Direct Enzymatic Branch-End Extension of Glycocluster-Presented Glycans: An Effective Strategy for Programming Glycan Bioactivity

A Journal of

Accepted Article

Title: Direct enzymatic branch-end extension of glycocluster-presented

glycans: An effective strategy for programming glycan bioactivity

Authors: Carlos Bayón, Ning He, Mario Deir-Kaspar, Pilar Blasco,

Sabine André, Hans-Joachim Gabius, Ángel Rumbero, Jesús

Jiménez-Barbero, Wolf-Dieter Fessner, and Maria J. Hernaiz

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content of this Accepted Article.

To be cited as: Chem. Eur. J. 10.1002/chem.201604550

Link to VoR: http://dx.doi.org/10.1002/chem.201604550

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Direct enzymatic branch-end extension of glycocluster-presented

glycans:

An effective strategy for programming glycan bioactivity

Carlos Bayón,^[a]Ning He,^[b]Mario Deir-Kaspar,^[a]Pilar Blasco,^[c]Sabine André,^[d]Hans-Joachim Gabius,^[d]

Ángel Rumbero,^[e]Jesús Jiménez-Barbero,^[c]Wolf-Dieter Fessner,^[b]and María J. Hernáiz^*[a]

Dedication ((optional))

Abstract: Both the sequence of

a

glycan and its topology of

their role as code words, i.e. their unsurpassed structural

versatility (in terms of sequence and topology), is an enormous

challenge for carbohydrate chemistry, that is to develop

synthesis tools matching the natural complexity in all aspects.^[2]

Having started with simple head groups, the chemical platform

of galactose conjugation onto various types of scaffolds has

disclosed the glycoside cluster effect for the hepatic

asialoglycoprotein receptor.^[3]This natural hallmark of a lectin

taught the lesson that complex-type N-glycans and their

enzymatic tailoring can markedly broaden the toolbox for

functional studies.^[4]In fact, the branch-end tailoring of glycans

serves as a molecular switch for various lectin families, turning

off/on binding affinity or modulating this property.^[5]Fittingly,

efforts have been invested into preparing oligosaccharides with

custom-made sequence and terminus, particularly by employing

glycosyltransferases owing to their regio- and stereospecificity

and high product yields.^[6]Using adequate spacer chemistry,

these chemoenzymatically prepared glycans were then attached

to a carrier (protein or synthetic matrix) to be used as probe for

lectin detection and monitoring of lectin affinity.^[7]

presentation team up to determine the specificity and selectivity of

recognition by saccharide receptors (lectins). Structure-activity

analysis would be furthered, if the glycan part of a glycocluster could

be efficiently elaborated in situ, keeping all other parameters

constant. Using a bacterial 2,6-sialyltransferase and a small library

of bi- to tetravalent glycoclusters, we here illustrate the complete

conversion of scaffold-presented lactoside units into two different

sialylated ligands based on N-acetyl/glycolyl-neuraminic acid

incorporation and ensuing effect on their bioactivity for a plant toxin,

together with an analysis of the non-covalent substrate binding

contacts that the added sialic acid moiety makes to the lectin.

Enzymatic diversification of a scaffold-presented glycan can thus be

brought to completion in situ, offering a versatile perspective for

rational glycocluster engineering.

Introduction

Carbohydrates, the third alphabet of life, have an unsurpassed

capacity to form biological signals. As part of cellular

glycoconjugates, the glycan chains can transmit their information

via recognition by sugar receptors (lectins), hereby affecting key

activities such as adhesion, apoptosis, effector release such as

pro-inflammatory cytokines, glycoprotein routing and delivery,

innate immunity, migration or proliferation.^[1]What is central to

As an alternative approach, processing of

a simple glycan

already attached to a scaffold to expand it into a sophisticated

glycocluster would likewise produce the same desired product.

However, this route will not only yield a single desired product

but enable the parallel synthesis and study of many analogous

products with identical chemical profiles (valency and relative

positioning of lectin-reactive sites), because, different from the

first approach, the head group constitution can be tailored by

design in a divergent combinatorial fashion, by using a suitable

[a]

[b]

[a]

Dr. C. Bayón, Prof. Dr. M. J. Hernáiz

combination of

a

specific glycosyltransferase and its

Department of Organic and Pharmaceutical Chemistry, Faculty of

Pharmacy, Complutense University

Plaza Ramón y Cajal s/n, 28040, Madrid, Spain

E-mail: mjhernai@ucm.es

Dr. N. He, Prof. Dr. W.-D. Fessner

Department of Organic Chemistry and Biochemistry, Technische

Universität Darmstadt

Alarich-Weiss-Str. 4, 64287 Darmstadt, Germany

Dr. P. Blasco and Prof. Dr. J. Jimenez-Barbero

Departamento de Ciencia de Proteínas, CIB-CSIC,

C/Ramiro denMaeztu 9, 28040 Madrid, Spain

Prof. Dr. H-J. Gabius, Dr. S. André

Institut für Physiologische Chemie, Tierärztliche Fakultät

LudwigMaximilians-Universität München, Veterinärstr. 13, 80539

München, Germany

corresponding sugar nucleotide substrate.^[8]In this report, we

illustrate the feasibility of this approach exemplarily by efficient

enzymatic sialylation of a small library of multivalent lactosylated

scaffolds using a bacterial 2,6-sialyltransferase (2,6-SiaT) and

two different naturally occurring neuraminic acid derivatives.

The potent plant toxin Viscum album agglutinin (VAA, viscumin),

a galactoside-specific lectin, has been described to tolerate

substitutions at the 6-OH of galactose, thus binding 2,6-

sialylated lactose/N-acetyllactosamine with equal or enhanced

affinity.^[7a,9]A series of lactoside-presenting bi- to tetravalent

glycoclusters based on an aromatic core such as 1, 3, 5-

triiodobenzene or phloroglucinol had previously been prepared

and shown to be active as inhibitors of toxin binding to

glycoproteins and cells.^[10]Here, we report on their diversification

by direct enzymatic sialylation for an activity assessment by

surface plasmon resonance (SPR)-based affinity measurements

and for NMR-spectroscopic interaction analysis. Molecular

[c]

[d]

[e]

Prof. Dr. Ángel Rumbero

Department of Organic Chemistry, Faculty of Science, Autonoma

University of Madrid, Spain

Supporting information for this article is given via a link at the end of

the document

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modeling was performed to provide structural information on the

role of the sialic acid residue in the binding interaction. The

proof-of-principle character of this study confirms the utility of a

post-synthetic enzymatic diversification of glycans following their

scaffold conjugation as an alternative strategy for the elaboration

of expanded glycoclusters and illustrates the preparation of pure

glycoclusters with different sialic acids for testing.^[8]

cancer metastasis, blood coagulation, inflammation, and

bacterial or viral infections, in several cases acting with different

affinity.^[16]

A requirement for enzymatic generation of multivalent sialylated

glycoclusters is the need for quantitative conversion of all

available reaction sites to avoid the formation of product

mixtures, and the purification of the multiply charged target

structures. Therefore, to evade effects from inhibition by side

products (e.g., released CMP), the sialoside extension of the

lactoside units was performed by first generating the nucleotide-

activated sialic acid donor separately, followed by its use as a

substrate for the sialylation step as outlined in Figure 1.

Following this strategy, in the first stage each of the two sialic

acids and CTP were converted to the corresponding CMP-

activated donor by using the CMP-sialate synthetase from

Neisseria meningitidis.^[17,18]The pyrophosphate side product was

Results and Discussion

Glycocluster synthesis

Six glycoclusters having different levels of multivalency,

molecular dimensions, structural flexibility and polarity were

designed for biochemical affinity studies. Their chemoenzymatic

synthesis was executed in three stages, namely (i) the chemical

synthesis of the multivalent core scaffolds, (ii) decoration with -

lactoside units by using click chemistry, and (iii) glycan

extension by enzymatic sialylation. One topological set was

composed of aromatic cores that were rigidly held by

ethynylidene connectors (G1 = 8, 12, and 15), while the other

set was constructed from phenol core structures that were

interconnected by conformationally flexible and more polar ether

linkages (G2 = 17, 19 and 21). The core scaffolds G1/G2

together with their synthetic route for installation of the proper

functionalization as terminal dialkyne, trialkyne and tetraalkyne

are shown in Scheme 1, and their decoration with peripheral -

lactoside units (24, 26, 28, 30, 32 and 34) is illustrated in

Scheme 1.

hydrolyzed in situ to inorganic phosphate by adding

a

pyrophosphatase to prevent inhibition. The reaction was carried

out in a pH-stat set-up to maintain the pH at 8.6, an optimal

value for maximum enzyme activity and high stability of the

nucleotide-activated products. Reaction progress was monitored

by TLC. Upon completion, crudes were removed by

centrifugation, the supernatant lyophilized and used without

further purification as substrate for the sialyltransferase

conversion.

