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\begin{document}

\title{Calcium iodide mediated coupling of complex fluorooligosaccharides to
phenolic peptide}



\author[1]{Pei Liu}%
\author[1]{Xing Quan}%
\author[1]{Fangyu Wei}%
\author[1]{Xianjun Deng}%
\author[1]{Jie Shen}%
\author[1]{Jiansong Cheng}%
\affil[1]{Nankai University}%


\vspace{-1em}



  
  \date{June 14, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Besides serving as therapeutic agents and building blocks for
glycoprotein synthesis, homogeneous synthetic glycopeptides also benefit
understanding function of specific glycoform. However, the selectivity
coupling of oligosaccharides to a given peptide remains challenging
despite the fact that the synthesis of structure-defined complex
oligosaccharides has been greatly facilitated by chemoenzymatic based
approaches. Herein, a Ca\textsuperscript{2+}-promoted glycosylation
approach was developed to exclusively modify phenolic peptide using a
panel of biologically important glycans.%
\end{abstract}%



\sloppy


\textbf{Cite this paper:}\emph{Chin. J. Chem.} \textbf{2023} , \emph{41}
, XXX---XXX. \textbf{DOI: 10.1002/cjoc.202300XXX}

Calcium iodide mediated coupling of complex fluorooligosaccharides to
phenolic peptide

Pei Liu,\emph{\textsuperscript{a}} Xing
Quan,\emph{\textsuperscript{a}}Fangyu
Wei,\emph{\textsuperscript{a}}Xianjun Deng,\emph{\textsuperscript{a}}
Jie Shen,*\textsuperscript{,\emph{a}} and Jiansong
Cheng*\textsuperscript{,\emph{a}}

\textsuperscript{a} State Key Laboratory of Medicinal Chemical Biology,
College of Pharmacy, Nankai university, Tianjin 300353, PR China\selectlanguage{english}
\begin{longtable}[]{@{}l@{}}
\toprule
\textbf{Keywords}\tabularnewline
\midrule
\endhead
Calcium iodide \textbar{} \selectlanguage{greek}α-\selectlanguage{english}fluoroglycoside \textbar{} glycosylation
\textbar{} coupling \textbar{} glycopeptide\tabularnewline
\textbf{Comprehensive Summary}\tabularnewline
\vtop{\hbox{\strut }\hbox{\strut Besides serving as therapeutic agents
and building blocks for glycoprotein synthesis, homogeneous synthetic
glycopeptides also benefit understanding function of specific glycoform.
However, the selectivity coupling of oligosaccharides to a given peptide
remains challenging despite the fact that the synthesis of
structure-defined complex oligosaccharides has been greatly facilitated
by chemo-enzymatic based approaches. Herein, a
Ca\textsuperscript{2+}-promoted glycosylation approach was developed to
exclusively modify phenolic peptide using a panel of biologically
important glycans.}}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

Background and Originality Content

Glycosylation is a ubiquitous post-translational modification and a key
mechanism in determining chemical diversity of natural
products.\textsuperscript{1, 2} Protein glycosylation involving the
attachment of complex sugar moieties (glycans) to amino acid side chains
plays important roles in protein folding and a diverse range of
biological processes.\textsuperscript{1, 3} Glycosylation can also
modulate the physiological properties of small molecules and peptides,
such as improved metabolic stability, subcellular localization,
ligand--target interactions and often their
bioactivity.\textsuperscript{4, 5}

Other than utilizing in the synthesis of large glycopeptides through
native chemical ligation (NCL),\textsuperscript{6}synthetic
glycopeptides are extant as important biochemical probes and therapeutic
agents.\textsuperscript{5, 7} Typically, glycopeptides are prepared
using solid-phase peptide synthesis through incorporating
protected-glycosyl residues. The selective glycosylation of unprotected
peptides in aqueous solvents, however, has been limited to `click'
S-glycosylation of cysteine,\textsuperscript{8, 9} aspartylation of
glycol-amide (such as
Man9GlcNAc2-NH\textsubscript{2})\textsuperscript{10}or enzymatical
introduction of glucose to a recognizable N- or O-glycosylation
site.\textsuperscript{11-13}

