahedron Lett. 1986,
27, 4623-4624. (d) Bosch, I.; Romea, P.; Urp´ı, F.; Vilarrasa, J .
Tetrahedron Lett. 1993, 34, 4671-4674. (e) Inazu, T.; Kobayashi, K.
Synlett 1993, 869-870. (f) Molina, P.; Vilaplana, M. J . Synthesis
(Stuttgart) 1994, 1197-1218. (g) Bosch, I.; Urp´ı, F.; Vilarrasa, J . J .
Chem. Soc., Chem. Commun. 1995, 91-92. (h) Shalev, D. E.; Chiacchi-
era, S. M.; Radkowsky, A. E.; Kosower, E. M. J . Org. Chem. 1996, 61,
1689-1701. (i) Bosch, I.; Gonzalez, A.; Urp´ı, F.; Vilarrasa, J . J . Org.
Chem. 1996, 61, 5638-5643. (j) Maunier, V.; Boullanger, P.; Lafont,
D. J . Carbohydr. Res. 1997, 16, 231-235. (k) Afonso, C. A. M. Synth.
Commun. 1998, 28, 261-276. (l) Tang, Z.; Pelletier, J . C. Tetrahedron
Lett. 1998, 39, 4773-4776. (m) Ariza X.; Urp´ı, F.; Viladomat, C.;
Vilarrasa J . Tetrahedron Lett. 1998, 39, 9101-9102. (n) Mizuno, M.;
Muramoto, I.; Kobayashi, K.; Yaginuma, H.; Inazu, T. Synthesis
(Stuttgart) 1999, 162-165. (o) Mizuno, M.; Haneda, K.; Iguchi, R.;
Muramoto, I.; Kawakami, T.; Aimoto, S.; Yamamoto, K.; Inazu, T. J .
Am. Chem. Soc. 1999, 121, 284-290. (p) Boullanger P.; Maunier, V.;
Lafont, D. Carbohydr. Res. 2000, 324, 97-106. (q) Velasco, M. D.;
Molina, P.; Fresneda, P. M.; Sanz, M. A. Tetrahedron 2000, 56, 4079-
4084. (r) Malkinson, J . P.; Falconer, R. A.; Toth, I. J . Org. Chem. 2000,
65, 5249-5252. (s) Ariza, X.; Pineda, O.; Urp´ı, F.; Vilarrasa, J .
Tetrahedron Lett. 2001, 42, 4995-4999.
‡ Department of Biochemistry.
(1) For recent reviews of peptide ligation methodology, see: (a) Tam,
J . P.; Yu, Q.; Miao, Z. Biopolymers 1999, 51, 311-332. (b) Dawson, P.
E.; Kent, S. B. H. Annu. Rev. Biochem. 2000, 69, 923-960. (c) Borgia,
J . A.; Fields, G. B. Trends Biotechnol. 2000, 15, 243-251. (d) Miranda,
L. P.; Alewood, P. F. Biopolymers 2000, 55, 217-226. (e) Tam, J . P.;
Xu, J .; Eom, K. D. Biopolymers 2001, 60, 194-205.
(2) Wieland, T.; Bokelmann, E.; Bauer, L.; Lang, H. U.; Lau, H.
Liebigs Ann. Chem. 1953, 583, 129-149. (b) Dawson, P. E.; Muir, T.
W.; Clark-Lewis, I.; Kent, S. B. H. Science 1994, 266, 776-779.
(3) Cysteine is uncommon, comprising only 1.7% of the residues in
proteins (McCaldon, P.; Argos, P. Proteins 1988, 4, 99-122). Modern
peptide synthesis is typically limited to peptides of e50 residues (ref
1). Hence, most proteins cannot be prepared by any method that allows
for the coupling of peptides only at cysteine residues.
(4) (a) Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. Org. Lett. 2000,
2, 1939-1941. (b) Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. Org.
Lett. 2001, 3, 9-12. For a review, see: (c) Gilbertson, S. Chemtractss
Org. Chem. 2001, 14, 524-528.
(5) (a) Staudinger, H.; Meyer, J . Helv. Chim. Acta 1919, 2, 635-
646. For
a review see: (b) Gololobov, Yu. G.; Kasukhin, L. F.
Tetrahedron 1992, 48, 1353-1406.
