Extending synthetic access to proteins with a removable acyl transfer auxiliary

Article abstract of DOI:10.1021/ja016731w

A chemo- and regioselective auxiliary-mediated peptide ligation has been developed that is effective under nonidealized conditions for the synthesis of proteins. This general amide bond ligation utilizes a removable auxiliary that is analogous to the role

Full text of DOI:10.1021/ja016731w

Published on Web 04/05/2002
Extending Synthetic Access to Proteins with a Removable
Acyl Transfer Auxiliary
John Offer, C. N. C. Boddy, and Philip E. Dawson*
Contribution from the Departments of Cell Biology and Chemistry, The Skaggs Institute for
Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road,
La Jolla, California 92037
Received July 30, 2001
Abstract: A chemo- and regioselective auxiliary-mediated peptide ligation has been developed that is
effective under nonidealized conditions for the synthesis of proteins. This general amide bond ligation utilizes
a removable auxiliary that is analogous to the role of cysteine in native chemical ligation, combining
chemoselective thioester exchange with efficient regioselective intramolecular acyl transfer. Acid lability
and improved ligation efficiency were introduced into the 2-mercaptobenzyl auxiliary by increasing the
electron density of the aromatic ring. The 62 amino acid SH3 domain from R-spectrin was synthesized
using the auxiliary-mediated ligation at a Lys-Gly sequence. The auxiliary was removed with TFA and
scavengers from the ligated product. This methodology enables unprotected peptides to be coupled at
noncysteine ligation sites expanding the scope of protein synthesis and semisynthesis.
Scheme 1. Mechanism of Peptide Ligations
Introduction
Native chemical ligation of a peptide thioester onto an NR-
terminal cysteine peptide is an established methodology for the
routine synthesis of many proteins using synthetic and expressed
peptides.1 The practical advantages of native chemical ligation
come from its ability to generate amide bonds in a single pot
reaction using unprotected peptides in neutral aqueous solution
at low concentration. Here we describe a method for the chemo-
and regioselective ligation of unprotected peptides at non-
cysteine ligation sites that maintains many of these useful
features (Scheme 1).
Intramolecular acyl transfer has been investigated by Kemp
and co-workers as a synthetic route to peptides2 and used as a
method to quantitatively acylate secondary amines during solid-
phase peptide synthesis (SPPS).3 A general amide bond ligation
strategy can be modeled after native chemical ligation by
utilizing an auxiliary that mimics cysteine, combining thioester
exchange with efficient intramolecular acyl transfer to the NR-
terminus of the peptide.
concentrations in denaturing aqueous buffer. These concentration
and solvent restrictions are a result of the unpredictable and
often poor solubility of large peptides. The final step must leave
a native amide bond at the ligation junction. Several NR-linked
auxiliaries based on cysteine have been investigated for the
synthesis of peptides.4 Our auxiliary, based on the 2-mercapto-
benzyl system,5 provisionally addressed these requirements for
a general synthesis of proteins.6 However, this preliminary study
was limited to a simple model system and did not demonstrate
the removal of the auxiliary. Herein we modify this original
system to improve ligation rates and introduce acid lability. This
The auxiliary must meet stringent specifications. There must
be a robust method for the introduction of the auxiliary onto
the NR-terminus of the peptide that is compatible with standard
SPPS. The ligation step must be rapid at ∼1 mM peptide
* Corresponding author. E-mail: dawson@scripps.edu.
(1) (a) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science
1994, 266, 776-779. (b) Dawson, P. E.; Kent, S. B. H. Annu. ReV. Biochem.
2000, 69, 923-960. (c) Tam, J. P.; Lu, Y.-A.; Liu, C. F.; Shao, J. Proc.
Natl. Acad. Sci. U.S.A. 1995, 92, 12485-12489. (d) Muir, T. W.; Sondhi,
D.; Cole, P. A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 6705-6710.
(2) (a) Kemp D. S.; Carey, R. I. J. Org. Chem. 1993, 58, 2216-2222. (b)
Kemp, D. S. Biopolymers 1981, 20, 1793-1804. (c) Coltart, D. M.
Tetrahedron 2000, 3449-3491.
(4) (a) Canne, L. E.; Bark, S. J.; Kent, S. B. H. J. Am. Chem. Soc. 1996, 118,
5891-5896. (b) Botti, P.; Carrasco, M. R.; Kent, S. B. H. Tetrahedron
Lett. 2001, 1831-1833. (c) Marinzi, C.; Bark, S. J.; Offer, J.; Dawson, P.
E. Bioorg. Med. Chem. 2001, 9, 2323-2328. (d) Low, D. W.; Hill, M. G.;
Carrasco, M. R.; Kent, S. B. H.; Botti, P. Proc. Natl. Acad. Sci. U.S.A.
