Modular Total Chemical Synthesis of HIV-1 Protease
A R T I C L E S
residues to the preloaded Boc-Arg(Tos)-OCH2-Pam resin before
coupling with â-mercaptopropionic acid and continuing with chain
assembly.19 The fluorogenic substrate and Cys95-99RTArg10 peptides
were synthesized on MBHA resin; the p24/p15 substrate peptide was
synthesized on Boc-Lys(2ClZ)-OCH2-Pam resin. Peptides were syn-
thesized on a 0.4 mmol scale by the manual Boc chemistry in situ
neutralization/HBTU protocol described previously,15 with slight
modifications. Briefly, NR-Boc removal was achieved with neat TFA,
via one rapid wash followed by a 2 min batch treatment. The
NR-deprotected peptide-resin was subject to a single flow wash with
DMF. Amino acids (2.2 mmol) were dissolved in 4 mL of 0.5M HBTU
(i.e., 2.0 mmol), were activated by addition of DIEA (1 mL, 6.6 mmol)
for 1 min, and then added to the peptide-resin; coupling was carried
out for 12 min, followed by a single DMF flow wash to remove excess
activated amino acid and soluble coproducts. After chain assembly was
complete, the NR-Boc group was removed as described above, and the
peptide-resin was washed with dichloromethane and dried by aspira-
tion. The peptide was cleaved from the resin and the side-chain
protecting groups were simultaneously removed by treatment with
anhydrous HF at 0 °C for 1 h, with 10% p-cresol added as a scavenger.
After removal of HF by evaporation, the peptide was precipitated,
washed with cold diethyl ether, dissolved in aqueous acetonitrile +
0.1% TFA, and then lyophilized. Isolated yields after HPLC purification,
based on the loading of the resin, were 14.1% for 1-27RCOSR, 13.3%
for Thz28-70RCOSR, 23.7% for Thz71-94RCOSR, and 46% for
Cys95-99.
HPLC. Analytical reversed-phase HPLC was performed on an
Agilent 1100 system with Microsorb C-4 (5 µm, 2.1 × 50 mm) silica
columns packed in-house. Peptides were eluted from the column with
a gradient of 5-65% acetonitrile/0.08% TFA versus water/0.1% TFA
at a flow rate of 0.5 mL/min. Preparative HPLC of crude peptides after
SPPS was performed on a Vydac C-18 (50 × 250 mm) column, and
preparative HPLC of 1-99RTArg10 after desulfurization was performed
on an Agilent C-3 (22 × 250 mm) column. Peptides were eluted from
the column with an appropriate shallow gradient of acetonitrile/0.08%
TFA versus water/0.1% TFA. Fractions containing the desired purified
peptide product were identified by analytical LC-MS, combined, and
lyophilized.
600 mg of NaBH4. The charged nickel was then washed in batch 3×
with 50 mL of water and added to the peptide solution. The reaction
was allowed to proceed under argon until complete as judged by LC-
MS of aliquots, after which the reaction solution was centrifuged and
the supernatant was decanted. The catalyst was washed 3× with 5 mL
of ligation buffer, with the final wash including 1 M imidazole to
displace any peptide adsorbed to the nickel. The supernatants were
pooled and the combined solution was acidified to pH 2 and then
purified by RP-HPLC as described above. The overall yield of purified
119 residue polypeptide for all ligations and other synthetic manipula-
tions was 26% based on the starting peptide segment Cys95-
99RTArg10.
Folding. 1-99RTArg10 polypeptide (15 mg) was dissolved in 12
mL of 6 M guanidine hydrochloride phosphate buffer (0.2 M, pH 7.4)
and folded by a three-step dialysis procedure against sodium acetate
buffer (pH 5.6) by use of a 3500 MW cutoff membrane. The first step
was against 2 L of 50 mM sodium acetate buffer (pH 5.6) for 3 h. The
second step was against 2 L of 25 mM sodium acetate buffer (pH 5.6)
for 2 h. The third step was against 2 L of 10 mM sodium acetate buffer
(pH 5.6) overnight at 4 °C. Final conditions were 10 mM sodium
acetate, pH 5.6. The final concentration of folded protein was
determined by absorbance at 280 nm with a calculated dimer extinction
coefficient of 25 120 M-1 cm-1
.