(6) For examples in which the acyl donor is attached covalently to
the phosphine, see: ref 4 and (a) Saxon, E.; Bertozzi, C. R. Science
2000, 287, 2007-2010. (b) Saxon, E.; Armstrong, J . I.; Bertozzi, C. R.
Org. Lett. 2000, 2, 2141-2143. (c) Kiick, K. L.; Saxon, E.; Tirrell, D.
A.; Bertozzi, C. R. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 19-24.
(8) For information on exploiting the chirality of R-amino acids,
see: Coppola, G. M.; Schuster, H. F. Asymmetric Synthesis; J ohn Wiley
& Sons: New York, 1987.
10.1021/jo025631l CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/14/2002
J . Org. Chem. 2002, 67, 4993-4996
4993

SCHEME 2
SCHEME 3
We then prepared several nonglycyl R-azido acids to
determine if epimerization occurs during the Staudinger
ligation. The azido benzamides of both the D and
L
enantiomers of phenylalanine, serine, and aspartic acid
were prepared (Scheme 3). The azido group was prepared
by diazo transfer;10 the benzamide was prepared by DCC/
HOBt coupling with benzylamine. Phenylalanine, aspar-
tic acid, and serine were chosen as being representative
of three distinct side chains and moderate (phenylala-
nine) to high (aspartate and serine) propensity to epimer-
ize during standard peptide couplings.16
Each of these azido acids was coupled with phos-
phinothioester 2117 (Table 1). The couplings were carried
out in THF/H2O (3:1) for 12 h at room temperature with
a 1:1 stoichiometry of starting materials. The resulting
peptides were purified by flash chromatography to give
a nearly quantitative yield of each product (Table 1). The
high yield of this equimolar reaction of phosphinothiol 1
with nonglycyl azides is consistent with those observed
previously with glycyl azides.4b
The coupling of thioesters in native chemical ligation,
which like the Staudinger ligation (Scheme 1) involves
transthioesterification followed by an S- to N-acyl shift,2,3
is known to proceed without detectable racemization.9
Because the Staudinger ligation has been demonstrated
previously only with glycyl azides, the propensity for
epimerization of the R-azido acid has not been assessed.
This issue is of concern because an R-carbanion could be
stabilized by inductive or resonance effects in azide 3 and
iminophosphorane 4.10
Here, we report the first use of the Staudinger ligation
to couple a peptide containing a nonglycyl azide. We also
search for epimerization11 during Staudinger ligation
reactions. Finally, we report an improved synthesis of
phosphinothiol 1 (Scheme 1), which is the most effective
known phosphinothiol for effecting the Staudinger liga-
tion of peptides.
The chirality of the Staudinger ligation products from
the reaction of the D and L R-azido acids was analyzed
by HPLC using a D-phenylglycine chiral column. The
chromatographic conditions enabled the baseline resolu-
tion of the two possible enantiomeric products (Figure
1).18 After reaction of the D epimer, there was no evidence
of product containing the L epimer, and vice versa. Thus,
the Staudinger ligation proceeds without detectable
epimerization of the R-carbon of the azido acid.19
We conclude that the Staudinger ligation can be used
to couple nonglycl azides in nearly quantitative yield and
without detectable epimerization. Thus, the Staudinger
ligation holds promise as a general method for the
convergent assembly of proteins from peptide fragments.
The previously reported synthesis of phosphinothiol 1
required four steps, two of which were problematic, with
an overall yield of only 39%.4b We have developed an
improved synthesis that uses air-stable borane protection
of the phosphine (Scheme 2).12 The synthesis is based on
the easily prepared alkylating agent 813 and the com-
mercially available borane-diphenylphosphine complex
9. Compound 9 is deprotonated by sodium hydride in
DMF followed by addition of 8 to give borane complex
10 (86% yield).14 Complex 10 is stable to air and moisture
and can be stored on the shelf at room temperature for
months without any sign of oxidation or decomposition.
The borane complex is disrupted by mild heating with
DABCO in toluene for 4 h (95% yield).15 The protecting
group of the resulting acyl phosphinothiol 11 is removed
as described previously4b to give phosphinothiol 1 (94%
yield). The overall yield for this three-step synthesis is
74%.
Exp er im en ta l Section
Reactions were monitored by thin-layer chromatography with
visualization by UV light or staining with ninhydrin or I2. Silica
gel used in flash chromatography had 230-400 mesh and 60 Å
(9) Lu, W.; Qasim, M. A.; Kent, S. B. H. J . Am. Chem. Soc. 1996,
118, 8518-8523.
