Coulombic effects on the traceless Staudinger ligation in water

Article abstract of DOI:10.1016/j.bmc.2008.02.047

The traceless Staudinger ligation can be mediated by phosphinothiols under physiological conditions. Proximal positive charges are necessary to achieve that transformation, presumably because those charges discourage protonation of the key iminophosphoran

Full text of DOI:10.1016/j.bmc.2008.02.047

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 17 (2009) 1055–1063
Coulombic effects on the traceless Staudinger ligation in water
Annie Tama and Ronald T. Rainesa,b,
*
aDepartment of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, WI 53706, USA
bDepartment of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706, USA
Received 12 December 2007; accepted 13 February 2008
Available online 16 February 2008
Abstract—The traceless Staudinger ligation can be mediated by phosphinothiols under physiological conditions. Proximal positive
charges are necessary to achieve that transformation, presumably because those charges discourage protonation of the key imino-
phosphorane intermediate. Here, a series of cationic phosphinothiols is used to probe Coulombic effects on the traceless Staudinger
ligation in aqueous buffers. The reagent bis(m-N,N-dimethylaminomethylphenyl)phosphinomethanethiol (3) is found to be superior
to others, both in its ability to mediate the traceless Staudinger ligation in water and in the efficiency of its synthesis.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
The advent of methodology for the chemoselective
ligation of peptide fragments has made proteins accessi-
ble targets for chemical synthesis. Many proteins have
already been assembled from synthetic peptides using
prior capture strategies. The most utilized method of
peptide ligation is ‘native chemical ligation’—the cou-
pling of a peptide (or protein) containing a C-terminal
thioester with another peptide containing an N-terminal
cysteine residue.1 An extension of native chemical
ligation, ‘expressed protein ligation’, employs an
engineered intein to access a polypeptide containing
the C-terminal thioester.2 Although this strategy for
protein thioester formation has expanded the utility of
Scheme 1. Putative mechanism of the traceless Staudinger ligation.
native chemical ligation for protein semisynthesis, it re-
tains the intrinsic requirement of a cysteine residue at
the ligation juncture.
mic ligation of peptides at virtually any residue.5 The
Staudinger ligation is chemoselective (especially regard-
Emerging strategies for protein assembly avoid the need
ing the relatively nonreactive azide),6 retains stereochem-
for a cysteine residue at the ligation site.3 The traceless
istry,7 and has been used in the orthogonal assembly of a
Staudinger ligation is one such strategy (Scheme 1).4 This
protein,8 and for the site-specific immobilization of
method is based on the Staudinger reduction, wherein a
peptides and proteins to a surface.9
phosphine reduces an azide via an iminophosphorane
intermediate. The iminophosphorane can be acylated
Recently, we reported on the development of water-sol-
to yield, ultimately, an amide. An important feature of
uble phosphinothiols that can mediate the traceless
this reaction is its potential to mediate the nonengram-
Staudinger ligation in water in moderate yields.10 The
most efficacious reagent, bis(p-N,N-dimethylaminoeth-
ylphenyl)phosphinomethanethiol (1) (Fig. 1), can also
evoke transthioesterification to generate phosphinothio-
Keywords: Coulombic effects; Iminophosphorane; Peptide ligation;
Phosphinothiol; Protein chemistry; Staudinger ligation.
esters at the C-terminus of proteins generated by
expressed protein ligation.
*
Corresponding author. Tel.: +1 608 262 8588; fax: +1 608 262 3453;
e-mail: rtraines@wisc.edu
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.047

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A. Tam, R. T. Raines / Bioorg. Med. Chem. 17 (2009) 1055–1063
group is closer to the reaction center by it being in the
meta rather than in the para position of the aryl ring
(2 and 3), and it having a methylene rather than an eth-
ylene linker (3). The N,N-dimethylaminoethyl substitu-
ent in the meta position is situated seven bonds away
from the key iminophosphorane nitrogen, compared to
the eight-bond distance of 1. The meta N,N-dimethyl-
aminomethyl substituent in phosphinothiol 3 is even clo-
ser, at a distance of six bonds. The ortho position was
not explored, as we feared that ortho substituents would
add steric encumbrance to the reaction center, and that
the protonated N,N-dimethylamino group could act as
an efficient intramolecular catalyst for deleterious pro-
tonation of the iminophosphorane nitrogen.
Figure 1. Bis(p-N,N-dimethylaminoethylphenyl)phosphinomethaneth-
iol (1), a reagent that mediates the traceless Staudinger ligation in
water.10
Water-soluble phosphinothiol 1 was designed to have
N,N-dimethylamino groups that could not only impart
water solubility but also (when protonated) serve to dis-
courage protonation of the iminophosphorane nitrogen,
which leads to the hydrolysis of the P–N bond prior to
S ! N acyl transfer (Scheme 1). Indeed, we have shown
that positive charges are favored over negative charges
in mediating the traceless Staudinger ligations in
water.10 Herein, we consider further the importance of
Coulombic effects on aqueous Staudinger ligations and
report on markedly improved reagents.
We also reasoned that the efficacy of the reagent in
mediating the traceless Staudinger ligation in water
would be enhanced by additional cationic charges. The
amplified Coulombic effects could have a more pro-
nounced effect on the protonation state of the imino-
phosphorane nitrogen. Phosphinothiol 4, which has
four rather than two meta N,N-dimethylaminomethyl
groups, was thus another target for synthesis and anal-
ysis (Fig. 2).
2. Results and discussion
The positively-charged amino groups of phosphinothiol
1 are eight bonds away from the key iminophosphorane
nitrogen in Scheme 1. Even at this distance, however,
through-space Coulombic effects can affect pKa values.
2.1. Synthesis of phosphinothiols 2–4
In phosphinothiols 1–4, tertiary amino groups, rather
than primary or secondary ones, were chosen to obviate
intramolecular S ! N acyl transfer in ensuing thioes-
ters. Due to the presence of nitrogen, phosphorus, and
sulfur in the phosphinothiol, several orthogonal protec-
tion/deprotection steps were necessary to effect its
synthesis. Accordingly, the route to 1 consisted of nine
linear steps.10 The purification of most intermediates
was possible by chromatography using simple silica
plugs, as the reactions were efficient and essentially
‘spot-to-spot’ conversions. Moreover, each reaction in
the route was readily scalable. Nonetheless, the time
required to generate fully-deprotected 1 from commer-
cial starting materials was approximately one week.
We sought to obtain an efficacious reagent in a shorter
time.