In the second stage, the two sets of glycoclusters were

subsequently sialylated by using the 2,6-sialyltransferase of

Photobacterium leiognathi (2,6SiaT)^{[17, 19]}as catalyst with either

the synthetic CMP-Neu5Ac or the CMP-Neu5Gc as donor

substrates, respectively. Reactions were carried out in Tris/HCl

buffer (50 mM, pH 8.6), supplemented with 50 mM MgCl₂as

cofactor, at 30 °C with careful pH control. Five equivalents of the

corresponding CMP-activated sialic acid were added as donor

for each glycocluster valency to ensure complete conversion of

each reactive site. The CMP released during the reaction, which

inhibits the sialyltransferase activity, was hydrolyzed in situ by

addition of an alkaline phosphatase to give cytidine and

inorganic phosphate (Figure 1).^[17]Reaction progress was

monitored by HPLC. Upon complete conversion, reaction

mixtures were centrifuged and lyophilized, and the products

were purified by gel permeation chromatography.

In the G1 series, compound

8 and 12 were prepared by

Sonogashira-Hagihara reaction (SH)^[11]with several aromatic

and aliphatic alkynes (see Supporting Information). The

synthesis of branched structure with four terminal acetylenes,

compound 15, started from 1,3,5-tribromobenzene by multistep

SH[^11]using the same procedure assisted by microwave

irradiation (MW) (Scheme 2 )

In the G2 series, the ether-type dialkyne 17, 19 and 21 were

synthesized

as previously reported

(see Supporting

Information).^[12,13]Ester-protected lactosyl azide 22 was

synthesized as previously reported,^[14]and the click reaction

[15]

with terminal acetylenes of scaffolds 8, 12, 15, 17, 19 and 21

was carried out using CuSO₄and sodium ascorbate in DMF-H₂O

by MW at 80 ºC for 15 min, to afford the compounds 23, 25, 27,

29, 31 and 33 with hydroxyl groups still protected. Deacetylation

was carried out with NaOMe in MeOH to yield the free

glycoclusters 24, 26, 28, 30, 32 and 34 (Scheme 1), which were

readily soluble in water.

Characterization

glycoclusters

of

the

sialyllactoside-containing

The bacterial 2,6SiaT was able to complete the sialylation

reactions with practically quantitative conversion of all lactosyl

moieties into 2,6-sialyllactosyl units (Table 1). Figure S1 (see

Supporting Information) shows the exemplary HPLC monitoring

of the reaction using the glycocluster 34 as the acceptor and

CMP-Neu5Ac as the donor prior to, during, and after complete

substrate processing. After two rounds of purification by BioGel

P-2 column chromatography, the isolated product yields

amounted to around 50%. All the twelve individual components

of the glycocluster library were fully characterized by one- and

two-dimensional NMR spectroscopy (¹H, ¹³C, HSQC) to verify

their structures as shown in Figure 2.

Enzymatic elaboration of sialylated glycoclusters

For the enzymatic diversification by -2,6-sialylation of the six bi-

to tetravalent glycoclusters in our test panel (Scheme 1) we

selected the naturally occurring N-acetyl-neuraminic and N-

glycolyl-neuraminic acids (Neu5Ac or Neu5Gc, respectively).

They are are key and differentially present elements of sialylated

glycans because of their participation in numerous

carbohydrate–protein interactions that mediate cellular

recognition phenomena, including fertilization, embryogenesis,

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Scheme 1. Synthesis of G1/G2-type glycoclusters 24, 26, 28, 30, 32 and 34: (i) CuSO₄.5H₂O, sodium ascorbate, DMF-H₂O, MW 80 ºC-20 min. Design elements

are color coded as the central aromatic scaffold (rigid G1 in blue, flexible G2 in red), a click linker (green) and the -lactoside as sugar head group for lectin

interaction (black).

Scheme 2. Synthesis of the rigid scaffold 15: i) PdCl₂(PPh₃)₂(5%), CuI (10%), NEt₃, THF dry, MW: 130ºC, 15 min.; ii) NaOH dry, toluene, reflux.

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Figure 1 Diversification of G1 and G2 glycoclusters using 2,6SiaT and two different CMP-activated sialic acids as donors

Figure 2. Structures of the sialyllactoside-presenting glycocluster sets G1 and G2 (35a/b-40a/b). Color coding used as in Scheme 1

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Lectin binding monitored by SPR

depicted. The strongest STD effect was detected for the sugar

protons for both Glc and Gal residues (with an absolute average

STD intensity of approximately 5%; see Figure 4C), strongly

suggesting that this region of the ligand is in close contact with

the protein. Additionally, several protons from the sialic acid

residue displayed STDs, around 4%, as identified in Fig. 4C.

Thus, this moiety apparently also is in contact to the lectin. The

aromatic linker also received saturation from the protein.

Figure 5 displays the STD analysis for ligand 40b, using the

same protocol. Similar to the observations for 40a, high STD

effects arose for all three sugar units and lower effects (<3%

absolute STD) for the aromatic linker protons. (Figure 5C). More

detailed analysis of the trisaccharide STD proton signals showed

the same effect for the Gal and Glc residues in the lactose

moiety (3% STD) but this time also an increased effect of the

Neu5Gc residue in the presence of viscumin (4% STD, Figure

5C). These results agree with the SPR results where the 5-Gc

modification in the sialic acid moiety appears to improve the

binding affinity to VAA, albeit not significantly (Table 2).

In these experiments, the lectin was covalently attached to the

CM5 sensor chip surface covered by

a dextran matrix

functionalized with carboxyl groups. The panel of sialylated

glycoclusters was then systematically tested under identical

conditions. Compounds 35-40 (a and b) were flowed over the

biochip containing immobilized lectin to estimate the binding

properties. Sensograms obtained showed increased interaction

profiles with the tetravalent glycoclusters 37a, 37b, 40a and 40b

(Figure 3), as noted before when using the corresponding

glycoclusters carrying only the unmodified lactoside as sugar

head group. Similar SPR measurements on compounds 35a,

35b, 36a, 36b, 38a, 38b, 39a and 39b showed low interactions

(data not shown). These experiments ascertained the bioactivity

of the products and the impact of valency.

Solutions with different concentrations of the selected

compounds were injected to establish an interaction model with

immobilized VAA. Processing of the results revealed that the

bivalent analyte model fits better to the experimental data than a

model assuming 1:1 binding, rendering the binding of one

glycocluster molecule to two lectin molecules as likely (Table 2).

The dissociation constant K_D(calculated as k_d1/k_a1) was found to

be in a nanomolar range, herewith in full agreement to the

affinity enhancement obtained by 2,6-sialylation when set in

relation to previously reported data on the lactose-presenting

glycoclusters.^[7a,9b,]The cluster 40b exhibited a slightly lower K_D,

but the observed differences in the interaction using the core

glycoclusters (Figure 2) and the two different sialic acids

(Neu5Ac and Neu5Gc) did not reach the threshold of statistical

significance.