More recently, an approach that enable aqueous O-glycosylation of
peptide-embedded tyrosine employing glycosyl fluorides has been
reported.\textsuperscript{14} Originally, the glycosylation reactions
have been discovered by Miller between \selectlanguage{greek}α-\selectlanguage{english}glycosyl fluorides and sucrosyl
acceptors in the presence of calcium ion and
trimethylamine.\textsuperscript{15} The formation of O-\selectlanguage{greek}β-\selectlanguage{english}glucosyl
products evidenced that both sucrose and Tyr O-glycosylation proceed via
a S\textsubscript{N}2-type substitution. In nature, GlcNAc and GalNAc
are most common hexosamines and initiating sugars of N- and mucin-type
O-glycans, respectively. More recently, GalNAc O-glycosylation of Tyr
residues were also identified in human and murine
proteins.\textsuperscript{16-18}It remains to be investigated whether
hexosamines can be installed onto Tyr residue in the same manner. Plus,
chemo-enzymatic methods, which employ a range of glycosyl transferases
to modify a synthetic oligosaccharide precursor, has greatly facilitated
the stereo controlled synthesis of complex carbohydrates in two
decades.\textsuperscript{19-22}Therefore, it is highly intriguing to
investigate whether the Ca\textsuperscript{2+}-promoted glycosylation
can be extended for coupling structurally well-defined oligosaccharides
with peptide-embedded Tyr (Scheme 1).

\textbf{Scheme 1} Tyr O-glycosylation in water
\emph{via}Ca\textsuperscript{2+} promotion of fluoride donors\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

Results and Discussion

We firstly synthesized \selectlanguage{greek}α-\selectlanguage{english}fluoroGlcNAc and \selectlanguage{greek}α-\selectlanguage{english}fluoroGalNAc, but found that
neither of them could give glycosylation product of leu-enkephalin,
being an opioid receptor ligand, under the promotion of
Ca(OH)\textsubscript{2}. Next, their fluoro-analogs with amine or azido
substitution at C-2 were evaluated for their potentials as glycosyl
donors (Figure 1). To our delight, GlcNH\textsubscript{2} modified
leu-enkephalin formed from \selectlanguage{greek}α-\selectlanguage{english}fluoroGlcNH\textsubscript{2} in a low yield
(10\%) although \selectlanguage{greek}α-\selectlanguage{english}fluoroGalNH\textsubscript{2}

\par\null

\textbf{Figure 1} Eighteen synthesized \selectlanguage{greek}α-\selectlanguage{english}fluoro donors of
monosaccharides

\textbf{Table} \textbf{1}Optimization of Ca\textsuperscript{2+}
promoter: focusing on anions\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Entry\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Ca\textsuperscript{2+} promoter\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Solvent\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
HPLC Yield (\%)\emph{\textsuperscript{a}}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Ca(OH)\textsubscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
H\textsubscript{2}O\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
10\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Ca-EDTA\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
4\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
3\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Ca-Levulinate\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
8\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
4\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Ca-Ascorbate\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
N.D.\emph{\textsuperscript{d}}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Ca-Glycerophosphate\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
45 wt\% NMe\textsubscript{3} in H\textsubscript{2}O\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
6\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
6\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Ca(ClO\textsubscript{4})\textsubscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
11\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
7\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Ca(OTf)\textsubscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
11\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
8\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Ca(H\textsubscript{2}PO\textsubscript{4})\textsubscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
4\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
9\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Ca(OAc)\textsubscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
13\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
10\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
CaCl\textsubscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
13\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
11\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
CaBr\textsubscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
19\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textbf{12}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textbf{CaI\textsubscript{2}}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textbf{37}\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{\emph{a}} Yields were calculated based on
\selectlanguage{greek}λ\selectlanguage{english}\textsubscript{214 nm}-peak areas of peptide\textbf{19} and
glyco-peptide \textbf{4P} in HPLC spectra.\emph{\textsuperscript{d}}
N.D. refers to not detected.

led to no product. Previous studies suggested that the glucosylation of
sucrose requires a special array of substrate hydroxyl groups (such as
OH-2, OH-1' and OH-3') that presumably engage both
Ca\textsuperscript{2+} and sucrose with multiple
contacts.\textsuperscript{15} OH-2 on glycosyl donors could also involve
an analogous interaction and in consequence affected the glycosylation
efficiency.

