Chemoselective reagents for covalent capture and display of glycans in microarrays

FULL PAPER

DOI: 10.1002/ejoc.200901234

Chemoselective Reagents for Covalent Capture and Display of Glycans in

Microarrays

Emiliano Cló,^[a]Ola Blixt,^[b]and Knud J. Jensen*^[a,c]

Dedicated to Professor Klaus Bock on the occasion of his 65th birthday

Keywords: Nanobioscience / Glycomics / Microarrays / Lectins / Carbohydrates / Chemoselectivity

Glycobiology has made very significant progress in the past

decades. However, further progress will significantly depend

on the establishment of novel methods for miniaturized,

high-throughput analysis of glycan–protein interactions. Ro-

bust solid-phase chemical tools and new, chemoselective rea-

gents for biologically meaningful display of surface-immobi-

lized glycans are likely to play a key role. Here we present

four new bifunctional linkers that allow highly chemoselec-

tive capture of unprotected glycans in solution to form gly-

can-linker conjugates for direct construction of glycan micro-

arrays (glycochips). The bifunctional linkers carry O-linked

aminooxy moieties, some with N-substituents at one end and

an amino group at the other. In addition, they contain a sub-

stituted benzene ring for UV traceability and improved puri-

fication of glycan-linker conjugates. NMR spectroscopic

studies in solution proved that N-substituted aminooxy link-

ers provided model glycan-linker conjugates with the β-gluc-

opyranoside configuration, i.e. the ring-closed form required

for biological recognition. Then an ensemble of glycan-linker

conjugates were assembled from mannobiose, lactose, and

N-acetyl-lactosamine and used for covalent printing of gly-

can microarrays. The stability of oximes were studied both in

solution and on-chip. In solution, two of the linkers provided

glycan-linker conjugates with a remarkable stability at pH 4

or higher, on-chip this relative stability was upheld. Two of

the linkers, with different properties, are recommended for

the glycobiology toolbox for the construction of glycan micro-

arrays from unprotected glycans.

ping of the glycome, new techniques that allow for a high-

throughput screening of carbohydrate probes with several

glycan-binding proteins (GBP) and glycan-processing en-

zymes (GPE) are important.

Introduction

In the post-genomic era the study of glycobiology has

attracted considerable attention, as most proteins in living

systems require post-translational modifications, most com-

monly glycosylation, for correct function.^[1]Glycans are es-

sential for life processes, including intra- and inter-cellular

signaling,^[2]host-guest interaction,^[3]cell adhesion,^[4]fertil-

ization^[5]and tumor proliferation.^[6]The term “glycomics”

covers the comprehensive study of all glycan structures of

a given cell type or organism.^[7]The absence of an apparent

direct flow of information from the genome to the glycome,

combined with the pronounced structural complexity of the

primary structure of glycans, makes the study of the gly-

come extremely complex. To facilitate the functional map-

Recently, a composite set of solid-phase tools for glyco-

biology specially devised to support high-throughput

screening has been emerging.^[8]One of the most prominent

of such tools is represented by glycan microarray technol-

ogy,^[9]in which a broad range of interaction types and mate-

rials have been used to anchor the glycan probes onto the

solid support.^[10]Ideally, glycan microarrays (i.e. glyco-

chips) should be able to present the glycans in a biologically

meaningful fashion. As GBPs typically require multival-

ency^[11]for efficient binding, presentation of the probe

structures should optimally occur in a clustered and direc-

tional fashion. Furthermore, it is desirable to present gly-

cans isolated from naturally occurring specimens on glyco-

chips. The attachment of glycans onto the array should use

methodologies that allow it to be robust and spatially de-

fined, such as covalent bonding. Several approaches to co-

valent glycochips have been described in recent years, in

which different types of attachment chemistries were suc-

cessfully applied, for example 1,3 dipolar cycloaddition be-

tween an azide and an alkyne,^[12]amide bond formation,^[13]

epoxide opening,^[14]Michael addition of thiols to malem-

[a] IGM, Faculty of Life Sciences, University of Copenhagen,

Thorvaldsensvej 40, 1871 Frederiksberg, Denmark

Fax: +45-3533-2398

E-mail: kjj@life.ku.dk

[b] Copenhagen Center for Glycomics, Department of Cellular and

Molecular Medicine, Faculty of Health Sciences, University of

Copenhagen,

Blegdamsvej 3, 2200 Copenhagen, Denmark

[c] Centre for Carbohydrate Recognition and Signaling, University

of Copenhagen,

Thorvaldsensvej 40, 1871 Frederiksberg, Denmark

Supporting information for this article is available on the

WWW under http://dx.doi.org/10.1002/ejoc.200901234.

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Glycans in Microarrays

ides^[15]and Staudinger ligation.^[16]However, most protocols

We reasoned that it would be desirable to develop a

require the carbohydrate probes to be constructed through methodology for covalent glycan microarray fabrication

multi-step synthesis in order to fashion them with the that could avoid extensive synthetic efforts and allow for

spacer moiety able to react with the solid support of choice. fast isolation of glycan-linker conjugates. The centerpiece

This process is time consuming, requires specialized skills of the linker design was the choice of α-nucleophile to pro-

in synthetic carbohydrate chemistry and makes the utiliza- vide stable conjugates and, ideally, retaining (at least par-

tion of glycans isolated from natural sources very difficult. tially) the cyclic nature of the sugar moiety at the reducing

Thus, the preparation of glycans for the glycochip is the end. The thermodynamic stability of substitution products

bottleneck of the whole process, preventing covalent glycan between aldehydes and α-nucleophiles is known to be the

microarrays from becoming a true high-throughput process. highest for hydroxylamine nucleophiles.^[22]Also, N-alkyl-

An attractive option to bypass extensive organic synthe- substituted hydroxylamines can direct the stereochemistry

sis for probe preparation is to make use of chemoselective at the saccharide reducing unit towards the retention of the

reactions between the native underivatized glycans and a ring-closed pyranose structure (either exclusively, as with

bifunctional linker. The presence of an aldehyde moiety at glucose, or predominantly, as with mannose and galac-

the reducing end of glycans can be used for chemoselective tose).^[23]The presentation of at least some of the glycan

couplings with strong α-nucleophiles such as hydrazines, hy- core structure in the ring-closed form has proven to be of

drazones, sulfonylhydrazides and hydroxylamines and sev- crucial importance for functional recognition of glycans by

eral examples of these glycoconjugates have been de- GBP and GPE, especially when interrogating short-chain

scribed.^[17]Some glycochips have already been designed to saccharides, as reported by Feizi et al. for neoglycolipids

take advantage of these chemoselective couplings. For ex- probes absorbed onto nitrocellulose microarrays^[18a]and re-

ample, oligosaccharides have been chemoselectively coupled cently highlighted by Thygesen^[24]et al. for gold nanopar-

with aminooxy-type linkers in solution, and the linker al- ticles. The only example available to date reporting the use

lowed specific, non-covalent absorption onto the chip sur- of a chemoselective bifunctional linker allowing solution-

face.^[18]Alternatively, the manufacture of slide surfaces phase glycan-linker coupling and subsequent covalent at-

coated with a suitable functional linker type exposing the tachment onto a reactive slide was provided by Blixt and

α-nucleophile to react with underivatized glycans spotted co-workers.^[25]This simple linear aliphatic linker comprised

onto it has also been described. The groups of Shin^[19]and both a N-methylhydroxylamine moiety, for chemoselective

Turnbull^[20]have both applied this concept to the construc- coupling with glycans, and a primary amine functionality

tion of hydrazide-coated microarrays starting from glass suitable for the attachment onto N-hydroxysuccinimide

and gold as solid supports, respectively. Moreover, conjuga- (NHS)-activated glass slides. However, this valuable linker

tion of free reducing oligosaccharides with a suitable spacer did not allow easy quantification of the glycoconjugates. In

moiety by imine formation followed by in situ reductive addition, precise information on the stability of glycan-

amination has been pursued in recent years. Cummings and linker conjugates constructed from α-nucleophiles is

co-workers^[21]reported a method to derivatize glycans with needed.

2,6-diaminopyridine linker (DAP) in order to generate co-

Here we present a panel of novel aminooxy-type bifunc-

valent microarrays onto NHS-activated glass slides. This tional linkers bearing different N,O-substitution patterns.

approach greatly reduced the synthetic manipulations To the best of our knowledge, no direct comparison of

needed to access derivatized carbohydrates, yet it exclusively structurally related aminooxy-type glycoconjugates, which

furnished open-chain structures of the core monosaccha- include both primary and secondary, N-alkylhydroxyl-

rides and had limited reactivity with the activated glass amines, is available to date for microarrays. Glycoconju-

slide.

gates with glucose as a model glycan were prepared in milli-

Scheme 1. Overview of three-step procedure involving glycan chemoselective coupling to the bifunctional linker, purification of the glyco-

conjugate and on-slide covalent fixation via amide bond formation.

