Paper
Organic & Biomolecular Chemistry
Preparation of Boc-Lys(AdeMeOBzl) and Boc-Dab(AdeMeOBzl)
After chain assembly was complete, the Nα-Boc group was
removed with TFA, and the peptide-resin was washed with
dichloromethane and dried by aspiration. 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 hour, with 5–10% (v/v) p-cresol added as a scaven-
ger. After thorough removal of HF by evaporation, the peptide
was precipitated and washed with cold diethyl ether, dissolved
in 50% aqueous acetonitrile + 0.1% TFA, and then lyophilized.
9-(4′-Methoxybenzyl)-6-chloropurine was prepared according to
a previously published literature procedure.19
Detailed procedures for the preparation of each modified
amino acid are described below.
9-(4′-Methoxybenzyl)-purine-(6-Nω)-Nα-(tert-butoxylcarbonyl)-
L-lysine. The mixture of 9-(4′-methoxybenzyl)-6-chloropurine
(1.00 g, 3.65 mmol), Nα-(tert-butoxylcarbonyl)-L-lysine (0.75 g,
3.05 mmol) and K2CO3 (0.8 g) in 20 mL of DMSO was stirred at
70 °C overnight. 250 mL of water and 20 mL of saturated
Na2CO3 were added and the aqueous solution was extracted
with EtOAc three times, in order to remove the excess 9-benzyl-
6-chloropurine. The pH of the aqueous layer was adjusted
to 6–7 by 1 N HCl and then extracted with EtOAc or CH2Cl2.
The combined organic layers were washed with water once and
dried over MgSO4. After filtration and removal of the solvent,
9-(4′-methoxybenzyl)-purine-(6-Nω)-Nα-(tert-butoxylcarbonyl)-
L-lysine (1.20 g) was obtained in 82% yield. (If it contains some
little impurity, impurity can be removed by addition of some
mixture solution of EtOAc and hexanes in a ratio of 1 : 10 and
filtration). 1H NMR (500 MHz, CDCl3) δ 9.57 (br, s, 0.30 H),
8.45 (s, 1 H), 8.10 (br, s, 0.20 H), 7.78 (br, s, 0.20 H), 7.64 (s,
1 H), 7.60 (br, s, 1 H), 7.23 (d, J = 8.0 Hz, 2 H), 6.86 (d, J =
8.0 Hz, 2 H), 5.65–5.50 (br, 1 H), 5.26 (s, 2 H), 4.33 (d, J =
6.5 Hz, 1 H); 3.78 (s, 3 H), 3.75 (br, s, 1 H), 3.48 (br, s, 1 H),
1.95 (br, s, 1 H), 1.85 (br, s, 1 H), 1.76 (s, 2 H), 1.70–1.50 (m,
2 H), 1.44 (s, 9 H); 13C NMR [see Fig. S1†] (125 MHz, CDCl3)
δ 176.78, 159.58, 155.51, 154.35, 153.65, 148.29, 138.59,
129.40, 126.86, 117.88, 114.31, 79.37, 55.15, 53.69, 46.80,
40.42, 32.05, 28.53, 28.27, 22.70. LCMS data are given in
Fig. S2.†
Peptide analysis and purification
Analytical reverse-phase HPLC was performed on an Agilent
1100 system with either a Microsorb C-18 (5 μm, 300 Å,
2.1 × 50 mm) silica column packed in-house or a Vydac C-4
(5 μm, 300 Å, 2.1 × 50 mm) silica column. Peptide masses were
obtained using on-line electrospray MS detection. Preparative
HPLC was performed on C-4 and C-18 (10 × 250, 22 × 250,
50 × 250 mm) columns from Vydac, depending on the peptide
quantities and retention characteristics. Peptides were eluted
from the column using an appropriate shallow gradient of
acetonitrile/0.08% TFA versus water/0.1% TFA. Fractions con-
taining the desired purified product were identified by analyti-
cal LC-MS, then combined and lyophilized.