For example, CH3ðCH2Þ NHþ3 has a pKa of 10.65.11
8
The addition þof
a
positive charge, as in
þH3NðCH2Þ NH , lowers the pKa to 10.10. Likewise,
CH3ðCH2Þ NHþ3 3and þH3NðCH2Þ NH3þ have pKa val-
8
5
5
ues of 10.63 and 9.74, respectively.11 Long-range Cou-
lombic effects were also evident in our attempts to
mediate the traceless Staudinger ligation in water.10
Based on these considerations, we reasoned that favor-
able Coulombic effects would be enhanced by increasing
the proximity of the positive charges.
We put forth compounds 2 and 3 to test our reasoning
(Fig. 2). In these compounds, the N,N-dimethylamino
Phosphinothiols 2–4 were synthesized by analogous
synthetic routes (Scheme 2). Although (3-bromoben-
zyl)dimethylamine (9) was available from commercial
sources, bromide 6 was accessible from 3-bromophen-
ethyl alcohol through the displacement of its mesylate
in 5 with dimethylamine. Bromide 8 was synthesized
from the bromination of 5-bromo-m-xylene with
N-bromosuccinimide and catalytic AIBN to give 7, fol-
lowed by benzylic displacement with dimethylamine.12
Double Grignard addition of the respective aryl
bromides with diethylphosphite gave the bis-adducts
10–12. Bromide 8 must be dried stringently under high
vacuum, and the addition of a few drops of 1, 2-dibro-
moethane was also advantageous for facilitating Grig-
nard initiation. Reduction with DIBAl-H and
subsequent protection with borane gave phosphine–bor-
ane complexes 13–15. The protected phosphinothioester
intermediates 17–19 were synthesized in a convergent
manner utilizing the easily-prepared bromide 16,13
Figure 2. Target phosphinothiols with enhanced Coulombic effects for
aqueous traceless Staudinger ligations.

A. Tam, R. T. Raines / Bioorg. Med. Chem. 17 (2009) 1055–1063
1057
Scheme 2. Reagents and conditions: (a) MsCl, NEt3, CH2Cl2; (b) HNMe2, THF; (c) NBS, cat. AIBN, CCl4; (d) HNMe2, benzene; (e) Mg(s), THF;
(f) (EtO)2P(O)H, THF; (g) DIBAl-H, CH2Cl2; (h) BH3ÆSMe2, CH2Cl2; (i) NaH, BrCH2SAc (16), DMF; (j) DABCO, toluene, 40 °C; (k) NaOH,
MeOH (l) 4 N HCl/dioxane.
which had been employed in the convergent synthesis of
(diphenylphosphino)methanethiol.7
2.2. Evaluation of phosphinothiols 2–4
To assess our reasoning on Coulombic effects on the
traceless Staudinger ligation in water, we used phosphi-
nothiols 2–4 to effect a model dipeptide coupling with
13C-labeled azido-glycine 2710 (Fig. 3). Phosphinothio-
esters 23–26 were synthesized by the reaction of the
respective phosphinothiol (1–4) with AcGlyOPfP in
the presence of DIEA. Equimolar amounts of the result-
ing phosphinothioester with 13C-labeled azido-glycine
27 were used in each reaction, and yields were deter-
mined by quantitative 13C NMR spectroscopy in so-
dium phosphate buffers of varying pH. As with
phosphinothioester 23 (derived from 1), phosphinothio-
esters 24–26 provided amide 28 with yields dependent on
the pH of the reaction mixture, peaking at pH 8.0. At
each pH, however, the ligation yield with 25 was supe-
rior to that with phosphinothiol 23, 24, and 26, as ex-
pected from a consideration of the proximity of
Coulombic interactions.
Phosphine–borane complexes 17–19 are stable to pro-
longed storage without decomposition. Removal of the
borane groups with DABCO, deprotection of the thiol
in basic methanol, and subsequent acidification with
4 N HCl/dioxane gave the chloride salts of phosphino-
thiols 2–4, which are easy-to-handle, odor-free white
solids that are soluble at >1 M in 0.4 M sodium-phos-
phate buffer (pH 7.8).
It was found that the synthesis of compound 2 was
comparable to its para counterpart 1. On the contrary,
the efficiency in the synthesis of benzylic compound 3
was dramatically enhanced, as most transformations
were completed in 20 min with excellent yields.
Although the overall yield of 4 was low at 5%, the over-
all yields of 2 and 3 were 15% and 41%, respectively.
Thus, the yield of 3 is markedly greater than that for
phosphinothiol 1 (17%).10 The commercial availability
of (3-bromobenzyl)dimethylamine (9) and shorter reac-
tion times reduced the synthetic route to a water-soluble
phosphinothiol from nine (1) to five (3) transformations,
increased the overall yield from 17% to 41%, and re-
duced the time period required for the synthesis from
7 to 2 1/2 days.
Interestingly, the additional cationic charges present in
the molecule, as in 26, did not improve yields further.
We reason that there are competing through-bond
effects that promote amine formation, which is the
only byproduct of this reaction.10 Unprotonated
N,N-dimethylaminomethyl
groups
are
slightly

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A. Tam, R. T. Raines / Bioorg. Med. Chem. 17 (2009) 1055–1063
that EtPPh2, which is a neutral mimic of HSCH2PPh2,
has a phosphorous pKa of 4.90.14 That value is much
larger than the phosphorous pKa of phosphinothiols
2–4, consistent with our hypothesis that through-space
Coulombic effects lowers the pKa of neighboring atoms,
and in particular discourages protonation of the key
iminophosphorane intermediate.
3. Conclusions
Careful consideration of the proximity of cationic
groups to the key iminophosphorane intermediate per-
mitted the development of improved phosphinothiol re-
agents for mediating the traceless Staudinger ligation in
water. With its positively-charged N,N-dimethylamino
groups closer to the iminophosphorane nitrogen, phos-
phinothiol 3 proved to be superior to our previous re-
agent (1), approaching yields of 70% near pH 8.0. The
synthesis of 3 is efficient and high-yielding, facilitating
its widespread use in peptide and protein chemistry.
An attempt to amplify the Coulombic effects with addi-
tional positive charges proved to be unsuccessful, pre-
sumably because of deleterious inductive effects. These
results show that an effective use of Coulombic interac-
tions can be used to tune pKa values, which are an
important consideration in aqueous traceless Staudinger
ligations. Studies are ongoing in our laboratory to use
such water-soluble phosphinothiols in the semisynthesis
of proteins.