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E. Cló, O. Blixt, K. J. Jensen

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gram amounts to assess both their stereochemistry and the hydroxy group with N-Boc-2-bromoethylamine in the

their hydrolytic stability. Furthermore, the applicability of

a three-step scheme (i.e. coupling, purification and on-chip

reaction) for the fabrication of glycan microarrays with

common saccharides (Scheme 1) was tested and the per-

formance of the different glycoconjugates with GBPs evalu-

ated.

presence of DBU as base in a microwave reactor at 65 °C

for 40 min, thus furnishing the fully Boc-protected target

linker Boc(B-AE) in 38% yield over two steps. Removal of

the acid-labile Boc groups was performed with HCl in diox-

ane, which gave the desired B-AE linker in 74% yield. The

use of acidic dioxane was preferred over neat trifluoroacetic

acid as the product upon deprotection became insoluble in

Results and Discussion

Preparation of Bifunctional Linkers and of Glucose-Linker

Conjugates

Linker Synthesis: The five linkers addressed by the pres-

ent study are presented in Figure 1; M-AE linker was origi-

nally developed by Blixt and co-workers^[25]and was used as

reference compound. The remaining four are novel struc-

tures which incorporate an aminooxy functionality for

chemoselective glycan capture, a primary amino group for

on-chip amide bond formation and an aromatic chromo-

phore moiety for UV detection of the target conjugates dur-

ing HPLC purification. The synthesis of N-benzylated O-

alkylated hydroxylamine linker B-AE made use of N-ben-

zylhydroxylamine as the central building block (Scheme 2,

Figure 1. Target linkers: 2-(methylaminooxy)ethanamine (M-AE),

2-(benzylaminooxy)ethanamine

(B-AE),

N-(2-aminoethyl)-4-

(aminooxymethyl)benzamide (AMB), N-(2-aminoethyl)-4-[(methyl-

aminooxy)methyl]benzamide (M-AMB), N-(2-aminoethyl)-4-

A). The nitrogen was first Boc-protected in order to react [(benzylaminooxy)methyl]benzamide (B-AMB).

Scheme 2. A: a) K₂CO₃, Boc₂O, THF, H₂O, room temp. (66%); b) N-Boc-2-bromoethylamine, DBU, MeCN, µw 65 °C (57%); c) HCl

4  in dioxane, room temp. (71%). B: d) N-Boc-ethylenediamine, EDC, DCM, room temp., (48%); e) N-hydroxyphthalimide, DBU,

MeCN, µw 65 °C (87%); f) hydrazine hydrate, DCM, MeCN, µw 65 °C (98%); g) N-Boc-N-methyl hydroxylamine or N-Boc-N-benzyl-

hydroxylamine (1), DBU, MeCN, µw 65 °C [Boc(M-AMB) 45%], [Boc(B-AMB) 90%]; h) HCl 4  in dioxane, room temp. (AMB 99%),

(M-AMB 83%), (B-AMB 94%).

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Glycans in Microarrays

dioxane, hence greatly facilitating its isolation as the HCl and their hydrolytic stability. We tested different reaction

mixtures, ranging from slightly acidic aqueous buffers to

mixtures of polar organic solvents and acetic acid. We

could notice that a change in solvent mixture had little or

no impact on the yield of conjugate formation. Further-

more, we could clearly observe that, given the same reaction

conditions, all the four N-substituted linkers consistently

gave lower amounts of conjugates compared AMB linker.

Ultimately, the conjugation reactions were performed in

milligram scale in mixtures of methanol and acetic acid at

room temperature. Intermediate peracetylation, in pyridine

once the conjugation reactions had reached equilibration,

served to facilitate the chromatographic separation of the

conjugates from the starting materials. Straightforward

Zemplén deacetylation allowed us to obtain the target glu-

cose conjugates Ac(B-AE)Glc, Boc(AMB)Glc, Ac(M-AMB)-

Glc and Ac(B-AMB)Glc in high purity (Figure 3, A).

Boc(AMB) was used for conjugation instead of AMB be-

cause peracetylation would also acetylate the linker amino

group. As the aliphatic M-AE linker structure is devoid of

UV-absorbing moieties, we had the need to tag its glucose

conjugate with a chromophore in order to later monitor

the hydrolytic process by HPLC fitted with online UV/Vis

detector. This was achieved by subjecting the crude glucose-

linker conjugate to one equivalent of N-(benzoyloxy)suc-

cinimide^[27](equimolar amounts to linker) in pyridine, thus

benzoylating the primary amine. Addition of excess acetic

salt by centrifugation. The synthesis of the linkers AMB,

M-AMB and B-AMB commenced with the preparation of

the common intermediate 2 by amide bond formation be-

tween 4-(chloromethyl)benzoic acid and N-Boc-ethylenedi-

amine^[26]in the presence of EDC as coupling reagent

(Scheme 2, B). The linker AMB was prepared following a

Gabriel-type synthesis scheme. The benzyl chloride 2 was

treated with N-hydroxyphthalimide in the presence of DBU

with microwave heating giving benzyl phthalic oxy-imide

intermediate, then treatment with hydrazine liberated the

insoluble phthalhydrazide, thus unmasking the aminooxy

moiety of the desired Boc(AMB) in high yield. Final Boc

removal from the primary amine in hydrochloric dioxane

furnished AMB as the hydrochloride. Only two steps led

from 2 to the remaining linkers M-AMB and B-AMB. First,

the chloride in 2 was substituted by N-Boc-N-methylhy-

droxylamine or N-Boc-N-benzylhydroxylamine (1) to ob-

tain the fully Boc-protected intermediates Boc(M-AMB)

and Boc(B-AMB), respectively. Finally, our standard Boc

deprotection scheme in HCl/dioxane allowed the isolation

of pure M-AMB and B-AMB linkers as the chloride salts

in high yields.

Model Glycoconjugates: Next, we focused on the prepa-

ration of glucose conjugates for all the five linkers presented

in Figure 1. These conjugates were chosen to serve as model

compounds in order to study their anomeric configuration

Scheme 3. Preparation of model glucose conjugates for studies of their hydrolytic stability. A: a) Glucose (5 equiv.), MeOH-AcOH (5:1),

room temp.; b) Ac₂O (excess), pyridine room temp.; c) MeONa, MeOH, room temp., [Ac(B-AE)Glc 59%, 3 steps], [Boc(AMB)Ac 74%,

3 steps], [Ac(M-AMB)Glc 49%, 3 steps], [Ac(B-AMB)Glc 35%, 3 steps]. B: d) Glucose (1.1 equiv.), MeOH/AcOH (5:1), room temp.; e)

2,5-dioxopyrrolidin-1-yl benzoate (1 equiv.), pyridine, room temp., 12 h then Ac₂O (excess); f) MeONa, MeOH, room temp. (Bz(M-AE)

Glc 22%, 3 steps). Note: for compound Boc(AMB)Glc only β-pyranose ring-chain tautomer is shown for simplicity.

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E. Cló, O. Blixt, K. J. Jensen

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anhydride led to peracetylation of the crude mixture en The hydrolysis of all the conjugates fitted well to a pseudo

route to isolation of pure Bz(M-AE)Glc in 22% overall first-order process (see Supporting Information) that al-

yield, as described previously for the other linker types lowed the calculation of the conjugate’s half-lives as re-

(Scheme 3, B). The yield of Bz(M-AE)Glc was compara- ported in Table 1 (at pH 7 all conjugates showed no detect-

tively low because the coupling reaction at equilibration still able hydrolysis throughout the course of the study).

contained significant amounts of free linker, which com-

Table 1. Stability (half-lifes) towards hydrolysis of glycan-linker

conjugates (study performed at room temperature).

peted for benzoylation with the desired conjugate.

The five glucose conjugates presented in Scheme 3 were

pH 3

t_(1/2)[h]

pH 4

t_(1/2)[h]

pH 5

t_(1/2)[h]

characterized in order to determine their configuration and

conformation. In accordance with previously reported spec-

troscopic data,^[18b,23,25]for glucose conjugates of N-methyl- Bz(M-AE)Glc

5.4

23

24

6.5

35

28

114

86

34

168

215

755

605

250

765

Ac(B-AE)Glc

hydroxylamines, we found that the conjugates Bz(M-AE)-

Glc and Ac(M-AMB)Glc were obtained in the C₁confor-

mation with complete diastereoselectivity as β-pyranose

forms. Also compound Boc(AMB)Glc, was in agreement

Boc(A-MB)Glc

4

Ac(M-AMB)Glc

Ac(B-AMB)Glc

with our prediction^[28]as NMR spectroscopic analysis re-

As expected, the rates of hydrolysis for all the conjugates

were pH-dependent, thus for a given specie, the lower the

pH the faster the hydrolysis. The hydrolysis of conjugates

formed with secondary N-methylhydroxylamines Bz(M-AE)-

Glc and Ac(M-AMB)Glc occurred four- to fivefold faster

than the oxime Ac(AMB)Glc, in the pH range investigated.

