Journal of the American Chemical Society
Article
CCL1 by a thioacid-mediated strategy using two peptide
segments (Lys1-Ala28-COSH 17 and Cys30(Npys)-Lys73 19)
prepared by SPPS. Although the native CCL1 sequence
contains Thr at the 30th position, we used a cysteine at the
30th position for TCL and a subsequent desulfurization
protocol to convert cysteine to alanine. We considered that
mutation of Thr to the resultant Ala may not have a negative
effect on folding processes because the N(glycan)-Thr-Ser
sequence is located near the flexible loop position.57
yielded glycopeptide-thioacid 24 in 34% (isolated yield).
Then, the second ligation was performed with 2 equiv of Ala1-
Asn16(glycan)-COSH 24 and peptide 25 (1.0 mM) by TCL in
a buffer solution (0.2 M sodium phosphate, pH 5.7) containing
6 M guanidine-HCl to obtain full-length IL3 peptide 26 (>90%
isolated yield). The full-length glycosyl IL3 Ala1-Asn16(glycan)-
Phe133 26 was subjected to deprotection of the formyl
protecting group and Fmoc protecting group with piperidine,
and then the removal of the internal phenacyl group with zinc
reduction66 afforded glycosyl IL3 polypeptide 27. The
oxidative folding of 27 employed stepwise dialysis conditions
under 6, 3, and 1 M guanidine-HCl solution and a final 10 mM
Tris-HCl buffer solution, where a 3 M guanidine-HCl solution
contained a mixture of 4.0 mM cysteine and 0.5 mM cystine
for disulfide bond formation.52,67 The folding processes were
monitored by LC-MS, and this condition successfully afforded
folded glycosyl IL3 28. After isolation of the folded glycosyl
IL3, CD spectra and a high-resolution mass (17268.3508,
average isotopes) were obtained.
According to the developed strategy, segments 17 and 19
were prepared by the Boc58 and Fmoc59 SPPS conditions
(average yield = 5%), respectively, and then the thioacid-
mediated strategy was examined. All internal peptide cysteines
were protected by the acetamidomethyl (Acm) protecting
group because their thiol groups can prevent essential disulfide
bond formation in DDC and TCL. Lys1-Asn29(glycan)-COSH
18 was obtained by the DDC of 2 equiv of peptide thioacid 17
and glycosyl asparagine thioacid 3 (15 mM), with a 27%
isolated yield. The reaction was monitored by LC-MS, and the
resultant product was confirmed by LC-MS without any
aggregation of peptide 17 or 18 in DMSO. The DMSO
experiments did not need denaturation conditions for short
peptide substrates. After isolation of 18, Lys1-Asn29(glycan)-
COSH 18 and 2 equiv of peptide 19 (1.0 mM) were coupled
by TCL in a buffer solution (0.2 M sodium phosphate, pH 5.7)
containing 6 M guanidine-HCl to yield the protected full-
length CCL1 peptide Lys1-Asn29(glycan)-Lys73 20 (>90%
isolated yield). Desulfurization of the 30th cysteine with a
The in vitro bioassay with glycosyl IL3 28 was performed
based on the cell proliferation of TF-1 cell,65 and the activity of
28 was confirmed to be similar to that of commercially
available nonglycosylated IL3 expressed in E. coli (Figure 6).
The biological assay and analytical data, such as the CD
spectrum and mass spectrum, supported that synthetic glycosyl
IL3 28 employed the native folded structure.
radical initiator60 and subsequent deprotection of the Acm-
61,62
protecting groups of cysteines with PdCl2
and the
phenacyl-protecting group of sialyloligosaccharide with piper-
idine and 2-mercaptoethanol (BME) were performed to yield
glycosyl CCL1 polypeptide 21. Finally, oxidative folding under
redox conditions55,56 at pH 8.0 yielded CCL1 22 with a
sialyloligosaccharide at the 29th position. After isolation of the
folded CCL1, enzyme digestion was performed to analyze
disulfide bond positions (Figure S26). The circular dichroism
(CD) spectrum and the high-resolution mass (10575.9479,
average isotopes) supported the correctly folded structure of
glycosyl CCL1 22. These results indicated that the feasibility of
synthetic processes dramatically improved compared with that
of previous synthesis.55
After successful synthesis of glycosyl CCL1 22, we set out to
synthesize IL3, a cytokine produced by T cells as a regulator of
hematopoiesis.63−65 Glycosylated IL3 is an ideal target to apply
our strategy, as there are no synthetic examples to date.
Normally, the IL3 protein is known to have a complex-type, N-
linked sialyloligosaccharide at the 15th asparagine63−65 (Figure
5A). Although the native IL3 sequence contains Val at the 14th
position, we used alanine at the 14th position to improve DDC
yield after observing a low yield (Figure 2B, entry 2:
compound 12). For the synthesis of IL3, we employed
glycosyl asparagine thioacid 3 and two peptides (Ala1-Ala15-
COSH 23 and Cys17(Npys)-Phe133 25), as shown in Figure
5B. Peptide 25 was prepared in >40% yield (total 4 steps) by
an E. coli expression system and several modification steps,
including pyridyl disulfide formation of the N-terminal thiol
protected by the phenacyl (Pac) protecting group because the
thiol group can prevent essential disulfide bond formation in
DDC and TCL. A short segment 23 was synthesized using safe
Boc SPPS conditions (Figure S28).58 The DDC of 2 equiv of
Ala1-Ala15-COSH 23 with glycosyl asparagine thioacid 3
Figure 6. TF-1 Cell proliferation assay. TF-1 cells were cultured for
72 h. Red and black color shows the activity of synthetic glycosylated
IL3 28 and IL3 expressed in E. coli., respectively. Error bars are SD (n
= 6). Relative proliferation was calculated by luminescence.
DISCUSSION
■
The crucial finding in developing this new chemoselective
convergent synthesis of glycoproteins was how to combine
glycosyl asparagine thioacid 3 with a peptide thioacid without
polymerization. We found that glycosyl asparagine thioacid 3
unexpectedly did not yield polymerization but afforded the
desired glycopeptide thioacid. This phenomenon was also
observed with asparagine thioacid with mono GlcNAc. Even
the monosaccharide did not yield any polymerization of
glycosyl asparagine thioacid. Therefore, the inhibition of
polymerization seemed to be due to the steric repulsion
between monosaccharides or oligosaccharides at the stage of
either the S−N acyl shift from the diacyl disulfide intermediate
31−32 or the formation of the diacyl disulfide intermediate 30
itself (Figure 7). In addition, we considered that fluctuation in
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J. Am. Chem. Soc. 2021, 143, 10157−10167