Chemoenzymatic synthesis of neoglycoproteins driven by the assessment of protein surface reactivity

Article abstract of DOI:10.1039/c4ra11131a

In this paper a series of 2-iminomethoxyethyl mannose-based mono- and disaccharides have been synthesized by a chemoenzymatic approach and used in coupling reactions with ε-amino groups of lysine residues in a model protein (ribonuclease A, RNase A) to give semisynthetic neoglycoconjugates. In order to study the influence of structure of the glycans on the conjugation outcomes, an accurate characterization of the prepared neoglycoproteins was performed by a combination of ESI-MS and LC-MS analytical methods. The analyses of the chymotryptic digests of the all neoglycoconjugates revealed six Lys-glycosylation sites with a the following order of lysine reactivity: Lys 1 Lys 91 ? Lys 31 > Lys 61 ? Lys 66. A computational analysis of the reactivity of each lysine residue has been also carried out considering several parameters (amino acids surface exposure and pK_a, protein flexibility). The in silico evaluation seems to confirm the order in lysine reactivity resulting from proteomic analysis.

Full text of DOI:10.1039/c4ra11131a

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Chemoenzymatic synthesis of neoglycoproteins
driven by the assessment of protein surface
reactivity†
Cite this: RSC Adv., 2014, 4, 56455
a
b
a
a
a
c
T. Bavaro,* M. Filice,* C. Temporini, S. Tengattini, I. Serra, C. F. Morelli,
a
a
G. Massolini and M. Terreni
In this paper a series of 2-iminomethoxyethyl mannose-based mono- and disaccharides have been
synthesized by a chemoenzymatic approach and used in coupling reactions with 3-amino groups of
lysine residues in a model protein (ribonuclease A, RNase A) to give semisynthetic neoglycoconjugates.
In order to study the influence of structure of the glycans on the conjugation outcomes, an accurate
characterization of the prepared neoglycoproteins was performed by a combination of ESI-MS and LC-
MS analytical methods. The analyses of the chymotryptic digests of the all neoglycoconjugates revealed
six Lys-glycosylation sites with a the following order of lysine reactivity: Lys 1 [ Lys 91 y Lys 31 > Lys
Received 24th September 2014
Accepted 23rd October 2014
6
1 y Lys 66. A computational analysis of the reactivity of each lysine residue has been also carried out
DOI: 10.1039/c4ra11131a
considering several parameters (amino acids surface exposure and pK , protein flexibility). The in silico
a
www.rsc.org/advances
evaluation seems to confirm the order in lysine reactivity resulting from proteomic analysis.
4
immunodiagnostic and therapeutic reagents can be derived.
Introduction
For example, conjugation of antigenic proteins from Mycobac-
Glycoproteins play a pivotal role in many physiological and terium tuberculosis with arabino-mannane polysaccharides has
1
pathological processes. Their function and versatility have long been proposed for developing highly immunogenic glyco-
3
suggested the power of these glycosylated biomolecules in conjugate vaccines against tuberculosis.
therapeutic strategies, with particular relevance in the devel-
Chemical routes to the synthesis of neoglycoproteins can
2,3
opment of carbohydrate-based vaccines. The glycosylation or involve random or site-selective modications of protein
other controlled chemical modications of proteins, such as surface expecting the nal covalent linkage of the glycans via
1,6
PEGylation and acylation, can dramatically improve their their reducing end interposed by a reactive spacer. Usually,
4
physical and biological properties. For instance, chemical methods used for the synthesis of neoglycoproteins permit to
conjugation with suitable glycans has been exploited for the conjugate the carbohydrate to a protein by the alkylation of
2
,7,8
production of therapeutic enzymes with improved activity and nucleophilic side chains of cysteine or lysine residues.
5
reduced in vivo metabolism. Neoglycoproteins, which contain Cysteine-based chemical ligation has been signicantly used to
unnatural linkages between protein and saccharides, can also fully synthesize several neoglycoproteins and in some cases the
8
provide carbohydrate antigens and immunogens from which incorporation, by site-directed mutagenesis, of one reactive
cysteine residue as a chemoselective tag at a preselected posi-
9
tion within the given protein was requested. A different
a
Department of Drug Sciences and Italian Biocatalysis Center, University of Pavia, via
strategy, known as indiscriminate glycosylation, relies on the
Taramelli 12, I-27100 Pavia, Italy. E-mail: teodora.bavaro@unipv.it
b
use of 2-iminomethoxyethyl thioglycosides (IME) and takes
Departamento de Biocatalisis, Instituto de Catalisis (ICP-CSIC), Marie Curie 2
Cantoblanco Campus UAM, 28049 Madrid, Spain. E-mail: marco.lice1@gmail.com
advantage from the high abundance of lysine residues on the
c
Department of Chemistry and Italian Biocatalysis Center, University of Milano, via protein surface, allowing the introduction of various saccharide
Golgi 19, I-20133 Milano, Italy
Electronic supplementary information (ESI) available: Regioselective hydrolysis
8
units for each molecule of protein. For example, Pearce and co-
†
workers employed this methodology to incorporate up to 20 000
of acetylated mannopyranosydes 1–3 catalyzed by immobilized Candida rugosa
lipase. Glycosylation study for the synthesis of disaccharides 5, 6 and 8 at
different reaction conditions. Different cycles of hydrolysis reaction of
10
glycan moieties onto the surface of adenovirus. Although
rstly introduced about 30 years ago, this simple and well-

compound 3 catalyzed by CRL-ECOD. Analysis of the solvent accessibility (ASA) established method can now be revalued in vaccine develop-
of the amino acids of RNase
localization of Lys residues of RNase A in monomeric form. H and C NMR
spectra of disaccharides 5, and 8. ESI-MS deconvoluted spectra of
A (PDB code 1FS3). Surface analysis and
ment in order to strengthen immune response by the use of
polyantigenic glycoconjugates prepared with a carrier protein
bearing a high number of lysines. A high glycosylation degree
1
13
6
11
n
neoglycoproteins 13–16. List of glycopeptides identied by LC-ESI-MS from
chymotryptic digestion of 13–16. See DOI: 10.1039/c4ra11131a
can also be useful in order to improve the therapeutic efficiency
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of bioactive proteins, but, in this case also the area of the
protein responsible of the biological activity could be involved
in the chemical modication.
Results and discussion
Synthesis of the target IME-saccharides for neoglycoproteins
preparation
The coupling reaction between oligosaccharides and a
protein by a non-selective glycosylation approach, allows the Mannopyranose acts as a pattern-recognition molecule in
19
formation of different randomly modied glycoforms, several human and bacterial glycoproteins. For example,
including the possible glycosylation of areas of the protein human immunoglobulins contain high mannose- and/or
surface associated with its biological activity. This implies the hybride-type oligosaccharides linked to asparagine or serine/
20
need of a detailed characterization of the conjugation products. threonine residues on the heavy chain of the protein, while,
In addition to the complex analysis required for the char- lipoarabinomannan (LAM), the major surface antigen of Myco-
acterization of glycoconjugates, it should be also considered bacterium tuberculosis, presents an arabinomannan core capped
21
that the development of new drugs based on complex structures with mono- , di- and trimannosides.
such as neoglycoproteins is still hampered by the complex
In this work, peracetylated mannose and derivatives thereof
multi-step strategies required for sugar preparation. Nowadays, have been thus subjected to enzymatic hydrolysis, in order to
different approaches are available to facilitate the access to pure obtain carbohydrate moieties bearing only one free hydroxyl
oligosaccharides, such as the automated solid-phase group in specic positions. The obtained compounds have been
2,12
synthesis.