In order to improve the product yield, a series of metal basic or
combinations of Ca\textsuperscript{2+} ion and aqueous amine bases were
screened (Table 1 and Table S1). Finally, a significant improved
conversion of GlcNH\textsubscript{2} modified leu-enkephalin (37\%) was
achieved under the optimized condition in the present of
CaI\textsubscript{2} and trimethylamine. O-GlcNacyl-leu-enkephalin was
then quantitatively obtained by one step acylation. Meanwhile,
CaI\textsubscript{2} exhibited good compatibility to other
fluoromonosaccharides lacking either OH-2 or OH-6, such as GlcNAc-F,
GalNH\textsubscript{2}-F and GlcA-F (Table 2). All monosaccharide
coupled leu-enkephalin were characterized by high resolution mass
spectroscopy (Supplementary pages 13-18). Only \selectlanguage{greek}β-\selectlanguage{english}anomer products were
observed when fluoromonosaccharides including \selectlanguage{greek}α-\selectlanguage{english}fluoro Glc, Gal,
derivatives of Glc and Gal, and Fuc were afforded. In contrast, \selectlanguage{greek}α-\selectlanguage{english}fluoro
Man and Rha involved reaction produced \selectlanguage{greek}α-\selectlanguage{english}anomer products that is
consistent with a proposed double inversion process involving the axial
C2-OH group.\textsuperscript{14}

\textbf{Table 2} Tyr O-glycosylation of \selectlanguage{greek}α-\selectlanguage{english}fluorides (\textbf{1-18} )
with YGGFL peptide: CaI\textsubscript{2} /NMe\textsubscript{3} vs.
Ca(OH)\textsubscript{2}

\par\null\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\selectlanguage{greek}α-\selectlanguage{english}fluoride donor\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\selectlanguage{greek}α-\selectlanguage{english}fluoride donor\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Glyco-peptide product\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Glyco-peptide product\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Glyco-peptide product\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Product structure\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
HPLC yield (\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
HPLC yield (\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
CaI\textsubscript{2}\emph{\textsuperscript{a}}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Ca(OH)\textsubscript{2}\emph{\textsuperscript{a}}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Glc- type\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\textbf{1}\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\selectlanguage{greek}β-\selectlanguage{english}Glc-YGGFL (\textbf{1P})\emph{\textsuperscript{b}}\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
100\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
85\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{2}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\selectlanguage{greek}β-\selectlanguage{english}GlcNAc-YGGFL (\textbf{2P})\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
22\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N.D.\emph{\textsuperscript{d}}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{3}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\emph{\textsuperscript{e}}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N.D.\emph{\textsuperscript{d}}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N.D.\emph{\textsuperscript{d}}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{4}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\selectlanguage{greek}β-\selectlanguage{english}GlcNH\textsubscript{2}-YGGFL (\textbf{4P})\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
37\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
10\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{5}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\selectlanguage{greek}β-\selectlanguage{english}GlcNH\textsubscript{2}-YGGFL(\textbf{4P})\emph{\textsuperscript{c}}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
31\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
30\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Gal- type\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\textbf{6}\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\selectlanguage{greek}β-\selectlanguage{english}Gal-YGGFL (\textbf{6P})\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
72\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
30\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{7}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\emph{\textsuperscript{e}}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N.D.\emph{\textsuperscript{d}}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N.D.\emph{\textsuperscript{d}}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{8}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\emph{\textsuperscript{e}}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N.D.\emph{\textsuperscript{d}}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N.D.\textsuperscript{d}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{9}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\selectlanguage{greek}β-\selectlanguage{english}GalNH\textsubscript{2}-YGGFL (\textbf{9P})\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
31\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N.D.\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{10}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\selectlanguage{greek}β-\selectlanguage{english}GalNH\textsubscript{2}-YGGFL(\textbf{9P})\emph{\textsuperscript{c}}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
26\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
11\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Man- type\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\textbf{11}\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\selectlanguage{greek}α-\selectlanguage{english}Man-YGGFL (\textbf{11P})\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
49\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
53\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{12}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\emph{\textsuperscript{e}}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N.D.\emph{\textsuperscript{d}}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N.D.\emph{\textsuperscript{d}}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{13}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\emph{\textsuperscript{e}}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N.D.\emph{\textsuperscript{d}}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N.D.\emph{\textsuperscript{d}}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{14}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N/A\emph{\textsuperscript{e}}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N.D.\emph{\textsuperscript{d}}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N.D.\emph{\textsuperscript{d}}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
deoxy type\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\textbf{15}\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
N/A\emph{\textsuperscript{e}}\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
N.D.\emph{\textsuperscript{d}}\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
N.D.\emph{\textsuperscript{d}}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{16}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\selectlanguage{greek}α-\selectlanguage{english}Rha-YGGFL(\textbf{16P})\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N.D.\emph{\textsuperscript{d}}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
42\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{17}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\selectlanguage{greek}β-\selectlanguage{english}Fuc-YGGFL (\textbf{17P})\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
69\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
57\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
GlcA\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{18}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\selectlanguage{greek}β-\selectlanguage{english}GlcA-YGGFL (\textbf{18P})\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
31\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
N.D.\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