Table 1. Yields obtained in the reactions of enzymatic glycocluster

functionalization using Neu5Ac-CMP or Neu5Gc-CMP as donors

α2,6SiaT

Yield (%)

35a

36a

37a

48

44

47

35b

36b

37b

53

54

52

38a

39a

40a

44

54

55

38b

39b

40b

43

52

54

Figure 3. Binding responses of different concentrations of the

sialylglycoclusters 40a (A) and 40b (B) with VAA immobilized on the CM5 chip,

showing association and dissociation phases. Responses were reference

Lectin interaction monitored by NMR spectroscopy

The contact pattern of the trisaccharide with the lectin was

measured by saturation transfer difference (STD) 1H NMR

spectroscopy. Figure 4 shows the STD results for 40a in the

presence of VAA, together with the assignment of the key

signals. Clear STD signals were observed, which are also

subtracted and blank corrected. Binding responses were fit to

interaction model.

a bivalent

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Lectin interaction by 3D modeling

X-ray crystal structure of the VAA complex with free lactose (pdb

code: 1PUU). Minimum conformation structures of the

The experimental observations were set in relation to a three-

dimensional model of the complex, which was built based on the

a]

Table 2. Binding constants for the sialylglycocluster–VAA interactions.^[

Sialylglycocluster

k_a1(M^-1s^-1)

k_d1(s^-1)

k_a2(RU^-1s^-1)

k_d2(s^-1)

K_D1(nM)

K_D2(RU)

^

37a

37b

40a

40b

9.05 (5.0) x 10³

8.28 (5.0) x 10³

2.75 (2.0) x 10⁴

1.20 (2.0) x 10⁵

5.35 (2.0) x 10^-3

6.22 (2.0) x 10^-3

2.11 (2.0) x 10^-2

2.06 (1.0) x 10^-2

1.54 (2.6) x 10^-3

2.84 (4.5) x 10^-3

7.71 (2.0) x 10^-3

8.64 (2.0) x 10^-3

2.37 (1.5) x 10^-2

3.52 (2.0) x 10^-2

4.85 (1.3) x 10^-2

3.91 (1.1) x 10^-2

591

751

767

171

102.00

91.10

39.00

35.50

3.65

4.09

2.63

1.50

^[a]Values in parentheses are standard errors.

OH

HO₂

O

C

HO₂

C

C)

HO

C

OH

O

H₃

NH

CH₃

HN

HO

O

HO

OH

O

HO

OH

O

OH

O

HO

OH

O

HO

OH

HO

~4%

~5%

O

OH

~5%

~4%

O

N

~5% OH

HO

~5%

N

O

N

CO₂Me

OH

O

HO

O

OH

O

HO₂

C

HO

H₃

OH

O

~4%

O

OH

~5%

HO₂

O

C

HO

HN

HO

O

HO

O

OH

NH

OH

O

CH₃

O

HO

OH

~4%

~5%

OH

HO

O

Figure 4. (A) STD spectra (bottom) and off-resonance or reference spectra (top) of the sample that contains 40a in the presence of VAA with a molar ratio 100:1.

The absolute STD percentages are given for the corresponding proton signals. (B) Extension of the proton sugar region with the corresponding STD absolute

values. (C) Schematic structure of 40a along with the average values of the absolute STD intensities.

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OH

HO₂

O

C

HO₂

C

C)

HO

OH

NH

O

HN

HO

O

HO

OH

HO

OH

O

OH

HO

OH

O

OH

O

HO

OH

O

HO

OH

HO

~4%

~3%

O

OH

~3%

~4%

O

N

~3% OH

HO

~3%

N

O

<3%

O

N

CO₂Me

OH

O

HO

O

OH

O

HO₂

C

HO

OH

O

~4%

O

OH

~3%

HO₂

O

C

HO

HN

HO

O

HO

O

OH

NH

OH

O

OH

HO

OH

~4%

~3%

OH

HO

O

Figure 5. (A) STD spectra (bottom) and off-resonance or reference spectra (top) of the sample that contains 40b in the presence of VAA with a molar ratio 100:1.

The absolute STD percentages are given for the corresponding proton signals. (B) Extension of the proton sugar region with the corresponding STD absolute

values. (C) Schematic structure of 40b along with the average values of the absolute STD intensities.

ASN256

GLN238

ASP235

TYR249

SER200

Figure 6. Docking model of the trisaccharide moiety of compound 40a showing protein residues with direct substrate contacts.

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glycoclusters were superimposed on the lactose binding

a Biotage Initiator Classic 2.5. NMR spectra were recorded at 293

K, using 500 MHz spectrometer (Bruker AC). Shifts are

site followed by

a

short molecular dynamics run and

a

energy minimization (see Experimental Section). Owing to

the differences in spatial accessibility of the two lectin sites

per B-chain at concentrations of µg/mL and higher (with

the Tyr site fully, the Trp site only partially accessible),^[20]

the Tyr site was analyzed, Figure 6 reveals that Ser200

and Gln238 form hydrogen bonds with 3-OH and the

carboxylic acid of the sialic acid residue, respectively. Of

course, these interactions arrest the flexibility of the 2,6-

sialylgalactosyl unit, counterbalancing their favorable

contribution to the thermodynamic balance sheet.

referenced relative to deuterated solvent residual peaks. Complete

signal assignments from 1D and 2D NMR spectroscopy were

based on COSY, HSQC and HMBC correlation experiments. Mass

spectra were recorded using an Applied Biosystems QSTAR XL.

Thin-layer chromatography (TLC) was carried out on aluminum

sheets coated with silica gel 60 F254 (Merck). TLC plates were

inspected by UV light ( = 254 nm) and developed by treatment

with phosphomolybdic acid hydrate in ethanol (10 %), followed by

heating. Silica gel column chromatography was performed with

silica gel (230-400 mesh).

When running docking protocols and DOSY experiments

to determine the diffusion coefficients of complexes, the

SPR sensor chips CM5 (carboxymethylated dextran) and others

reagents used in SPR experiments were purchased from GE

Healthcare. SPR experiments were performed using a Biacore

3000.

tetravalent compound was found to associate with

a

maximum of two lectin molecules (data not shown). The

presence of the terminal sialic acid thus did not affect the

complex stoichiometry. However, an extension of the

glycan chain to the natural complex-type N-glycan can do

so, with each branch then serving as docking site in

triantennary N-glycans of the glycoprotein asialofetuin.^[21]

Neu5Ac and CTP were purchased from R&S Pharmchem (China)

and Carl Roth (Germany), respectively. Neu5Gc was synthesized

18]

as previously described.^[17,

CMP-Neuraminic acid synthetase

and and the bacterial 2,6 SiaT were produced and purified as

previously described.^[17,18]

VAA was purified by affinity chromatography on lactosylated

Sepharose 4B, obtained after activation by divinyl sulfone,

checked for purity by one- and two-dimensional gel electrophoresis

and for activity by haemagglutination, solid-phase assays and cell

toxicity.^[25]

Conclusions

Glycoclusters offer an enormous potential to gain robust

insights into the specificity and selectivity of lectins,

depending on sugar sequence and topology of

presentation. The exemplary post-synthetic in situ

decoration of a set of model glycoclusters by enzymatic

2,6-sialylation of the -lactoside sugar head group

provides proof-of-principle evidence for this route of glycan

elaboration after their scaffold conjugation, as naturally

occurring in vivo in the Golgi apparatus for glycoproteins

and glycolipids. Due to the growing availability of

5-Bromo-1,3-bis(3-hydroxyl-3-methylbutynyl)benzene (13)

1,3,5-Tribromobenzene (4 g, 12.74 mmol), Pd(PPh₃)2Cl₂(447 mg,

0.637 mmol), CuI (243 mg, 1.28 mmol) and 2-methyl-3-butyn-2-ol

(2.13 g, 25.4 mmol), gave 13 (2.57 g, 63%). Flash chromatography

on silica (hexane-AcOEt 2:1). ¹H NMR:  7.59 (s, 1H), 7.34 (s, 2H),

1.59 (s, 12H).