2001, 98, 6554-6559.
(5) Offer, J.; Dawson, P. E. Org. Lett. 2000, 2, 23-26.
(6) This auxiliary does not introduce a new stereogenic center and its associated
difficulties into the peptide.4b-d
(3) (a) Sheppard, R. C. In Peptides 1994, Proceedings of the 23rd European
Peptide Symposium; Maia, H. S. L., Ed.; ESCOM Science Publishers B.
V.: Leiden, The Netherlands, 1995; pp 3-17. (b) Johnson, T.; Quibell,
M.; Sheppard, R. C. J. Pept. Sci. 1995, 1, 11-25. (c) Offer, J.; Johnson,
T.; Quibell, M. Tetrahedron Lett. 1997, 38, 9047-9050.
9
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J. AM. CHEM. SOC. 2002, 124, 4642-4646
10.1021/ja016731w CCC: $22.00 © 2002 American Chemical Society

Synthesis of Proteins with a Removable Acyl Transfer Auxiliary
A R T I C L E S
Table 1. Half-Lives for the Ligation of Unprotected Peptide Segments from the SH3 Domain of R-Spectrin and Protein G B1
Domain (PGB1)
N-terminal thioester piecea
C-terminal piecea
ligation junction
t1/2
obsd mass (calcd)/Dab
R-spectrin[1-28]
R-spectrin[1-27]
R-spectrin[1-27]
R-spectrin[1-28]
PGB1[1-23]
Dmb R-spectrin[28-62]
Dmb R-spectrin[28-62]
Tmb R-spectrin[28-62]
Dmb PGB1[24-56]
Gly/Gly
Lys/Gly
Lys/Gly
Gly/Ala
Ala/Ala
0.2 h
2.0 h
2.0 h
5.0 h
no rxn
7459.0 ( 1.0 (7458.6)
7400.0 ( 1.0 (7401.5)
7432.0 ( 1.0 (7431.6)
7139.0 ( 1.0 (7139.9)
Dmb PGB1[24-56]
a Concentrations are approximately 1 mM. b Characterized by ESI-MS.
Scheme 2. General Synthetic Strategya
Scheme 3. Synthesis of the
2,3,4-Trimethoxyl-6-(4-methylbenzylthio)benzylaminea
a This general approach should also be compatible with Fmoc SPPS.
The auxiliary is acid stable until the amine is converted to the amide by
ligation.
a Key: (a) NaNO2, HCl, 0 °C then KS2COEt, 65 °C (56%). (b) NaOH,
65 °C (90%). (c) 4-MeBnBr, NaH, PPh3, (97%). (d) POCl3, DMF, 150 °C
(97%). (e) NH2OH, LiAlH4 (45%).
new removable acyl transfer auxiliary is used to synthesize the
62 amino acid SH3 domain from R-spectrin.
Results and Discussion
commercially available material. The similarly protected Tmb
amine was synthesized in five steps from 3,4,5-trimethoxyaniline
as shown in Scheme 3.
Auxiliary Design, Synthesis, and Introduction. Acid lability
and improved ligation efficiency were introduced into the
original 2-mercaptobenzyl auxiliary5 by increasing the electron
density of the aromatic ring. Methoxy substitution at the 4- and
6-positions of the benzyl group increases its acid lability.
Methoxy substitution at the 5-position raises the nucleophilicity
of the thiol and increases the efficiency of thioester exchange,
the putative rate-limiting step.5,7,8 Of the possible substitution
patterns, we initially investigated the 4,5-dimethoxy-2-mercapto-
benzyl (Dmb) (subsequently reported for a ligation at a Gly-
Gly ligation site on a small peptide)9 and the 4,5,6-trimethoxy-
2-mercaptobenzyl (Tmb) auxiliaries. The Tmb auxiliary was
expected to combine the ligation properties of Dmb with greater
acid lability.