(10) Zaloom, J .; Roberts, D. C. J . Org. Chem. 1981, 46, 5173-5176.
(11) Because the reaction products herein are enantiomers and not
diastereomers, we actually probe “racemization” (Reist, M.; Testa, B.;
Carrupt, P.-A.; J ung, M.; Schurig, V. Chirality 1995, 7, 396-400). We
use the term “epimerization” to focus attention on the fate of the
chirality at the R-carbon of the azide in couplings of peptides that
contain multiple stereogenic centers.
(12) For reviews of borane protection of phosphines, see: (a) Brunel,
J . M.; Faure, B.; Maffei, M. Coord. Chem. Rev. 1998, 180, 665-698.
(b) Carboni, B.; Monnier, L. Tetrahedron 1999, 55, 1197-1248.
(13) Farrington, G. K.; Kumar, A.; Wedler, F. C. Org. Prep. Proced.
Int. 1989, 21, 390-392.
(16) Romoff, T. T.; Goodman, M. J . Pept. Res. 1997, 49, 281-292.
(17) The preparation of phosphinothioester 21 was modified from
our previous reports, in which coupling using DCC alone led to lower
yields and several undesired products (ref 4a and 4b). Pretreatment
of N-acetylglycine (8) with HOBt and DCC followed by addition of
phosphinothiol 1 improved the yield dramatically. See the Experimen-
tal Section for details.
(18) Material was injected onto a D-phenylglycine analytical chiral
HPLC column and eluted with 30% (v/v) 2-propanol in hexanes
(isocratic) for 20 min followed by a shallow gradient to 50% (v/v)
2-propanol for 40 min.
(19) We estimate the detection limit of the HPLC chromatographic
analysis to be e0.5%, so that the Staudinger ligation proceeds with
g99.5% retention of chirality. This latter value is greater than values
for analogous intermolecular acyl transfer reactions to an iminophos-
phorane (ref 7g).
(14) Imamoto, T.; Oshiki, T.; Onozawa, T.; Kusumoto, T.; Sato, K.
J . Am. Chem. Soc. 1990, 112, 5244-5252.
(15) Brisset, H.; Gourdel, Y.; Pellon, P.; Le Corre, M. Tetrahedron
Lett. 1993, 34, 4523-4526.
4994 J . Org. Chem., Vol. 67, No. 14, 2002

TABLE 1. Sta u d in ger Liga tion of AcGlySCH2P P h 2 (21)
a n d Non -Glycyl r-Azid o Acid s
were referenced against an external standard of deuterated
phosphoric acid (0 ppm). Mass spectra were obtained with
electrospray ionization (ESI) techniques.
Bor an e-Th ioacetic Acid S-[(Diph en ylph osph an yl)m eth -
yl] Ester Com p lex (10). Borane-diphenylphosphine complex
9 (10.33 g, 51.6 mmol) was dissolved in dry DMF under Ar(g)
and cooled to 0 °C. NaH (1.24 g, 51.6 mmol) was added slowly,
and the mixture was stirred at 0 °C until bubbling ceased.
Alkylating agent 813 (8.73 g, 51.6 mmol) was then added, and
the mixture was allowed to warm to room temperature and
stirred for 12 h. Solvent was removed under reduced pressure,
and the residue was purified by flash chromatography (silica
gel, 10% v/v EtOAc in hexanes). Compound 10 was isolated as
1
a colorless oil in 86% yield. H NMR (300 MHz, CDCl3) δ 7.74-
7.67 (m, 4 H), 7.54-7.41 (m, 6 H), 3.72 (d, J ) 6 Hz, 2 H), 2.23
(s, 3 H), 1.51-0.53 (broad m, 3 H) ppm; 13C NMR (75 MHz,
CDCl3) δ 192.94, 132.26 (d, J ) 9.2 Hz), 131.61 (d, J ) 2.3 Hz),
128.71 (d, J ) 10.2 Hz), 127.43 (d, J ) 55.4 Hz), 29.87, 23.59 (d,
J ) 35.5 Hz) ppm; 31P NMR (121 MHz, CDCl3) 19.40 (d, J )
59.3 Hz) ppm; MS (ESI) m/z 311.0806 (MNa+ [C15H18BOPSNa]
) 311.0807).