Synthesis of RNase A analogues by native chemical ligation
The synthesis of the full length 124 amino acid polypeptide
chains of the RNase A analogues were performed using two
sets of sequential one-pot ligations similar to that described
previously.16 Briefly, equimolar amounts of purified, lyophi-
lized RNase A(95-124) and RNase A(Thz84-94)-αCOSR were dis-
solved to a concentration of approximately 4 mM in 6 M
guanidine hydrochloride, 0.1 M Na phosphate pH 7.0 contain-
ing 200 mM MPAA and 20 mM TCEP (ligation buffer). Upon
reaction completion, the Thz was converted to cysteine by
addition of 0.2 M methoxylamine HCl, adjustment of pH to
4.0, and overnight reaction. The next ligation was performed
by re-adjusting to pH 7.0, adding 1.3 equivalents of
9-(4′-Methoxybenzyl)-purine-(6-Nγ)-Nα-(tert-butoxylcarbonyl)-
L-diaminobutyric acid. The reaction of 9-(4′-methoxybenzyl)-6-
chloropurine (1.48 g, 5.40 mmol), Nα-(tert-butoxylcarbonyl)-
L-2,4-diaminobutyric acid (1.09 g, 5.00 mmol) and K2CO3
(1.38 g) afforded 1.83 g (81%) of 9-(4′-methoxybenzyl)-purine-
(6-Nω)-Nα-(tert-butoxylcarbonyl)-L-diaminobutyric
acid.
1H
[D83A]RNase A(Thz65-83)-αCOSR′ (containing
a C-terminal
NMR (500 MHz, CDCl3) δ 9.69 (br, s, 0.25 H), 8.42 (s, 1 H), 7.79
(br, s, 0.25 H), 7.60 (br, s, 0.25 H), 7.28 (s, 1 H), 7.18 (d, J =
8.0 Hz, 2 H), 6.85 (d, J = 8.0 Hz, 2 H), [6.22 (br, s, 0.33 H), 5.97
(br, s, 0.67 H)], [5.26 (br, s, 0.50 H), 5.08 (s, 1.50 H)], 4.44 (s, 1
H), 4.31 (0.25 H), 3.96 (br, s 1 H), 3.75 (s, 3 H), 3.65 (br, s, 1 H),
2.48 (br, s 1 H), 2.16 (br, s, 1 H), 1.44 (s, 9 H); 13C NMR [see
Fig. S3†] (125 MHz, CDCl3) δ 176.44, 159.74, 155.56, 154.42,
153.38, 148.39, 138.52, 129.42, 126.87, 117.84, 114.43, 79.53,
55.24. LCMS data are given in Fig. S4.†
6-Arg tag in the thioester leaving group and one of the
described un-natural amino acids) and adding an additional
20 mM TCEP. Following completion of the ligation reaction,
the Thz protection was removed overnight by lowering the pH
of the reaction to 4.0 without additional methoxylamine HCl.
The product, polypeptide RNase A(65-124), containing one of
the two described unnatural amino acids, was purified by
preparative HPLC.
The second series of one-pot ligations was initiated by dis-
solving RNase A(65-124) containing one of the modified amino
acids and RNase A(Thz40-64)-αCOSR to approximately 2.5 mM
Peptide synthesis
Peptide-αCOSCH2CH2COLeu (-αCOSR) thioesters were syn- in 0.1 M sodium phosphate pH 7.0 ligation buffer containing
thesized on HCOSCH2CH2COLeu-OCH2-Pam-resin, as 200 mM MPAA and 20 mM TCEP. After the reaction was com-
a
described previously.20 Arginine-tagged segment 65–83 was plete, Thz deprotection was performed overnight by adding
synthesized on Boc-Arg-OCH2-Pam-resin to which six arginines 0.2 M methoxylamine HCl at pH 4.0 as described above. The
and a thioester moiety were coupled. Peptides were syn- ligation of RNase A(Thz26-39)-αCOSR was initiated by re-adjust-
thesized on a 0.5 mmol scale, using the manual Boc chemistry ing the reaction pH to 7.0, adding the peptide, and an
“in situ neutralization”/HBTU protocol described previously.21 additional 20 mM TCEP. After the reaction was complete,
Org. Biomol. Chem.
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