The submonomer approach was used to introduce the
auxiliaries onto the N-terminus of the C-terminal peptide
fragments. The displacement of an R-bromo-peptide resin with
a primary amine results in inversion of configuration at the
R-carbon4c,10 (Scheme 2). As expected no epimerization (<2%)
of the N-terminal CR carbon was observed in small model
peptides using this preparative method. Protection of the thiol
group was necessary to prevent S-alkylation. The S-4-methyl-
benzyl protected Dmb amine11 was synthesized directly from
Peptide Ligation. To demonstrate the effectiveness of this
methodology we used the auxiliary-mediated ligation to syn-
thesize the 62 amino acid SH3 domain from R-spectrin at a
Lys-Gly sequence (entry 3, Table 1). Since the rate of ligation
is putatively controlled by thioester exchange,5,7 the nature of
the C-terminal amino acid of the thioester peptide has a major
effect on the ligation rate. For example, thioester exchange with
glycine and histidine thioester peptides is relatively rapid while
the â-branched amino acids exchange slowly.4,7 These extreme
cases are not representative of the ligation properties of the
majority of amino acids such as leucine and lysine. As a result,
the Lys-Gly ligation junction was selected to best represent a
typical ligation site in a protein.7
To perform the ligation reaction, a single equivalent of SH3
R-spectrin[1-27] thiophenol thioester13,14 was added to the Tmb
N-terminally modified [28-62] peptide (Figure 1).15,16 The
major product peak possessed the expected mass of the target
material. Preparative HPLC of the ligation mixture yielded the
SH3 sequence with attached auxiliary in 66% yield. The
successful construction of the SH3 Lys-Gly bond demonstrates
the broad scope of our method.
(11) Available from Acros Organics. For synthesis see: (a) Vinkler, E.; Szabo´,
J. Acta Chim. Hung. 1955, 6, 323-333. (b) Szabo´, J.; Vinkler, E. Acta
Chim. Hung. 1962, 34, 447-454.
(12) HPLC conditions: Vydac C18 (100 × 4.6 mm) column, 25-50% B over
30 min (1 mL min-1). Buffer A ) 0.1% aqueous TFA. Buffer B ) 10%
water and 0.09% TFA in acetonitrile.
(7) Hackeng, T. M.; Griffin, J. H.; Dawson, P. E. Proc. Natl. Acad. Sci. U.S.A.
1999, 96, 10068-10073.
(8) Model studies suggest that the 5-methoxy substitution increases ligation
rates (data not shown).
(9) Kawakami, T.; Akaji, K.; Aimoto, S. Org. Lett. 2001, 3, 1403-1405.
(10) (a) Koppenhoefer, B.; Schurig, V. Organic Syntheses; Wiley: New York,
119-123; Collect. Vol. 8. (b) Briggs, M. T.; Morley, J. S. J. Chem. Soc.,
Perkin Trans. 1 1979, 2138-2143. (c) Baca, M.; Kent, S. B. H. Proc.
Natl. Acad. Sci. U.S.A. 1993, 90, 11638-11642.
(13) Muir, T. W.; Dawson, P. E.; Kent, S. B. H. Methods Enzymol. 1997, 289,
266-298. Available from Fmoc/t-Bu chemistry: Ingenito, R.; Bianchi, E.;
Fattori, D.; Pessi, A. J. Am. Chem. Soc. 1999, 121, 11369-11374.
(14) Ligation was observed for Gly-Gly (8 h) and Lys-Gly (48 h) with C-terminal
alkyl thioester in the absence of thiol additives.
(15) Ligation can be completely suppressed by the addition of thiols.
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A R T I C L E S
Offer et al.
Figure 2. (A) Typical analytical HPLC12 of crude R-spectrin SH3 after
removal of ligation auxiliary. Tmb: The lyophilized crude ligation mixture
was treated with TFA and 5% triisopropylsilane for 2 h at 25 °C. Dmb:
The ligation product was treated with 1% v/v p-cresol and 1% v/v
ethanedithiol in HF for 0.5 h at 0 °C. (B) Reconstructed mass of the SH3
domain.
Figure 1. (A) Analytical HPLC12 of the time-course for a ligation reaction
between SH3 R-spectrin[1-27] thiophenol thioester (a, 4.0 mg, 1.2 mM)
and SH3 R-spectrin Tmb[28-62] (b, 5.0 mg, 1.18 mM) with TCEP (5 mg,
17.4 mM) in degassed 200 mM NaH2PO4 and 6 M GdmCl (1 mL) at pH
7.0. The product was isolated in 66% yield (c, 5.8 mg). (B) Reconstructed
mass of product c.
The scope of this ligation reaction was further investigated
by attempting ligation reactions at three additional ligation sites,
Gly-Gly, Gly-Ala, and Ala-Ala. Because of facile thioester
exchange onto C-terminal glycine thioester peptides, the half-
life for the Gly-Gly coupling was very rapid (entry 1, Table 1).
Ligation onto non-glycine sites has been particularly challeng-
ing.4 The successful ligation of the sterically demanding
N-terminal Dmb-alanine with the synthetically convenient
C-terminal glycine thioester (entry 4, Table 1) was thus an
important result. However, no product was observed for the
more challenging alanine thioester to Dmb-alanine ligation
(entry 5, Table 1). Table 1 shows the range of the auxiliary-
mediated ligation indicating the broad applicability of this
methodology and its limitations.