Alternative enzymatic or chemoenzymatic then used as key building blocks for the synthesis of mannose-
approaches has also gained popularity because of intrinsic based disaccharides. First, the hydrolyses of the peracetylated a-
properties of the biocatalysts. In this vein, the lipase-assisted mannopyranoside (1) (Scheme 1), catalyzed by different lipases
chemoenzymatic synthesis of biologically relevant di- and adsorbed on hydrophobic support, were studied (Table S1, see
13–15
trisaccharides has been recently reported.
In this work, we propose a useful multidisciplinary
ESI†).
Among all the enzymes tested (lipases from Candida rugosa,
approach for a rapid preparation and analytical characteriza- Pseudomonas uorescens, Aspergillus niger, Pseudomonas cepa-
tion of complex neoglycoproteins obtained by indiscriminate cia, Pancreas porcine and Rhizomucor miehei), only the lipase
glycosylation of lysine 3-amino groups of ribonuclease A from Candida rugosa (CRL) showed both a good activity and a
(
RNase A), selected as model protein. For the synthesis of the useful selectivity, affording 1,2,3,4-tetra-O-acetyl-a-D-man-
reactive glycans used for conjugation with the protein, the nopyranoside (1a), deriving from the hydrolysis of the acetyl
enzymatic regioselective deprotection of peracetylated man- group at the 6 position, as the only product, according to the
1
3–15
nopyranose derivatives 1–3 has been studied. The compound 3 results previously obtained with other pyranoses.
The
carries a thiocyanomethyl group at the C-1 position as catalytic performances of the other enzymes tested were
precursor of the reactive IME. The products 3a,b obtained by negligible (data not shown) and, consequently, CRL was
hydrolysis of the thiocyanomethyl mannopyranoside 3 have selected as the biocatalyst for the hydrolysis of acetylated a-O-
been then considered as intermediates for the synthesis of methyl mannopyranoside (2) and 1-thio-(S-cyanomethyl)-a-
peracetylated
dimannopyranoside
and
arabino- mannopyranoside (3).
mannopyranosides 5, 6 and 8, according to the general che-
CRL, adsorbed onto two supports with different grade of
1
3–15
moenzymatic strategy previously reported.
The nal IME- hydrophobicity such as octyl-sepharose (OAg) or decaoctyl-
22
saccharides (9–12) have been used for the glycosylation of sepabeads (ECOD), afforded different results depending on
RNase A. Flow Injection Analysis (FIA) of intact glycoproteins the substrate (Table S1, see ESI†). Great differences can be
by MS is generally carried out to establish product identity and observed in term of selectivity, depending on the functional
integrity of natural glycoproteins, as well as to assess the group linked at the anomeric position of the sugar (acetoxy,
1
6–18
relative abundance of individual glycoforms.
In this work, methoxy or thiocyanomethyl group). In particular, a complete
this MS analytical approach has been used also for the char- selectivity was achieved toward the primary position when 1 and
acterization of the neoglycoproteins in order to determine the 2 were used as substrate, while the presence of thiocyanomethyl
number and position of glycans introduced. Accordingly, the group at the anomeric position strongly inuenced the regio-
glycosylation degree was monitored by direct infusion of intact selectivity. In fact, when compound 3 was subjected to enzy-
proteins in a linear ion trap mass spectrometer (ESI-LIT-MS), matic
hydrolysis,
2,3,4-tri-O-acetyl-1-thio-(S-cyanomethyl)-
while a detailed characterization of glycosylation sites and a-D-mannopyranoside 3a (deprotected at the C-6 position)
their relative abundance was performed by liquid together with 3,4,6-tri-O-acetyl-1-thio-(S-cyanomethyl)-a-D-man-
chromatography-mass spectrometry peptide mapping aer an nopyranoside 3b (deprotected at the C-2 position) were
appropriate proteolytic cleavage of the synthesised neo- obtained in almost equal amounts (Scheme 1 and Table S1†) In
glycoproteins. Moreover, a computational study of the reac- addition, the selected biocatalyst (CRL-ECOD) resulted
tivity of the different lysines on the RNase A surface has been completely stable aer ve cycles (Fig. S1, see ESI†).
performed. The in silico evaluation of selected physico-
The hydrolysis of substrate 3 was scaled up to 1.65 L
chemical parameters that could correlate with the reactivity affording 2.2 g and 1.8 g of products 3a and 3b, respectively. The
of the different glycosylation sites (lysines) on the protein C-6 and C-2 monodeprotected products 3a and 3b were used as
surface, could be useful tools for the design, preparation and building blocks to prepare the saccharides 5, 6 and 8 (Scheme 2)
characterization of neoglycoconjugates.
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Scheme 1 Regioselective hydrolysis of acetylated mannopyranosydes 1–3 catalyzed by immobilized Candida rugosa lipase. The best
percentage conversions of monodeprotected compounds 1a–3a and 3b are reported. Experimental conditions: 50 mM phosphate buffer with
ꢀ1
20% acetonitrile; pH 4; room temperature; [substrate] ¼ 5 mM, *10 mM; volume ¼ 25 mL; CRL-OAg ¼ CRL-ECOD ¼ 100 IU, IU ¼ mmol min
.
by Schmidt glycosylation reaction (Lewis acid-catalyzed glyco- 70% and 62% yields, respectively using the mild Lewis acid
13
sylation using glycosyl trichloroacetimidate as the donor).
boron triuoride etherate as the promoter (Scheme 2). The
The reaction was optimized with respect to nature and synthesis of the peracetylated disaccharide Ara(1-2)Man (8)
concentration of glycosylation promoter, time and temperature required instead the use of the stronger promoter tert-butyldi-
(Table S2, see ESI†) and the acetylated thiocyanomethyl disac- methylsilyl triuoromethanesulfonate, to afford product 8 in
charides Man(1-6)Man (5) and Ara(1-6)Man (6) were obtained in 50% yield (Table S2†).
ꢁ
3 2 2 2
Scheme 2 Synthesis of disaccharides 5, 6 and 8 via glycosylation reaction. (a) 3a, BF $OEt , CH Cl , 0 C, MS 4 A˚ , 5 h, 70% yield; (c) 3b, TBSOTf,
ꢁ
ꢁ
0
C, Et
2
O, MS 4 A˚ , 1 h; 50% yield; (b) 3a, BF
3
$OEt
2
, CH
2
Cl
2
, 25 C, MS 4 A˚ , 1 h, 62% yield. MS ¼ molecular sieves, DMF ¼ N,N-dimethylformamide,
TBSOTf ¼ tert-butyldimethylsilyl trifluoromethanesulfonate, DBU ¼ 1,8-diazabicyclo[5.4.0]undec-7-ene.