1,4-Bis[{3,5-bis(3-hydroxyl-

3methylbutynyl)phenyl}ethynyl]benzene (14)

glycosyltransferases,

this

strategy

of

structural

13 (2.44 g, 7.59 mmol ), Pd(PPh₃)2Cl₂(266 mg, 0.38 mmoles), CuI

(144 mg, 0.76 mmol) and 3 (319 mg, 2.53 mmol), gave 14 (600 mg,

40%). Flash chromatography on silica (hexane-AcOEt 1:2). ¹H

NMR:  7.61 (s, 2H), 7.57 (s, 4H), 7.32 (s, 4H).

diversification of lectin ligands in vitro enables a systematic

analysis of the importance of sequence at constant

topology of presentation. Using this diversification tool, the

conjugation of engineered sialic acid derivatives^[8,17]makes

a straightforward extension possible to determine its effect

on lectin binding affinities with our test set of glycoclusters

1,4-Bis[{3,5-bis(ethynyl)phenyl}ethynyl]benzene (15)

or other suitable scaffolds,^[2]for VAA or other biomedically

14 (500 mg, 0.82 mmol), NaOH (13.2 mg, 0.34 mmol) and 15 mL

dry toluene, gave15 (193 g, 63%). Flash chromatography on silica

(hexane-AcOEt 20:1). ¹H NMR:  7.61 (s, 4H), 7.56 (s, 2H), 7.49 (s,

4H), 3.09 (s, 4H); ¹³C NMR (CDCl₃):  135.27, 135.13, 131.69,

123.81, 123.00, 90.32, 89.41, 81.73, 78.60. The spectroscopic

properties were consistent with those published.^[26]

22]

relevant lectins.^[5,

Remarkably, the method can be

applied to spherical glyconanoclusters,^[23]facilitating work

with vesicle-presented glycodendrimers to yield

programmable cell surfaces and natural or rationally

engineered lectins.^[24]

Click reaction (General procedure)

The scaffold (8, 12, 15, 18, 19 and 21), CuSO₄.5H₂O (0.3

equiv/alkyne) and sodium ascorbate (0.3 eqiv/alkyne) was

dissolved in DMF/H₂O (9:1). The mixture was stirred until the

complete dissolution of the reactants, then the sugar azide (22)

(1.5 equiv/alkyne) was added dropwise and the mixture reaction

was stirred several minutes. Afterwards, the reaction mixture was

Experimental Section

Materials and reagents

All reagents were commercial and used without further purification.

Reactions under microwave (MW) activation were performed using

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transferred into a reactor microwave (Biotage Initiator Classic 2.5)

and the resulting mixture was heated at 80 ºC for 20 minutes. After

being cooled to room temperature, the mixture was dissolved in

ethyl acetate and washed with a solution of 1% EDTA in water

(2x10 ml) and brine (2x10 ml). The combined organic layers were

dried over anhydrous magnesium sulfate, filtered and concentrated

under reduced pressure. The residue was purified by flash

chromatography on silica (230-400 mesh, ethyl acetate-ethanol

50:1).

Five equiv. of CMP-activated sialic acid were added per valency.

Reactions were started by adding solutions containing 2,6SiaT (60

μL) and alkaline phosphatase (20 μL) at 30ºC. The pH was

adjusted periodically to 8.6 with 2.0 M NaOH. After 24 h, the crude

mixture was centrifuged at 10.000 r.p.m for 10 min.

The course of the reaction was followed by TLC (mobile phase

butanol/acetone/acetic acid/water 32:33:7:28) and HPLC, using a

Shimadzu equipment with diode-array detector on an XBridge

column (C₁₈silica, 3,5 μm; 0,3 x 15 cm) using a mobile phase

composed of solvent A (water + 0,1% TFA) and solvent B (95%

acetonitrile, 4,9% water, 0,1% TFA) with the gradient conditions

described in Table 3.

Compounds 23, 25 and 27. Flash chromatography on silica (ethyl

acetate-ethanol 50:1). For NMR data, see Table in Supporting

information.

Table 3. Gradient conditions used for the HPLC analysis of

Compounds 29, 31 and 33. Flash chromatography on silica (ethyl

acetate-ethanol 50:1). For NMR data, see Table in Supporting

information.

sialylglycoclusters.

Solvent

t (min)

A (%)

B (%)

Deacetylation (General Procedure)

The peracetylated glycoconjugate (23, 25, 27, 29, 31 and 33) were

dissolved in anhydrous methanol and NaOMe (1M) was added,

and the reaction was stirred until TLC showed full deacetylation,

then neutralized (Amberlist-15, H+) and filtered. The filtrate was

concentrated under reduced pressure, and the solid precipitate

was washed with anhydrous methanol without any further

purification.

0

100

55

0

25

30

35

45

100

0

100

Compound 24. NMR data (see Table S1) MALDI C₄₈H₆₄N₆O₂₂[M ⁺

Na]⁺1099.5 (Supporting Information)

Purification of the sialylated glycoclusters

+

Sialylated products obtained from the sialylation reactions were

purified by molecular exclusion chromatography on a 170 x 2 cm

column using BioGel P-2 Gel (fine, 45-90 μm, exclusion limits 100-

1800; BioRad) as stationary phase and water as the mobile phase.

Fractions were analyzed by TLC using butanol/acetone/acetic

acid/water 32:33:7:28 as the eluent. Product containing fractions

were concentrated by rotary evaporation and the purification

process was repeated. Tubes containing the pure compound were

concentrated by rotary evaporation, lyophilized and stored at -20ºC.

Samples were analyzed by NMR spectroscopy.

Compound 26. NMR data (see Table S2) MALDI C₈₁H₉₉N₉O₃₃[M

Na]⁺1748.8 (Supporting Information)

Compound 28. NMR data (see Table S3). MALDI C₉₀H₁₂₂N₁₂O₄₄

[M ⁺Na]⁺2097.7 (Supporting Information)

Compound 30. NMR data (see Table S4). MALDI : C₄₄H₆₆N₆O₂₆[M

+ Na]⁺1117.5 (Supporting Information)

Compound 32. NMR data (see Table S5). MALDI : C₉₄H₁₃₆N₁₂O₅₂

[M ⁺Na]⁺1538.6 (Supporting Information)

Compound 34. NMR data (see Table S6). MALDI C₆₀H₉₃N₉O₃₆[M

⁺Na]⁺2287.9 (Supporting Information)

For original ¹H NMR and ¹³C NMR spectra of compounds 35a/b

through 40a/b (see Supporting information).

Sialic acid activation

The reaction was carried out with Neu5Ac (504 mg, 1 equiv.) and

cytidine triphosphate (945 mg, 1.2 equivalents) in Tris/HCl buffer

(16 mL; 50 mM, pH 8.6), containing 50 mM MgCl₂. After adding 60

mg of CMP-sialate synthetase from Neisseria meningitidis^[17,18]and

40 µL alkaline phosphatase, the reaction was allowed to proceed

with magnetic stirring at 37ºC for 24h in a pH-stat (Metrohm 665

Dosimat), keeping a constant pH of 8.6 using 2.0 M NaOH. The

35a

¹H NMR (500 MHz, DMSO-d6)  1.34 (4H, t, J = 12 Hz, H-2´´´B),

1.89 (6H, s, H-11´´´), 2.15 (4H, m, H-18), 2.63 (4H, dd, J = 12 and

5 Hz, H-2´´´A), 3.11 (2H, dd, J = 9.5 and 7.9 Hz, H-2´), 3.20 (2H,

H-7´´´), 3.31 (2H, H-4´), 3.31 (2H, H-2´´), 3.32 (2H, H-5´), 3.33 (2H,

H-5´´´), 3.36 (2H, H-4´´´), 3.37 (2H, H-3´), 3.34 (4H, H-6´´B), 3.48

(4H, H-8´´´), 3.49 (4H, H-19B), 3.52 (2H, H-3´´´), 3.56 (2H, H-5´´),

3.59 (4H, H-6´´A), 3.60 (2H, H-4´´), 3.60 (2H, H-6´´´), 3.62 (4H, H-

6´B), 3.76 (4H, H-6´A), 3.82 (4H, H-19A), 4.21 (2H, d, J = 7.5 Hz,

H-1´´), 4.24 (2H, d, J = 7.9 Hz, H-1´), 4.54 (4H, t, J = 6.6 Hz, H-17),

7.66 (4H, d, J = 7.3 Hz, H-3,H -5, H-10, H-14), 7.92 (4H, d, J = 7.3

Hz, H-2, H-6, H-11, H-13), 8.64 (2H, s br, H-16); ¹³C NMR (125

reaction progress was followed by TLC (Merck silica 60 GF₂₅₄

using n-propanol/H₂O/NH₃8:2:1 as mobile phase. The crude

mixture was centrifuged at 13.000 r.p.m for min, and the

)

3

supernatant was lyophilized and stored at -20ºC. The same

protocol was used for Neu5Gc activation.

Sialyltransferase reactions

MHz, DMSO-d6)

 23.02 (C11´´´), 30.38 (C18), 42.17 (C2´´´),

Reactions were carried out with 30 mg of glycocluster (1 equiv.) in

1.5 mL of 50 mM tris/HCl buffer containing 50 mM MgCl₂, at pH 8.6.