Th ioa cetic Acid S-[(Dip h en ylp h osp h a n yl)m eth yl] Ester
(11). Compound 10 (4.00 g, 13.9 mmol) was dissolved in toluene
(0.14 L) under Ar(g). DABCO (1.56 g, 13.9 mmol) was added,
and the mixture was heated at 40 °C for 4 h. Solvent was
removed under reduced pressure, and the residue was dissolved
in CH2Cl2 and washed with both 1 N HCl and saturated brine.
The organic layer was dried over MgSO4(s), and the solvent was
removed under reduced pressure. Compound 11 was isolated in
95% yield and was used without further purification. Spectral
data was the same as that reported previously.4b
2(S)-Azid o-N-ben zyl-3-p h en ylp r op ion a m id e (18-L). N3-
(L)PheOH (15-L) was synthesized from L-phenylalanine es-
sentially by the procedure of Lundquist and Pelletier.20 N3(L)-
PheOH (1.08 g, 5.7 mmol) was dissolved in anhydrous DMF (40
mL). HOBt (0.87 g, 5.7 mmol) was then added, followed by DCC
(1.17 g, 5.7 mmol). Once precipitate was observed in the reaction,
benzylamine (0.62 mL, 5.7 mmol) was added. The reaction was
allowed to stir under Ar(g) for 3 h. The resulting precipitate
(DCU) was removed by filtration, and the filtrate was concen-
trated under reduced pressure to give a yellow oil. This oil was
purified by flash chromatography (silica gel, 35% v/v ethyl
acetate in hexanes). N3(L)PheNHBn (18-L) was isolated as an
off-white solid in 90% yield. The procedure was repeated with
D-phenylalanine to give N3(D)PheNHBn (18-D) as a white solid
in 92% yield. The spectral data for both N3PheNHBn products
a
Reaction conditions: THF/H2O (3:1) at room temperature for
b
12 h. Isolated yield of product after purification by flash chro-
matography.
1
(D and L enantiomers) are identical. N3(L)P h eNHBn (18-L) H
NMR (500 MHz, CDCl3) δ 7.31-7.12 (m, 8 H), 7.11 (m, 2 H),
6.55 (bs, 1 H), 4.38 (m, 2 H), 4.22 (dd, J ) 7.8, 4.6 Hz, 1 H), 3.34
(dd, J ) 14.0, 4.5 Hz, 1 H), 3.07 (dd, J ) 14.1, 7.5 Hz, 1 H) ppm;
13C NMR (125 MHz, CDCl3) δ 168.30, 137.35, 135.96, 129.51,
128.61, 128.66, 128.61, 128.55, 127.70, 127.68, 127.66, 127.57,
127.16, 65.40, 43.41, 38.41 ppm; MS (ESI) m/z 303.1235 (MNa+
[C16H16N4ONa] ) 303.1222).
3(S)-Azid o-N-ben zylsu ccin a m ic Acid Meth yl Ester (19-
L). Benzyl-protected L-aspartate was used in the procedure of
Lundquist and Pelletier20 to give N3(L)Asp(OMe)OH (16-L).
Under these conditions, we observed transesterification to give
the methyl ester product as opposed to the benzyl ester. N3(L)-
Asp(OMe)OH (16-L) was produced as a yellowish oil in 78%
yield. N3(L)Asp(OMe)OH (16-L) was then coupled with benzy-
lamine as above to give N3(L)Asp(OMe)NHBn (19-L) as a
yellowish oil in 90% yield (70% overall, two steps). The procedure
above was repeated with benzyl-protected D-aspartate to give
N3(L)Asp(OMe)NHBn (19-D) as a yellowish oil in 67% overall
yield. The spectral data for both N3Asp(OMe)OH (D and
L
enantiomers) and both N3Asp(OMe)NHBn (D and L enantiomers)
products are identical. N3(L)Asp (OMe)OH (16-L) 1H NMR (500
MHz, CDCl3) δ 10.24 (bs, 1H), 4.47 (dd, J ) 7.4, 5.3 Hz, 1 H),
3.76 (s, 3 H), 2.91 (dd, J ) 16.9, 5.1 Hz, 1 H), 2.79 (dd, J ) 16.8,
7.6 Hz) ppm; 13C NMR (125 MHz, CDCl3) δ 174.68, 170.12, 50.09,
F IGURE 1. HPLC elution profile of AcGly(D)SerNHBn,
AcGly(L)SerNHBn, and a mixture of the two enantiomers.18
pore size. Chiral HPLC was performed with a D-phenylglycine
analytical chiral column. NMR spectra were obtained with a 500
or 300 MHz spectrometer at the University of Wisconsin nuclear
magnetic resonance facility. Carbon-13 and phosphorus-31 NMR
spectra were both proton-decoupled, and phosphorus-31 spectra
(20) Lundquist, J . T., IV.; Pelletier, J . C. Org. Lett. 2001, 3, 781-
783.