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Compounds 3, 5, 6 and 8 were subsequently submitted to
23
Zempl ´e n deacetylation reaction with the double purpose to
remove the acetyl esters protecting groups and to functionalize
the C-1 position as 2-iminomethoxyethyl (IME) group in single a
synthetic step (Scheme 3). In all the cases, the IME-saccharides
9–12 were obtained in about 50% yield, as evaluated by ESI-MS.
Conjugation of these glycans was performed with the
commercially available ribonuclease A (124 amino acids,
molecular mass 13.681 Da) containing 10 Lys residues (Fig. 1
and Scheme 3).
First, the reaction of Man-IME (9) with RNase A was studied
as a model reaction in order to optimize the experimental
conditions for the formation of a covalent bond involving the
Fig. 1 Surface analysis and localization of Lys residues belonging to
each monomer of RNase A dimer.
3-amino groups of the lysine residues (Table 1). The effects of
the buffer, pH and temperature on the reaction outcomes have
been investigated through ESI-MS analyses carried out by direct
infusion (see Experimental section).
The nature of the buffer and the temperature seem to poorly
inuence the outcome of the reaction (Entries 1–3, 5 and 6). On
the contrary, surprisingly increasing the pH from 9.5 to 10.5
resulted in a strong reduction of the yield from 94% to 58% of
the glycosylated protein (Entries 3 and 4), while a decrease of pH
value below 9.0 poorly inuenced the amount of conjugated
protein.
The glycosylation of RNase was also performed with activated
disaccharides 10–12 at the optimal reaction conditions
ꢁ
(Na
2
B
4
O
7
buffer, pH 9.5, 37 C) (Table 2).
Generally, the glycosylation yields ranged between excellent
(93% for Man-IME 9) and good reaction yields (78% for Ara(1-2)
Man-IME 12). These values were calculated as the ratio between
of the sum of the relative abundance of all the glycoforms over
the total ions, including the non-glycosylated protein (Table 2).
Under a qualitative point of view, the glycosylation proles
varied depending on the glycoside used. In particular, when the
monosaccharide 9 was used, the glycosylation ratio (the number
of saccharides bound to each molecule of RNase) was 2.3.
Similar results have been obtained with IME-disaccharides
The neoglycoprotein 13 is composed of a mixture of glyco-
forms with a common peptide backbone but bearing a different
number of sugar units. In particular, the most abundant glyco-
forms are those bearing two or three mannose units (Table 1).
9–11, while a lower ratio (about 1.0) was observed when
Scheme 3 Synthesis of neoglycoproteins 13–16 by conjugation reaction of IME-saccharides 9–12 with ribonuclease A. (a) 3, 5, 6 and 8, MeONa/
ꢁ
MeOH, room temperature, 24 h, 50% yield; (b) 9–12, sodium tetraborate buffer, pH 9.5, 37 C, molar ratio glycosidic reagent/ribonuclease A
1
00 : 1, 24 h.
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Table 1 Optimization of experimental conditions for the conjugation reaction of RNase A with Man-IME (9)
N incorporated Man (% relative abundance)
Mannose bound/protein
(mol/mol)
Conversion
(%)
ꢁ
Entry
T ( C)
pH
8
Buffer
0
1
2
3
4
5
6
7
1
2
3
4
5
6
37
37
37
37
37
25
Na
2
Na
2
Na
2
Na
2
Na
2
Na
2
B
B
B
B
4
4
4
4
O
O
O
O
7
7
7
7
15
5
7
42
6
—
35
16
19
38
9
25
29
29
20
28
11
16
26
24
—
29
18
7
1
7
5
—
8
—
1
1
—
—
14
—
—
—
—
—
9
1.7
2.6
2.4
0.8
2.7
4.0
85
95
93
58
94
8.5
16
15
—
20
25
9.5
10.5
9.5
CO
3
9.5
B
4
O
7
3
20
100
ꢁ
Table 2 Synthesis of neoglycoproteins 13–16. Experimental conditions: 100 mM sodium tetraborate buffer, pH 9.5, 37 C, molar ratio glycosidic
reagent/ribonuclease A 100 : 1, 24 h
N incorporated glycan (% relative abundance)
Glycan bound/protein (mol/mol)
Conversion (%)
Reagent
Product
0
1
2
3
4
5
6
ꢂ SD
ꢂ SD
9
13
14
15
16
7
8
9
19
24
21
39
29
27
31
27
24
21
24
13
15
14
11
—
5
6
4
—
1
2.3 ꢂ 0.3
2.3 ꢂ 0.0
2.2 ꢂ 0.1
1.3 ꢂ 0.1
93 ꢂ 3
92 ꢂ 3
91 ꢂ 2
78 ꢂ 6
10
11
12
—
—
—
21
Ara(1-2)Man-IME (12) was used as the glycosylating agent (Table to unambiguously identify the actual glycosylation site.
). In the last case, the presence of a glycosidic bond in position However, taken together the evidences on which the K7 looks on
C-2 of mannose seems to reduce the efficiency of the reaction, to the dimeric interface (Fig. 1), it might be assumed that the
2
probably as a consequence of a steric hindrance.
lysine conjugated with the glycans is the K1.
The other positions involved in the reactions are almost
equally distributed (about 20% each) among K91 and K31, while
a minor amount of RNase was found to be glycosylated at K61-
K66.
Interestingly, the same reactivity order of surface lysines was
observed when RNase was coupled with either mono- or disac-
charides (9–12), notwithstanding the lower glycosylation extent
reached with the latter, markedly evident with Ara(1-2)Man-IME
Characterization of neoglycoproteins
The analytical characterization of the obtained neo-
glycoproteins (13–16) was carried out by applying an LC-MS
method previously developed for natural glycoproteins, which
includes selective proteolysis and analysis of the glycopeptides
24,25
obtained in order to locate the glycosylation sites.
In order to have a direct evidence of the exact positions
where the observed multi-site glycosylation has occurred and a
quantitative estimate of the glycosylation relevance at each site,
all the neoglycoproteins were hydrolyzed by chymotrypsin and
the released glycopeptides were selectively extracted, separated
1
2 (Table 2).
Finally, in attempt to modulate the reactivity of the different
lysine residues, the conjugation reaction with Man-IME was
performed at lower pH value (8.5 and 8). Reducing the pH of the
reaction medium from 9.5 to 8, an improvement of the reactivity
of K1 from 40% up to 55% of the total conjugated lysines was
observed, while reducing below the 20% the glycosylation extent
of K91 which was the second residue in reactivity order (Fig. 3).
n
and identied by ESI-MS . In detail, glycopeptides were selec-
tively enriched by on-line SPE on hypercarb trap column (PGC-
online SPE), and subsequently separated on an Amide 80
column by HILIC-MS/MS. The obtained results (Fig. 2) indicate
that the vast majority of conjugation reactions implicate those
lysine residues not implicated in the interface of the dimeric
In silico analysis of RNase A lysine reactivity
form of the protein (Fig. 1), regardless the glycan used. This In order to strengthen the experimental evidence a computa-
result conrms that RNase A in solution is mainly present in the tional study has been carried out considering different physico-
dimeric complex. Indeed, only a minor part (<10%) of lysine chemical parameters of RNase A to gain a theoretical biophys-
residues included in the interface of the dimeric aggregate, ical correlation of the reactivity of the different glycosylation
namely K98 and K104, react with mannose and other glycans, as sites of the protein.
probable consequence of the reaction of a minor portion of
monomeric protein in equilibrium with the dimeric form.