The results in Table 1 are consistent with known data. The
order of magnitude rate increase between lysine (entry 2 and
3) and glycine thioesters (entry 1) is consistent with known
thioester exchange behavior7 and supports thioester exchange
as the rate-limiting step for these ligations. The dramatic rate
decrease between Dmb-glycine and Dmb-alanine ligations with
glycine thioester (entries 1 and 4) suggests a change in the rate-
determining step from thioester exchange to acyl migration. This
hypothesis is consistent with the known acyl migration behavior
of the related 2-hydroxybenzylamines where secondary amines
of glycine rearrange much faster than secondary amines of other
amino acids.2,3 On the basis of these preliminary observations
we propose that thioester exchange is the rate-limiting step for
ligation reactions where the auxiliary is attached to a glycine
residue. These results also suggest that the rate-limiting step
for the ligation reaction changes to acyl migration when non-
glycine residues are modified with the auxiliary.
Figure 3. Equilibrium urea denaturation of folded R-spectrin SH3 in 0.1
M phosphate at pH 7.0 (intrinsic fluorescence was monitored at 315 nm
with an excitation wavelength of 280 nm).
used for peptide cleavage and side chain deprotection (Scheme
2). The Dmb and Tmb auxiliaries were completely stable to
anhydrous HF prior to their conversion from amine to amide
substituents. Ligation transforms the benzylamine into a ben-
zylamide, which is more electron withdrawing. This increases
the leaving group propensity of the system rendering it acid
sensitive.3b,4,5,17 The Tmb auxiliary can be removed with TFA
and scavengers, while the Dmb auxiliary can be removed with
either TFMSA9 or anhydrous HF (Scheme 2 and Figure 2). In
our hands TFMSA did not give clean results on large peptides.
The practical difficulties associated with TFMSA or HF
significantly limit the use of the Dmb auxiliary. The Tmb
auxiliary has the same ligation properties and has a simple and
general cleavage.
To complete the synthesis of the SH3 domain of R-spectrin,
it was necessary to remove the auxiliary. This was easily
achieved under acidic conditions (Figure 2) providing the final
product. The CD spectrum of the folded material was consistent
with the published spectrum of the SH3 domain of R-spectrin.18
The denaturation profile (Figure 3) was determined by monitor-
ing the fluorescence of the two Trp residues as a function
of urea concentration; ∆G°H O ) 16 ( 0.46 kJ mol-1
,
2
Cleavage of the Auxiliary. Because amines are poor leaving
groups, benzylamines are stable to the strong acid conditions
(17) (a) Baumgarten, R. J. J. Org. Chem. 1968, 33, 234-237. (b) Tolborg, J.
F.; Jensen, K. J. Chem. Commun. 2000, 2, 147-148. (c) Alsina, J.; Jensen,
K. J.; Albericio, F.; Barany, G. Chem. Eur. J. 1999, 5, 2787-2795.
(18) Viguera, A. R.; Martinez, J. C.; Filimonov, V. V.; Mateo, P. L.; Serrano,
L. Biochemistry 1994, 33, 2142-2150.
(16) This ligation was also carried our using the Dmb auxiliary. The time course
for ligation was identical with that of the Tmb auxiliary as monitored by
HPLC (entry 2, Table 1).
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Synthesis of Proteins with a Removable Acyl Transfer Auxiliary
A R T I C L E S
m ) 2.83 ( 0.09 kJ mol-1 M-1, consistent with published
values.18 The presence of both Trp and Met residues in the final
product, known to be sensitive to chemical modification during
acid deprotection,19 made this target a good test for the removal
of the 2-mercaptobenzyl auxiliary.
MHz, CDCl3) δ 214.0, 154.2, 140.4, 125.4, 113.0, 71.2, 61.8, 57.1,
14.6; HRMS (MALDI-FTMS) calcd for C12H16O4S2Na (M + Na+)
311.0382, found 311.0382.
Thiophenol 3: Xanthate 2 (7.90 g, 0.02739 mol) was dissolved in
EtOH (100 mL). A solution of sodium hydroxide (3 M, 100 mL) was
added and the reaction mixture was heated to 65 °C for 2 h. The reaction
mixture was cooled to 25 °C and acidified to pH 5 by the addition of
10% aqueous HCl. The resulting mixture was extracted with EtOAc
(3 × 200 mL) and the combined organic extracts were washed with
brine (200 mL), dried (Na2SO4), and concentrated in vacuo to provide
thiophenol 3 (4.91 g, 90%), which was used without further purification.
