RIJKERS ET AL.
Staudinger ligation [5–8], oxime/hydrazone ligation [9], and the
Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition [10,11]. We
have applied the chemoselective reaction of a thio acid and a
sulfonyl azide developed by Williams and coworkers [12–14] to
peptide-based thio acids and amino acid–derived sulfonyl azides
as a novel chemical ligation reaction to provide an entry toward
large densely functionalized peptide mimic systems with a high
degree of chemical diversity [15] (3, Scheme 1A). In addition, we
have explored its use as a novel approach for the preparation of
the N-acyl sulfonamide linker 5 (Kenner’s safety catch linker [16]),
as shown in Scheme 1B [17].
The thio acid/sulfonyl azide amidation reaction, which we have
denoted as ‘sulfo-click’, requires a sulfonyl azide (e.g. HCl.H-
Gly-ꢀ[CH2SO2]-N3 6 or its DOTA-conjugated derivative 7) and
a suitable thio acid (e.g. dipeptide thio acid 2, coumarin thio
acid 9 as fluorescent label, and biotin thio acid derivative 10) or
a protected precursor (e.g. 8) as building blocks (Figure 1). The
sulfonyl azides 1 and 6 were prepared from their N-protected
amino acid derivatives as previously described [18]. The thio
acids could be prepared via the corresponding succinimidyl ester
followed by reaction with NaHS [19], via a BOP-mediated coupling
with NaHS [17], or via the S-(2,4,6-trimethoxybenzyl) ester of the
desired acid followed by acidolysis with TFA [20]. In a typical
experiment, the sulfonyl azide was mixed with 2,6-lutidine in a
suitable solvent (DMF, CHCl3, aqueous buffer) and the thio acid
derivative was added. The thio acid and sulfonyl azide reacted
smoothly at room temperature and the reaction was complete
within 15 min. The resulting acyl sulfonamides could be isolated
in good to excellent yield. Also, in more complex systems [21,22]
with unprotected peptide fragments (i.e. 12 and 14), the thio
acid/sulfonylazideamidationreactionallowedthechemoselective
and site-specific incorporation of suitably labeled chemical entities
(peptides, fluorophores and metal chelators) as shown in Schemes
2 and 3.
28.4, 19.4, 18.3; ESMS calcd. for C29H40N4O8S: 604.26, found:
m/z 605.55 [M + H]+, 627.50 [M + Na]+ (All ESMS data
as reported in this protocol were measured on a Shimadzu
LCMS-QP8000 single quadrupole bench-top mass spectrometer
operating in a positive ionization mode). Elemental analysis: calcd
for C29H40N4O8S: C, 57.60; H, 6.67; N, 9.27, found C, 57.52; H, 6.65;
N, 9.21.
Example 2 (Scheme 1B)
A portion of Tentagel S NH2 amino methyl resin (3 g, 0.8 mmol)
was allowed to swell in NMP for 1 h. Subsequently, the resin was
washed with 50% HOAc in NMP (10 ml; 2 × 10 min), NMP (10 ml;
2 × 2 min), i-PrOH (10 ml; 1 × 2 min), NMP (10 ml; 2 × 2 min), 20%
DiPEA in NMP (10 ml; 1 × 2 min), NMP (10 ml; 3 × 2 min), i-PrOH
(10 ml; 1 × 2 min) and finally NMP (10 ml; 3 × 2 min). A solution of
succinic anhydride (801 mg, 8 mmol, 10 equiv) and DiPEA (1.4 ml,
8 mmol, 10 equiv) in NMP (10 ml) was added to the resin and the
resulting mixture was shaken for 1 h before the resin was filtered
and washed with NMP (10 ml; 3 ×2 min), i-PrOH (10 ml; 1 ×2 min),
and NMP (10 ml; 3 × 2 min). Completion of the reaction was
confirmed by a negative Kaiser test [24] and a positive Malachite
green test [25] for the acid-functionalized resin. Then, a solution
of HCl.H-Gly-ꢀ[CH2SO2]-N3 6 (597 mg, 3.2 mmol, 4 equiv), BOP
(1.4 g, 3.2 mmol, 4 equiv), and DiPEA (1.1 ml, 6.4 mmol, 8 equiv)
in NMP (10 ml) was added to the resin, and the obtained reaction
mixture was shaken for 16 h. Finally, the resin was filtered and
washed with NMP (10 ml; 3 × 2 min), i-PrOH (10 ml; 1 × 2 min),
and CH2Cl2 (10 ml; 3 × 2 min) and dried under vacuum. A portion
of dried sulfonyl azide functionalized resin 4 (185 mg, 50 µmol,
1 equiv) was allowed to swell in DMF (1 ml) for 1 h before the
solvent was drained. To the swollen resin, 2,6-lutidine (465 µl,
4 mmol, 80 equiv) and the Nα-protected amino thio acid (4 equiv)
in DMF (800 µl) were added and the obtained reaction mixture
was gently shaken for 3–6 h. The resin was subsequently washed
with DMF (1 ml; 3 × 2 min), i-PrOH (1 ml; 1 × 2 min), and CH2Cl2
(1 ml; 4 × 2 min) and dried to give resin 5. See reference 17 for
more specific details.