To this scope, various critical parameters have been
considered: intermolecular protein–protein interactions, mul-
The most reactive area of the protein (almost 40% of glyco- timeric conformations or tendency to aggregation in solution,
sylated form) involved K1/K7 amino group. Unfortunately, the solvent accessibility for each amino acid side chain, exibility of
glycopeptide obtained by enzymatic digestion contains two Lys different surface area and ionization of the reactive amino
2
3
residues and the generated MS and MS spectra did not allow acids. By using the PDB structure, we assessed of the probable
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Fig. 2 Glycosylation sites involved in conjugation reaction of IME-
saccharides 9–12 with RNase A.
Fig. 4 Analysis of the solvent accessibility (ASA) of the lysines of RNase
A (PDB code 1FS3).
Fig. 3 Glycosylation sites involved in conjugation reaction of IME-Man
(9) with RNase A at different pH values.
a
Fig. 5 pK values of lysine groups in RNase A based on its 3D structure.
conformation in solution using the Protein Interfaces, Surfaces
and Assemblies (PISA) service at the European Bioinformatics
Thanks to the analysis of exibility in biomolecules and
Institute (https://www.ebi.ac.uk/pdbe/pisa/). The results showed networks service it is possible to estimate the space of vibration
a tendency of this protein to assembly in a dimeric form in for each residues. The results obtained in this analysis suggest
solution. The monomeric form of RNase A exhibited on its that the most exible areas of RNase are centered on the resi-
surface all lysine residues (Fig. S2, see ESI†). However, only few dues 66–70, 1, 88–93 and 30–39 of the protein sequence (Fig. 6).
of them (K1, K31, K61, K66 and K91) were completely exposed Especially the rst residue range comprised between amino
2
˚
on the area of the protein external at the interface (Fig. 1) while acids 66–70 showed a great uctuation variation up to 17 A .
others lysines are placed at the limit of the protein surface Accordingly to the evaluation this parameter the most reactive
involved in the dimeric complex, and thus, probably poorly lysine residues could be outlined (K1, K66, K61, K91 or K31).
exposed towards the reaction medium. Accordingly, the acces-
sible surface area (ASA) of the different lysines has been ana-
Experimental
lysed including also the amino acids involved in the interface
26
Lipase from Candida rugosa and octyl agarose (Octyl Sepharose
CL-4B) was purchased from Sigma-Aldrich (Milano, Italy).
Decaoctyl-Sepabeads (Sepabeads EC-OD/S) was kindly provided
by Resindion s.r.l. - Mitsubishi Chemical Corporation (Binasco,
Milano, Italy). Other reagents and chemicals were purchased
from Fluka, Aldrich, Pharmacia Biotech (Uppsala, Sweden) and
used without further purication. Solvents were dried by stan-
dard methods.
between the two monomers. With the combined PISA and ASA
analyses a ranking of the different lysines related to their
surface exposition has been obtained. As reported in Fig. 4 and
S3 (see ESI†), the most accessible Lys resulted the K1, followed
by K66 z K91 > K61 z K98 > K31.
a
Fig. 5 reports the pK analysis of the Lys of the protein. The
lowest pK values are comprised between 8 and 8.5 (K41 and
a
K66). It is noteworthy that, in the dimeric form of the protein,
a
for K66 the pK value decrease from about 10.5 in the mono-
meric form to about 8.5 in the dimer. Another important Thin layer chromatography (TLC)
parameter to be considered is the protein exibility. In fact,
Analytical TLC was performed on silica gel F254 precoated
protein structure varies among different conformations
dened by the amino acidic backbone, and the more exible
zones could theoretically better react during a chemical
modication.
aluminum sheets (0.2 mm layer, Merck, Darmstadt, Germany).
Products were detected by spraying with 5% H SO in ethanol,
followed by heating to ca. 150 C. Silica gel 60 (40–63 mm,
Merck) was used for ash chromatography.
2
4
ꢁ
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Anzola Amilia (BO) Italy), eluent phosphate buffer 10 mM-
ꢀ
1
acetonitrile 6 : 4, pH 4, ow rate 1.0 mL min , UV detector l
ꢁ
2
20 nm, temperature 25 C.
Mass spectrometry (MS) analyses
MS experiments were carried out on a LTQ-MS (Thermo Elec-
tron, San Jose, CA) with an electrospray ionization (ESI) source
controlled by X-calibur soware 1.4 (Thermo Finnigan, San
Jose, CA). The mono- and disaccharide samples were dissolved
in methanol and introduced into the mass spectrometer with a
ꢀ
1
syringe pump at a ow rate of 5 mL min . Full scan intact MS
experiments were carried out under the following instru-
mental conditions: positive ion mode, mass range 150–1000
m/z, source voltage 4.6 kV, capillary voltage 36 V, sheath gas 6,
ꢁ
auxiliary gas 1, capillary temperature 250 C, tube lens voltage
8
0 V. The intact glycoprotein and glycopeptide samples were
prepared and analyzed according to the procedures below
reported.
Protein determination
Protein concentration was determined by the Bradford assay on a
Shimadzu UV spectrophotometer 1601 (UV Probe 1.0 Shimadzu).
Enzymatic activity
Enzymatic activities were evaluated by hydrolysis of ethyl buty-
rate. The hydrolytic activity was quantied titrimetrically at
pH 7.0 and room temperature using a pH-Stat (Metrohm 718
STAT Tritino, Herisau, Switzerland).
Fig. 6 Flexibility analysis of RNase A structure (PDB: 1FS3) [http://
biocomp.chem.uw.edu.pl/CABSflex/]. (a) Fluctuation variation of
entire protein sequence, (b) Fluctuation variation ranked by amino
acids.
In silico analysis of RNase A
Different bioinformatic tools are freely offered by various
research groups working in this fast growing topic (http://
at 400.1 and www.expasy.org/). It has been considered the protein data
Nuclear magnetic resonance (NMR)
1
13
H and C NMR spectra were recorded in CDCl
3
1
00.6 MHz, respectively, on a Bruker Avance 400 Spectrom- bank (PDB) structure of bovine pancreatic RNase A, 1FS3. The
eter (Bruker, Karlsruhe, Germany) equipped with a Topspin pK of each lysine residue was estimated using a bioinformatic
a
soware package on a workstation running Windows oper- tool developed to predict the pK values of ionisable groups in
a
ating system. Chemical shis (d) are given in ppm and were proteins based on the 3D structure PROPKA (http://
referenced to the solvent signals (dH 7.26, dC 77.00). Signal propka.ki.ku.dk/). The space of vibration for each lysine
multiplicities are abbreviated as follows: s, singlet; d, residue was evaluate by the analysis of exibility and networks
1
3
doublet; t, triplet; m, multiplet; b, broad. C NMR signal service (http://biocomp.chem.uw.edu.pl/CABSex/). The anal-
multiplicities were based on attached proton test (APT) ysis of the solvent accessibility (ASA) was performed according
24
spectra. Structures assignment were performed by means of to the literature.