4. Experimental
4.1. General
Reagent chemicals were obtained from commercial sup-
pliers, and reagent grade solvents were used without fur-
ther purification. Procedures were performed at room
temperature (<23 °C) unless indicated otherwise. Reac-
tions were monitored by thin-layer chromatography
with visualization by ultraviolet light or staining with
KMnO4, ninhydrin, PMA, or I2. Compound purifica-
tion was carried out with flash chromatography on a sil-
ica gel, which had a mesh of 230–400 (ASTM) and a
Figure 3. Comparison of amide yield in the traceless Staudinger
ligations mediated by phosphinothiols 1–4 in an aqueous buffer. (A)
Phosphinothioesters 23–26 were synthesized by the reaction of the
respective phosphinothiol with AcGlyOPfP. (B) Dependence of amide
yield on the solution pH. Reactions were performed with equimolar
amounts of phosphinothioesters and azide 27 (60 mM) in 0.40 M
sodium phosphate buffers of varying pH. Data are mean values
(SE = 2%) from two experiments, and yields were determined by 13C
NMR spectroscopy.10 Data for phosphinothioester 23 are from Ref. 10.
˚
pore size of 60 A. The removal of solvents and other vol-
atile materials ‘under reduced pressure’ refers to the use
of a rotary evaporator at water-aspirator pressure
(<20 torr) and a water bath of <40 °C.
electron-donating substituents and would increase elec-
tron density on the phosphorus of the iminophosphor-
ane, rendering the nitrogen more susceptible to
protonation. Such an inductive effect is apparent in the
phosphorous pKa values of (p-OMe-C6H4)3P (4.57),
Ph3P (2.73), and (p-F-C6H4)3P (1.97) in water.14
4.1.1. Instrumentation. NMR spectra were acquired at
ambient temperature with a Bruker AC-300 spectrome-
ter (1H, 300 MHz; 13C, 75 MHz; 31P, 121 MHz) at the
University of Wisconsin Chemistry Department Nuclear
Magnetic Resonance Facility or a Bruker DMX-400
Avance spectrometer (1H, 400 MHz; 13C, 100.6 MHz;
31P, 161 MHz) or Bruker Avance DMX-500 spectrome-
ter (1H, 500 MHz; 13C, 125.7 MHz; 31P, 202 MHz) at
the National Magnetic Resonance Facility at Madison
(NMRFAM) or a Varian Inova 500 (1H, 500 MHz;
13C, 125.7 MHz; 31P, 202 MHz) spectrometer at the
University of Wisconsin Nuclear Magnetic Resonance
Facility. Carbon-13 and phosphorus-31 spectra were
We sought to determine the pKa values of phosphinothi-
ols 2–4 so as to quantitate the impact of Coulombic
effects. Using 31P NMR spectroscopy, we found that
the phosphorus atoms of all positively-charged phosphi-
nothiols remained largely unprotonated at pH P 1.0 in
water (data not shown). Thus, the phosphorous pKa va-
lue of our cationic phosphinothiols appears to be below
the limit of experimental detection. We note, however,

A. Tam, R. T. Raines / Bioorg. Med. Chem. 17 (2009) 1055–1063
1059
proton-decoupled, and phosphorus-31 spectra were ref-
erenced against an external standard of deuterated phos-
phoric acid (0 ppm).
to generate the Grignard reagent. In a separate
flamed-dried flask, diethyl phosphite (237 lL,
1.84 mmol) was dissolved in anhydrous THF (1.0 mL),
and cooled to 0 °C with an ice bath. The solution of
Grignard reagent was added dropwise to this solution,
and the resulting solution was allowed to warm to room
temperature and stirred overnight. The reaction mixture
was then quenched with water (1 mL), and the solvent
was removed under reduced pressure. The residue was
dissolved in CH2Cl2, and the resulting solution was
washed with water and brine. The combined organic ex-
tracts were dried over anhydrous MgSO4(s) and filtered,
and the solvent was removed under reduced pressure.
The residue was purified by flash chromatography (silica
gel, 20% v/v MeOH in CH2Cl2) to give phosphine oxide
Mass spectrometry was performed with a Micromass
LCT (electrospray ionization, ESI) in the Mass Spec-
trometry Facility in the Department of Chemistry.
4.2. Synthesis
4.2.1. m-Br-C6H4CH2CH2OMs (5). Triethylamine
(5.2 mL, 37.3 mmol) was added to a solution of 4-
bromophenethyl alcohol (5.0 g, 24.9 mmol) in CH2Cl2
(200 mL), and the resulting solution was cooled to
0 °C with an ice bath. Methanesulfonyl chloride
(2.7 mL, 34.8 mmol) was added dropwise to the reaction
mixture, and the resulting solution was allowed to warm
slowly to room temperature overnight. The solution was
washed with 0.1 N HCl and brine, and the combined or-
ganic extracts were dried over anhydrous MgSO4(s) and
filtered, and the solvent was removed under reduced
pressure. The crude yellow solid was purified by flash
chromatography (silica gel, 70% v/v hexanes in CH2Cl2)
1
10 as a colorless oil in 66% yield. H NMR (CDCl3,
400 MHz) d 8.06 (d, J = 1.2 Hz, 1H), 7.69 (s, 1H), 7.66
(s, 1H), 7.58 (t, J = 7.5 Hz, 2H), 7.54 (dd, J = 8.9,
1.2 Hz 2H), 7.45 (dd, J = 7.6, 3.1 Hz 2H), 3.45 (s, 4H),
2.22 (s, 12H) ppm; 13C NMR (CDCl3, 100.6 MHz) d
140.22 (d, J = 10.7 Hz), 133.45, 132.15, 131.31 (d,
J = 12.2 Hz), 129.67 (d, J = 11.2 Hz), 129.14 (d,
J = 12.2 Hz), 64.06, 45.58 ppm; 31P NMR (CDCl3,
161 MHz) d 22.02 ppm; MS (ESI) m/z 288.0393 (MH+
[C18H25N2OPH+] = 288.0383).
1
to give mesylate 5 as a white solid in 98% yield. H
NMR (CDCl3, 400 MHz) d 7.42–7.40 (m, 2H), 7.21–
7.16 (m, 2H), 4.41 (t, J = 6.9 Hz, 2H), 3.03 (t,
J = 6.7 Hz, 2H), 2.90 (s, 3H) ppm; observed 13C NMR
(CDCl3, 100.6 MHz) d 138.87, 132.25, 130.51, 127.87,
123.92, 69.79, 37.67, 35.46 ppm; MS (ESI) m/z
300.9519 (MNa+ [C9H11BrO3SNa+] = 300.9595).