Thioether 4: Thiophenol 3 (1.29 g, 6.45 mmol) was dissolved in
THF (50 mL) and MeOH (50 mL). A 60% dispersion of NaH in mineral
oil (0.70 g, 17.5 mmol) was added slowly to the reaction and the mixture
was stirred for 5 min. 4-Methylbenzyl bromide (1.31 g, 7.08 mmol)
and triphenylphosphine (1.71 g, 6.53 mmol) were then added to the
reaction. The mixture was stirred for 15 min and then concentrated in
vacuo. Flash column chromatography of the residue (silica gel, 10%
EtOAc in hexanes) afforded thioether 4 (1.90 g, 97%). 4: Rf ) 0.38
(silica gel, 20% EtOAc in hexanes); IR (thin film) νmax 2935, 2831,
1578, 1498, 1406, 1307, 1233, 1179, 1124, 1007 cm-1; 1H NMR (600
MHz, CDCl3) δ 7.13 (d, J ) 7.9 Hz, 2 H, ArH), 7.06 (d, J ) 7.4 Hz,
2 H, ArH), 6.50 (s, 2 H, ArH), 4.01 (s, 2 H, SCH2), 3.79 (s, 3 H,
OCH3), 3.73 (s, 6 H, OCH3), 2.29 (s, 3 H, CH3); 13C NMR (150 MHz,
CDCl3) δ 154.0, 138.1, 137.6, 135.5, 131.6, 130.0, 129.8, 109.1, 61.7,
56.9, 40.7, 21.9; HRMS (MALDI-FTMS) calcd for C17H20O3S (M+)
304.1128, found 304.1136.
Summary
The 4,5,6-trimethoxy-2-mercaptobenzyl (Tmb) auxiliary func-
tions as a cysteine mimic for the ligation of polypeptides. Our
results suggest that this auxiliary will be applicable to ligation
sites containing a glycine at either side of the ligation junction.
This added flexibility in synthetic design is crucial for the
synthesis of larger, more challenging, biologically important
targets.20 A variety of approaches for the synthesis of non-
cysteine ligation sites have recently been disclosed.4,5,9 The
strength of our approach is that it combines simple auxiliary
construction and introduction, practical ligation rates at low
concentrations, and removal of the auxiliary by TFA. We
anticipate that this approach can be extended to the chemose-
lective synthesis of a range of amide-containing molecules such
as natural products, materials, and bioconjugates.
Experimental Section
Synthesis of 2,3,4-Trimethoxy-6-(4-methyl benzylthio)benzyl-
amine (6). General Techniques. All reactions were carried out under
an argon atmosphere with dry, freshly distilled solvents under anhydrous
conditions, unless otherwise noted. Tetrahydrofuran (THF) was distilled
from sodium benzophenone; methylene chloride (CH2Cl2) was freshly
distilled from calcium hydride. Yields refer to chromatographically and
spectroscopically (1H NMR) homogeneous materials, unless otherwise
stated.
Reagents were purchased at the highest commercial quality and used
without further purification, unless otherwise stated. Reactions were
monitored by thin-layer chromatography (TLC) carried out on 0.25
mm E. Merck silica gel plates (60F-254) using UV light as a visualizing
agent and 7% ethanolic phosphomolybdic acid or p-anisaldehyde
solution and heat as developing agent. E. Merck silica gel (60, particle
size 0.040-0.063 mm) was used for flash column chromatography.
NMR spectra were recorded on Bruker DRX-600 or DRX-500
instruments and calibrated using residual undeuterated solvent as an
internal reference. The following abbreviations were used to explain
the multiplicities: s ) singlet, d ) doublet, t ) triplet, q ) quartet,
sept ) septet, m ) multiplet, b ) broad, bs ) broad singlet. IR spectra
were recorded on a Perkin-Elmer 1600 series FT-IR spectrometer. High-
resolution mass spectra (HRMS) were recorded on a IONSPEC FTMS
spectrometer (MALDI) with DHB as matrix.
Aldehyde 5: Thioether 4 (1.82 g, 5.98 mmol) and N,N-dimethyl-
formamide (0.70 mL, 9.04 mmol) were disolved in CH2Cl2 (10 mL) at
0 °C. Phosphorus oxychloride (0.95 mL, 10.2 mmol) was added
dropwise over 10 min. The reaction was heated to 150 °C for 2 h
generating a dark red oil. H2O (100 mL) was added to the oil and the
mixture was refluxed for 1 h. The resulting mixture was extracted with
EtOAc (3 × 200 mL) and the combined organic extracts were washed
with brine (200 mL), dried (Na2SO4), and concentrated in vacuo. Flash
column chromatography of the residue (silica gel, 20% EtOAc in
hexanes) afforded aldehyde 5 (1.92 g, 97%). 5: Rf ) 0.37 (silica gel,
30% EtOAc in hexanes); IR (thin film) νmax 2936, 2850, 1664, 1580,
1546, 1488, 1369, 1299, 1240, 1117, 1018 cm-1; 1H NMR (500 MHz,
CDCl3) δ 10.41 (s, 1 H, CHO) 7.39 (d, J ) 8.1 Hz, 2 H, ArH), 7.19
(d, J ) 7.7 Hz, 2 H, ArH), 6.65 (s, 1 H, ArH), 4.18 (s, 2 H, SCH2),
4.04 (s, 3 H, OCH3), 3.90 (s, 3 H, OCH3), 3.87 (s, 3 H, OCH3), 2.39
(s, 3 H, CH3); 13C NMR (125 MHz, CDCl3) δ 189.4, 158.7, 158.5,
153.5, 140.2, 138.7, 137.6, 133.5, 129.8, 129.1, 120.5, 105.0, 62.8,
61.5, 56.5, 37.3, 21.5; HRMS (MALDI-FTMS) calcd for C18H21O4S
(M + H+) 333.1155, found 333.1150.