By contrast, the stabilities of the N-benzyl hydroxylamine-

based conjugates were much higher, even slightly higher

than Ac(AMB)Glc. Furthermore, given the same substitu-

ent at nitrogen, there was marginal, yet noticeable increased

stability in favor of the O-benzyl vs. the O-alkyl conjugates

throughout the pH range investigated [Bz(M-AE)Glc vs.

Ac(M-AMB)Glc and Ac(B-AE)Glc vs. Ac(B-AMB)Glc].

One possible explanation could be that it is due to the dif-

ferent electronic effects that the different substituents exert

upon the linker aminooxy moiety, both on nitrogen and

oxygen. Substituents on nitrogen though, have a more pro-

nounced impact on the conjugate stability as opposed to

the ones on oxygen. In view of this, the remarkable stability

of glucose conjugates of N-benzyl linkers could be ascribed

to the slightly negative inductive effect of the benzyl moiety,

as opposed to the methyl moiety, which, on the other hand

imparts a slightly positive inductive effect. In accordance to

what was reported recently by Nitz and co-workers,^[31]we

assume that upon exposure of the conjugates to acid envi-

ronment, an equilibrium between neutral and nitrogen pro-

tonated forms is established. Protonation opens the conju-

gate yielding an oxyiminium cation, in which C₁is joined

to the positively charged nitrogen via a double bond. The

oxyiminium ion is the intermediate susceptible of nucleo-

philic attack from a water molecule in the rate-determining

step of the hydrolytic process. N-benzyl conjugates are more

electron deficient, thus less prone to protonation compared

to their N-methyl analogues, which in turn makes them

more resilient towards hydrolysis.

vealed the presence of both open-chain and ring-closed tau-

tomers as follows: (E)-oxime/(Z)-oxime/β-pyranose ring-

chain in 60:20:20 ratio respectively. No structural charac-

terization has been previously reported in the literature con-

cerning conjugates between carbohydrates and N-benzylhy-

droxylamines. HPLC-MS analysis of both crude Ac(B-AE)-

Glc and Ac(B-AMB)Glc showed a single product peak,

which bode well for the formation of single tautomer with

complete diastereoselectivity. Further analysis by 1D and

2D NMR spectroscopy corroborated the presence of the

conjugates with ⁴C₁conformation exclusively as β-pyranose

forms (see Supporting Information).

Hydrolytic Stability of Glucose-Linker Conjugates

In recent years the chemoselective coupling between the

reducing end of glycans and N-alkyl hydroxylamines has

been exploited for different types of applications that ex-

tend beyond glycochip fabrication.^[29]Nevertheless, data

concerning the stability towards hydrolysis of N-substituted

aminooxy-based glycoconjugates is scantily present in the

literature.^[30]During the preparation of this paper, Nitz and

co-workers published a survey addressing the hydrolytic sta-

bility of glycoconjugates of xylose, glucose and N-acetylglu-

cosamine with two different types of N-methylhydroxyl-

amine derivatives which showed that conjugates formed

from relatively electron-rich monosaccharides hydrolyzed

more rapidly.^[31]Our goal was to perform a thorough map-

ping of the hydrolytic stability of linker-glucose conjugates

shown in Scheme 3 in solutions ranging from pH 3–5 at one

pH unit intervals. The glycoconjugate concentration

throughout the experiment was set at 1 m and the solu-

tions were incubated at room temperature. The hydrolytic

events were monitored at intervals by HPLC with UV/Vis

detection over 4 d. UV absorption of the linkers was the

same as their corresponding conjugates (see Supporting In-

formation). We monitored the peak areas [at 230 nm for all

but Ac(B-AE)Glc for which 215 nm was used instead] to

estimate the ratio between conjugate and linker as percent

Preparation of Glycan-Liker Conjugates for Microarray

Fabrication

The results obtained for the chemoselective couplings of

the five linkers with glucose bode well for their applicability

of conjugate remaining according to the formula [A_conjugate

/

(A_conjugate+ A_linker)] for each of the three buffer systems. to other reducing glycans. We aimed to devise a methodol-

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Glycans in Microarrays

ogy that could allow straightforward purification of the gly- AMB with all the glycans prepared. This lower reactivity

coconjugates. To this end, we decided to focus on the three could be due to a combination of steric crowding and nega-

linkers of the O-benzylic series, AMB, M-AMB and B- tive inductive effect exerted onto the aminooxy moiety by

AMB as they proficiently absorb UV light, which allowed the benzyl group. Third, disaccharides showed the following

tracing during chromatography and the linkers’ aromatic order of reactivity with the N-substituted linkers M-AMB

moiety could aid retention of the conjugates in reverse- and B-AMB: lactose Ͼ N-acetyllactosamineϾϾ1,2-α-man-

phase columns, which are normally not well suited for nobiose. These observations are in accordance with the re-

highly hydrophilic compounds such as glycans. Moreover, sults reported by Peri et al.^[23]for the formation of N-meth-

as the linkers share the same core structure, it would be ylhydroxylamine conjugates. Interestingly, the N-unsubsti-

possible to rationalize their different performances (in tuted AMB linker, gave approximately the same product

terms of glycoconjugate formation, purification and on- conversions with 1,2-α-mannobiose and lactose, while the

chip behavior towards GBPs probing) to the substitution N-acetyllactosamine was the least reactive.

pattern at the nitrogen of the aminooxy moiety.

The use of buffered eluents (10 m NH₄OAc, pH 5.8)

We then selected lactose, N-acetyllactosamine and 1,2-α- with mixed-mode HILIC column operated in RP (capable

mannobiose to produce a small ensemble of conjugates with of polar and hydrogen bonding interactions) proved to be

each of the selected linkers. We focused on disaccharides, as particularly well suited both for the monitoring of the cou-

the binding event with GBPs would occur in direct proxim- pling reactions as well as purification of glycoconjugates. In

ity of the attachment point between glycan and linker (i.e. the instances when conjugation reactions were not proceed-

the reducing end), thus we would be able to determine the ing with high isomeric selectivity, it became often possible

role of steric effects as well as possible anomeric hetero- to neatly observe well resolved multiple product peaks on

geneity. Higher order oligosaccharide could potentially the chromatogram. No attempts were made to isolate sepa-

level off any differences as the binding events typically oc- rately different isomers (ring-chain tautomers) of the target

cur further away from the anchoring point. Furthermore, conjugates, if present. Furthermore, as only few micromol

N-acetyllactosamine presents N-acetylglucosamine as re- of material were required from each analyte for the prepara-

ducing-end hexose, thus serving as a model substrate for N- tion of several microarray slides, we achieved proficient pu-

linked glycoproteins and glucoaminoglycans. Mannobiose, rification of sufficient amount of conjugates by using the

on the other hand, could serve as model compound for, e.g. same analytical HPLC columns employed for reaction

membrane glycoproteins, which have a “core oligosaccha- monitoring (Figure 2).

ride” structure which includes α-linked mannose residues.