47.14 (C17), 53.43 (C4´´´), 59.72 (C8´´´), 60.94 (C6´), 63.82 (C6´´),

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65.79 (C19), 67.78 (C3´´´), 68.68 (C4´´), 69.49 (C7´´´), 70.99

(C2´´), 71.82 (C6´´´), 73.31 (C5´´´), 73.4 (C3´´), 73.57 (C2´), 74.27

(C5´´), 75.34 (C3´), 75.34 (C5´), 81.09 (C4´), 90.48 (C7, C8),

100.71 (C1´´´), 102.96 (C1´), 104.21 (C1´´), 121.88 (C4, C9),

122.84 (C16), 125.79 (C2, C6, C11, C13), 131.54 (C1, C12),

132.48 (C3, C5, C10, C14), 145.97 (C15), 171.65 (C9´´´), 172.70

(C10´´´). HRMS m/z calcd for C₇₀H₉₈N₈O₃₈Na [M + Na]⁺1681.5880,

found 1681.5873

¹H NMR (500 MHz, DMSO-d6)  1.32 (6H, t, J = 12 Hz, H-2´´´B),

2.15 (6H, m, H-18), 2.66 (6H, dd, J = 12 and 5 Hz, H-2´´´A), 3.10

(3H, dd, J = 9.5 and 7.9 Hz, H-2´), 3.18 (3H, H-7´´´), 3.30 (3H, H-

2´´), 3.31 (3H, H-4´), 3.31 (3H, H-5´), 3.32 (6H, H-6´´B), 3.36 (3H,

H-3´), 3.40 (3H, H-4´´´), 3.40 (6H, H-8´´´), 3.44 (3H, H-5´´´), 3.49

(6H, H-19B), 3.55 (3H, H-5´´), 3.61 (6H, H-6´B), 3.65 (3H, H-3´´´),

3.58 (6H, H-6´´A), 3.62 (3H, H-4´´), 3.61 (3H, H-6´´´), 3.76 (6H, H-

6´A), 3.81 (6H, H-19A), 3.88 (6H, s, H-11´´´), 4.20 (3H, d, J = 7.5

Hz, H-1´´), 4.25 (3H, d, J = 7.9 Hz, H-1´), 4.54 (6H, t, J = 6.6 Hz,

H-17), 7.70 (6H, d, J = 7.3 Hz, H-10, H-14), 7.82 (3H, s, H-2, H-4,

H-6), 7.95 (6H, d, J = 7.3 Hz, H-11, H-13), 8.71 (2H, s br, H-16);

¹³C NMR (125 MHz, DMSO-d6)  30.32 (C18), 42.50 (C2´´´), 47.09

(C17), 53.11 (C4´´´), 58.83 (C8´´´), 61.00 (C6´), 61.74 (C11´´´),

64.01 (C6´´), 65.56 (C19), 67.61 (C3´´´), 68.59 (C4´´), 69.62

(C7´´´), 71.01 (C2´´), 71.88 (6´´´), 73.39 (C5´´´), 73.51 (C3´´),

73.71 (C2´), 74.20 (C5´´), 75.34 (C3´), 75.34 (C5´), 81.15 (C4´),

88.57 (C7), 91.41 (C8), 100.98 (C1´´´), 102.95 (C1´), 104.21 (C1´´),

121.29 (C9), 122.86 (C16), 124.23 (C1, C3, C5), 125.83 (C11,

C13), 131.97 (C12), 132.72 (C10, C14), 134.19 (C2, C4, C6),

145.86 (C15), 171.19 (C9´´´), 175.10 (C10´´´).

35b

¹H NMR (500 MHz, DMSO-d6)  1.35 (4H, t, J = 12 Hz, H-2´´´B),

2.15 (4H, m, H-18), 2.66 (4H, dd, J = 12 and 5 Hz, H-2´´´A), 3.15

(2H, dd, J = 9.5 and 7.9 Hz, H-2´), 3.20 (2H, H-2´´), 3.21 (2H, H-

7´´´), 3.32 (2H, H-5´), 3.33 (2H, H-4´), 3.34 (4H, H-6´´B), 3.38 (2H,

H-3´), 3.43 (2H, H-4´´´), 3.48 (2H, H-5´´´), 3.50 (4H, H-8´´´), 3.51

(4H, H-19B), 3.57 (2H, H-5´´), 3.61 (4H, H-6´´A), 3.62 (4H, H-6´B),

3.62 (2H, H-6´´´), 3.64 (2H, H-4´´), 3.68 (2H, H-3´´´), 3.78 (4H, H-

6´A), 3.82 (4H, H-19A), 4.07 (4H, s, H-11´´´), 4.22 (2H, d, J = 7.5

Hz, H-1´´), 4.26 (2H, d, J = 7.9 Hz, H-1´), 4.55 (4H, t, J = 6.6 Hz,

H-17), 7.66 (4H, d, J = 7.3 Hz, H-3, H-5, H-10, -14), 7.92 (4H, d, J

= 7.3 Hz, H-2,H -6, H-11, H-13), 8.69 (2H, s br, H-16); ¹³C NMR

(125 MHz, DMSO-d6)  30.35 (C18), 42.43 (C2´´´), 47.13 (C17),

53.08 (C4´´´), 59.80 (C8´´´), 60.99 (C6´), 61.72 (C11´´´), 63.98

(C6´´), 65.77 (C19), 67.63 (C3´´´), 68.54 (C4´´), 69.58 (C7´´´),

71.00 (C2´´), 71.88 (C6´´´), 73.01 (C5´´´), 73.37 (C3´´), 73.59 (C2´),

74.20 (C5´´), 75.34 (C3´), 75.34 (C5´), 81.06 (C4´), 90.48 (C7, C8),

100.93 (C1´´´), 102.97 (C1´), 104.20 (C1´´), 121.88 (C4, C9),

122.90 (C16), 125.79 (C2, C6, C11, C13), 131.55 (C1, C12),

132.48 (C3, C5, C10, C14), 145.96 (C15), 171.16 (C9´´´), 175.12

(C10´´´). HRMS m/z calcd for C₇₀H₉₈N₈O₄₀Na [M + Na]⁺1713.5778,

found 1713.5763

HRMS m/z calcd for C₁₁₄H₁₅₀N₁₂O₆₀Na [M + Na]⁺2669.8953, found

2669.8941.

37a

¹H NMR (500 MHz, DMSO-d6)  1.33 (8H, t, J = 12 Hz, H-2´´´B),

1.89 (12H, s, H-11´´´), 2.18 (8H, m, H-26), 2.64 (8H, dd, J = 12 and

5 Hz, H-2´´´A), 3.10 (4H, dd, J = 9.5 and 7.9 Hz, H-2´), 3.20 (4H,

H-7´´´), 3.32 (4H, H-2´´), 3.32 (4H, H-4´), 3.33 (4H, H-5´), 3.34 (4H,

H-4´´´), 3.34 (8H, H-6´´B), 3.35 (4H, H-5´´´), 3.38 (4H, H-3´), 3.47

(8H, H-8´´´), 3.51 (4H, H-3´´´), 3.53 (8H, H-27B), 3.55 (4H, H-5´´),

3.58 (8H, H-6´´A), 3.59 (4H, H-6´´´), 3.62 (4H, H-4´´), 3.63 (8H, H-

6´B), 3.76 (8H, H-6´A), 3.82 (8H, H-27A), 4.21 (4H, d, J = 7.5 Hz,

H-1´´), 4.26 (4H, d, J = 7.9 Hz, H-1´), 4.56 (8H, t, J = 6.6 Hz, H-25),

7.72 (4H, s, H-10, H-11, H-13, H-14), 8.02 (3H, s, H-2, H-4, H-18,

H-22), 8.46 (6H, d, J = 7.3 Hz, H-6, H-20), 8.79 (2H, s br, H-24);

¹³C NMR (125 MHz, DMSO-d6)  23.02 (C11´´´), 30.32 (C26),

42.15 (C2´´´), 47.39 (C25), 53.47 (C4´´´), 59.80 (C8´´´), 60.94

(C6´), 63.89 (C6´´), 65.94 (C27), 67.74 (C3´´´), 68.6 (C4´´), 69.52

(C7´´´), 71.01 (C2´´), 71.80 (C6´´´), 73.38 (5´´´), 73.60 (C3´´),

73.70 (C2´), 74.21 (C5´´), 75.32 (C3´), 75.32 (C5´), 81.07 (C4´),

89.65 (C8, C15), 91.16 (C7, C16), 100.70 (C1´´´), 102.98 (C1´),

104.34 (C1´´), 122.30 (C6, C2), 122.8 (C24), 122.91 (C3, C17),

123.00 (C9, C12), 127.59 (C2, C4, C18, C22), 132.38 (C10, C11,

C13, C14), 132.59 (C1, C5, C19, C21), 145.61 (C23), 171.65

(C9´´´), 172.70 (C10´´´). HRMS m/z calcd for C₁₃₄H₁₉₀N₁₆O₇₆Na [M

+ Na]⁺3262.1392, found 3262.1404.