Amine 6: Aldehyde 5 (0.68 g, 2.05 mmol) was disolved in CH2Cl2
(25 mL). To this was added hydroxylamine (0.24 g, 3.45 mmol) and
triethylamine (1.0 mL, 13.6 mmol). The reaction mixture was stirred
for 18 h and then quenched via the addition of H2O (50 mL). The
resulting mixture was extracted with EtOAc (3 × 100 mL) and the
combined organic extracts were washed with brine (100 mL), dried
(Na2SO4), and concentrated in vacuo. The resulting oil was dissolved
in THF (30 mL) and cooled to 0 °C. A solution of lithium aluminum
hydride in THF (1.0 M, 6.0 mL) was added dropwise over 5 min. The
reaction was heated to reflux for 1 h and then cooled to 0 °C and
quenched via the addition of H2O (0.25 mL), 3 M aqueous NaOH (0.25
mL), and H2O (0.75 mL). The mixture was dilluted with EtOAc (300
mL) and filtered and concentrated in vacuo. Flash column chromatog-
raphy of the residue (silica gel, gradient elution 0f20% MeOH in
EtOAc) afforded amine 6 (0.31 g, 45%). 6: Rf ) 0.21 (silica gel, 25%
MeOH in EtOAc); IR (thin film) νmax 3344, 2934, 1583, 1482, 1397,
Xanthate 2: Aniline 1 (9.97 g, 0.0544 mol) was dissolved in MeOH
(10 mL) and 10% aqueous HCl and was then cooled to 0 °C. A solution
of sodium nitrite (5.0 g, 0.0725 mol) in H2O (20 mL) was added
dropwise over 1 h. The reaction mixture was stirred at 0 °C for an
additional 15 min at which time the solution was added to a solution
of potassium ethyl xanthate (17.3 g, 0.108 mol) in H2O (50 mL) at 65
°C. After the mixture was stirred for 15 min, the reaction was cooled
to 25 °C. The resulting mixture was extracted with EtOAc (3 × 200
mL) and the combined organic extracts were washed with brine (200
mL), dried (Na2SO4), and concentrated in vacuo. Flash column
chromatography of the residue (silica gel, 10% EtOAc in hexanes)
afforded xanthate 2 (8.80 g, 56%). 2: Rf ) 0.36 (silica gel, 20% EtOAc
in hexanes); IR (thin film) νmax 2936, 1580, 1496, 1455, 1405, 1306,
1
1234, 1125, 1024 cm-1; H NMR (600 MHz, CDCl3) δ 6.72 (s, 2 H,
ArH), 4.60 (q, J ) 7.1 Hz, 2 H, OCH2), 3.86 (s, 3 H, OCH3), 3.84 (s,
6 H, OCH3), 1.33 (t, J ) 7.0 Hz, 3 H, OCH2CH3); 13C NMR (150
1
1303, 1237, 1195, 1108, 1021 cm-1; H NMR (500 MHz, CDCl3) δ
(19) (a) Stachel, S. J.; Habeeb, R. L.; Van Vranken, D. L. J. Am. Chem. Soc.
1996, 118, 1225-1226. (b) Guy, C. G.; Fields, G. B. Methods Enzymol.
1997, 289, 67-83.
7.14 (s, 4 H, ArH), 6.74 (s, 1 H, ArH), 4.05 (s, 2 H, SCH2), 3.95 (s, 3
H, OCH3), 3.93 (s, 3 H, OCH3), 3.89 (s, 2 H, NCH2), 3.83 (s, 3 H,
OCH3), 2.38 (s, 3 H, CH3); 13C NMR (125 MHz, CDCl3) δ 152.6,
(20) McCaldan, P.; Argos, P. Protein 1988, 4, 99-122.