D-COSY (Correlation Spectroscopy), HSQC (Heteronuclear
2
Single Quantum Correlation) experiments and HMBC (Hetero-
nuclear Multiple Bond Correlation). Spectra analyses were carried
out using iNMR reader soware (http://www.inmr.net) on an Apple
computer.
Ribonuclease A glycosylation with 2-iminomethoxyethyl (IME)
thioglycosides
27,28
According to B.G. Davis' protocol
with some minor modi-
cations, the glycosylation reactions were carried out in sodium
tetraborate buffer 100 mM, pH 9.5. Ribonuclease A was dis-
solved in the buffer at a concentration of 1 mg mL , then to the
High performance liquid chromatography (HLPC)
ꢀ1
A Merck-Hitachi L-7100 liquid chromatograph equipped with a L- solution was added IME-thioglycosides to a nal glycoside/
7
400 UV detector (E. Merck, Darmstad, Germany) was used for protein molar ratio of 100/1. The reaction mixtures were vor-
ꢁ
analytical HPLC. Chromatographic conditions: column Pheno- texed for 1 minute and incubated for 24 hours at 37 C under
menex Gemini C18 250 ꢃ 4.6 mm (5 mm) (Chemtek Analitica, continuous stirring.
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Glycoproteins purication
Glycopeptides were identied on the basis of fragmentation
2
3
ions in MS and MS spectra by in silico screening with Bioworks
Browser (Thermo Electron, revision 3.1) setting the glycan
moieties as optional modications of lysine residues. Only the
identication with a X-corr greater than 1 were consider and to
Aer glycosylation, the reaction mixtures were puried in order to
make them suitable to direct infusion in ESI-MS. All the samples
were submitted to ultracentrifugation using lter with loading
capacity of 500 mL and Nominal Molecular Weight Limit (NMWL)
of 3 kDa. The purication protocol consists of seven centrifuga-
2
3
avoid false positive MS and MS spectra of all species recog-
nized as glycopeptides were manually conrmed.
For quantitative measurements, the area of each glycopep-
tide was determined by integrating the peak in the respective
single ion current chromatogram.
ꢁ
tion steps of 20 min at 13 000g and 4 C using deionized water as
washing solvent. Aer purication, the glycoproteins were
ꢁ
collected in deionized water and stored at ꢀ20 C. For long period
storage, ammonium bicarbonate 10 mM, pH 7.4 was used.
Glycosylation yield determination
1-Thio-S-cyanomethyl-2,3,4,6-tetra-O-acetyl-a-D-
mannopyranoside (3)
Glycosylation yields and relative abundance of glycoforms were
determined by direct infusion on a ESI-LTQ-MS (Thermo Elec- Synthesis of 1-thio-S-cyanomethyl-2,3,4,6-tetra-O-acetyl-a-D-
tron, San Jose, CA). A small rate of puried reaction mixture mannopyranoside (3) was performed according to the proce-
27
from glycosylation reaction was diluted to a nal concentration dure previously reported.
ꢀ
1
of 0.3 mg mL in H O : acetonitrile, 50 : 50 containing 0.05%
2
TFA and introduced into the mass spectrometer with a syringe
General procedure for enzymatic hydrolysis
ꢀ
1
pump at a ow rate of 5 mL min . Full scan intact MS experi-
ments were carried out under the following instrumental
conditions: positive ion mode, mass range 700–2000 m/z, source
The enzymatic hydrolyses of the mannose derivatives 1–3
(between 5 and 20 mM) were carried out in 25 mM phosphate
buffer containing 20% acetonitrile for complete solubilisation
of the substrates under mechanical stirring. The reactions
started aer addition of the immobilized enzyme preparation.
During the reactions, pH was maintained constant by automatic
titration. The course of the hydrolyses was monitored by HPLC
and products were quantied using authentic standards. Aer
complete or almost complete consumption of the substrates,
the reactions were stopped by biocatalyst ltration and the
obtained products were isolated by extraction with ethyl acetate
followed by purication using ash chromatography, and were
voltage 4.5 kV, capillary voltage 35 V, sheath gas 15, auxiliary gas
ꢁ
2, capillary temperature 220 C, tube lens voltage 140 V.
The spectra were deconvoluted with Bioworks Browser
(Thermo Electron, revision 3.1). The identities of ribonuclease A
and its glycoforms were assigned on the basis of the average
molecular weight in the deconvoluted spectra and their relative
abundance determined by the relative intensities of the corre-
sponding peaks.
Digestion with chymotrypsin
1
characterized by H and COSY 2D NMR.
The reaction mixtures were digested with chymotrypsin as follows:
to a 100 mL of protein solution 100 mM in water was added 90 mL of
ammonium bicarbonate 100 mM pH 8.5 and 10 mL of dithio-
1
,2,3,4-Tetra-O-acetyl-a-D-mannopyranose (1a)
1
threitol (DTT) solution (100 mM in ammonium bicarbonate
H NMR (400 MHz, CDCl
3
): d ¼ 6.1 (s, 1H, H1), 5.40 (dd, 1H, J ¼
ꢁ
1
00 mM pH 8.5). The solution was heated at 60 C for 30 minutes 3.3, 1.7 Hz, H-2), 5.30 (dd, J ¼ 3.4, 1.4 Hz, 1H, H-3), 5.27 (m, 1H,
in order to reduce the disulde bridges. Enzyme was then added H-4), 3.70 (dd, J ¼ 11.9, 6.5 Hz, 1H, H-6B), 3.61 (dd, J ¼ 11.6, 6.4
to a nal protein/enzyme ratio of 100 : 1. The solution was incu- Hz, 1H, H-6A), 3.83 (m, 1-H, H-5), 2.15 (s, 3H), 2.10 (s, 3H), 2.05
ꢁ
bated overnight at 37 C under continuous stirring. The digestion (s, 3H), 2.00 (s, 3H), CH
COO.
3
was stopped by inactivating the chymotrypsin with TFA 2.5% (v/v).
Methyl 2,3,4-tri-O-acetyl-a-D-mannopyranoside (2a)
On-line PGC-Solid-Phase Extraction and HPLC-MS analysis
1
H NMR (400 MHz, CDCl
.6 Hz, 1H, H-2), 5.25 (m, 1H, H-4), 4.98 (dd, J ¼ 3.6, 1.3 Hz, 1H,
H-3), 4.14 (m, 1-H, H-5), 3.80 (dd, J ¼ 11.4, 6.8 Hz, 1H, H–6B),
.66 (dd, J ¼ 11.2, 6.1 Hz, 1H, H-6A), 3.30 (s, 3H, OCH ), 2.20
3
): d ¼ 5.90 (s, 1H, H1), 5.55 (dd, J ¼ 3.2,
Glycopeptides obtained by chymotrypsin digestion were selec-
1
tively on-line extracted rearranging a method of Temporini
25
et al. The nal conditions provide 10 minutes of desorption
3
3
from the trap column with 80% A (acetonitrile with TFA 0.05%)/
(
s, 3H), 2.15 (s, 3H) and 2.07 (s, 3H), CH COO.