4.2.6. HP(O)(C6H4-m-CH2CH2NMe2)2 (11). Dimethyl-
amine 6 (1.70 g, 7.47 mmol) was dissolved in anhydrous
THF (18 mL) under Ar(g) in a flame-dried round-bot-
tomed flask equipped with a reflux condenser. To facil-
itate generation of the Grignard reagent, a catalytic
amount of I2(s) was added to the solution. Crushed
magnesium turnings (272 mg, 11.2 mmol) were then
added, and the resulting solution was heated at reflux
for 2 h to generate the Grignard reagent. In a separate
4.2.2. m-Br-C6H4CH2CH2NMe2 (6). Mesylate 5 (6.68 g,
23.9 mmol) was dissolved in anhydrous THF (60 mL)
under Ar(g), and dimethylamine (2 M in THF, 47 mL,
94 mmol) was added to this solution. The resulting solu-
tion was heated at 45 °C for 30 h. The white solid that
formed was removed by filtration, and the solvent was
removed under reduced pressure. The residue was puri-
fied by flash chromatography (silica gel, 10% v/v MeOH
in CH2Cl2) to give dimethylamine 6 as a yellow oil in
63% yield. 1H NMR (CDCl3, 400 MHz) d 7.30–7.26
(m, 2H), 7.21–7.17 (m, 2H), 2.80–2.75 (m, 2H), 2.55–
2.51 (m, 2H), 2.30 (s, 6H) ppm; observed 13C NMR
(CDCl3, 100.6 MHz) d 140.64, 128.87, 128.87, 128.61,
126.28, 61.86, 45.73, 34.65 ppm; MS (ESI) m/z
228.0387 (MNa+ [C10H14BrNNa+] = 228.0388).
flamed-dried
flask,
diethylphosphite
(289 lL,
2.24 mmol) was dissolved in anhydrous THF (1.5 mL),
and cooled to 0 °C with an ice bath. The solution of
Grignard reagent was added dropwise to this solution,
and the resulting solution was allowed to warm to room
temperature and stirred overnight. The reaction mixture
was then quenched with water (1 mL), and the solvent
was removed under reduced pressure. The residue was
dissolved in CH2Cl2, and the resulting solution was
washed with water and brine. The combined organic ex-
tracts were dried over anhydrous MgSO4(s) and filtered,
and the solvent was removed under reduced pressure.
The residue was purified by flash chromatography (silica
gel, 20% v/v MeOH in CH2Cl2) to give phosphine oxide
4.2.3. 1-Br-3,5-bis(CH2Br)C6H3 (7). Bromide 7 was syn-
thesized according to reports published previously.12
Spectral data were as reported previously.12
1
11 as a colorless oil in 69% yield. H NMR (CDCl3,
400 MHz) d 8.03 (d, J = 1.2 ppm, 1H), 7.62 (s, 1H),
7.59 (s, 1H), 7.44–7.41 (m, 6H), 2.84–2.80 (m, 4H),
2.55–2.51 (m, 4H), 2.28 (s, 12H) ppm; 13C NMR
(CDCl3, 100.6 MHz) d 141.62 (d, J = 14.0 Hz), 133.21,
132.09, 131.10 (d, J = 11.4 Hz), 129.13 (J = 15.4 Hz),
128.53 (d, J = 14.0 Hz), 61.26, 45.62, 34.33 ppm; 31P
NMR (CDCl3, 161 MHz) d 22.14 ppm; MS (ESI) m/z
345.2079 (MNa+ [C20H29N2OPNa+] = 345.2096).
4.2.4. 1-Br-3,5-bis(CH2NMe2)C6H3 (8). Bromide 8 was
synthesized according to reports published previously.12
Spectral data were as reported previously.12
4.2.5. HP(O)(C6H4-m-CH2NMe2)2 (10). Dimethylamine
9 (1.31 g, 6.13 mmol) was dissolved in anhydrous THF
(15 mL) under Ar(g) in a flame-dried round-bottomed
flask equipped with a reflux condenser. To facilitate gen-
eration of the Grignard reagent, a catalytic amount of I2
was added to the solution. Crushed magnesium turnings
(223 mg, 9.2 mmol) were then added to this solution,
and the resulting solution was heated at reflux for 2 h
4.2.7. HP(O)(C6H3-bis-m,m-CH2NMe2)2 (12). Bromide
8 (2.88 g, 10.6 mmol) was dissolved in anhydrous THF
(30 mL) under Ar(g) in a flame-dried round-bottomed
flask equipped with a reflux condenser. To facilitate

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A. Tam, R. T. Raines / Bioorg. Med. Chem. 17 (2009) 1055–1063
generation of the Grignard reagent, two drops of 1,
2-dibromoethane was added to the solution. Crushed
magnesium turnings (387 mg, 16.0 mmol) were then
added to this solution, and the resulting solution was
heated at reflux for 2 h to generate the Grignard reagent.
In a separate flamed-dried flask, diethyl phosphite
(256 lL, 3.19 mmol) was dissolved in anhydrous THF
(15 mL), and cooled to 0 °C with an ice bath. The
solution of Grignard reagent was added dropwise to this
solution, and the resulting solution was allowed to warm
to room temperature and stirred overnight. The reaction
mixture was then quenched with water (1 mL), and the
solvent was removed under reduced pressure. The
residue was dissolved in CH2Cl2, and the resulting
solution was washed with water and brine. The com-
bined organic extracts were dried over anhydrous
MgSO4(s) and filtered, and the solvent was removed
under reduced pressure. The residue was purified by
flash chromatography (silica gel, 20% v/v MeOH in
CH2Cl2 with 1% v/v NEt3) to give phosphine oxide 12
as a yellow oil in 50% yield. 1H NMR (CDCl3,
400 MHz) d 8.09 (d, J = 1.2 ppm, 1H), 7.58 (s, 2H),
7.54 (s, 2H), 7.51 (s, 2H), 3.44 (s, 8H), 2.21 (s, 24H)
ppm; 13C NMR (CDCl3, 100.6 MHz) d 140.00 (d,
J = 13.4 Hz), 133.91, 131.36, 129.98 (d, J = 11.6 Hz),
63.75, 45.33 ppm; 31P NMR (CDCl3, 161 MHz) d
4.2.9. BH3ÆPH(C6H4-m-CH2CH2NMe2)2 (14). A solu-
tion of phosphine oxide 11 (288 mg, 0.84 mmol) in anhy-
drous CH2Cl2 (1.8 mL) was added dropwise slowly to a
solution of DIBAl-H (1 M in CH2Cl2, 4.18 mL,
4.18 mmol) under Ar(g) in a flame-dried three-neck
round-bottomed flask. The resulting solution was stirred
for 20 min, then cooled to 0 °C with an ice bath. The
solution was then diluted with CH2Cl2 (10 mL), and a
sparge needle of Ar(g) was allowed to blow through
the solution for 5 min. A solution of 2 N NaOH
(5 mL) was added dropwise slowly to the reaction mix-
ture (Caution! Gas evolution!) followed by a saturated
solution of Rochelle’s salt (5 mL) to dissipate the emul-
sion that forms. The resulting biphasic solution was
transferred to a separatory funnel, and the organic layer
was separated, dried over anhydrous MgSO4(s), filtered,
and concentrated under reduced pressure to ꢀ8 mL. The
resulting solution was cooled to 0 °C with an ice bath
under Ar(g), and boraneÆdimethyl sulfide complex
(10 M, 268 lL, 2.68 mmol) was added dropwise. The
resulting reaction mixture was allowed to warm slowly
to room temperature overnight. The solvent was re-
moved under reduced pressure, and the crude oil was
purified by flash chromatography (silica gel, CH2Cl2)
to give phosphine–borane complex 14 as a white solid
1
in 69% yield. H NMR (CDCl3, 400 MHz) d 7.54–7.50
22.48 ppm;
MS
(ESI)
m/z
431.2946
(MH+
(m, 4 H), 7.44–7.37 (m, 4H), 6.30 (dq, J = 380.2 Hz,
1H), 3.14–3.09 (m, 4H), 2.97–2.93 (m, 4H), 2.66 (s,
12H), 2.15–0.70 (m, 9H) ppm; 13C NMR (CDCl3,
[C24H39N4OPH+] = 431.2935).