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152.5, 142.3, 137.4, 135.0, 132.2, 129.6, 129.4, 129.2, 112.7, 61.7,
61.3, 56.4, 41.1, 39.3, 21.5; HRMS (MALDI-FTMS) calcd for C18H23-
NO3SNa (M + Na+) 356.1291, found 356.1304.
to the peptide-resin. The peptide-resin was washed with DMF, and
2,3,4-trimethoxy-6-(4-methylbenzylthio)benzylamine (3 equiv) dis-
solved in a minimum of DMF was added and left for 18 h. Inspection
of analytical HPLC of peptide showed quantitative addition of Dmb
and Tmb to N-terminal bromoacetylated peptide-resin with no loss of
Dmb or Tmb with HF treatment. Similarly addition of Dmb to (R)-2-
bromopropionyl terminal peptide proceeded with an estimated unop-
timized yield of 50%. Peptides were purified by semipreparative HPLC
after cleavage from resin.
Peptide Synthesis. General Methods. Electrospray ionization mass
spectrometry (ESI-MS) was performed on an API-III triple quadrupole
mass spectrometer (PE-Sciex). Peptide masses were calculated from
the experimental mass to charge (m/z) ratios from all the observed
protonation states of a peptide using MacSpec software (Sciex).
Theoretical masses of peptides and proteins were calculated using
MacProMass software (Beckman Research Institute, Duarte, CA).
HPLC. Analytical reversed-phase HPLC was performed on a
Hewlett-Packard HPLC 1050 system using Vydac C18 columns (5 µm,
0.46 × 15 cm). Semipreparative reversed-phase HPLC was performed
on a Rainin HPLC system using a Vydac C-18 column (10 µm, 1.0 ×
25 cm). Linear gradients of acetonitrile in water/0.1% TFA were used
to elute bound peptides. The flow rates used were 1 mL/min (analytical)
and 5 mL/min (semipreparative).
Reagents. Boc-amino acids for peptide synthesis were from Midwest
Biotech (Fishers, IN), 2-(1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-
uronium hexafluorophosphate (HBTU) from Quantum-Appligene (Carls-
bad, CA), diisopropylethylamine from Applied Biosystem (Foster City,
CA), TFA from Halocarbon (River Edge, NJ), and DMF from EM-
Science (Gibbstown, NJ).
Solid-Phase Peptide Synthesis (SPPS). Peptides were prepared by
manual solid-phase synthesis using the in situ neutralization/HBTU
activation for Boc chemistry as previously described;21 couplings were
carried out with a 5-fold excess of activated amino acid for a minimum
of 15 min and monitored by quantitative ninhydrin test.22 C-Terminal
peptides were synthesized on the appropriate Boc-aminoacyl-OCH2-
Pam preloaded resin (Applied Biosystems).
After chain assembly was complete the peptide-resin was treated
with HF containing 5% (v/v) p-cresol for 1 h at 0 °C. After evaporation
of HF, the crude peptide was precipitated in anhydrous Et2O, dissolved
in HPLC buffer, and lyophilized.
SH3 r-spectrin Dmb[28-62]: DmbGDILTLLNSTNKDWWKV-
EVNDRQGFVPAAYVKKLD-OH, observed mass 4216 ( 1 Da,
calculated 4216.8 Da.
SH3 r-spectrin Tmb[28-62]: TmbGDILTLLNSTNKDWWKV-
EVNDRQGFVPAAYVKKLD-OH, observed mass 4246.2 ( 1 Da,
calculated 4246.9 Da.
PGB1 Dmb[24-56]: DmbATAEKVFKQYANDNGVDGEWTY-
DDATKTFTVTE-OH, observed mass 3898.0 ( 0.3 Da, calculated
3898.2 Da.
Ligation of Peptide Segments. 6 M guanidine‚HCl, 200 mM sodium
phosphate, pH 8.5, was degassed under vacuum and flushed with argon.
TCEP (5 mg mL-1) was added. Typically peptides were added to
ligating buffer at concentrations of approximately 5 mg mL-1 of each
peptide. The pH of the ligation mixture after the addition of peptides
was 7.0. The ligation reactions were monitored to completion by
analytical HPLC.
SH3 r-spectrin 28-N-Dmb: H-MDETGKELVLALYDYQEKSP-
REVTMKK-DmbG-DILTLLNSTNKDWWKVEVNDRQGFVPAAY-
VKKLD-OH, observed mass 7400 ( 1.0 Da, calculated 7401.5 Da.
SH3 r-spectrin 28-N-Tmb: H-MDETGKELVLALYDYQEKSP-
REVTMKK-TmbG-DILTLLNSTNKDWWKVEVNDRQGFVPAAY-
VKKLD-OH, observed mass 7432 ( 1.0 Da, calculated 7431.6.