36a

¹H NMR (500 MHz, DMSO-d6)  1.32 (6H, t, J = 12 Hz, H-2´´´B),

1.89 (9H, s, H-11´´´), 2.15 (6H, m, H-18), 2.64 (6H, dd, J = 12 and

5 Hz, H-2´´´A), 3.11 (3H, dd, J = 9.5 and 7.9 Hz, H-2´), 3.19 (3H,

H-7´´´), 3.31 (3H, H-4´), 3.30 (3H, H-2´´), 3.32 (3H, H-5´), 3.33 (6H,

H-6´B), 3.33 (6H, H-6´´B), 3.35 (3H, H-5´´´), 3.35 (3H, H-4´´´), 3.37

(3H, H-3´), 3.48 (6H, H-8´´´), 3.49 (6H, H-19B), 3.50 (3H, H-3´´´),

3.56 (3H, H-5´´), 3.58 (6H, H-6´´A), 3.62 (3H, H-4´´), 3.60 (3H, H-

6´´´), 3.76 (6H, H-6´A), 3.81 (6H, H-19A), 4.21 (3H, d, J = 7.5 Hz,

H-1´´), 4.25 (3H, d, J = 7.9 Hz, H-1´), 4.55 (6H, t, J = 6.6 Hz, H-17),

7.70 (6H, d, J = 7.3 Hz, H-10, H-14), 7.82 (3H, s, H-2, H-4, H-6),

7.95 (6H, d, J = 7.3 Hz, H-11, H-13), 8.70 (2H, s br, H-16); ¹³C

NMR (125 MHz, DMSO-d6)  23.02 (C11´´´), 30.35 (C18), 42.35

(C2´´´), 47.07 (C17), 53.48 (C4´´´), 59.92 (C8´´´), 60.97 (C6´),

63.91 (C6´´), 65.68 (C19), 67.91 (C3´´´), 68.59 (C4´´), 69.56

(C7´´´), 71.00 (C2´´), 71.80 (C6´´´), 73.27 (C5´´´), 73.39 (C3´´),

73.59 (C2´), 74.25 (C5´´), 75.33 (C3´), 75.33 (C5´), 81.09 (C4´),

88.60 (C7), 91.50 (C8), 100.93 (C1´´´), 102.94 (C1´), 104.19 (C1´´),

121.36 (C9), 122.85 (C16), 124.24 (C1, C3, C5), 125.83 (C11,

C13), 132.01 (C12), 132.72 (C10, C14), 134.20 (C2, C4, C6),

145.87 (C15), 171.36 (C9´´´), 172.70 (C10´´´). HRMS m/z calcd for

C₁₁₄H₁₅₀N₁₂O₅₇Na [M + Na]⁺2621.9105, found 2621.9091.

37b

¹H NMR (500 MHz, DMSO-d6)  1.33 (8H, t, J = 12 Hz, H-2´´´B),

2.18 (8H, m, H-26), 2.66 (8H, dd, J = 12 and 5 Hz, H-2´´´A), 3.09

(4H, dd, J = 9.5 and 7.9 Hz, H-2´), 3.19 (4H, H-7´´´), 3.32 (4H, H-

2´´), 3.32 (4H, H-4´), 3.32 (8H, H-6´´B), 3.33 (4H, H-5´), 3.35 (4H,

H-5´´´), 3.38 (4H, H-3´), 3.41 (4H, H-4´´´), 3.45 (8H, H-8´´´), 3.53

(8H, H-27B), 3.54 (4H, H-5´´), 3.59 (8H, H-6´´A), 3.60 (4H, H-6´´´),

3.62 (8H, H-6´B), 3.63 (4H, H-4´´), 3.67 (4H, H-3´´´), 3.76 (8H, H-

6´A), 3.83 (8H, H-27A), 3.87 (8H, s, H-11´´´), 4.22 (4H, d, J = 7.5

Hz, H-1´´), 4.27 (4H, d, J = 7.9 Hz, H-1´), 4.55 (8H, t, J = 6.6 Hz,

36b

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H-25), 7.72 (4H, s, H-10, H-11, H-13, H-14), 8.02 (3H, s, H-2, H-4,

H-18, H-22), 8.46 (6H, d, J = 7.3 Hz, H-6, H-20), 8.79 (2H, s br, H-

24); ¹³C NMR (125 MHz, DMSO-d6)  30.32 (C26), 42.23 (C2´´´),

47.28 (C25), 53.10 (C4´´´), 60.00 (C8´´´), 60.85 (C6´), 61.77

(C11´´´), 63.96 (6´´), 65.97 (C27), 67.60 (C3´´´), 68.60 (C4´´),

69.58 (C7´´´), 71.01 (C2´´), 71.85 (C6´´´), 73.40 (C5´´´), 73.62

(C3´´), 73.71 (C2´), 74.25 (C5´´), 75.32 (C3´), 75.32 (C5´), 81.05

(C4´), 89.60 (C8, C15), 91.10 (C7, C16), 100.90 (C1´´´), 102.99

(C1´), 104.33 (C1´´), 122.10 (C3, C17), 122.23 (C2, C6), 122.70

(C9, C12), 122.83 (C24), 127.50 (C2, C4, C18, C22), 132.38 (C10,

C11, C13, C14), 132.60 (C1, C5, C19, C21), 145.67 (C23), 171.13

(C9´´´), 175.10 (C10´´´). HRMS m/z calcd for C₁₃₄H₁₉₀N₁₆O₈₀Na [M

+ Na]⁺3326.1189, found 3326.1201

39a

¹H NMR (500 MHz, D₂O)  1.69 (6H, t, J = 12 Hz, H-2´´´B), 1.97

(9H, s, H-11´´´), 2.15 (6H, m, H-11), 2.66 (6H, dd, J = 12 and 5 Hz,

H-2´´´A), 3.28 (3H, dd, J = 9.5 and 7.9 Hz, H-2´), 3.45 (3H, H-5´),

3.46 (6H, H-12B), 3.51 (3H, H-2´´), 3.60-3.80 (3H, H-5´´´), 3.55

(3H, H-3´), 3.55 (3H, H-4´), 3.60 (3H, H-7´´´), 3.60 (6H, H-6´´B),

3.70 (6H, H-8´´´), 3.75 (3H, H-3´´´), 3.75 (3H, H-5´´), 3.75 (6H, H-

6´B), 3.79 (6H, H-12A), 3.80 (3H, H-4´´´), 3.80 (3H, H-6´´´), 3.80

(6H, H-6´´A), 3.86 (6H, H-6´A), 3.90 (3H, H-4´´), 4.27 (3H, d, J =

7.9 Hz, H-1´), 4.36 (3H, d, J = 7.5 Hz, H-1´´), 4.51 (6H, t, J = 6.5

Hz, H-10), 5.15 (6H, s, H-7), 6.31 (3H, s, H-2, H-4, H-6), 8.04 (3H,

s br, H-9); ¹³C NMR (125 MHz, D₂O)  22.09 (C11´´´), 29.49 (C11),

40.10 (C2´´´), 47.16 (C10), 51.83 (C4´´´), 59.36 (C8´´´), 60.25

(C6´), 61.51 (C7), 62.67 (C6´´), 66.12 (C12), 68.26 (C3´´´), 68.40

(C7´´´), 68.55 (C4´´), 70.81 (C2´´), 71.79 (C6´´´), 72.42 (C5´´´),

72.57 (C3´´), 72.73 (C2´), 73.71 (C5´´), 74.59 (C3´), 74.59 (C5´),

79.62 (C4´), 96.63 (C2, C4, C6), 100.28 (C1´´´), 102.02 (C1´),

103.27 (C1´´), 125.54 (C9), 143.06 (C8), 159.40 (C1, C3, C5),

173.41 (C9´´´), 175.08 (C10´´´).