4.2.8. BH3ÆPH(C6H4-m-CH2NMe2)2 (13). A solution of
phosphine oxide 10 (383 mg, 1.21 mmol) in anhydrous
CH2Cl2 (2.6 mL) was added dropwise slowly to a solu-
tion of DIBAl-H (1 M in CH2Cl2, 6.05 mL, 6.05 mmol)
under Ar(g) in a flame-dried three-neck round-bottomed
flask. The resulting solution was stirred for 20 min, then
cooled to 0 °C with an ice bath. The solution was then
diluted with CH2Cl2 (10 mL), and a sparge needle of
Ar(g) was allowed to blow through the solution for
5 min. A solution of 2N NaOH (5 mL) was added drop-
wise slowly to the reaction mixture (Caution! Gas evolu-
tion!) followed by a saturated solution of Rochelle’s salt
(5 mL) to dissipate the emulsion that forms. The result-
ing biphasic solution was transferred to a separatory
funnel, and the organic layer was separated, dried over
anhydrous MgSO4(s), filtered, and concentrated under
reduced pressure to ꢀ12 mL. The resulting solution
was cooled to 0 °C with an ice bath under Ar(g), and
boraneÆdimethyl sulfide complex (10 M, 387 lL,
3.87 mmol) was added dropwise. The resulting reaction
mixture was allowed to warm slowly to room tempera-
ture overnight. The solvent was removed under reduced
pressure, and the crude oil was purified by flash chroma-
tography (silica gel, CH2Cl2) to give phosphine–borane
100.6 MHz) d 139.50 (d, J = 9.8 Hz), 133.20 (d,
J = 10.4 Hz), 132.48, 131.59 (d, J = 8.8 Hz), 129.77 (d,
J = 56.7 Hz), 126.57 (d, J = 58.7 Hz), 65.89, 52.12 (d,
J = 9.4 Hz), 30.92 ppm; 31P NMR (CDCl3, 161 MHz)
d
1.64 ppm;
MS
(ESI)
m/z
388.3372
ðMNHþ4 ½C20H36B3N2PNHþ4 ꢁ ¼ 388:3391Þ.
4.2.10. BH3ÆPH(C6H3-bis-m,m-CH2NMe2)2 (15). A solu-
tion of phosphine oxide 12 (200 mg, 0.46 mmol) in anhy-
drous CH2Cl2 (1.5 mL) was added dropwise slowly to a
solution of DIBAl-H (1 M in CH2Cl2, 2.32 mL,
2.32 mmol) under Ar(g) in a flame-dried three-neck
round-bottomed flask. The resulting solution was stirred
for 20 min, then cooled to 0 °C with an ice bath. The
solution was then diluted with v (10 mL), and a sparge
needle of Ar(g) was allowed to blow through the solu-
tion for 5 min. A solution of 2 N NaOH (3 mL) was
added dropwise slowly to the reaction mixture (Caution!
Gas evolution!) followed by a saturated solution of Ro-
chelle’s salt (5 mL) to dissipate the emulsion that forms.
The resulting biphasic solution was transferred to a
separatory funnel, and the organic layer was separated,
dried over anhydrous MgSO4(s), filtered, and concen-
trated under reduced pressure to ꢀ5 mL. The resulting
solution was cooled to 0 °C with an ice bath under
Ar(g), and boraneÆdimethyl sulfide complex (10 M,
241 lL, 2.40 mmol) was added dropwise. The resulting
reaction mixture was allowed to warm slowly to room
temperature overnight. The solvent was removed under
reduced pressure, and the crude oil was purified by flash
chromatography (silica gel, 5% v/v EtOAc and 25% v/v
hexanes in CH2Cl2) to give phosphine–borane complex
15 as a white solid in 50% yield. 1H NMR (CDCl3,
400 MHz) d 7.85 (s, 2H), 7.82 (s, 2H), 7.70 (s, 2H),
1
complex 13 as a white solid in 87% yield. H NMR
(CDCl3, 400 MHz) d 7.73–7.70 (m, 4H), 7.55–7.49 (m,
4H), 6.38 (dq, J = 381.3 Hz, 7.1 Hz, 1H), 3.95 (s, 4H),
2.53 (s, 6H), 2.52 (s, 6H), 2.15–0.80 (m, 9H) ppm; 13C
NMR (CDCl3, 100.6 MHz) d 137.01 (d, J = 8.4 Hz),
135.91, 133.80 (d, J = 8.5 Hz), 132.85 (d, J = 10.2 Hz),
129.41 (d, J = 56.7 Hz), 126.67 (d, J = 55.9 Hz), 67.39,
50.76 (d, J = 21.6 Hz) ppm; 31P NMR (CDCl3,
161 MHz)
ðMNHþ4 ½C18H34B3N2PNHþ4 ꢁ ¼ 360:3078Þ.
d 0.98 ppm; MS (ESI) m/z 360.3095

A. Tam, R. T. Raines / Bioorg. Med. Chem. 17 (2009) 1055–1063
1061
6.47 (dq, J = 0.963 ppm, 7.1 Hz, 1H), 3.98–3.91 (m, 8H),
2.56 (s, 12H), 2.53 (s, 12H), 1.80–0.80 (m, 15H) ppm;
13C NMR (CDCl3, 100.6 MHz) d 140.27, 137.53 (d,
J = 9.8 Hz), 133.05 (d, J = 10.0 Hz), 125.69 (d,
J = 56 Hz), 67.12, 51.38 (d, J = 50.9 Hz) ppm; 31P
NMR (CDCl3, 161 MHz) d ꢂ1.12 ppm; MS (ESI) m/z
507.4447 (MNa+ [C24H54B5N4PNa+] = 507.4444).
