2 of 5
PERKINS ET AL.
FTICR-MS, a hybrid mass spectrometer. After ionization, the ions fly
through a quadrupole and a hexapole before detection in an ion
cyclotron resonance cell.
2.2
|
General procedure A: Diester 1
Diol 7 (1 equiv), Boc-Xxx-OH (8) (3.7 equiv), EDCꢀHCl (4.9 equiv), and
DMAP (10 mol%) were combined and placed under argon. THF (5 ml)
and DCM (15 ml) were added, and the reaction was stirred for 1–6 h.
The reaction mixture was transferred to a separatory funnel using
EtOAc and DCM and the organics washed once each with H2O, sat.
NaHCO3, and sat. brine, dried over MgSO4, filtered, and concentrated
in vacuo. Purification by flash column chromatography (gradient from
10% to 40% EtOAc-hexane) afforded diester 1.
FIGURE 1 Our latent thioester substituted (twice) with a generic,
protected amino acid; unmasking yields a thioester, which can be
isolated or used in situ in ligation with cysteine
2.3
|
General procedure B: Thioester 2
To diester 1 (1 equiv) dissolved in a combination of DCM, DMF, and
THF (total 100 μl) were added TEA (ꢁ5 equiv) and ethanethiol
(ꢁ20 equiv), and the reaction was placed under argon, sealed tightly
(to retain the ethanethiol), stirred vigorously, and allowed to run
until completion as determined by TLC (about 24 h). The reaction
mixture was concentrated in vacuo. Purification by flash column
chromatography (EtOAc-hexane eluent) afforded thioester 2.
2.4
|
General procedure C: Cys-dipeptide 6
To diester 1 (1 equiv) and cysteine methyl ester hydrochloride
(H-L-Cys-OMeꢀHCl) (ꢁ2.5 equiv) in DMF (190 μl) were added TEA
(ꢁ5 equiv) and ethanethiol (ꢁ20 equiv), and the reaction was placed
under argon, sealed tightly (to retain the ethanethiol), stirred
vigorously, and allowed to run until completion as determined by TLC
(about 24 h). Reduction of any disulfides was accomplished by
addition of TCEPꢀHCl (1 equiv) and water (ꢁ50 equiv) and stirring for
an additional 1–2 h. The reaction mixture was concentrated in vacuo,
then transferred to a separatory funnel with EtOAc and H2O. The
organics were washed with 0.5 M KHSO4 (three times), H2O, sat.
NaHCO3, and sat. brine, dried over MgSO4, filtered, and concentrated
in vacuo. Purification by flash column chromatography (gradient from
40% to 70% EtOAc-hexane) afforded dipeptide 6.
FIGURE 2 Unmasking a latent thioester (1) and further reactions.
With an appropriate thiol (here, ethanethiol), thioester 2 can be
isolated. In the presence of an N-terminal Cys residue, NCL ensues to
give thioester 5, then ligated 6
reported aqueous conditions. As seen below, these organic
reaction conditions can be employed to convert protected latent
thioesters into thioesters or to enter directly into chemoselective
ligations, if desired.
Latent thioester 1 comprises a disulfide-protected sulfur atom
positioned two carbon atoms away from an ester oxygen atom, which
enables an entropically favorable 5-exo-trig O ! S acyl transfer when
unmasked.25 (In fact, 1 contains two such functional groups, symmet-
rically disposed. The solid-phase linker version of this material con-
3
|
RESULTS AND DISCUSSION
The latent thioester in Figure 1 meets all of the desired criteria:
compatibility with Fmoc SPPS and both aqueous and organic
unmasking conditions. We have already demonstrated its suitability
for Fmoc/t-Bu SPPS and subsequent aqueous ligation;23,24 here, we
establish that our latent thioester also functions under conditions
compatible with hydrophobic materials, in addition to the previously
tains one latent thioester along with a carboxylic acid moiety.24
)
Though this intramolecular acyl transfer may favor the ester
rather than the thioester, the steady-state concentration of
thioester appears great enough to allow the reaction to proceed.
For this type of latent thioester to work, as noted in Figure 2,
the reaction conditions must (a) cleave the intramolecular disulfide