The fractions containing the target conjugates were then

Finally, lactose bears glucose as reducing unit, which was pooled, freeze-dried and taken up with print buffer in order

our model glycan for the hydrolysis studies outlined above to furnish the stock solutions to be arrayed on chip. The

but also serve as a model for glycolipids (Glc-Cer).

concentration of the different stock solutions was conve-

The chemoselective coupling reactions between linkers niently estimated via calibration curves built from pure

and glycans were carried out in aqueous ammonium acetate linker. The purification of the reaction mixtures containing

buffer at pH 4.5, in order to maximize the reaction rate.^[32]AMB was particularly challenging due to the poor resolu-

The equilibrium constants for such kind of condensations tion between product and free linker peaks. We could con-

are moderate,^[31,32]and high yields are often obtained by veniently circumvent the problem by capping the excess of

performing the reactions under concentrated conditions. linker as the acetone oxime.^[33]The addition of acetone

However, this cannot be used for complex glycans derived (10 equiv.) to crude reaction mixture at equilibration re-

from natural sources, as they are difficult and expensive to sulted in the appearance of a new peak at longer retention

isolate in large amounts. To this end, we preferred to oper- times within 1 h (identified as AMB-acetone oxime by ESI-

ate with excess of linker and to minimize the reaction vol- MS), without having any detectable deleterious effect upon

umes. In our standard protocol, the reaction volumes were the target glycan conjugates (Figure 2, C).

kept between 100 and 200 µL, the glycan concentration was

The ease of use of our linkers for glycan microarray fab-

set at 20 m whilst the linkers were present in either three- rication would improve further if pure glycan-linker conju-

fold (M-AMB and B-AMB) or 1.5-fold (AMB) excess and gates were obtained without the need of HPLC chromatog-

gentle heating was applied (37 °C). Thus, only a few micro- raphy. We reasoned that purification by extraction of the

mol of glycans were actually used per reaction.

excess of linkers with water-immiscible organic solvents

Monitoring of the coupling reactions by HPLC high- could be a viable option as the linkers might display a suf-

lighted differences in reactivity among the three linkers, ficient degree of lipophilicity to allow partition into the or-

which were analyzed qualitatively. First, reactions involving ganic phase. In such case, the crude reaction mixture would

the unsubstituted aminooxy linker AMB reached thermo- eventually contain only the desired conjugate and some un-

dynamic equilibrium faster (within 24 h vs. 48–72 h) and reacted glycan molecules. The latter can be considered irrel-

consistently provided higher conversions of conjugate prod- evant as most glycans neither affect concentration estimate

uct (given the same glycan) compared to the N-substituted of the target conjugate (i.e. residual free glycan is devoid of

variants M-AMB and B-AMB. Second, in line with the re- UV absorption) nor react with NHS-coated slides, because

sult obtained with the glucose conjugates, the B-AMB no primary amines are present. To test our hypothesis, the

linker gave lower conversions of conjugate compared to M- reaction mixtures at equilibration were freeze-dried and the

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E. Cló, O. Blixt, K. J. Jensen

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Figure 3. RP-HPLC separations with Jupiter C4 (Phenomenex)

column (recorded at 254 nm) of the water phase after five (top) and

eight (middle) DCM extractions. Only B-AMB linker was detected

in the DCM phase (bottom). Peak assignment corroborated by on-

line ESI-MS.

On-Chip Glycan-Linker Conjugates Assay with Lectins

With our ensemble of disaccharide-linker conjugates in

hand, we probed their ability to interact with biologically

relevant lectins in a microarray. The conjugates were spot-

ted with a robotic microarrayer on Nexterion Slides H at

four different concentrations, with a fivefold dilution gap

from one another, namely 250, 50, 10 and 2 µ. Each slide

housed 16 identical arrays, which displayed the sugar

probes in four replicate per dilution.

The on-chip behavior of the printed conjugates was as-

sessed by means of four biotin-labeled lectins, Con A, RCA

I, ECA and WGA, all applied at 10 µg/mL. Visualization of

bound lectins was then achieved by treatment with Strept-

Figure 2. RP-HPLC chromatograms (230 nm) with a mixed-mode

Acclaim HILIC (Dionex) column of the conjugation reaction be-

tween N-acetyllactosamine (LacNAc) and (A) M-AMB, (B) B-

AMB and (C) AMB at equilibration (top trace in each panel) and

after HPLC purification (bottom trace in each panel). In panel C,

three product peaks are visible corresponding to different isomers

of (AMB)LacNAc, as corroborated by online ESI-MS analysis.

residue taken up in 100 m NH₄OAc buffer, pH 9 so that avidin-Alexa488 conjugate; in Figure 4 the histograms rep-

the linkers primary amino group could be rendered pre- resenting the average relative fluorescence units (RFU) re-

dominantly in the neutral form. Then, the organic solvent corded from microarray scanning are shown. Rewardingly,

was applied in iterated rounds of extractions. HPLC analy- all the lectins tested were able to successfully bind only to

sis of both water and organic phase showed that it was in- their cognate ligands, while otherwise only fluorescence sig-

deed possible to remove excess of B-AMB linker from all nals down to background levels were recorded. This ruled

the three reactions containing the different disaccharides out the occurrence of non-specific binding events between

and, most importantly, no trace of target conjugates was lectins and linker moieties on microarray. M-AMB and B-

detected in the organic phase. Dichloromethane (DCM) AMB conjugates displayed very similar performances with

was found to be more suited for extraction, also by virtue the α-mannose binding lectin Con A,^[34]the β-galactose

of its limited wettability. Thus, the amount of free B-AMB binding lectin RCA I^[35]and the LacNAc-binding lectin

left in the water phase was down to impurity level. On the ECA.^[36]Moreover, all the batches of B-AMB conjugates

other hand, excess of M-AMB and AMB linkers were only purified by extraction proved to be properly spotted and

marginally removed from the respective reaction mixtures recognized by lectins, hence furnishing fluorescence read-

even when the extractions were iterated several times (Ͼ10) outs which were in line with their HPLC-purified counter-

and for many minutes (Ͼ60) each time.

parts.

Interestingly, when the glycochips were assayed with

In order to evaluate the extraction procedure, a conden-

sation reaction containing 1 m of N-acetyllactosamine WGA,^[37]in order to elicit binding with N-acetylglucos-

and 50 m of B-AMB was set up. Upon equilibration and amine, which was internally presented in N-acetyllactos-

switch to the basic buffer, repeated extractions were under- amine conjugates, only (M-AMB)LacNAc was successfully

taken. To our delight, even with such a high excess of B- recognized while (B-AMB)LacNAc was not detected. The

AMB present, the extraction procedure proved to be suc- chitopentaose conjugate of AMB, (AMB)(GlcNAc)₅, was

cessful. As shown in Figure 3, after five extraction rounds, prepared in the same fashion described above for the other

the amount of free B-AMB was greatly reduced and after conjugates, and was spotted as a positive control; as ex-

eight extractions it was just above detection levels. More- pected clear binding with WGA was recorded. Then, the

over, control analysis of the organic phases showed no isomeric nature of the linkage between B-AMB and N-ace-

traces of the target conjugate.

tyllactosamine was investigated by preparing milligram

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Glycans in Microarrays

Figure 4. Microarray image after fluorescent scanning (top) and histograms representing average relative fluorescence units (RFU) re-

corded from microarray scanning (bottom). The fluorescence values (the mean fluorescence value arising from each spot was used) are

the average of 16 spot replica per conjugate on a single slide and no background subtraction was performed. The spot size was determined

by ScanArray Express analysis program with the adaptive circle method (nominal diameter set at 130 µm, minimum and maximum spot

diameter 60% and 160% of nominal, respectively). All conjugates printed at 250, 50, 10 and 2 µ (left to right), except (B-AMB)LacNAc

Extr. printed in at 50, 10 and 2 µ (50 µ printed on two rows in image) and (B-AMB)Man2 Extr. printed in at 100, 50, 10 and 2 µ

(in histograms 100 µ not shown).

amounts of pure Ac(B-AMB)GlcNAc conjugate (see Sup-

It is remarkable that all the different AMB conjugates

porting Information). NMR spectroscopic characterization exhibit lower fluorescent signals, i.e. lower binding, upon

revealed that the conjugate was in pyranose form with β- probing with lectins compared to respective M-AMB and

conformation, the same as reported for GlcNAc conjugates B-AMB conjugates throughout the dilution range. These

with N-methylhydroxlyamine-type linkers such as M- differences are less apparent with Con A, but become quite

AMB.^[23]Accordingly, we speculate that the lack of binding clear with the remaining three lectins. As an example, if

between (B-AMB)LacNAc and WGA is related to the pres- we consider the signal arising from the spots at 250 µ, a

ence of N-benzyl substituent in direct proximity with the concentration at which it is reasonable to assume reaction

target glycan ligand. Such bulky and hydrophobic moiety of all the reactive-NHS ester sites on Nexterion Slide H

might somehow prevent the ligand to achieve efficient by the glycan conjugates,^[38]AMB conjugates approximately

docking into the binding pocket of this specific lectin.

provide only 20% of the signal in three instances [i.e.