3
ꢀ
1
20% B (H
2
O with TFA 0.05%) at 100 mL min . The HPLC
gradient analysis was from 30% to 57% B in 11 minutes with a
ꢀ1
Cyanomethyl 2,3,4-tri-O-acetyl-a-D-thiomannopyranoside (3a)

ow rate of 200 mL min . Mass spectra were generated in
1
positive ion mode under the following instrumental conditions:
H NMR (400 MHz, CDCl
3
): d ¼ 5.50 (s, 1H, H-1), 5.40 (dd, J ¼
source voltage 4.5 kV, capillary voltage 31 V, sheath gas ow 40 3.4, 1.7 Hz, 1H, H-2), 5.35 (t, 1H, H-4), 5.27 (dd, J ¼ 3.2, 1.2 Hz,
(
arbitrary units), auxiliary gas ow 10 (arbitrary units), sweep 1H, H-3), 4.10–4.18 (m, 1H, H-5), 3.82 (dd, J ¼ 11.2, 6.2 Hz, 1H,
ꢁ
gas ow 0 (arbitrary units), capillary temperature 250 C, tube H-6B), 3.69 (dd, J ¼ 11.6, 6.4 Hz, 1H, H-6A), 3.49 (d, J ¼ 17.22 Hz,
2
3
lens voltage 95 V. MS and MS spectra were obtained by CID 1H, HA SCH
2
CN), 3.35 (d, J ¼ 17.23 Hz, 1H, HB SCH
COO.
2
CN), 2.19
with normalized collision energy 35.0. (s, 3H), 2.12 (s, 3H) and 2.03 (s, 3H), CH
3
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Cyanomethyl 3,4,6-tri-O-acetyl-a-D-thiomannopyranoside (3b)
(0.500 g), and the solution was le to stir under nitrogen. Aer
stirring for 1 hour, the reaction mixture was neutralized by the
addition of triethylamine (0.018 mL). The solvent was removed
under reduced pressure and The solvent was removed under
reduced pressure and the residue was puried by ash column
chromatography (hexane–ethyl acetate, 1 : 1) affording 6 (white
1
H NMR (400 MHz, CDCl ): d ¼ 5.55 (s, 1H, H-1), 5.42 (dd, J ¼
3
9
4
.7, 9.8 Hz, 1H, H-4), 5.18 (dd, J ¼ 3.2, 1.2 Hz, 1H, H-3), 4.30–
.40 (m, 2H, H-5, H-6A), 4.15–4.22 (m, 2H, H-2, H-6B), 3.48 (d, J
¼
17.2 Hz, 1H, HA SCH
2
CN), 3.35 (d, J ¼ 17.2 Hz, 1H, HB
SCH CN), 2.16 (s, 3H), 2.12 (s, 3H) and 2.06 (s, 3H), CH
2
3
COO.
amorphous solid, 0.170 g, 62% yield).
1
H-NMR (400 MHz; CDCl
3
): d ¼ 5.45 (d, J ¼ 1.4 Hz, 1H, H-1),
Recycling of the biocatalysts
5
.34 (dd, J ¼ 3.5, 1.4 Hz, 1H, H-2), 5.30 (dd J ¼ 9.8, 9.8 Hz, 1H, H-
0
Recycling of CRL immobilized on decaoctyl Sepabeads was 4), 5.24 (ddd, J ¼ 3.5, 3.5, 1.7 Hz, 1H, H-4 ), 5.20 (dd, J ¼ 9.8, 3.5
0
performed by evaluating the hydrolysis of substrate 2 in the Hz, 1H, H-3), 5.17 (dd, J ¼ 9.2, 6.7 Hz, 1H, H-2 ), 5.04 (dd, J ¼ 9.2,
0
0
same conditions above described. When the highest conversion 3.5 Hz, 1H, H-3 ), 4.51 (d, J ¼ 6.7 Hz, 1H, H-1 ), 4.24 (ddd J ¼ 9.8,
0
was achieved, the reaction mixture was ltered under reduced 6.5, 2.4 Hz, 1H, H-5), 4.04 (dd, J ¼ 13.0, 3.5 Hz, 1H, H-5 A), 3.96
pressure and the immobilized biocatalyst was re-used for the (dd, J ¼ 11.7, 6.5 Hz, 1H, H-6A), 3.66 (dd, J ¼ 11.7, 2.4 Hz, 1H, H-
0
next reaction.
6B), 3.62 (dd, J ¼ 13.0, 1.7 Hz, 1H, H-5 B), 3.54 (d, J ¼ 17.2 Hz,
1
3
H, HA SCH
2
CN), 3.28 (d, J ¼ 17.2 Hz, 1H, HB SCH
2
CN), 2.17 (s,
H), 2.13 (s, 3H), 2.09 (s, 3H), 2.06 (s, 3H), 2.04 (s, 3H) and 1.99
Cyanomethyl (2,3,4,6-tetra-O-acetyl-a-D-mannopyranosil)-
(
s, 3H), CH COO.
3
(
1/6)-2,3,4-tri-O-acetyl-a-D-thiomannopyranoside (5)
1
3
C-NMR (101 MHz; CDCl ): d ¼ 170.46, 170.30, 169.99,
3
2,3,4,6-Tetra-O-acetyl-a-D-mannopyranose trichloroacetimidate
1
(
3
2
69.87, 169.70 and 169.58, CH COO, 116.04 (SCH CN), 100.78
3 2
4
(0.136 g; 0.276 mmol) and cyanomethyl 2,3,4-tri-O-acetyl-a-D-
0
0
C-1 ), 81.50 (C-1), 70.94 (C-5), 70.05 (C-3 ), 69.74 (C-2), 69.53 (C-
thiomannopyranoside 3a (0.050 g; 0.138 mmol) were dissolved
0
0
0
), 69.15 (C-2 ), 67.60 (C-4 ), 66.88 (C-6), 66.47 (C-4), 63.23 (C-5 ),
ꢁ
in anhydrous CH
2
Cl
2
(5 mL) and the solution was cooled to 0 C.
1.08, 21.00, 20.89 and 20.80, CH
3
2
COO, 15.22 (SCH CN).
Subsequently, boron triuoride diethyl etherate (0.01 mL) was
˚
+
added in the presence of activated 4 A molecular sieves (0.300
MS: m/z ¼ 642.33 [M + Na] (calcd 642.58).
g), under magnetic stirring under a nitrogen atmosphere. Aer
6
hours, the reaction mixture was neutralised with the addition
of triethylamine (0.006 mL). The solvent was removed under Cyanomethyl (2,3,5-tri-O-acetyl-a-D-arabinofuranosyl)-(1/2)-
reduced pressure and the residue was puried by ash column 3,4,6-tri-O-acetyl-a-D-thiomannopyranoside (8)
chromatography (hexane–ethyl acetate, 1 : 1) affording 5 (white
2
,3,5-Tri-O-acetyl-a-D-arabinofuranosyl trichloroacetimidate 7
amorphous solid, 0.067 g, 70% yield).