J . Org. Chem, Vol. 67, No. 14, 2002 4995

52.44, 35.84 ppm; MS (ESI) m/z 196.0340 (MNa+ [C5H7N3O4-
Na] ) 196.0334). N3(L)Asp (OMe)NHBn (19-L) 1H NMR (500
MHz, CDCl3) δ 7.38-7.27 (m, 5 H), 6.83 (bs, 1 H), 4.54 (m, 3 H),
3.75 (s, 3 H), 3.18 (dd, J ) 17.1, 3.7 Hz, 1 H), 2.75 (dd, J ) 17.3,
8.7 Hz, 1 H) ppm; 13C NMR (125 MHz, CDCl3) δ 170.77, 167.90,
137.35, 128.81, 127.80, 127.77, 60.32, 52.24, 43.71, 37.00 ppm;
MS (ESI) m/z 285.0953 (MNa+ [C12H12N4O3Na] ) 285.0964).
2(S)-Azid o-N-b en zyl-3-b en zyloxyp r op ion a m id e (20-L).
Benzyl-protected L-serine was used in the procedure above to
give N3(L)Ser(Bzl)NHBn (20-L) as a yellowish oil in 93% yield.
The procedure was repeated with benzyl-protected D-serine to
give N3(D)Ser(Bzl)NHBn (20-D) as a yellowish oil in 90% yield.
The spectral data for both N3Ser(Bzl)NHBn products (D and L
enantiomers) are identical. N3(L)Ser (Bzl)NHBn (20-L) 1H
NMR (500 MHz, CDCl3) δ 7.36-7.23 (m, 10 H), 6.86 (bs, 1 H),
4.57 (s, 2 H), 4.43 (m, 2 H), 4.25 (dd, J ) 6.9, 3.5 Hz, 1 H), 4.01
(dd, J ) 10.3, 3.5 Hz, 1 H), 3.83 (10.1, 6.7 Hz, 1 H) ppm; 13C
NMR (125 MHz, CDCl3) δ 166.81, 137.40, 137.22, 128.65, 128.40,
127.81, 127.59, 127.54, 73.45, 70.54, 63.28, 43.38 ppm; MS (ESI)
m/z 333.1337 (MNa+ [C17H18N4O2Na] ) 333.1327).
(Acetyla m in o)th ioa cetic Acid S-[(Dip h en ylp h osp h a n yl)-
m eth yl] Ester (21). 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, phosphi-
nothiol 1 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. This solid was dissolved
in ethyl acetate and purified by flash chromatography (silica gel,
ethyl acetate). Compound 21 was isolated in 96% yield. Spectral
data was the same as that reported previously.4b
2(S)-(2-(Acet yla m in o)a cet yla m in o)-N-b en zyl-3-p h en yl-
pr opion am ide (22-L). N-Acetylglycylphosphinothioester 9 (0.166
g, 0.5 mmol) and N3(L)PheNHBn (18-L) (0.140 g, 0.5 mmol) was
dissolved in THF/H2O (3:1, 4 mL), and the mixture was stirred
at room temperature for 12 h. Solvent was removed under
reduced pressure, and the residue was purified by flash chro-
matography (silica gel, 5% v/v methanol in dichloromethane).