\textsuperscript{\emph{a}} Yields were calculated based on
\selectlanguage{greek}λ\selectlanguage{english}\textsubscript{214 nm}-peak areas of peptide \textbf{19} and
corresponding product in HPLC spectra.\emph{\textsuperscript{b}}Reaction
time=10 min. \textsuperscript{\emph{c}} For donors \textbf{5}and
\textbf{10} , NTFA group was converted to amino group under the reaction
conditons, and therefore their glycosylation products were the same to
donors \textbf{4} and \textbf{9} respectively.\textsuperscript{\emph{d}}
N.D. refers to not detected.\textsuperscript{\emph{e}} N/A refers to not
applicable.

\textbf{F} \textbf{igure 2} Synthesis of oligosaccharide coupled
leu-enkephalin\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image9/image9}

\end{center}
\end{figure}

\textbf{Scheme 2} Synthesis of \selectlanguage{greek}α-\selectlanguage{english}fluoroLacNAc (\textbf{35} ) and
\selectlanguage{greek}α-\selectlanguage{english}fluoroLewis X (\textbf{36} )

Reaction conditions: (c) One-pot multienzyme: Gal, SpGalK, SpGalU and
NmlgtB; (d) Ac\textsubscript{2}O and Pyridine; (e) NIS, DAST and DCM;
(f) NaOMe and MeOH; (g) One-pot multienzyme: Fuc, BfFKP and Hp3/4FT.

With the optimized condition established, a number of biologically
important glycans were ready to prepared by using \selectlanguage{greek}α-\selectlanguage{english}fluorolactosyl or
suitable \selectlanguage{greek}α-\selectlanguage{english}fluoromonosaccharides as starting materials.
Glycosyltransferases that catalyze glycosidic bond formation in high
regio- and stereo-selectivity often remain relative strict substrate
specificity as well. In this study, most enzymes except for BiGalHexNAcP
and LgtB can well recognize \selectlanguage{greek}α-\selectlanguage{english}fluoroglycosides. BiGalHexNAcP, being a
galactosyl \selectlanguage{greek}β\selectlanguage{english}1,3-N-acetyl-hexosamine phosphorylase
from\emph{Bifidobacterium infantis} , is capable of reversely taking
both GlcNAc and GalNAc as acceptors to form \selectlanguage{greek}β\selectlanguage{english}1,3-linked
galactosides.\textsuperscript{23} Herein, we unexpectedly found that
neither \selectlanguage{greek}α-\selectlanguage{english}fluoroGlcNH\textsubscript{2} nor
\selectlanguage{greek}α-\selectlanguage{english}fluoroGalNH\textsubscript{2} was acceptable for BiGalHexNAcP. However,
\selectlanguage{greek}α-\selectlanguage{english}fluoroGlcNTFA and \selectlanguage{greek}α-\selectlanguage{english}fluoroGalNTFA that bearing a larger
trifluoroacetamido group at C2 were able to be efficiently glycosylated
by the enzyme.