(0.650 g, 1.544 mmol), and cyanomethyl 3,4,6-tri-O-acetyl-a-D-
1
H NMR (400 MHz, CDCl
3
): d ¼ 5.48 (d, J ¼ 1.4 Hz, 1H, H-1),
thiomannopyranoside 3b (0.280 g, 0.775 mmol) were coevapo-
rated with anhydrous toluene and dried under reduced pressure
overnight. The mixture was dissolved in Et
0
0
0
5
.39 (dd, J ¼ 3.2, 1.6 Hz, 1H, H-2), 5.35–5.21 (m, 5H, H-4 , H-3 ,
0
0
H-2 , H-4, H-3), 4.85 (d, 1H, J ¼ 1.4 Hz, H-1 ), 4.29–4.25 (m, H-5,
2
O (25 mL), cooled to
C and tert-butyldimethylsilyl triuoromethanesulfonate
2
1
1
3
2
3
H, H-6A), 4.13 (dd, J ¼ 11.2, 6.1 Hz, 1H, H-6B), 4.05–4.00 (m,
ꢁ
0
0
H, H-5 ), 3.85 (dd, J ¼ 11.6, 6.8 Hz, 1H, H-6 B), 3.58 (dd, J ¼
˚
(0.178 mL) was added in the presence of activated 4 A molecular
0
1.6, 3.2 Hz, 1H, H-6 A), 3.54 (d, J ¼ 17.3 Hz, 1H, HA SCH CN),
2
sieves (0.500 g). The reaction solution was le to stir under
nitrogen to 0 C. Aer stirring for 3 hours the reaction mixture
.36 (d, J ¼ 17.3 Hz, 1H, HB SCH
2
CN), 2.19 (s, 3H), 2.16 (s, 3H),
ꢁ
.11 (s, 3H), 2.08 (s, 3H), 2.06 (s, 3H), 2.00 (s, 3H) and 1.99 (s,
H), CH COO.
C NMR (101 MHz, CDCl
69.86, 169.68 (CH
was neutralized by the addition of triethylamine (0.130 mL). The
solvent was removed under reduced pressure and The solvent
was removed under reduced pressure and the residue was
puried by ash column chromatography (hexane–ethyl
acetate, 1 : 1) affording 8 (white amorphous solid, 0.240 g, 50%
3
1
3
3
): d ¼ 170.20, 170.03, 169.97,
0
1
3
COO), 115.74 (SCH
2
0
CN); 97.36 (C-1 ), 81.55
(
5
2
C-1), 70.63 (C-5), 69.56 (C-2), 69.46 (C-3 ), 69.08 (C-3), 68.97 (C-
0
0
0
), 66.52 (C-4), 66.21 (C-6), 65.87 (C-4 ), 62.51 (C-6 ), 21.01,
0.94, 20.85, 20.81, 20.71 (CH COO), 15.47 (SCH CN).
yield).
1
3
2
H-NMR (400 MHz; CDCl
3
): d ¼ 5.59 (d, J ¼ 1.3 Hz, 1H, H-1),
0
5
5
.30 (t, J ¼ 10.0 Hz, 1H, H-4), 5.28–5.24 (m, 2H, H-4 and H-4 ),
+
MS: m/z ¼ 714.29 [M + Na] (calcd 714.63).
.04 (dd, J ¼ 3.2, 1.2 Hz, 1H, H-3), 5.02 (dd, J ¼ 3.0, 1.3 Hz, 1H,
0
H-3 ), 4.43 (dd, J ¼ 12.5, 4.0 Hz, 1H, H-6A), 4.38 (d, J ¼ 7.0 Hz,
0
1
3
H, H-1 ), 4.25(ddd, J ¼ 10.1, 4.0, 2.0 Hz, 1H, H-5), 4.16 (dd, J ¼
Cyanomethyl (2,3,5-tri-O-acetyl-a-D-arabinofuranosyl)-(1/6)-
.2, 1.3 Hz, 1H, H-2), 4.14–4.09 (m, 2H, H-2 and H-6B), 4.02 (dd,
2,3,4-tri-O-acetyl-a-D-thiomannopyranoside (6)
0
J ¼ 13.1, 3.1 Hz, 1H, H-5 A), 3.59 (dd, J ¼ 13.1, 1.6 Hz, 1H, H-5B),
2
(
,3,5-Tri-O-acetyl-a-D-arabinofuranosyl trichloroacetimidate 7 3.45 (d, J ¼ 17.2 Hz, 1H, HA CH CN), 3.30 (d, J ¼ 17.2 Hz, 1H, HB
2
0.373 g, 0.886 mmol) and cyanomethyl 2,3,4-tri-O-acetyl-a-D- CH CN), 2.17 (s, 3H), 2.16 (s, 3H), 2.15 (s, 3H), 2.07 (s, 3H), 2.04
2
thiomannopyranoside 3a (0.160 g, 0.443 mmol) were dissolved (s, 3H) and 2.03 (s, 3H), CH COO.
3
1
3
in anhydrous CH Cl (20 mL) and the mixture was stirred to
C-NMR (101 MHz; CDCl ): d ¼ 171.03, 170.43, 170.38,
2
2
3
room temperature. Boron triuoride diethyl etherate (0.028 mL) 170.20, 169.69 and 169.38 (CH
3
COO), 116.05 (SCH
2
CN), 102.65
˚
0
0
was added in the presence of activated 4 A molecular sieves (C-1 ), 84.45 (C-1), 76.30 (C-2), 71.34 (C-3), 69.87 (C-3 ), 69.80
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0 0 0
C-5), 68.75 (C-2 ), 67.51 (C-4), 65.63 (C-4 ), 63.55 (C-5 ), 61.56 (C- selective glycosylation of these two residues can be obtained,
(
6
), 29.84, 21.08, 20.87 and 20.76 (CH COO), 16.02 (SCH CN).
probably because at low pH, the reactivity of all lysines is
depressed (as demonstrated by the reduction of the percentage
of glycosylated protein), and only the most exposed and/or
3
2
+
MS: m/z ¼ 642.50 [M + Na] (calcd 642.58).

exible residues maintain a good reactivity.
The possibility to drive the conjugation with glycans toward
General Procedure for synthesis of IME mono- and disaccharides
–12
dened areas of the protein surface can be very important in
case of antigenic and other bioactive proteins, in order to avoid
modication of the residues involved in the biological activity.
To this scope and to widen the possible application areas,
studies involving antigenic protein structures are ongoing.
9
Compounds 3–8 (1.0 mmol) were dissolved in 5 mL of dry
methanol and 1 wt% of sodium methoxide (1.6 mmol) was
added. The reaction was reacted for 48 h under nitrogen, aer
which time the reaction mixture was concentrated in vacuo and
the solid formed was characterized by MS analysis.
Acknowledgements
+
Man-IME (9) MS: m/z ¼ 290.33 [M + Na] (calcd 290.29).