SH3 r-spectrin [1-28-TmbG-29-62]: H-MDETGKELVLALY-
DYQEKSPREVTMKKG-TmbG-DILTLLNSTNKDWWKVEVNDRQG-
FVPAAYVKKLD-OH, observed mass 7459 ( 1.0 Da, calculated
7458.6 Da.
Peptide-r-thioesters. Thio acid peptides were synthesized on the
appropriate Boc-aminoacyl-S-resins.23 Alternatively N-terminal â-mer-
captopropionic acid-leucine (MPAL) thioester peptides were synthesized
according to a published procedure7 and converted to thio acids by
dissolving the crude peptide in 6 M guanidine‚HCl, 0.2 M phosphate,
pH 7.2, containing ammonium sulfide. The conversion was monitored
to completion by analytical HPLC. Peptide-COSC6H5 esters were
prepared by dissolving the peptide thio acid in 6 M guanidine‚HCl,
0.1 M sodium acetate, pH 4.5, and adding an excess of Ellman’s reagent
(5,5′-dithiobis(2-nitrobenzoic acid)). The mixture was vortexed briefly
and after 20 min thiophenol (2% v/v) was added. The exchange reaction
was monitored by analytical HPLC to completion.
Condensation product of SH3 r-spectrin[1-28] and PGB1 Dmb-
[24-56]: H-MDETGKELVLALYDYQEKSPREVTMKKG-DmbA-
TAEKVFKQYANDNGVDGEWTYDDATKTFTVTE-OH, observed
mass 7139 ( 1 Da, calculated 7139.9 Da.
Removal of Auxiliary. The peptide was placed in a 15 mL Falcon
tube with p-cresol (10 µL) and ethanedithiol (10 µL). After brief
centrifugation the Falcon tube was inserted into a Teflon reaction vessel
and HF (approximately 1 mL) was condensed into the Falcon tube.
The cleavage was left for 30 min at 0 °C. HF was evaporated and
peptide precipitated with ice-cold anhydrous Et2O (10 mL) and left in
the CO2/acetone bath for 5 min. Et2O was decanted off and the Et2O
wash was repeated three times.
SH3 r-spectrin[1-27] thiophenol thioester: H-MDETGKELV-
LALYDYQEKSPREVTMKK-SC6H5, observed mass 3294 ( 0.6 Da,
calculated 3294.7 Da.
SH3 r-spectrin[1-28] thiophenol thioester: H-MDETGKELV-
LALYDYQEKSPREVTMKKG-SC6H5, observed mass 3351 ( 0.3 Da,
calculated 3351.8 Da.
Protein G B1 domain (PGB1[1-23] thiophenol thioester: H-M-
TYKLILNGKTLKGETTTEAVDA-SC6H5, observed mass 2590 ( 0.6
Da, calculated 2589.9.
The Tmb auxiliary was removed by treating the lyophilized crude
ligation reaction product with a cocktail containing 5% (v/v) triiso-
propylsilane in TFA for 2 h at 23 °C. The bulk of TFA was removed
by rapid sparging with a stream of dry argon. The peptide was
precipitated with ice-cold anhydrous Et2O and washed as for the HF
cleavage.
Attachment of NrTmb and NrDmb to Peptides. After chain
assembly was completed the NR-Boc group was removed and the
peptide-resin neutralized with DIEA, washed with DMF and CH2Cl2.
The appropriate R-bromo symmetric anhydride prepared from the
R-bromo acid (bromoacetic acid for N-terminal glycine and (R)-2-
bromopropionic acid for N-terminal (S)-alanine) in CH2Cl2 was added
SH3 r-spectrin: H-MDETGKELVLALYDYQEKSPREVTMKK-
GDILTLLNSTNKDWWKVEVNDRQGFVPAAYVKKLD-OH, ob-
served mass 7219 ( 1 Da, calculated 7219.3 Da.
Acknowledgment. We thank Chiara Marinzi for her generous
assistance in synthesizing the peptides and Jesse Zalatan for
careful proof-reading of the manuscript. This work was finan-
cially supported by the Skaggs Institute for Chemical Biology,
the Alfred P. Sloan Foundation, and the NIH MH62261 or
GM59380.
(21) Schno¨lzer, M.; Alewood, P.; Jones, A.; Alewood, D.; Kent, S. B. H. Int. J.
Pept. Protein Res. 1992, 40, 180-193.
(22) Sarin, V. K.; Kent S. B. H.; Tam, J. P.; Merrifield, R. B. Anal. Biochem.
1981, 117, 147-157.
(23) Canne, L. E.; Walker, S. M.; Kent, S. B. H. Tetrahedron Lett. 1995, 36,
1217-1220.
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4646 J. AM. CHEM. SOC. VOL. 124, NO. 17, 2002