At the same time, we found that CaI\textsubscript{2}-mediated
glycosylation reactions that employ either \selectlanguage{greek}α-\selectlanguage{english}fluorohexosamines or their
\selectlanguage{greek}α-\selectlanguage{english}fluoro N-trifluoroacetyl derivatives performed in similar conversions
and generated identical products, namely, hexosamine modified
leu-enkephalins (Table 2). Therefore, carbohydrate determinants of TF
(\textbf{29} ), sialyl TF (\textbf{32} ), Sialyl-Tn (\textbf{31} ),
Lacto-N-biose I (\textbf{33} ), Lewis a (\textbf{34} ) and H antigen
(type 3, \textbf{30} ) were prepared with \selectlanguage{greek}α-\selectlanguage{english}fluoroGlcNTFA or
\selectlanguage{greek}α-\selectlanguage{english}fluoroGalNTFA as starting materials (Supplementary pages 27-32).
Because \emph{Neisseria meningitidis} derived \selectlanguage{greek}β\selectlanguage{english}1,4-galactosyltransferase
LgtB can only recognize \selectlanguage{greek}β-\selectlanguage{english}STolGlcNTFA but \selectlanguage{greek}β-\selectlanguage{english}STolGlcNH\textsubscript{2},
\selectlanguage{greek}α-\selectlanguage{english}fluoroGlcNTFA or \selectlanguage{greek}α-\selectlanguage{english}fluoroGlcNH\textsubscript{2}, the
Gal\selectlanguage{greek}β\selectlanguage{english}1,4GlcNTFA\selectlanguage{greek}β\selectlanguage{english}STol disaccharide was firstly synthesized and then
converted to Gal\selectlanguage{greek}β\selectlanguage{english}1,4GlcNTFA\selectlanguage{greek}α\selectlanguage{english}fluoride (\textbf{35} ) which can be further
fucosylated to form Lewis X fluoride (Scheme 2). Other Kdn, Neu5Ac or
Neu5Gc containing sialosides were synthesized starting from
\selectlanguage{greek}α-\selectlanguage{english}fluorolactose (Supplementary pages 23-27). All reactions were carried
out in a one-pot multienzyme fashion where sugar nucleotides were
generated \emph{in situ} by using nucleotidyltransferases and/or
glycokinases and transferred to individual acceptors by corresponding
glycosyltransferases.\textsuperscript{24}

As shown in Figure 2, both \selectlanguage{greek}α-\selectlanguage{english}fluorolactose (\textbf{20} ) and
\selectlanguage{greek}α-\selectlanguage{english}fluoroGb3 (\selectlanguage{greek}α\selectlanguage{english}F-Gal\selectlanguage{greek}α\selectlanguage{english}1,4Lac, \textbf{24} ) involved glycosylation
proceeded in excellent yields in the presence of
Ca(OH)\textsubscript{2}. However, for \selectlanguage{greek}α-\selectlanguage{english}fluorosialosides,
Ca(OH)\textsubscript{2} and CaI\textsubscript{2}-promoted glycosylation
showed distinct compatibility or efficiency. With
Ca(OH)\textsubscript{2}, \selectlanguage{greek}α\selectlanguage{english}2,6KDN- and \selectlanguage{greek}α\selectlanguage{english}2,6Neu5Gc-modified Lactosyl
leu-enkephalins were obtained in relative low yields (19\% and 39\%,
respectively). \selectlanguage{greek}α-\selectlanguage{english}fluorosialosides containing Neu5Ac even failed to
generate glycosyl products regardless of the sialyl linkages. However,
exchanging Ca(OH)\textsubscript{2} with CaI\textsubscript{2} led to near
complete conversion of leu-enkephalin to GM3 (\textbf{25} ) modified
glycopeptide. \selectlanguage{greek}α\selectlanguage{english}2,6sialylated lactosyl leu-enkephalins decorated by all
three natural forms including KDN (\textbf{23} ), Neu5Ac (\textbf{21} )
and Neu5Gc (\textbf{22} ) also formed in moderate conversations
(43\%-54\%) under the promotion of CaI\textsubscript{2}. Apparently, the
hydrogen-bonding network of substrate is more dependable for
Ca(OH)\textsubscript{2}-mediated glycosylation. Notably,
\selectlanguage{greek}α\selectlanguage{english}2,6Neu5Ac(\selectlanguage{greek}α\selectlanguage{english}2,3Neu5Ac)lacosyl fluoride (\textbf{28} ) was an efficient
glycosyl donor in comparison of totally inactive
\selectlanguage{greek}α\selectlanguage{english}2,8Neu5Ac\selectlanguage{greek}α\selectlanguage{english}2,3Neu5Aclacosyl fluoride (\textbf{27} ) under the optimized
condition, suggesting that stereo-configuration of glycosyl donor might
also affect the glycosyl transfer.