This work was funded by Regione Lombardia, Italy (VATUB
project, Project Framework agreement Lombardy Region
Universities-DGR 9139) and by Fondazione Banca del Monte di
Lombardia (Italy) FBML. M. F. thanks to CSIC for a JAE-Doc
contract (“Junta para la Ampliacion de estudios”) cofounded
by ESF (European Social Fund).
Man(1-6)Man-IME (10) MS: m/z ¼
+
52.75 [M + Na] (calcd 452.64).
4
Ara(1-6)Man-IME (11) MS: m/z ¼
22.33 [M + Na] (calcd 422.40).
+
4
Ara(1-2)Man-IME (12) MS: m/z ¼
22.33 [M + Na] (calcd 422.40).
+
4
Notes and references
1
R. D. Astronomo and D. R. Burton, Nat. Rev. Drug Discovery,
010, 9, 308.
2
Conclusions
2
G. J. L. Bernardes, B. Castagner and P. H. Seeberger, ACS
Chem. Biol., 2009, 4, 703.
In this work we presented a convenient and integrated approach
to the synthesis and characterization of neoglycoproteins
prepared by non-selective glycosylation of lysine 3-amino groups
of RNase A, chosen as a model protein. More in detail, by means
of a chemo-biocatalytic approach, different activated mannose-
based glycans were synthesized in good or excellent yields and
efficiently coupled to the model protein via reaction with 2-
iminomethoxyethyl reactive group generated on mannose
glycans. With the aim to depict a reactivity map of protein
surface and to support the analytical characterization of newly
generated glycoproteins, an investigation of different biophys-
ical parameters of this protein was conducted. Thanks to suit-
able LC-MS analyses the reacting lysines of RNase A were
detected demonstrating a good correlation between the most
reactive conjugation sites and the study of the solvent accessi-
bility and exibility of the different areas of the protein surface
containing the lysine residues. By this way, the feasibility of this
proposed approach to support the design and characterization
of neoglycoproteins has been clearly conrmed. In particular,
among all reactive lysines of RNase A only those exposes outside
the interface of the dimeric complex can react with the glycans.
3 G. Kallenius, A. Pawlowski, B. Hamasur and S. B. Svenson,
Trends Microbiol., 2008, 16, 456.
4 S. Frokjaer and D. E. Otzen, Nat. Rev. Drug Discovery, 2005, 4,
298.
5 D. G. Ilyushin, I. V. Smirnov, A. A. Belogurov,
I.
A.
Dyachenko,
T.
I.
Zharmukhamedova,
T. I. Novozhilova, E. A. Bychikhin, M. V. Serebryakova,
O. N. Kharybin, A. N. Murashev, K. A. Anikienko,
E. N. Nikolaev, N. A. Ponomarenko, D. D. Genkin,
G. M. Blackburn, P. Masson and A. G. Gabibov, Proc. Natl.
Acad. Sci. U. S. A., 2005, 110, 1243.
6 M. Monsigny, A. C. Roche, E. Duverger and O. Srinivas,
Neoglycoproteins. Comprehensive Glycoscience, From
Chemistry to Systems Biology, Elsevier, Amsterdam, 2007, p. 477.
7 B. G. Davis, Chem. Rev., 2002, 102, 579.
8 D. P. Gamblin, E. M. Scanlan and B. G. Davis, Chem. Rev.,
2009, 109, 131.
9 E. J. Grayson, G. J. L. Bernardes, J. M. Chalker, O. Boutureira,
J. R. Koeppe and B. G. Davis, Angew. Chem., Int. Ed., 2011, 50,
4127.
The solvent accessibility and the exibility of the different 10 O. M. T. Pearce, K. D. Fisher, J. Humphries, L. W. Seymour,
reactive lysines exposed externally to the interface seems to be
the most relevant parameters regulating the reactivity of the
A. Smith and B. G. Davis, Angew. Chem., Int. Ed., 2005, 44,
1057.
different residues. The ionization of the different residues (pK
seems instead to have a very poor contribution in the differen-
tiation of the reactivity of the different lysines. Accordingly, by
a
)
11 J. Zhu, Q. Wan, D. Lee, G. Yang, M. K. Spassova, O. Ouerfelli,
G. Ragupathi, P. Damani, P. O. Livingston and
S. J. Danishefsky, J. Am. Chem. Soc., 2009, 131, 9298.
modulating the pH of the reaction medium, the preferential 12 C. H. Hsu, S. C. Hung, C. Y. Wu and C. H. Wong, Angew.
reactivity of lysines (K1 and K31) can be exalted and an almost Chem., Int. Ed., 2011, 50, 11872.
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1
1
1
3 M. Filice, J. M. Palomo, P. Bonomi, T. Bavaro, R. Fernandez- 21 A. Holemann, B. L. Stocker and P. H. Seeberger, J. Org.
Lafuente, J. M. Guisan and M. Terreni, Tetrahedron, 2008, 64,
286.
4 M. Filice, T. Bavaro, R. Fernandez-Lafuente, M. Pregnolato,
Chem., 2006, 71, 8071.
22 J. M. Palomo, M. Filice, R. Fernandez-Lafuente, M. Terreni
and J. M. Guisan, Adv. Synth. Catal., 2007, 349, 1969.
9
J. M. Guisan, J. M. Palomo and M. Terreni, Catal. Today, 23 Z. Wang, Comprehensive Organic Name Reactions and
009, 140, 11. Reagents, John Wiley & Sons, 2010, p. 3123.
5 M. Filice, J. M. Guisan, M. Terreni and J. M. Palomo, Nat. 24 C. Temporini, E. Perani, E. Calleri, L. Dolcini, D. Lubda,
2
Protoc., 2012, 7, 1783.
6 Y. J. Kim and M. L. Doyle, Anal. Chem., 2010, 82, 7083.
7 K. Sandra, P. Sandra and I. Vandenheede, LC GC Eur., 2013,
G. Caccialanza and G. Massolini, Anal. Chem., 2007, 79, 355.
25 C. Temporini, P. Bonomi, I. Serra, A. Tagliani, T. Bavaro,
D. Ubiali, G. Massolini and M. Terreni, Biomacromolecules,
2010, 11, 1623.
1
1
26, 10.
1
1
2
8 A. Staub, D. Guillarme, J. Schappler, J. L. Veuthey and 26 C. R. Søndergaard, M. H. M. Olsson, M. Rostkowski and
S. Rudaz, J. Pharm. Biomed. Anal., 2011, 55, 810. J. H. Jensen, J. Chem. Theory Comput., 2011, 7, 2284.
9 L. East and C. M. Isacke, Acta Biochim. Biophys. Sin., 2002, 27 C. W. Chang, S. S. Chang, C. S. Chao and K. K. T. Mong,
572, 364. Tetrahedron Lett., 2009, 50, 4536.
0 J. N. Arnold, M. R. Wormald, R. B. Sim, P. M. Rudd and 28 B. G. Davis, S. I. Van Kasteren, D. Anthony and N. Sibson,
R. A. Dwek, Annu. Rev. Immunol., 2007, 25, 21. Imaging agent, International Patent, WO 2007/020450 A2,
2 February 2007.
1
2
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