AcGly(L)PheNHBn (22-L) was obtained in as a white solid in
90% yield. The procedure was repeated with N3(D)PheNHBn (18-
D) to give AcGly(D)PheNHBn (22-D) in 93% yield. The spectral
data for both dipeptide products (D and L enantiomers) are
identical. AcGly(L)P h eNHBn (22-L) 1H NMR (300 MHz,
CDCl3:CD3OD 1:1) δ 7.30-7.22 (m, 6 H), 7.19-7.16 (m, 2 H),
7.16-7.11 (m, 2H), 4.63 (t, J ) 7.3 Hz, 1 H), 4.33 (dd, J ) 31.1,
14.6 Hz, 2 H), 3.79 (dd, J ) 33.1, 16.7 Hz, 2 H), 3.12 (dd, J )
13.8, 7.2 Hz, 1 H), 2.98 (dd J ) 13.7, 7.2 Hz, 1 H), 1.98 (s, 3 H)
ppm; 13C NMR (125 MHz, CDCl3:CD3OD 1:1) δ 171.98, 170.93,
169.33, 137.29, 136.00, 128.76, 128.05, 127.97, 127.03, 126.75,
126.41, 54.16, 42.79, 42.37, 37.52, 21.56 ppm; MS (ESI) m/z
376.1624 (MNa+ [C20H23N3O3Na] ) 376.1637).
3(S )-(2-(Ace t yla m in o)a ce t yla m in o)-N -b e n zylsu ccin -
a m ic Acid Meth yl Ester (23-L). N3(L)Asp(OMe)NHBn (19-L)
was used in the procedure above to give AcGly(L)Asp(OMe)-
NHBn (23-L) as a white solid in 91% yield. The procedure was
repeated with N3(D)Asp(OMe)NHBn (19-D) to give AcGly(D)Asp-
(OMe)NHBn (23-D) as a white solid in 95% yield. The spectral
data for both AcGlyAsp(OMe)NHBn products (D and L enanti-
omers) are identical. AcGly(L)Asp (OMe)NHBn (23-L) 1H NMR
(500 MHz, CDCl3:CD3OD 1:1) δ 7.34-7.23 (m, 5 H), 4.84 (t, J )
5.7 Hz, 1 H), 4.34 (s, 2 H), 3.84 (q, J ) 16.6 Hz, 2H), 3.69 (s, 3
H), 2.87 (m, 2 H), 2.01 (s, 3 H) ppm; 13C NMR (125 MHz, CDCl3:
CD3OD 1:1) δ 177.24, 176.43, 175.18, 174.58, 142.56, 133.17,
133.03, 131.93, 131.78, 56.46, 54.13, 47.91, 47.81, 47.67, 40.11,
26.59 ppm; MS (ESI) m/z 358.1388 (MNa+ [C16H21N3O5Na] )
358.1379).
2(S )-(2-(Ace t yla m in o)a ce t yla m in o)-N -b e n zyl-3-b e n z-
yloxyp r op ion a m id e (24-L). N3(L)Ser(Bzl)NHBn (20-L) was
used in the procedure above to give AcGly(L)Ser(Bzl)NHBn (24-
L) as a white solid in 92% yield. The procedure was repeated
with N3(D)PheNHBn (20-D) to give AcGly(D)Ser(Bzl)NHBn (24-
D) as a white solid in 99% yield. The spectral data for both
AcGlySer(Bzl)NHBn products (D and L enantiomers) are identi-
cal. AcGly(L)Ser (Bzl)NHBn (24-L) 1H NMR (500 MHz, CDCl3:
CD3OD 1:1) δ 7.34-7.21 (m, 10 H), 4.60 (t, J ) 4.4 Hz, 1 H),
4.43 (dd, J ) 23.9, 14.9 Hz, 2 H), 3.85 (m, 3 H), 3.69 (dd, J )
9.6, 4.6 Hz, 1 H), 1.98 (s, 3 H) ppm; 13C NMR (125 MHz, CDCl3:
CD3OD 1:1) δ 172.19, 169.86, 169.61, 137.49, 127.87, 127.31,
127.23, 127.76, 126.59, 72.88, 69.07, 52.93, 42.71, 42.49, 21.38
ppm; MS (ESI) m/z 406.1750 (MNa+ [C21H25N3O4Na] ) 406.1743).
Ack n ow led gm en t. We are grateful to Prof. L. L.
Kiessling for contributive discussions, and to Dr. R. J .
Hinklin and Y. He for advice on the improved synthesis
of phosphinothiol 1. This work was supported by Grant
GM44783 (NIH). B.L.N. was supported by the Abbott
Laboratories Fellowship in Synthetic Organic Chemis-
try (University of WisconsinsMadison).
Su p p or tin g In for m a tion Ava ila ble: 1H, 13C, and 31P
(where applicable) spectra for all novel compounds; chiral
HPLC traces for each peptide in Table 1. This material is
available free of charge via the Internet at http://pubs.acs.org.
J O025631L
4996 J . Org. Chem., Vol. 67, No. 14, 2002