All \selectlanguage{greek}α-\selectlanguage{english}fluorooligosaccharides initiating with \selectlanguage{greek}α-\selectlanguage{english}fluoroGlcNTFA or
\selectlanguage{greek}α-\selectlanguage{english}fluoroGalNTFA underwent glycosylation with CaI\textsubscript{2} in
spite of relative low yields. The moieties directly linked to
leu-enkephalins were identified as GlcNH\textsubscript{2} or
GalNH\textsubscript{2}. The hexosamine containing glycosyl
leu-enkephalins were further acylated to give the final products almost
quantitatively. Thus, TF (\textbf{29} ), sialyl TF (\textbf{32} ),
sialyl Tn (\textbf{31} ), Lacto-N-biose I (\textbf{33)} , Lewis a
(\textbf{34} ) and H antigen (type 3, \textbf{30} ) coupled
leu-enkephalins were synthesized in two sequential steps. All
oligosaccharide coupled glycopeptides were characterized by high
resolution mass spectroscopy (Supplementary pages 32-39). \selectlanguage{greek}α-\selectlanguage{english}fluoroGM2
(\textbf{26} ) that is a branched ganglioside with residues of Neu5Ac
and GalNAc at the non-reducing end was not an active glycosyl donor in
the Ca\textsuperscript{2+}-promoted reaction.

Besides most abundant Asn N-glycosylation and Ser/Thr O-glycosylation,
natural glycosyl modification also occurs at Trp, Tyr and hydroxylated
lysine. Enzymes that catalyze peptide glycosylation have mostly evolved
stringent selectivity on both glycosyl donor and acceptor. For example,
a N-glycosyltransferase derived from \emph{Actinobacillus
pleuropneumoniae} (ApNGT) is capable of transferring Glc but substituted
GlcNH\textsubscript{2}, GlcN\textsubscript{3} and GlcNAc to an
asparagine residue in the conserved Asn-X-Ser/Thr (where X [?] Pro)
sequon.\textsuperscript{25} The Glc-tagged peptide/protein can be
further remodelled by various endo-\selectlanguage{greek}β-\selectlanguage{english}N-acetylglucosaminidases (ENGases)
to exclusively add a preassembled high-mannose type or complex type
N-glycan.\textsuperscript{12} Another example is \emph{Legionella}
effector SetA that site-specifically introduces O-glucose onto a
Ser/Thr-X-Leu-Pro/Gly sequence motif.\textsuperscript{13} In contrast,
CaI\textsubscript{2}-mediated glycosylation exhibits excellent tolerance
toward an array of complex glycosyl donors, especially for Neu5Ac
attached glycosyl fluorides. Glycopeptides conjugated with
N-acetyl-hexosamine initializing carbohydrates can alternately achieved
by employing its hexosamine or N-trifluoroacetyl hexosamine derivatives
as donors followed by an acetylation reaction. Thus,
Ca\textsuperscript{2+}-promoted glycosylation was able to be further
generalized and turned out to be an alternate and facile glycosylation
mean other than chemical or enzymatic formation of Cys-O-, Asn-N- or
Ser/Thr-O-glycosides. CaI\textsubscript{2}-mediated coupling approach
offers the exceptional flexibility between glycosyl donors and peptides,
while the regio- and stereo-selectivity within oligosaccharides was well
defined by glycosyltransferases. In addition, optimized reaction
condition with CaI\textsubscript{2} instead of Ca(OH)\textsubscript{2}
allowed to lower the equivalent of glycosyl donors used in reaction. For
example, reducing lactosylfluoside by half did not obviously impair the
yield (Supplementary page 33).

Conclusions

Though the synthesis of structure-defined complex oligosaccharides has
been greatly facilitated by chemo-enzymatic based approaches, the
selectively coupling of these oligosaccharides to a given peptide
remains challenging. Herein, a series of \selectlanguage{greek}α-\selectlanguage{english}fluorooligosaccharides were
readily produced in a one-pot multienzyme fashion, and 14 of them
excepting for GD3 (\textbf{27} ), GM2 (\textbf{26} ) and Lewis X
(\textbf{36} ) fluoroglycosides were successfully used to generate their
corresponding glycopeptides in Ca\textsuperscript{2+}-promoted
glycosylation reaction.