The thio acid/sulfonyl azide amidation reaction was found to
be extremely versatile as a metal-free click reaction for the incor-
poration of metal chelators like 1,4,7,10-tetraazacyclododecane-
1,4,7,10-tetraacetic acid (DOTA)-derivative 7 to obtain DOTA-
conjugated peptides (16) for tumor imaging and targeting [23].
Example 3 (Scheme 2)
To a solution of H-Gly-Arg-Arg-Arg-Arg-Ser-Val-Glu(Gly-ꢀ[CH2
SO2N3])-Trp-Cys(Acm)-Ala-NH2 12 (10 mg, 6 µmol) (analysis data:
Rt 26.35 min [Alltech Prosphere C18, 300 Å, 5 µm, 250 × 4.6 mm,
using a linear gradient of 100% buffer A (0.1% TFA in H2O/CH3CN
95 : 5 v/v) to 30% buffer B (0.1% TFA in CH3CN/H2O 95 : 5 v/v)
in 40 min at 1 ml/min]; ESMS calcd for C61H104N30O16S2:
1578.80, found: m/z 791.38 [M + 2H]2+ave) in an aqueous
25 mM HEPES buffer (2 ml, pH 7.4), Boc-Val-Gly-SH (4 mg,
14 µmol) was added as a single portion. After 1 h of stirring
at room temperature, the reaction was complete, according
to HPLC analysis. The reaction mixture was loaded onto a
preparative HPLC [Alltech Adsorbosphere C8, 90 Å, 10 µm,
250 × 22 mm, using a linear gradient of 100% buffer A (0.1%
TFA in H2O/CH3CN 95 : 5 v/v) to 100% buffer B (0.1% TFA in
CH3CN/H2O 95 : 5 v/v) in 40 min at 5 ml/min] and peptide 13
was isolated in 80% yield (8.7 mg, 4.8 µmol) and characterized
by mass spectrometry. Rt 35.45 min [Alltech Prosphere C18,
300 Å, 5 µm, 250 × 4.6 mm, using a linear gradient of 100%
buffer A (0.1% TFA in H2O/CH3CN 95 : 5 v/v) to 30% buffer B
(0.1% TFA in CH3CN/H2O 95 : 5 v/v) in 40 min at 1 ml/min]; ESMS
calcd for C73H126N30O20S2: 1809.10, found: m/z 905.47 [M +
Experimental Procedure
Example 1 (Scheme 1A)
To a mixture of Cbz-Phe-ꢀ[CH2SO2]-N3 1 (64 mg, 0.17 mmol)
and 2,6-lutidine (26 µl, 0.22 mmol, 1.3 equiv) in CHCl3 (2.5 ml),
Boc-Val-Gly-SH 2 (64 mg, 0.22 mmol) was added as a single
portion. The reaction mixture was stirred for 15 min at room
temperature, after which it was evaporated to dryness. The residue
was purified by crystallization from EtOAc/hexane to give the
peptidyl sulfonamide 3 as a white solid in nearly quantitative
yield (102 mg, 0.17 mmol). Rf 0.48 (Merck silica gel 60-F254 plate,
EtOAc/hexane/HOAc, 49.5 : 49.5 : 1 v/v/v); Rt 17.08 min [Alltech
Adsorbosphere XL C8, 90 Å, 5 µm, 250 × 4.6 mm, using a linear
gradient of 100% buffer A (0.1% TFA in H2O) to 100% buffer B
(0.1% TFA in CH3CN/H2O 95 : 5 v/v) in 20 min at 1 ml/min]; Mp
191 ◦C; [α]D −29.9 (c 0.1 DMF); 1H NMR (300 MHz, DMSO-d6):
23
δ 11.89 (s, 1H), 8.16 (m, 1H), 7.53 (d, J = 9.1 Hz, 1H), 7.34–7.17
(m, 10H) 6.75 (d, J = 9.1 Hz, 1H), 4.98 (m, 2H), 4.18 (m, 1H),
3.84 (m, 3H), 3.59 (m, 2H), 2.90 (m, 1H), 2.78 (m, 1H), 1.99 (m,
1H), 1.38, (s, 9H), 0.86 (m, 6H); 13C NMR (75 MHz, DMSO-d6)
δ 172.1, 169.2, 155.7, 155.4, 137.9, 137.3, 129.4, 128.5, 127.9,
127.6, 127.2, 126.6, 78.2, 65.3, 60.0, 59.7, 56.1, 48.4, 42.3, 30.6,
2H]2+
.
ave
c
Copyright ꢀ 2009 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2010; 16: 1–5