38a

¹H NMR (500 MHz, D₂O)  1.70 (4H, t, J = 12.0 Hz, H-2'''B), 1.99

(6H, s, H-11´´´), 2.17 (4H, m, H-13), 2.68 (4H, dd, J = 12.0 and 5.0

Hz, H-2´´´A), 3.28 (2H, dd, J = 9.5 and 7.9 Hz, H-2´), 3.45 (2H, H-

5´), 3.45 (2H, H-2´´), 3.45 (4H, H-14B), 3.53 (2H, H-5´´´), 3.53 (2H,

H-3´´´), 3.57 (2H, H-3´), 3.57 (2H, H-4´), 3.58 (2H, H-7´´´), 3.60

(4H, H-6´´B), 3.68 (4H, H-8´´´), 3.75 (2H, H-5´´), 3.75 (4H, H-14A),

3.76 (4H, H-6´B), 3.80 (2H, H-4´´´), 3.80 (2H, H-6´´´), 3.80 (4H, H-

6´´A), 3.85 (3H, s, H-8), 3.88 (4H, H-6´A), 3.90 (2H, H-4´´), 4.25

(2H, d, J = 7.9 Hz, H-1´), 4.37 (2H, d, J = 7.5 Hz, H-1´´), 4.55 (4H, t,

J = 6.6 Hz, H-12), 5.25 (4H, s, H-9), 6.88 (1H, H-4), 7.25 (2H, s, H-

2, H-6), 8.08 (2H, s br, H-11); ¹³C NMR (125 MHz, D₂O)  22.08

(C11´´´), 29.46 (C13), 40.14 (C2´´´), 47.13 (C12), 51.82 (C4´´´),

52.95 (C8), 59.37 (C8´´´), 60.25 (C6´), 61.73 (C9), 62.68 (C6´´),

66.24 (C14), 68.4 (C3´´´), 68.56 (C4´´), 68.56 (C7´´´), 70.82 (C2´´),

71.83 (C6´´´), 72.36 (C5´´´), 72.44 (C3´´), 72.72 (C2´), 73.73 (C5´´),

74.61 (C3´), 74.61 (C5´), 79.64 (C4´), 100.32 (C1´´´), 102.03 (C1´),

103.27 (C1´´), 108.00 (C4), 109.67 (C2, C6), 125.62 (C11), 131.8

(C1), 142.90 (C10), 158.61 (C3, C5), 168.00 (C7), 173.51 (C9´´´),

174.94 (C10´´´). HRMS m/z calcd for C₆₆H₁₀₀N₈O₄₂Na [M + Na]⁺

1699.5833, found 1699.5825

HRMS m/z calcd for C₉₃H₁₄₄N₁₂O₆₀Na [M + Na]⁺2411.8483, found

2411.8490

39b

¹H NMR (500 MHz, D₂O)  1.71 (6H, t, J = 12 Hz, H-2´´´B), 2.17

(6H, m, H-11), 2.70 (6H, dd, J = 12 and 5 Hz, H-2´´´A), 3.28 (3H,

dd, J = 9.5 and 7.9 Hz, H-2´), 3.43 (6H, H-12B), 3.50 (3H, H-5´),

3.50 (3H, H-7´´´), 3.57 (3H, H-3´), 3.57 (3H, H-4´), 3.60 (3H, H-2´´),

3.60-3.80 (3H, H-5´´´), 3.63 (6H, H-6´´B), 3.72 (6H, H-8´´´), 3.72

(3H, H-3´´´), 3.73 (6H, H-6´B), 3.75 (6H, H-12A), 3.78 (3H, H-5´´),

3.82 (6H, H-6´´A), 3.83 (3H, H-6´´´), 3.87 (6H, H-6´A), 3.88 (3H, H-

4´´), 3.90 (3H, H-4´´´), 4,07 (6H, s, H-11´´´), 4.28 (3H, d, J = 7.9 Hz,

H-1´), 4.37 (3H, d, J = 7.5 Hz, H-1´´), 4.51 (6H, t, J = 6.5 Hz, H-10),

5.15 (6H, s, H-7), 6.30 (3H, s, H-2, H-4, H-6), 8.02 (3H, s br, H-9);

¹³C NMR (125 MHz, D₂O)  29.50 (C11), 40.17 (C2´´´), 47.16

(C10), 51.54 (C4´´´), 59.90 (C8´´´), 60.25 (C6´), 61.51 (C11´´´),

61.73 (C7), 62.64 (C6´´), 66.29 (C12), 68.11 (C3´´´), 68.36 (C7´´´),

68.56 (C4´´), 70.81 (C2´´), 71.91 (C6´´´), 72.29 (C5´´´), 72.42

(C3´´), 72.74 (C2´), 73.74 (C5´´), 74.61 (C3´), 74.61 (C5´), 79.64

(C4´), 96.60 (C2, C4, C6), 100.30 (C1´´´), 102.03 (C1´), 103.31

(C1´´), 125.53 (C9), 143.06 (C8), 159.40 (C1, C3, C5), 173.46

(C9´´´), 175.68 (C10´´´).