Enzyme Kinetics. The fluorogenic substrate Abz-TI(Nle)F(NO2)-
QR-CONH, where Abz ) 2-aminobenzoyl, Nle ) norleucine, and
F(NO2) ) p-nitrophenylalanine, was incubated at various concentrations
with the enzyme (100 nM), and initial velocities were determined by
the initial rate of increase in fluorescence after substrate cleavage.25
Final assay conditions were 50 mM sodium acetate buffer, pH 5.6,
and 1% dimethyl sulfoxide (DMSO), 37 °C. Assays were performed
in triplicate for each substrate concentration. kcat and Km were
determined after fitting of the data points to the Michaelis-Menten
equation by use of a nonlinear least-squares fitting program in
Origin 7.0.
Crystallization. Crystals were grown at room temperature by the
hanging drop vapor diffusion method from a well solution consisting
of 0.1 M citrate, 0.2 M sodium phosphate, 30% saturated ammonium
sulfate, and 10% DMSO, pH 6.0. Protein solution (aliquot of
concentrated folding solution) (∼0.5 mM enzyme) was preincubated
with a 20-fold molar excess of (S)-JG-365 and was then mixed in a
2:1 (v/v) ratio with well solution. Crystals grew within 5 days and were
frozen in liquid nitrogen with a cryoprotectant of 30% glycerol.
MS Analysis. Peptide masses were obtained on an Agilent 1100
LC-MS with on-line electrospray ion trap detection. MALDI-TOF MS
analysis of the folding reaction was performed by mixing a sample of
the reaction mixture and a sample of sinapinic acid (saturated in aqueous
acetonitrile + 0.1% TFA) in a 1:1 mixture. MALDI spectra were
acquired on a Perseptive Biosystems Voyager-DE in linear mode.
Structure Determination and Refinement. Diffraction data were
collected at 19-BM station in the Advanced Photon Source (APS) at
Argonne National Laboratory and processed with HKL2000.26 PHASER27
was used to obtain the initial phases, by use of the model of HIV-1
protease in complex with JG-365 (PDB code 7HVP). The starting model
was initially refined by the CNS program28 with data between 50 and
1.5 Å. After free R-factor convergence,29 the model was fed into
SHELX9730 for full anisotropic temperature factor refinement, with
data to the highest resolution shell and with low-resolution cutoff at
10 Å. Iterative model building and refinement were performed with
COOT31 and SHELXL30 until the final Rcryst/Rfree values converged to
14.36/18.37%. Coordinates for the refined model have been deposited
in the Protein Data Bank with accession code 2JE4. Model analysis
Peptide Ligations. Ligations were performed on a 5 µmol scale
(2.5 mM) in 6 M guanidine hydrochloride phosphate buffer (0.2 M) at
pH 7.0, with 200 mM 4-mercaptophenylacetic acid (MPAA) added as
a catalyst and 20 mM Tris(2-carboxyethyl)phosphine hydrochloride
(TCEP) added as a reducing agent. MPAA was repurifed from the
commercial source by HPLC before use. After each ligation, the Thz
was converted into cysteine with 0.2 M methoxylamine HCl at pH
4.0. After Thz conversion was complete, the crude peptide products
were subjected to solid-phase extraction (SPE) on C-4 silica SPE
columns and recovered in 50% aqueous acetonitrile and lyophilized.
After the final ligation was complete, cysteine (1 M) was added and
the solution was made 20% in piperidine for 20 min to remove
formyl protection on tryptophan. The solution was then acidified with
HCl, purified by SPE to remove all nonpeptidic material, and then
lyophilized.
(25) Toth, M. V.; Marshall, G. R. Int. J. Pept. Protein Res. 1990, 36, 544-550.
(26) Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307-326.
(27) Read, R. J. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2001, 57, 1373-
1382.
Desulfurization. Lyophilized crude peptide 1-99RTArg10 (67 mg)
was dissolved in 10 mL of 6 M guanidine hydrochloride phosphate
buffer (0.2 M, pH 7.4) with 20 mM TCEP. Raney nickel was prepared
by dissolving 3.6 g of nickel acetate in 24 mL of H2O and slowly adding
(28) Brunger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros, P.;
Grosse-Kunstleve, R. W.; Jiang, J. S.; Kuszewski, J.; Nilges, M.; Pannu,
N. S.; Read, R. J.; Rice, L. M.; Simonson, T.; Warren, G. L. Acta
Crystallogr., Sect. D: Biol. Crystallogr. 1998, 54, 905-921.
(29) Brunger, A. T. Nature 1992, 355, 472-475.
(30) Sheldrick, G. M.; Schneider, T. R. Methods Enzymol. 1997, 277, 319-
343.
(24) Hackeng, T. M.; Griffin, J. H.; Dawson, P. E. Proc. Natl. Acad. Sci. U.S.A.
1999, 96, 10068-10073.
(31) Emsley, P.; Cowtan, K. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004,
60, 2126-2132.
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