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547

E. Cló, O. Blixt, K. J. Jensen

FULL PAPER

(AMB)Lac with RCA I, (AMB)LacNAc with ECA and compared to the lactose ones (see M-AMB and B-AMB

WGA] compared to the corresponding M-AMB and B- conjugates at 24 h in Figure 5), indication of more pro-

AMB conjugates. We believe that the observed limited inter- nounced stability of the LacNAc conjugates to hydrolysis.

action of AMB conjugates with lectins, as opposed to M- Comparing linkers, (B-AMB)LacNAc conjugates displayed

AMB and B-AMB conjugates, are due to the fact that the superior stability compared to (M-AMB)LacNAc, as it can

glycans are presented by the former linker with anomeric easily be visualized in Figure 5 from the probing of the 24 h

heterogeneity (oxime tautomer), where the open-chain iso- incubation subarrays: (M-AMB)LacNAc signal is greatly

mer is the one most represented (approximately 75% for diminished while (B-AMB)LacNAc furnished readouts in

both lactose and N-acetyllactosamine^[39]and close to 100% line with the subarrays incubated in print buffer. As this is

with mannose^[40]) whereas the latter give rise only to ring- only an indirect measure of the degree of hydrolysis on-

closed conjugates.^[41]Such rationalization is in line with fin- chip, comparable levels of fluorescence output between a

dings of Shin et al.^[42]who, after solution NMR spectro- given conjugate in a subarray incubated at pH 3 and one

scopic and on-slide reactivity studies, related higher fluores- incubated at pH 8.6 did not necessarily mean that all the

cence signals upon lectin binding for glycans applied to hy- conjugates were left unchanged from acid treatment, but

drazide vs. hydroxylamine coated glass slides to the nature rather that on-slide the conjugate density was still sufficient

of the anomeric linkage established on the surface. The to guarantee that the coupled assay applied for probing (i.e.

preferential formation of ring-closed conjugates with the RCA I/Strptavin-Alexa488) furnished very similar fluori-

hydrazide linking moieties resulted in higher signals. metric readout.

Furthermore, Cummings and co-workers recently reported

that when lactose was conjugated to a bifunctional linker

via reductive amination (thus forming exclusively open-

chain structure at the glucose unit) and subsequently spot-

ted onto Nexterion Slide H, no binding was observed when

probing with RCA I.^[43]

As a final experiment, it was investigated how the sur-

face-bound glycoconjugates responded to hydrolytic condi-

tions. After the glycochip was printed (same setup as above)

and blocked, identical subarrays were subjected to either

phosphate buffer 100 m, pH 3 or to print buffer, pH 8.6,

and incubated. After 6 h, pH 3 buffer was removed from

some subarrays, which were backfilled with print buffer. Af-

ter 24 h of incubation, all the wells were emptied and the

slide washed and treated with RCA I. Subsequent treatment

with Streptavin-Alexa488 conjugate and fluorescent scan-

ning allowed us to assess the response of the glycoconjugate

Figure 5. Histogram of the on-chip hydrolysis study at pH 3. The

to hydrolytic conditions. RCA I was chosen for probing to

obtain information from the lactose glycoconjugates with

M-AMB and B-AMB, which present glucose at the reduc-

ing end, to see how these related to the hydrolysis study in

solution. Thus the lactose conjugates could be compared

with N-acetyllactosamine conjugates (with N-acetylglucos-

amine at the reducing end), as solution studies^[31]had found

N-methylhydroxylamine conjugates with N-acetylglucos-

amine to be more stable than with glucose. In Figure 5 the

values for each conjugate type (250 uM spots) were normalized to

their respective mean fluorescent readouts collected upon incu-

bation with print buffer for 24 h. Values are the mean of two set

of identical experiments, standard deviation between 4% and 7%.

Conclusions

We have studied five bifunctional aminooxy-type linkers,

results from microarray scanning of the 250 µ print are four of which were new, for the chemoselective capture of

presented as bar graph; for each conjugate the data pre- unprotected oligosaccharides in solution followed by con-

sented was normalized to its respective fluorescent readout struction of covalent microarrays. The linkers displayed a

collected after lectin probing from the subarrays incubated diversified N- and O-substitution pattern at the aminooxy

in print buffer for 24 h, conditions at which we assume the moiety, where different combinations of O- and N-alkyl and

hydrolysis to be negligible. The results are in agreement benzyl substituents were in place. Conjugates with glucose

with the solution studies, since the overall higher stability as a model system were characterized by NMR spec-

of B-AMB vs. M-AMB conjugates was observed on-chip as troscopy, and their hydrolytic stability in solution (pH 3–5)

well. If we consider the lactose series, (M-AMB)Lac ap- was assessed. Conjugates with N-benzyl-substituted ami-

pears to be hydrolyzed nearly to completion already after nooxy-type linkers, B-AE and B-AMB, were characterized

6 h, whereas RCA I binding to (B-AMB)Lac is unchanged for the first time and proved to yield glucose conjugates in

on the 6 h subarray and reduced down to 20% on the 24 h closed-ring β-pyranose form with high diastereoselectivity.

subarray. Furthermore, given the same linker, N-acetyllac- The hydrolytic events fitted well with pseudo-first order ki-

tosamine conjugates provided higher normalized signals netics, thus the half-lives of the conjugates were calculated.

548

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Eur. J. Org. Chem. 2010, 540–554

Glycans in Microarrays

1.0 mL/min linear gradient flow of B (MeCN, 0.1% formic acid)

in A (H₂O, 0.1% formic acid) 12 min; analytical columns used were

either Phenomenex Gemini^TMC-18, 50ϫ4.60 mm, 3 µ, 110 Å or

Phenomenex Jupiter^TMC-4, 150ϫ4.60 mm, 5 µ, 300 Å. Elution

Condition 2: 1.0 mL/min linear gradient flow of D (9:1, v/v, MeCN/

NH₄OAc 100 m, pH 5.8) in C (NH₄OAc 10 m, pH 5.8) over

15 min; analytical columns used were either Phenomenex Hydro^TM

150ϫ3.0 mm, 4 µ, 80 Å or Dionex Acclaim^TMMixed-Mode

HILIC-1 150ϫ5 mm, 5 µ, 120 Å. Unless otherwise stated, yields

refer to chromatographically isolated and spectroscopically pure

N-methyl-substituted conjugates, Bz(M-AE)Glc and Ac(M-

AMB)Glc, were the least stable, whereas N-benzyl-substi-

tuted conjugates, Ac(B-AE)Glc and Ac(B-AMB)Glc, dis-

played stabilities similar or superior to that of the oxime-

type conjugate Boc(AMB)Glc throughout the pH range

studied. The structurally related and UV-active linkers

AMB, M-AMB, and B-AMB were selected, and each one

was conjugated with lactose, N-acetyllactosamine, and 1,2-

α-mannobiose for microarray fabrication. Convenient puri-

fication was achieved with RP HPLC (mixed-mode HILIC materials. Commercially available starting materials were used as

received without further purification. Dry-column chromatography

was performed using Merck silica gel 60 (230–400 mesh). Microar-

ray printing was performed by robotic contact printing with a Bio-

Robotics Microgrid II (Genomics Solutions) fitted with Stealth 3B

Micro Spotting pins (Telechem International ArrayIt Division).

Source plates were prepared with BD Falcon Microtest^TM384-well

30 µL assay plates (BD Biosciences, Le Pont De Claix, France).

Microarrays were visualized in a ProScanArray HT Microarray

Scanner (Perkin–Elmer) and image analysis performed with Pro-

ScanArray Experess 4.0 (Perkin–Elmer).

column) and by DCM extraction (B-AMB linker only). Fi-

nally, microarrays fabricated on NHS-activated slides were

probed with four lectins, Con A, RCA I, ECA, and WGA.

All conjugates were able to give efficient binding exclusively

to their cognate lectin. Remarkably, conjugates of the N-

substituted linkers M-AMB and B-AMB gave similar fluo-

rescent readouts throughout the whole range of concentra-

tions spotted with Con A, RCA I, and ECA, but not the

N-acetylglucosamine binding lectin WGA, which bound

proficiently to (M-AMB)LacNAc yet not to (B-AMB)-

LacNAc. We hypothesize this to be due to the deleterious

presence of the benzyl substituent in direct proximity to the

WGA binding epitope. AMB conjugates upon lectin bind-

ing provided fluorescent signals consistently inferior com-

pared to those of the M-AMB and B-AMB linkers for all

the four lectins probed and throughout the spotted concen-

tration range. Given that the linkers share the same struc-

tural framework, the immobilization efficiency on slide are

N-Boc-N-Benzylhydroxylamine (1): N-Benzylhydroxylamine hydro-

chloride (1000 mg, 6.26 mol) was placed in a round-bottom flask

and dissolved in a 1:1 mixture of THF and water (10 mL). After

cooling on ice bath, potassium carbonate (432 mg, 3.13 mol) was

slowly added to the solution and gas evolution was observed. Once

gas evolution ceased, a solution of di-tert-butyl dicarbonate

(1500 mg, 6.9 mol) in THF (15 mL) was added dropwise to the

mixture and stirring was continued for 2 h at 0 °C and then at room

temperature overnight. Then the solution was concentrated in

likely to be the same, and therefore we conclude that the vacuo and the residue taken up in DCM (50 mL) and washed with

water (3ϫ800 mL) and brine (1ϫ80 mL). The organic solution

was dried with MgSO₄, the solvent removed in vacuo and the re-

sulting residue absorbed onto celite. Purification with vacuum-col-

umn chromatography (EtOAc 0 to 50% in heptane) gave the pure

product 6 in 66% yield as colorless crystalline solid. ¹H NMR

(300 MHz, CDCl₃): δ = 7.35–7.33 (m, 5 H, ArH), 5.98 (s, 1 H,

NH), 4.65 (s, 2 H, CH₂Ar), 1.50 [s, 9 H, (CH₃)₃-C] ppm. ¹³C NMR

(75 MHz, CDCl₃): δ = 156.6, 136.3, 128.5, 128.1, 127.6, 82.3, 53.9,

28.3 ppm.

weaker readouts from AMB conjugates are due to their

structural heterogeneity at the anomeric center. The open

chain form was the predominant isomer with all the glycans

tested, whereas both M-AMB and B-AMB furnished

closed-ring conjugates. Our work highlights that, when ad-

dressing glycoconjugates with short oligosaccharides, it is

crucial to produce structures that mimic their parent natu-

ral compounds. Loss of structural integrity, in form of

open-chain oxime conjugates or the presence of bulky sub-

stituents at the anomeric center (such as the benzyl moiety),

can in this system hamper the occurrence of the desired

binding event.