38b

¹H NMR (500 MHz, D₂O)  1.73 (4H, t, J = 12 Hz, H-2´´´B), 2.19

(4H, m, H-13), 2.71 (4H, dd, J = 12 and 5 Hz, H-2´´´A), 3.30 (2H,

dd, J = 9.5 and 7.9 Hz, H-2´), 3.50 (4H, H-14B), 3.74 (2H, H-3´´´),

3.58 (2H, H-3´), 3.58 (2H, H-4´), 3.58 (2H, H-7´´´), 3.60 (2H, H-2´´),

3.60-3.80 (2H, H-5´´´), 3.65 (4H, H-6´´), 3.71 (4H, H-8´´´), 3.80 (2H,

H-5´´), 3.80 (2H, H-5´), 3.80 (2H, H-6´´´), 3.78 (4H, H-6´B), 3.82

(4H, H-14A), 3.87 (4H, H-6´´A), 3.90 (3H, s, H-8), 3.90 (4H, H-6´A),

3.92 (2H, H-4´´), 3.93 (2H, H-4´´´), 4.09 (4H, s, H-11´´´), 4.27 (2H,

d, J = 7.9 Hz, H-1´), 4.39 (2H, d, J = 7.5 Hz, H-1´´), 4.54 (4H, t, J =

6.6 Hz, H-12), 5.26 (4H, s, H-9), 6.88 (1H, H-4), 7.26 (2H, s, H-2,

H-6), 8.09 (2H, s br, H-11); ¹³C NMR (125 MHz, D₂O)  29.49

(C13), 40.23 (C2´´´), 47.16 (C12), 51.58 (C4´´´), 52.99 (C8), 59.41

(C8´´´), 60.29 (C6´), 61.49 (C11´´´), 61.76 (C9), 62.68 (C6´´),

66.27 (C14), 68.15 (C3´´´), 68.41 (C7´´´), 68.60 (C4´´), 70.85

(C2´´), 71.90 (C6´´´), 72.32 (C5´´´), 72.47 (C3´´), 72.76 (C2´),

73.79 (C5´´), 74.64 (C3´), 74.64 (C5´), 79.66 (C4´), 100.37 (C1´´´),

102.06 (C1´), 103.30 (C1´´), 108.51 (C4), 109.70 (C2, C6), 125.64

(C11), 131.85 (C1), 142.96 (C10), 158.65 (C3, C5), 168.10 (C7),

173.52 (C9´´´), 175.72 (C10´´´).HRMS m/z calcd for

C₆₆H₁₀₀N₈O₄₄Na [M + Na]⁺1731.5731, found 1731.5719

HRMS m/z calcd for C₉₃H₁₄₄N₁₂O₆₃Na [M + Na]⁺2459.8331, found

2459.8339

40a

¹H NMR (500 MHz, D₂O)  1.69 (8H, t, J = 12 Hz, H-2´´´B), 1.98

(12H, s, H-11´´´), 2.09 (8H, m, H-20), 2.68 (8H, dd, J = 12 and 5

Hz, H-2´´´A), 3.28 (4H, dd, J = 9.5 and 7.9 Hz, H-2´), 3.45 (4H, H-

5´), 3.45 (8H, H-21B), 3.50 (4H, H-2´´), 3.52 (4H, H-3´´´), 3.57 (4H,

H-3´), 3.57 (4H, H-4´), 3.60 (4H, H-5´´´), 3.60 (8H, H-6´´B), 3.65

(4H, H-7´´´), 3.70 (8H, H-8´´´), 3.73 (8H, H-6´B), 3.69 (8H, H-21A),

3.80 (4H, H-5´´), 3.80 (3H, s, H-8), 3.80 (4H, H-4´´´), 3.80 (4H, H-

6´´´), 3.80 (8H, H-6´´A), 3.87 (8H, H-6´A), 3.90 (4H, H-4´´), 4.34

(4H, d, J = 7.9 Hz, H-1´), 4.37 (4H, d, J = 7.5 Hz, H-1´´), 4.37 (8H, t,

J = 6.5 Hz, H-19), 4.60 (4H, s br, H-9), 4.84 (8H, s br, H-16), 6.41

(4H, s, H-4), 6.41 (4H, s, H-11, H-15), 6.41 (2H, H-13), 6.81 (3H, s,

H-2, H-6), 7.85 (3H, s br, H-18); ¹³C NMR (125 MHz, D₂O)  22.04

10.1002/chem.201604550

Chemistry - A European Journal

FULL PAPER

(11´´´), 29.59 (C20), 40.11 (C2´´´), 47.14 (C19), 51.85 (C4´´´),

52.76 (C8), 59.36 (C8´´´), 60.26 (C6´), 60.96 (C16), 62.67 (C6´´),

66.30 (C21), 68.28 (C3´´´), 68.41 (C7´´´), 68.56 (C4´´), 69 (C9),

70.81 (C2´´), 71.79 (C6´´´), 72.45 (C5´´´), 72.57 (C3´´), 72.74 (C2´),

73.68 (C5´´), 74.62 (C3´), 74.62 (C5´), 79.61 (C4´), 100.31 (C1´´´),

101.10 (C13), 102.03 (C1´), 103.31 (C1´´), 106.74 (C4), 106.74

(C11, C15), 108.41 (C2, C6), 125.00 (C18), 131.41 (C1), 139.27

(C10), 143.12 (C17), 158.91 (C3, C5), 158.91 (C12, C14), 167.52

(C7), 173.44 (C9´´´), 174.90 (C10´´´). HRMS m/z calcd for

C₁₃₈H₂₀₄N₁₆O₈₄Na [M + Na]⁺3452.2081, found 3452.2096

The NMR study of 40a and 40b in water solution was performed

using mM samples in D₂O. Standard 2D-methods, such as

1

TOCSY (with mixing times of 30 and 80 ms), ROESY (300 ms),

NOESY (400 and 600 ms), and HSQC experiments were

employed to assign the NMR chemical shifts, using BrukerAvance

500 and 600 MHz (equipped with a triple channel cryoprobe) NMR

spectrometers.

VAA solutions for STD experiments were prepared in deuterated

phosphate-buffered saline (10mM NaH₂PO₄, 137 mM NaCl and 7

mM KCl₂; pH 7.3) using ligand/lectin ratios of 1:100. The

temperature was 298 K. Data from one-dimensional STD

experiments were acquired at 600 and 500 MHz using a Gaussian

pulse (49 ms) cascade separated by 1 ms delays. In all cases, the

on-resonance frequency was set at the aliphatic regions (0.5 ppm),

and the experiments were recorded with a saturation time of 2 s.

The off-resonance frequency was always set at 100 ppm.

40b

¹H NMR (500 MHz, D₂O)  1.70 (8H, t, J = 12 Hz, H-2´´´B), 2.05

(8H, m, H-20), 2.66 (8H, dd, J = 12 and 5 Hz, H-2´´´A), 3.30 (4H,

dd, J = 9.5 and 7.9 Hz, H-2´), 3.42 (8H, H-21B), 3.43 (4H, H-5´),

3.51 (4H, H-2´´), 3.50 (4H, H-7´´´), 3.52 (4H, H-3´), 3.52 (4H, H-4´),

3.60 (8H, H-6´´B), 3.69 (8H, H-21A), 3.70 (8H, H-6´B), 3.70 (4H,

H-3´´´), 3.72 (8H, H-8´´´), 3.72 (4H, H-5´´´), 3.73 (3H, s, H-8), 3.73

(4H, H-5´´), 3.80 (4H, H-6´´´), 3.80 (8H, H-6´´A), 3.85 (8H, H-6´A),

3.88 (4H, H-4´´), 3.89 (4H, H-4´´´), 4.07 (12H, s, H-11´´´), 4.30 (4H,

d, J = 7.9 Hz, H-1´), 4.35 (4H, d, J = 7.5 Hz, H-1´´), 4.38 (8H, t, J =

6.5 Hz, H-19), 4.59 (4H, s br, H-9), 4.82 (8H, s br, H-16), 6.41 (4H,

s, H-4), 6.41 (4H, s, H-11, H-15), 6.41 (2H, H-13), 6.82 (3H, s, H-2,

H-6), 7.85 (3H, s br, H-18); ¹³C NMR (125 MHz, D₂O)  29.61

(C20), 39.93 (C2´´´), 47.18 (C19), 51.54 (C4´´´), 52.75 (C8), 59.92

(C8´´´), 60.28 (C6´), 61.05 (C16), 61.72 (C11´´´), 62.72 (C6´´),

66.30 (C21), 67.90 (C3´´´), 68.37 (C7´´´), 68.54 (C4´´), 69 (C9),

70.79 (C2´´), 71.98 (C6´´´), 72.37 (C5´´´), 72.46 (C3´´), 72.76 (C2´),

73.66 (C5´´), 74.60 (C3´), 74.60(C5´), 79.69 (C4´), 100.01 (C1´´´),

101.18 (C13), 102.02 (C1´), 103.32 (C1´´), 106.73 (C4), 106.73

(C11, C15), 108.40 (C2, C6), 125.03 (C18), 131.41 (C1), 139.27

(C10), 143.08 (C17), 158.96 (C3, C5), 158.96 (C12, C14), 167.52

(C7), 172.93 (C9´´´), 175.67 (C10´´´).

Molecular Modeling

The templates for the modeling procedures were built based on

crystallographic information of complexes of VAA with lactose (pdb

code: 1PUU). Structures of the glycoclusters were superimposed

in the TYR249 binding site working with the most populated

conformation found for the free state (according to a standard

NOE/molecular modeling approach). The complex structure was

then submitted to a short molecular dynamics run, followed by

energy minimization with a low gradient convergence threshold

(0.05) in 5000 steps. In all cases, the OPL2005 force field^[28]was

employed, as integrated in the Schroedinger MAESTRO suite of

programs.^[29]

Acknowledgements

HRMS m/z calcd for C₁₃₈H₂₀₄N₁₆O₈₈Na [M + Na]⁺3516.1877, found

This work was supported by the Ministry of Economy and

Competitiveness of Spain (MINECO, Grants CTQ2012-

32042 and CTQ2015-66206-C2-1-R) and by the State of

Hessia.

3516.1889.

SPR experiments

SPR experiments were performed at 25 ºC using Biacore 3000

(GE Healthcare). PBST (10 mM phosphate pH 7.40, 150 mM NaCl

and 0.005% v/v surfactant P20) was used as running buffer for

CM5 experiments. Solution with VAA was adjusted to 1 mg/mL in

10 mM phosphate pH 5.50 buffer and the lectin was immobilized in

flow cell 2 of a CM5 sensor chip following the amine coupling

Keywords: Carbohydrate-protein interaction • Enzymatic

synthesis • Glycodendrimer • Molecular recognition •

Sialyltransferase

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10.1002/chem.201604550

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Carlos Bayón, Ning He, Mario Deir-

Kaspar, Pilar Blasco, Hans-Joachim

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Lectin

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Direct enzymatic branch-end

extension of glycocluster-presented

glycans:

An effective strategy for

programming glycan bioactivity

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Direct Enzymatic Branch-End Extension of Glycocluster-Presented Glycans: An Effective Strategy for Programming Glycan Bioactivity

DOI: 10.1002/chem.201604550

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