Experimental

One pot multienzyme synthesis of \selectlanguage{greek}α-\selectlanguage{english}fluorooligosaccharides: A mixture of
the glycosyl donor, acceptor of \selectlanguage{greek}α-\selectlanguage{english}fluorosaccharide, nucleoside
5'-triphosphate, a suitable glycokinase, nucleotidyltransferase and
glycosyltransferase was buffered with Tris-HCL and incubated at 37 \selectlanguage{ngerman}°C.
The product formation was monitored by TLC. The reaction was stopped by
adding the same volume of ice-cold ethanol and incubated at 4 °C for 30
min. The mixture was centrifuged and the precipitates were removed. The
supernatant was concentrated and passed through a silica gel column
followed by a Bio-Gel P-2 gel filtration column purifcation to give the
product.

General synthesis of glycopeptides procedure: To a flask were
sequentially added YGGFL peptide (0.3 \selectlanguage{greek}μ\selectlanguage{english}mol, 1.0 equiv), a fluoro donor
(0.3 mmol, 1000 equiv.), CaI\textsubscript{2} (0.3 mmol, 1000 equiv.)
and 45 wt\% NMe\textsubscript{3}/H\textsubscript{2}O (300 \selectlanguage{greek}μ\selectlanguage{english}L). The flask
was sealed with a rubber septum, and the reaction mixture was stirred at
room temperature for 12 h. The resulting mixture was then centrifuged at
12000 rpm for 10 min. Supernatant was analyzed by HPLC (Hypersil Gold
C18 5 \selectlanguage{greek}μ\selectlanguage{english}m 4.8 mm \selectlanguage{ngerman}× 250 mm, 1 mL/min flow rate; gradient = 5 \% B for 5
min, linear gradient of 5 \% B to 50 \% B in 11.5 min, held at 50 \% for
a further 5 min; buffer A = 0.5 \selectlanguage{english}%0 TFA--H\textsubscript{2}O, buffer B =
0.5 \selectlanguage{english}%0 TFA--CH\textsubscript{3}CN). Alternatively,
Ca(OH)\textsubscript{2} (0.3 mmol, 1000 equiv.) in 300 \selectlanguage{greek}μ\selectlanguage{english}L water was used
to replace CaI\textsubscript{2} in 45 wt\%
NMe\textsubscript{3}/H\textsubscript{2}O.

Supporting Information

The supporting information for this article is available on the WWW
under https://doi.org/10.1002/cjoc.2023xxxxx.

Acknowledgement

We are grateful for financial support from the National Key R\&D Program
of China (2018YFE0200501) and the National Natural Science Foundation of
China (21977055).

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  Xu, Y.; Wu, Z.; Zhang, P.; Zhu, H.; Zhu, H.; Song, Q.; Wang, L.; Wang,
  F.; Wang, P. G.; Cheng, J. A novel enzymatic method for synthesis of
  glycopeptides carrying natural eukaryotic N-glycans. \emph{Chem.
  Commun.} \textbf{2017}, \emph{53}, 9075-9077.
\end{enumerate}\selectlanguage{english}
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\toprule
Manuscript received: XXXX, 2023 Manuscript revised: XXXX, 2023
Manuscript accepted: XXXX, 2023 Accepted manuscript online: XXXX, 2023
Version of record online: XXXX, 2023\tabularnewline
\bottomrule
\end{longtable}

The Authors\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
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& After acceptance, please insert a group photo of the authors taken
recently. &\tabularnewline
& \textbf{Left to Right:} Authors Names &\tabularnewline
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\textbf{Entry for the Table of Contents}\selectlanguage{english}
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\textbf{Calcium iodide mediated coupling of complex
fluorooligosaccharides to phenolic peptide} Pei Liu, Xing Quan, Fangyu
Wei, Xianjun Deng, Jie Shen,* and Jiansong Cheng* \emph{Chin. J. Chem.}
\textbf{2023}, \emph{41}, XXX---XXX. \textbf{DOI:
10.1002/cjoc.202300XXX}\tabularnewline
\selectlanguage{greek}α-\selectlanguage{english}fluorooligosaccharides were chemo-enzymatically synthesized and
further coupled with peptide-embedded Tyr under the promotion of
CaI\textsubscript{2} in water.\tabularnewline
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