under reduced pressure, and the residue was purified
by flash chromatography (silica gel, 10% v/v ethyl ace-
tate and 20% v/v hexanes in CH2Cl2) to give phos-
phine–borane complex 19 as a white solid in 56%
yield. 1H NMR (CDCl3, 400 MHz)
d 7.91 (d,
J = 1.1 Hz, 2H), 7.88 (d, J = 1.1 Hz, 2H), 7.74 (s, 2H),
3.93 (q, J = 13.7 Hz, 8H), 3.73 (d, J = 6.3 Hz, 2H),
2.58 (s, 12H), 2.57 (s, 12H), 2.23 (s, 3H), 2.00–1.50 (m,
15H) ppm; 13C NMR (CDCl3, 100.6 MHz) d 192.70,
140.46, 137.24 (d, J = 9.5 Hz), 132.84 (d, J = 10.2 Hz),
126.98 (d, J = 54.9 Hz), 67.94, 52.04, 51.09, 30.28
24.21 (d, J = 34.9 Hz) ppm; 31P NMR (CDCl3,
4.2.11. BH3ÆAcSCH2P(C6H4-m-CH2NMe2)2 (17). Phos-
phine–borane complex 13 (81 mg, 0.24 mmol) was dis-
solved in dry DMF (2.5 mL) under Ar(g) and cooled
to 0 °C. NaH (5.7 mg, 0.24 mmol) was added slowly,
and the mixture was stirred at 0 °C until bubbling
ceased. Bromide 16 (40 mg, 0.24 mmol) was then added,
and the resulting 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, 2% v/v ethyl acetate
and 28% hexanes in CH2Cl2) to give phosphine–borane
161 MHz) d 18.74 ppm; MS (ESI) m/z 590.4409
(MNa+ [C27H58B5N4OPSNa+] = 590.4427).
4.2.14. AcSCH2P(C6H4-m-CH2NMe2)2 (20). Phos-
phine–borane complex 17 (100 mg, 0.23 mmol) was dis-
solved in toluene (2 mL) under Ar(g). DABCO (84 mg,
0.74 mmol) was added, and the resulting solution was
heated to 40 °C for 4 h. The solvent was removed under
reduced pressure, and the residue was purified by flash
chromatography (silica gel, 20% v/v MeOH in CH2Cl2)
to give phosphinothioester 20 as a colorless oil in 95%
yield. 1H NMR (CDCl3, 400 MHz) d 7.38–7.29 (m,
8H), 3.53 (d, J = 3.7 Hz, 2 H), 3.41 (s, 4H), 2.28 (s,
3H), 2.22 (s, 12H) ppm; 13C NMR (CDCl3,
100.6 MHz) d 194.74, 138.88 (d, J = 4.7 Hz), 136.91 (d,
J = 13.4 Hz), 133.83 (t, J = 19.2 Hz), 131.56, 130.17 (d,
J = 29.9 Hz), 128.63 (d, J = 32.2 Hz), 64.15, 45.63,
30.76, 20.96 (d, J = 23.5 Hz) ppm; 31P NMR (CDCl3,
161 MHz) ꢂ15.23 ppm.
1
complex 17 as a white solid in 78% yield. H NMR
(CDCl3, 400 MHz) d 7.78–7.73 (m, 4H), 7.57–7.49 (m,
4H), 3.95 (d, J = 2.4 Hz, 4H), 3.73 (d, J = 6.3 Hz, 2H),
2.53 (d, J = 6.3 Hz, 12H), 2.25 (s, 3H), 2.20–0.60 (m,
9H) ppm; 13C NMR (CDCl3, 100.6 MHz) d 192.88,
136.42 (d, J = 9.5 Hz), 135.93, 133.41 (d, J = 9.4 Hz),
132.53 (d, J = 9.9 Hz), 129.03 (d, J = 10.0 Hz), 127.73
(d, J = 54.6 Hz), 67.45, 53.65, 30.18, 23.70 (d,
J = 35.7 Hz) ppm; 31P NMR (CDCl3, 161 MHz) d
19.97 ppm; MS (ESI) m/z 453.2595 (MNa+
[C21H38B3N2OPSNa+] = 453.2614).
4.2.12. BH3ÆAcSCH2P(C6H4-m-CH2CH2NMe2)2 (18).
Phosphine–borane complex 14 (100 mg, 0.27 mmol)
was dissolved in dry DMF (3 mL) under Ar(g) and
cooled to 0 °C. NaH (6.5 mg, 0.27 mmol) was added
slowly, and the mixture was stirred at 0 °C until
bubbling ceased. Bromide 16 (46 mg, 0.27 mmol) was
then added, and the resulting 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, 2%
v/v ethyl acetate and 28% hexanes in CH2Cl2) to give
phosphine–borane complex 18 as a white solid in 75%
4.2.15. AcSCH2P(C6H4-m-CH2CH2NMe2)2 (21). Phos-
phine–borane complex 18 (38 mg, 0.096 mmol) was dis-
solved in toluene (1 mL) under Ar(g). DABCO (35 mg,
0.30 mmol) was added, and the resulting solution was
heated to 40 °C for 4 h. The solvent was removed under
reduced pressure, and the residue was purified by flash
chromatography (silica gel, 20% v/v MeOH in CH2Cl2)
to give phosphinothioester 21 as a colorless oil in 95%
yield. 1H NMR (CDCl3, 400 MHz) d 7.28–7.20 (m,
8 H), 3.50 (d, J = 3.4 Hz, 2H), 2.80–2.76 (m, 4H),
2.57–2.53 (m, 4H), 2.32 (s, 12H), 2.30 (s, 3H) ppm;
13C NMR (CDCl3, 100.6 MHz) d 194.87, 140.54 (d,
J = 7.7 Hz), 136.96 (d, J = 14.2 Hz), 133.25 (d,
J = 22.8 Hz), 130.48 (d, J = 16 Hz), 129.70, 128.78 (d,
J = 4.9 Hz), 64.26, 46.94, 45.47, 34.25, 25.99 (d,
J = 23.4 Hz) ppm; 31P NMR (CDCl3, 161 MHz) d
ꢂ14.99 ppm.
yield. 1H NMR (CDCl3, 400 MHz)
d 7.56–7.52
(m, 4H), 7.45–7.38 (m, 4H), 3.71 (d, J = 6.8 Hz, 2H),
3.13–3.09 (m, 4H), 2.97–2.92 (m, 4H), 2.67 (d,
J = 2.7 Hz, 12H), 2.28 (s, 3H), 2.40–0.60 (m, 9H) ppm;
13C NMR (CDCl3, 100.6 MHz) d 193.34, 139.26 (d,
J = 9.9 Hz), 132.77 (d, J = 11.7 Hz), 132.58, 130.96 (d,
J = 8.3 Hz), 129.54 (d, J = 10.0 Hz), 128.23 (d,
J = 55.9 Hz), 65.83, 52.05, 30.95, 30.32, 23.81 (d,
J = 35.3 Hz) ppm; 31P NMR (CDCl3, 161 MHz) d
19.65 ppm; MS (ESI) m/z 481.2918 (MNa+
[C23H42B3N2OPSNa+] = 481.2927).