2-(N-Boc-N-Benzylaminooxy)-N-Boc-ethanamine [Boc(B-AE)]: An

oven-dry microwave vial provided with magnetic flea was loaded

with

N-Boc-N-benzylhydroxylamine

(400 mg,

1.78 mmol,

1.0 equiv.) and tert-butyl 2-bromoethylcarbamate (460 mg,

2.0 mmol, 1.15 equiv.) under Ar. Acetonitrile (5 mL) was added,

rapidly giving a clear solution. Finally DBU (330 µL, 2.23 mmol,

1.3 equiv.) was added dropwise via syringe, the vial was sealed and

microwave irradiation was undertaken for 40 min at 65 °C. The vol-

atiles were then removed in vacuo and the resulting residue taken

up in 30 mL of DCM and extracted 2ϫ30 mL of HCl 0.5 ,

2ϫ30 mL of NaOH 1  and 1ϫ30 mL of saturated NaHCO₃

aqueous solution. The organic layer was concentrated to dryness

and the residue absorbed on celite. Purification with vacuum-col-

umn chromatography (EtOAc 0 to 50% in heptane) gave the pure

product 7 (371 mg, 57%) as a colorless oil. ¹H NMR (300 MHz,

CDCl₃): δ = 7.34–7.31 (m, 5 H, ArH), 5.28 (br. s, 1 H, NH) 4.58

(s, 2 H, CH₂Ar), 3.75 (t, J = 4.8 Hz, 2 H, CH₂ON), 3.24 (dd, J =

4.8, 10 Hz, 2 H, CH₂NH), 1.49 [s, 9 H, (CH₃)₃-C], 1.42 [s, 9 H,

(CH₃)₃-C-] ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 156.8, 155.9,

136.6, 128.4, 127.6, 82.1, 79.1, 77.2, 74.1, 53.6, 38.9, 28.3, 28.2 ppm.

Experimental Section

Materials and Analysis: All chemicals were purchased from Sigma–

Aldrich Denmark. Biotin labelled lectins purchased from Vector

Labs (Peterborough, UK) and Streptavis-Alexa488 conjugate pur-

chased from Invitrogen Denmark. MilliQ water was used for all

aqueous preparations. All solvent ratios are v/v. ¹H, ¹³C, APT,

HSQC, and COSY NMR spectra were recorded on a Bruker

Avance 300 spectrometer with a BBO probe. The chemical shifts

are referenced to the residual solvent signal. Assignments were

aided by COSY, HSQC, and APT experiments. Mass determination

(high resolution MS, HR-MS) was performed on a Micromass

LCT instrument with an ESI probe. Analytical HPLC was per-

formed on a Dionex Ultimate 3000 system with Chromeleon

6.80SP3 software. Unless otherwise stated, two sets of reverse- HR-MS (ESI): calcd. for C₁₉H₃₀N₂O₅[M + Na]⁺, 389.2047; found

phase (RP) elution conditions were used. Elution Condition 1: 389.2052.

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549

E. Cló, O. Blixt, K. J. Jensen

N-(2-Boc-Aminoethyl)-4-(chloromethyl)benzamide (2): 4-Chloro yl)carbamate (120 mg, 0.52 mmol) were treated as described for

FULL PAPER

toluic acid (510 mg, 3.0 mmol) and EDC hydrochloride (632 mg,

3.3 mmol) were placed in a round-bottomed flask and covered in

DCM (5 mL). The flask was closed with a septum and tert-butyl

2-aminoethylcarbamate (530 mg, 3.3 mmol), dissolved in DCM

(2 mL), was added dropwise via syringe. Few seconds after comple-

tion of the addition, the reaction mixture became homogeneous

whereupon the vial was sealed and placed in the microwave reactor

cavity and irradiated at 60 °C for 10 min. Once cooled to room

compound Boc(B-AE). The crude product was absorbed on celite

and purified with vacuum-column chromatography (EtOAc 0 to

50% in heptane) gave the pure product Boc(B-AMB) as colorless

1

amorphous solid (197 mg, 90%). H NMR (300 MHz, CDCl₃): δ

= 7.75 (d, J = 8.2 Hz, 2 H, ArH), 7.30 (m, 7 H), 7.19 (br. s, 1 H,

NHCO), 4.98 [br. s, 1 H, NHCO(O)], 4.71 (s, 2 H, CH₂Ar), 4.54

(s, 2 H, CH₂Ar), 3.53 (dd, J = 5, 10 Hz, 2 H, CH₂NHCO), 3.40

[dd, J = 5, 10 Hz, 2 H, CH₂NHCO(O)], 1.49 [s, 9 H, (CH₃)₃-C],

temp. the reaction mixture was diluted with DCM (20 mL) and 1.42 [s, 9 H, (CH₃)₃-C] ppm. ¹³C NMR (75 MHz, CDCl₃): δ =

extracted with 5% citric acid solution (2ϫ30 mL), NaOH 1 

(1ϫ20 mL) and NaHCO₃(1ϫ20 mL) then dried with MgSO₄, re-

duced to dryness in vacuo and the resulting residue absorbed onto

celite. Purification with vacuum-column chromatography (EtOAc 0

to 100% in heptane) gave the pure product 2 (448 mg, 48%) as a

white solid. H NMR (300 MHz, CDCl₃): δ = 7.81 (d, J = 8.2 Hz,

2 H, ArH), 7.45 (d, J = 8.2 Hz, 2 H, ArH), 7.23 (br. s, 1 H, NHCO),

4.94 [br. s, 1 H, NHCO(O)], 4.61 (s, 2 H, CH₂Ar), 3.58 (dd, J = 5,

167.3, 156.6, 138.9, 136.7, 134.0, 129.2, 128.6, 128.4, 127.5, 127.0,

81.7, 80.0, 76.6, 54.1, 42.2, 39.9, 28.3, 28.2 ppm. HR-MS (ESI):

calcd. for C₂₇H₃₇N₃O₆[M + H]⁺, 500.2755; found 500.2761.

2-(Benzylaminooxy)ethanamine Hydrochloride (B-AE): N-Benzyl-

O-2-aminoethylhydroxylamine (135 mg, 0.37 mmol) was placed in

a reaction vial and dissolved in HCl 4  in dioxane (4 mL) where-

upon a white crystalline precipitate quickly appeared. After 1 h

stirring at room temp. the heterogeneous reaction mixture was di-

luted with diethyl ether and the precipitate was separated from the

supernatant by centrifugation. The precipitate was washed with

fresh ethyl ether (2ϫ20 mL) and then dried under an air stream at

first and finally under reduced pressure to yield the desired product

1

10 Hz,

2 H, CH₂NHCO), 3.54 [dd, J = 5, 10 Hz, 2 H,

CH₂NHCO(O)], 1.43 [s, 9 H, (CH₃)₃-C] ppm. ¹³C NMR (75 MHz,

CDCl₃): δ = 167.0, 141.0, 134.1, 129.1, 128.6, 127.4, 45.4, 42.3,

39.9, 28.3 ppm. MS (ESI): calcd. for C₁₅H₂₁ClN₂O₃[M + H]⁺,

312.1; found 312.1.

1

B-AE (102 mg, 93%) as a white brittle solid. H NMR (300 MHz,

D₂O): δ = 7.51 (s, 5 H), 4.47 (s, 2 H, CH₂Ar), 3.53 (t, J = 5 Hz, 2

H, CH₂ON), 3.31 (t, J = 5, 10 Hz, 2 H, CH₂N) ppm. ¹³C NMR

(75 MHz, D₂O): δ = 130.2, 129.6, 129.1, 69.6, 53.8, 37.8 ppm. HR-

MS (ESI): calcd. for C₉H₁₄N₂O [M + H]⁺, 167.1179; found

167.1180.