4.2.16.
AcSCH2P(C6H3-bis-m,m-CH2NMe2)2
(22).
Phosphine–borane complex 19 (59 mg, 0.10 mmol) was
dissolved in toluene (1.5 mL) under Ar(g). DABCO
(60 mg, 0.54 mmol) was added, and the resulting solu-
tion was heated to 40 °C for 4 h. The solvent was re-
moved under reduced pressure, and the residue was
purified by flash chromatography (silica gel, 20% v/v
MeOH in CH2Cl2 with 1% v/v NEt3) to give phosphino-
4.2.13. BH3ÆAcSCH2P(C6H3-bis-m,m-CH2NMe2)2 (19).
Phosphine–borane complex 15 (86 mg, 0.18 mmol) was
dissolved in dry DMF (2 mL) under Ar(g) and cooled
to 0 °C. NaH (4.3 mg, 0.18 mmol) was added slowly,
and the mixture was stirred at 0 °C until bubbling
ceased. Bromide 16 (30 mg, 0.18 mmol) was then added,
and the resulting mixture was allowed to warm to room
temperature and stirred for 12 h. Solvent was removed
1
thioester 22 as a colorless oil in 91% yield. H NMR
(CDCl3, 400 MHz) d 7.28–7.21 (m, 6H), 3.53 (d,
J = 3.7 Hz, 2H), 3.39 (m, 8H), 2.27 (s, 3H), 2.21 (s,
24H) ppm; 13C NMR (CDCl3, 100.6 MHz) d 194.82,
139.07 (d, J = 7.4 Hz), 136.88 (d, J = 14.6 Hz), 132.48

1062
A. Tam, R. T. Raines / Bioorg. Med. Chem. 17 (2009) 1055–1063
(d, J = 18.8 Hz), 131.01, 64.12, 45.39, 30.43, 26.05 ppm;
31P NMR (CDCl3, 161 MHz) d ꢂ15.32 ppm.
J = 24.8 Hz),
161 MHz) d ꢂ14.48 ppm.
23.25 ppm;
31P
NMR
(CDCl3,
4.2.17. HSCH2P(C6H4-m-CH2NMe2)2Æ2 HCl (3). Phos-
phinothioester 20 (35 mg, 0.09 mmol) was dissolved in
degassed MeOH (1 mL), and NaOH (3.7 mg) was added
to this solution under Ar(g). The resulting solution was
stirred at room temperature for 1.5 h. The solvent
was removed under reduced pressure, and the residue
was acidified with 4 N HCl in dioxane and filtered to
give hydrochloride salt of phosphinothiol 3 as a white
4.2.20. AcGlySCH2P(C6H4-m-CH2NMe2)2 (25). Phos-
phinothioester 20 (72 mg, 0.21 mmol) was dissolved in
degassed MeOH (2 mL), and NaOH (8.3 mg) was
added to this solution under Ar(g). The resulting solu-
tion was stirred at room temperature for 1.5 h. The sol-
vent was removed under reduced pressure, and the
residue was acidified with 4 N HCl in dioxane and fil-
tered to give the hydrochloride salt of phosphinothiol 3
as a white solid in quantitative yield. The residue was
used without further purification. N-Acetyl glycine pen-
tafluorophenol ester (62 mg, 0.22 mmol) was dissolved
in anhydrous DMF (2 mL) under Ar(g). A solution
of the above phosphinothiol in DMF (1 mL) was
added to the mixture. DIEA (144 lL, 0.83 mmol)
was added to the resulting solution, and the mixture
was stirred for 4 h at room temperature under Ar(g).
The solvent was removed under reduced pressure,
and the resulting crude oil was purified by flash chro-
matography (silica gel, 20% v/v MeOH in CH2Cl2) to
give phinothioester 25 as a colorless oil in 95% yield.
1H NMR (CDCl3, 400 Hz) d 7.40–7.28 (m, 8H), 6.68
(bs, 1H), 4.11 (d, J = 6.0 Hz, 2H), 3.53 (d, J = 4.4 Hz,
2H), 3.48 (s, 4H), 2.27 (s, 12H), 2.04 (s, 3H) ppm;
13C NMR (CDCl3, 100.6 MHz) d 196.63, 170.75,
1
solid in quantitative yield. H NMR (CD3OD, 400 Hz)
d 7.81 (d, J = 5.9 Hz, 2H), 7.61–7.56 (m, 4H), 7.49 (d,
J = 7.4 Hz, 2H), 4.37 (s, 4H), 3.27 (d, J = 2.6 Hz, 2H),
2.84 (s, 12H) ppm; 13C NMR (CD3OD, 100.6 MHz) d
140.34 (d, J = 17.4 Hz), 136.25 (d, J = 17.2 Hz), 135.45
(d, J = 23.1 Hz), 133.02, 132.18, 130.65 (d, J = 7.0 Hz),
61.99, 54.95, 43.16 ppm; 31P NMR (CDCl3, 161 MHz)
d ꢂ8.59 ppm.
4.2.18. HSCH2P(C6H3-bis-m,m-CH2NMe2)2Æ4 HCl (4).
Phosphinothioester 22 (47 mg, 0.09 mmol) was dissolved
in degassed MeOH (1 mL), and NaOH (3.7 mg) was
added to this solution under Ar(g). The resulting solu-
tion was stirred at room temperature for 1.5 h. The sol-
vent was removed under reduced pressure, and the
residue was acidified with 4 N HCl in dioxane and fil-
tered to give hydrochloride salt of phosphinothiol 4 as
137.01
(d,
J = 14.2 Hz),
136.38,
134.18
(d,
1
a white solid in quantitative yield. H NMR (CDCl3,
J = 18.8 Hz), 132.65 (d, J = 19.8 Hz), 130.93, 128.96
(d, J = 6.2 Hz), 63.37, 49.15, 44.65, 25.32, 23.11 ppm;
31P NMR (CDCl3, 161 MHz) d ꢂ14.33 ppm.
400 Hz) d 7.26–7.24 (m, 6H), 4.05 (d, J = 5.2 Hz, 2H),
3.38, (s, 8H), 3.07 (m, 2H), 2.20 (s, 24H) ppm; 13C
NMR (CDCl3, 100.6 MHz) d 139.20 (d, J = 6.3 Hz),
137.12 (d, J = 14.4 Hz), 132.48 (d, J = 17.0 Hz),
130.99, 64.20, 45.48, 21.14 (d, J = 23.3 Hz) ppm; 31P
NMR (CDCl3, 161 MHz) d ꢂ7.71 ppm.
4.2.21. AcGlySCH2P(C6H3-bis-m,m-CH2NMe2)2 (26).
N-Acetyl glycine pentafluorophenol ester (27.7 mg,
0.1 mmol) was dissolved in anhydrous DMF (1 mL) un-
der Ar(g). The hydrochloride salt of phosphinothiol 4
(41.4 mg, 0.09 mmol) was added, followed by DIEA
(129 lL, 0.74 mmol). The resulting solution was stirred
for 4 h at room temperature under Ar(g). The solvent
was removed under reduced pressure, and the resulting
crude oil was purified by flash chromatography (silica
gel, 20% v/v MeOH in CH2Cl2 with 1% v/v NEt3) to give
phosphinothioester 26 as a mixture with unreacted 4.
This mixture was dissolved in CH2Cl2 (2 mL) and 2-
chlorotrityl chloride resin (200 mesh) (3 equiv) and
TBD-methyl polystyrene resin (5 equiv) was added and
gently stirred at RT for 30 min. The resin was filtered
and washed with CH2Cl2. The filtrate was concentrated
under reduced pressure to give 26 as a colorless oil in
4.2.19. AcGlySCH2P(C6H4-m-CH2CH2NMe2)2 (24).
Phosphinothioester 21 (40 mg, 0.10 mmol) was dis-
solved in degassed MeOH (1 mL), and NaOH
(3.8 mg) was added to this solution under Ar(g). The
resulting solution was stirred at room temperature for
1.5 h. The solvent was removed under reduced pres-
sure, and the residue was acidified with 4 N HCl in
dioxane and filtered to give hydrochloride salt of phos-
phinothiol 2 as a white solid in quantitative yield. The
residue was used without further purification. N-Acetyl
glycine pentafluorophenol ester (28.3 mg, 0.1 mmol)
was dissolved in anhydrous DMF (1 mL) under
Ar(g). The hydrochloride salt of phosphinothiol 2 from
above was added, followed by DIEA (67 lL,
0.38 mmol). The resulting solution was stirred for 4 h
at room temperature under Ar(g). The solvent was re-
moved under reduced pressure, and the resulting crude
oil was purified by flash chromatography (silica gel,
20% v/v MeOH in CH2Cl2) to give phosphinothioester
1
72% yield. H NMR (CDCl3, 400 Hz) d 7.27–7.22 (m,
6H), 6.50 (bs, 1H), 4.09 (d, J = 5.7 Hz, 2H), 3.54 (d,
J = 4.6 Hz, 2H), 3.37 (s, 8H), 2.20 (s, 24H), 2.02 (s,
3H) ppm; observed 13C NMR (CDCl3, 100.6 MHz) d
196.41, 170.35, 139.19 (d, J = 7.5 Hz), 136.51 (d,
J = 13.6 Hz), 132.51 (d, J = 18.2 Hz), 64.22, 53.61,
49.20, 45.51, 25.29 (d, J = 26.5 Hz) ppm; 31P NMR
(CDCl3, 161 MHz) d ꢂ13.68 ppm.
1
24 as a colorless oil in 75% yield. H NMR (CDCl3,
400 Hz) d 7.30–7.22 (m, 8 H), 4.19 (d, J = 6.2 Hz,
2H), 3.54 (d, J = 3.9 Hz, 2H), 2.80–2.76 (m, 4H),
2.55–2.51 (m, 4H), 2.30 (s, 12H), 2.05 (s, 3H) ppm;
13C NMR (CDCl3, 100.6 MHz) d 196.48, 170.50,
140.66 (d, J = 6.9 Hz), 136.78 (d, J = 13.4 Hz), 133.37
(d, J = 20.0 Hz), 130.66 (d, J = 18.0 Hz), 129.92,
128.93, 61.38, 49.35, 45.62, 34.43, 25.60 (d,
4.3. Staudinger ligations of 23 + 27, 24 + 27, 25 + 27, and
26 + 27
The respective phosphinothioester (36 lmol) and azide
27 (3.6 mg, 36 lmol) were dissolved in 0.40 M sodium

A. Tam, R. T. Raines / Bioorg. Med. Chem. 17 (2009) 1055–1063
1063
2. Muir, T. W. Annu. Rev. Biochem. 2003, 72, 249–289.
3. Nilsson, B. L.; Soellner, M. B.; Raines, R. T. Annu. Rev.
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phosphate buffers (0.6 mL) of various pH. The resulting
reaction mixtures were stirred for 16 h and the reaction
was monitored with quantitative 13C NMR spectros-
copy. Amide yields were obtained by the integration of
all normalized product 13C NMR signals.
4.4. pKa Determination of phosphinothiols
5. Soellner, M. B.; Tam, A.; Raines, R. T. J. Org. Chem.
2006, 71, 9824–9830.
Phosphinothioesters 20–22 (10 mg) were each dissolved
in sodium phosphate buffer containing 20% v/v D2O.
The solution was adjusted with a solution of either
6 N HCl or 2 N NaOH, and chemical shifts at each
pH were determined by 31P NMR spectroscopy. An
external standard (D3PO4) was used to calibrate the
chemical shifts. A control experiment using tris(2-car-
boxyethyl)phosphine (TCEP) gave a pKa value of 7.7,
which is similar to the reported value of 7.66.15
6. Soellner, M. B.; Nilsson, B. L.; Raines, R. T. J. Am. Chem.
Soc. 2006, 128, 8820–8828; Agard, N. J.; Baskin, J. M.;
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8. Nilsson, B. L.; Hondal, R. J.; Soellner, M. B.; Raines, R.
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Acknowledgments
This paper is dedicated to Professor Jonathan A. Ellman
on the occasion of his winning the 2006 Tetrahedron
Young Investigator Award. We acknowledge E. L.
Myers for comments on this manuscript. This work
was supported by Grant GM044783 (NIH).
10. Tam, A.; Soellner, M. B.; Raines, R. T. J. Am. Chem. Soc.
2007, 129, 11421–11430.
11. Brown, H. C.; McDaniel, D. H.; Ha¨fliger, O. In Determi-
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Supplementary data
NMR spectra of the novel compounds reported herein.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2008.02.047.
13. Farrington, G. K.; Kumar, A.; Wedler, F. C. Org. Prep.
Proced. Int. 1989, 21, 390–392.
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