N-(2-Boc-Aminoethyl)-4-[(1,3-dioxoisoindolin-2-yloxy)methyl]benz-

amide: An oven-dry microwave vial provided with a magnetic flea

was loaded with N-hydroxyphthalimide (232 mg, 1.43 mmol) and

3 mL of acetonitrile. Upon dropwise addition of DBU (200 µL,

1.43 mmol) the mixture became a red homogeneous solution. A

solution of tert-butyl 2-[4-(bromomethyl)phenylamido]ethylcarba-

mate (300 mg, 0.84 mmol) in 4 mL of acetonitrile was added, the

vial was sealed and microwave-heated for 30 min at 65 °C. After

irradiation a white precipitate appeared in the reaction mixture.

The precipitate was filtered by suction and washed with HCl 0.5 

(2ϫ30 mL), NaOH 1  (2ϫ30 mL) and water (2ϫ30 mL) and fi-

nally dried in the oven at 145C for 1 h thus giving the title com-

pound in 87% yield. ¹H NMR (300 MHz, [D₆]DMSO): δ = 8.49

(br. t, J = 5.2 Hz, 1 H, NHCO), 7.85 (m, 6 H), 7.59 (d, J = 8.2 Hz,

2 H, ArH), 6.91 [br. t, J = 5.6 Hz, 1 H, NHCO(O)], 5.22 (s, 2 H,

CH₂Ar), 3.28 (dd, J = 6, 12 Hz, 2 H, CH₂NHCO), 3.11 [dd, J =

6, 12 Hz, 2 H, CH₂NHCO(O)], 1.36 [s, 9 H, (CH₃)₃-C-] ppm. ¹³C

NMR (75 MHz, [D₆]DMSO): δ = 165.8, 162.9, 155.6, 137.0, 134.8,

134.7, 129.1, 128.3, 127.2, 123.1, 78.4, 77.5, 28.1 ppm. HR-MS

(ESI): calcd. for C₂₃H₂₅N₃O₆[M + Na]⁺, 462.16; found 462.25.

N-(2-Boc-Aminoethyl)-4-(aminooxymethyl)benzamide [Boc(AMB)]:

tert-Butyl 2-{4-[(1,3-dioxoisoindolin-2-yloxy)methyl]phenyl-

amido}ethyl carbamate (205 mg, 0.57 mmol) was placed in a micro-

wave vial and covered with a 1:1 mixture of DCM and acetonitrile

(10 mL) whereupon hydrazine hydrate (430 µL, 8.54 mmol) was

added. The vial was sealed, placed in the microwave cavity and

irradiated at 65 °C for 15 min; after irradiation a white glossy pre-

cipitate was formed. The reaction mixture was filtered by suction

on a pad of Celite with the aid of fresh acetonitrile (10 mL). The

filtrate was concentrated in vacuo yielding the pure product

Boc(AMB) as white solid (180 mg, 98 %). ¹H NMR (300 MHz,

CDCl₃): δ = 7.81 (d, J = 8.2 Hz, 2 H, ArH), 7.40 (d, J = 8.2 Hz, 2

H, ArH), 7.21 (br. s, 1 H, NHCO), 5.45 (br. s, 2 H, NH₂), 5.02 [br.

s, 1 H, NHCO(O)], 4.72 (s, 2 H, CH₂Ar), 3.54 (m, 2 H,

CH₂NHCO), 3.41 [m, 2 H, CH₂NHCO(O)], 1.42 [s, 9 H, (CH₃)₃-

C] ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 167.4, 141.1, 133.6,

128.1, 127.1, 80.0, 77.2, 42.1, 39.9, 28.3 ppm. HR-MS (ESI): calcd.

for C₁₅H₂₃N₃O₄[M + Na]⁺, 332.1581; found 332.1555.

N-(2-Boc-Aminoethyl)-4-[(N-Boc-N-methylaminooxy)methyl]benz-

amide [Boc(M-AMB)]: tert-Butyl 2-[4-(bromomethyl)phenylamido]-

ethylcarbamate (171 mg, 0.48 mmol) and tert-butyl hydroxy(methyl)-

carbamate (82 mg, 0.55 mmol) were treated as described for com-

pound Boc(B-AE). The crude product was absorbed on Celite and

purified with vacuum-column chromatography (EtOAc 0 to 100%

in heptane) gave the pure product Boc(M-AMB) as colorless

N-(2-Aminoethyl)-4-(aminooxymethyl)benzamide Hydrochloride

(AMB): tert-Butyl 2-[4-(aminooxymethyl)phenylamido]ethyl carba-

mate (50 mg, 0.12 mmol) was deprotected as described for B-AE,

giving the desired product AMB (33 mg, 99%) as a white powder.

¹H NMR (300 MHz, D₂O): δ = 7.85 (d, J = 8.4 Hz, 2 H, ArH),

7.60 (d, J = 8.4 Hz, 2 H, ArH), 5.15 (s, 2 H, CH₂Ar), 3.72 (t, J =

6 Hz, 2 H, CH₂NHCO), 3.27 (t, J = 6 Hz, 2 H, CH₂N) ppm. ¹³C

NMR (75 MHz, D₂O): δ = 170.9, 136.8, 134.1, 129.4, 127.7, 76.1,

39.3, 37.3 ppm. HR-MS (ESI): calcd. for C₁₀H₁₅N₃O₂[M + H]⁺,

210.1237; found 210.1250.

1

amorphous solid (90 mg, 45%). H NMR (300 MHz, CDCl₃): δ =

7.81 (d, J = 8.2 Hz, 2 H, ArH), 7.45 (d, J = 8.2 Hz, 2 H, ArH),

7.23 (br. s, 1 H, NHCO), 5.00 [br. s, 1 H, NHCO(O)], 4.86 (s, 2 H,

CH₂Ar), 3.54 (dd, J = 5, 10 Hz, 2 H, CH₂NHCO), 3.40 [dd, J =

5, 10 Hz, 2 H, CH₂NHCO(O)], 3.03 (s, 3 H, ONCH₃), 1.49 [s, 9

H, (CH₃)₃-C], 1.42 [s, 9 H, (CH₃)₃-C] ppm. ¹³C NMR (75 MHz,

CDCl₃): δ = 167.3, 156.9, 139.1, 134.1, 129.1, 127.1, 81.4, 80.0,

75.8, 42.2, 39.9, 37.0, 28.3, 28.2 ppm. HR-MS (ESI): calcd. for

C₂₁H₃₃N₃O₆[M + Na]⁺, 446.2262; found 446.2283.

N-(2-Aminoethyl)-4-[(methylaminooxy)methyl]benzamide Hydro-

chloride [M-AMB]: Compound Boc(M-AMB) was deprotected as

described for compound B-AE, giving the desired product M-AMB

1

N-(2-Boc-Aminoethyl)-4-[(N-Boc-N-benzylaminooxy)methyl]benz-

amide [Boc(B-AMB)]: tert-Butyl 2-[4-(bromomethyl)phenylamido]-

ethylcarbamate (160 mg, 0.45 mmol) and tert-butyl hydroxy(benz-

(66 mg, 83%) as a white powder. H NMR (300 MHz, D₂O): δ =

7.85 (d, J = 8.5 Hz, 2 H, ArH), 7.60 (d, J = 8.5 Hz, 2 H, ArH),

5.20 (s, 2 H, CH₂Ar), 3.72 (t, J = 6 Hz, 2 H, CH₂NHCO), 3.27 (t,

550

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Eur. J. Org. Chem. 2010, 540–554

Glycans in Microarrays

J = 6 Hz, 2 H, CH₂N), 3.04 (s, 3 H) ppm. ¹³C NMR (75 MHz, ent condition 1, 5 to 100% of B. MS (ESI): calcd. for C₂₅H₃₄N₂O₁₁

D₂O): δ = 170.9, 136.6, 134.1, 129.4, 127.8, 75.1, 39.3, 37.3, 35.3 (M + H)⁺, 539.2; found 539.2]. The intermediate product was then

ppm. HR-MS (ESI): calcd. for C₁₁H₁₇N₃O₂[M + H]⁺, 224.1394;

found 224.1404.

dissolved in methanol (3 mL) in an oven-dried reaction vial and

treated with NaOMe 0.1  in methanol (0.3 mL). The reaction

mixture was stirred at room temp. for 12 h, whereupon Amberlite-

IR 120 was added to neutral pH. The solids were filtered off and

the volatiles removed in vacuo yielding the pure product Ac(B-AE)-

Glc (37 mg, 59%) as a white solid. ¹H NMR (300 MHz, D₂O): δ =

7.49–7.38 (m, 5 H, ArH), 4.22 (d, J = 13.0 Hz, 1 H, ArCH_2aN),

4.16 (d, J = 9.0 Hz, 1 H, 1-H), 4.06 (d, J = 13.0 Hz, 1 H,

ArCH_2bN), 3.96 (dd, J = 12.3, 2.0 Hz, 1 H, 6a-H), 3.75 (dd, J =

12.3, 5.3 Hz, 1 H, 6b-H), 3.70 (t, J = 5.0 Hz, 1 H, CH₂NO), 3.60

(t, J = 9.0 Hz, 1 H, 2-H), 3.57 (t, J = 5.0 Hz, 1 H, CH₂NO), 3.47

(t, J = 9.0 Hz, 1 H, 3-H), 3.43 (t, J = 9.0 Hz, 1 H, 4-H), 3.38–35

(m, 1 H, 5-H, from COSY), 3.16 (t, J = 5.4 Hz, 2 H, CH₂NHCO),

1.92 (s, 3 H, NHCOCH₃) ppm. ¹³C NMR (75 MHz, D₂O): δ =

174.4, 137.0, 130.7, 129.1, 128.5, 92.2, 78.1, 77.8, 72.9, 70.4, 70.0,

61.5, 57.2, 38.9, 22.5 ppm. HR-MS (ESI): Calcd. for C₁₇H₂₆N₂O₇

[M + Na]⁺, 393.1638; found 393.1649.

N-(2-Aminoethyl)-4-[(benzylaminooxy)methyl]Benzamide Hydro-

chloride [B-AMB]: Compound Boc(B-AMB) was deprotected as de-

scribed for compound B-AE, giving the desired product B-AMB

1

(113 mg, 94%) as a white powder. H NMR (300 MHz, D₂O): δ =

7.79 (d, J = 8.5 Hz, 2 H, ArH), 7.49–7.46 (m, 7 H), 5.12 (s, 2 H,

CH₂Ar), 4.50 (s, 2 H, CH₂Ar), 3.70 (t, J = 6 Hz, 2 H, CH₂NHCO),

3.25 (t, J = 6 Hz, 2 H, CH₂N) ppm. ¹³C NMR (75 MHz, D₂O): δ

= 170.9, 136.9, 133.9, 130.5, 129.8, 129.2, 129.0, 128.8, 127.6, 75.3,

53.5, 39.2, 37.3 ppm. HR-MS (ESI): calcd. for C₁₇H₂₁N₃O₂[M +

H]⁺, 300.1707; found 300.1711.

N-[N-Methyl-O-(benzamidoethyl)hydroxylamino] β-D-Glucopyran-

oside [Bz(M-AE)Glc]: The aminooxy linker M-AE (24 mg,

0.15 mmol) and glucose (30 mg, 0.17 mmol) were placed in a reac-

tion vial provided with stirrer and dissolved in a 5:1 mixture of

methanol and acetic acid (5 mL), whereupon the vial was sealed

and stirred at room temp. for 72 h. The reaction mixture was then

reduced to dryness in vacuo and the residue taken up in pyridine

(4 mL) and N-(benzoyloxy)succinimide (49 mg, 0.225 mmol) was

added, then the resulting reaction mixture stirred at room temp.

After 24 h the acetic anhydride (0.8 mL) was added and stirring

was continued for a further 12 h at room temp. The volatiles were

removed under vacuum and the residue taken up in DCM (10 mL)

and extracted with saturated NaHCO₃(1ϫ20 mL) and brine. Puri-

fication with vacuum-column chromatography (EtOAc 0 to 100%

in heptane, then methanol 0 to 5% in EtOAc) of the reaction crude

after treatment with acetic anhydride gave a single peracetylated

glucose conjugate, as characterized by HPLC-MS [eluent condition

2, 5 to 50% of D. MS (ESI): calcd. for C₂₄H₃₂N₂O₁₁(M + H)⁺,

525.2; found 525.1]. The intermediate product was then dissolved

in methanol (2 mL) in an oven-dried reaction vial and treated with

NaOMe 0.1  in methanol (0.2 mL). The reaction mixture was

stirred at room temp. for 12 h whereupon Amberlite-IR 120 was

added to neutral pH. The solids were filtered off and the volatiles

removed in vacuo yielding the pure glucose conjugate Bz(M-AE)-

Glc (11 mg, 21%) as a white solid. ¹H NMR (300 MHz, D₂O): δ =

7.78 (dt, J = 7.0, 1.5 Hz, 2 H, ArH ortho), 7.64 (tt, J = 7.0, 1.5 Hz,

1 H, ArH para), 7.54 (tt, J = 7.0, 1.5 Hz, 2 H, ArH meta), 4.15 (d,

J = 9.1 Hz, 1 H, 1-H), 4.0 (t, J = 5.4 Hz, 2H: CH₂NO), 3.91 (dd,

J = 12.3, 2.2 Hz, 1 H, 6a-H), 3.72 (dd, J = 12.3, 5.3 Hz, 1 H, 6b-

H), 3.63 (t, J = 5.4 Hz, 2 H, CH₂NHCO), 3.55–3.46 (m, 2 H, 2-H,

3-H), 3.40–3.30 (m, 2 H, 4-H, 5-H), 2.77 (s, 3 H, NCH₃) ppm. ¹³C

NMR (75 MHz, D₂O): δ = 171.0, 133.5, 132.1, 128.8, 127.0, 93.2,

77.4, 77.0, 70.9, 69.9, 69.4, 60.8, 38.9, 38.5 ppm. HR-MS (ESI):

Calcd. for C₁₆H₂₄N₂O₇[M + Na]⁺, 379.1481; found 379.1486.

D

-Glucose O-{4-[(2-Boc-Aminoethyl)aminocarbonyl]benzyl}oxime

[Boc(AMB)Glc]: The aminooxy linker AMB (100 mg, 0.32 mmol)

was treated with glucose in the same two-step procedure as de-

scribed for Ac(B-AE)Glc. Purification with vacuum-column

chromatography (EtOAc 0 to 100% in heptane, then methanol 0

to 5% in EtOAc) of the reaction crude after treatment with acetic

anhydride gave a mixture of open- (major) and closed-ring (minor)

peracetylated glucose conjugates, as characterized by HPLC-MS

[eluent condition 1, 5 to 100% of B. MS (ESI): calcd. for the closed-

ring form C₂₉H₄₁N₃O₁₃(M + H)⁺, 640.3; found 640.2; Calcd. for

open-ring form C₃₁H₄₃N₃O₁₄(M + H)⁺, 682.3; found 682.2]. Fol-

lowing deacetylation, the pure product Boc(AMB)Glc (112 mg,

74%, isomeric mixture) was obtained as a white solid. ¹H NMR

(300 MHz, D₂O): δ = 7.79–7.70 (m, 2 H, ArH), 7.57 (d, J = 6.7 Hz,

0.6 H, E-oxime 1-H), 7.52–7.44 (m, 2 H, ArH), 6.85 (d, J = 6.1 Hz,

0.2 H, Z-oxime 1-H), 5.16 (s, 0.4 H, OCH₂Ar Z-oxime) 5.14 (s, 1.2

H, OCH₂Ar E-oxime), 4.96 (t, J = 6.1 Hz, 0.2 H, Z-oxime 2-H),

4.88 (d, J = 9.0 Hz, 0.2 H, OCH_2aAr), 4.84 (d, J = 9.0 Hz, 0.2 H,

OCH_2bAr, partly under H₂O signal), 4.33 (t, J = 6.9 Hz, 0.6 H, E-

oxime 2-H), 4.23 (d, J = 9.0 Hz, 0.2 H, β-pyranose 1-H), 3.96–3.23

(m, 9.3 H), 1.32 [s, 9 H, (CH₃)₃-C] ppm. ¹³C NMR (75 MHz, D₂O):

δ = 170.5, 158.2, 153.0, 152.1, 141.1, 133.2, 128.7, 128.2, 127.3,

95.8, 90.2, 80.8, 77.1, 76.8, 76.7, 76.0, 75.9, 75.7, 75.1, 74.8, 74.1,

71.4, 71.4, 71.0, 70.8, 70.8, 70.2, 70.1, 69.9, 69.5, 69.3, 66.4, 63.0,

62.7, 60.7, 46.6, 39.8, 39.3, 37.3, 27.5 ppm. HR-MS (ESI): Calcd.

for C₂₁H₃₃N₃O₉[M + Na]⁺, 494.2114; found 494.2118.

N-(N-Methyl-O-{4-[(2-acetamidoethyl)aminocarbonyl]benzyl}-N-

hydroxylamino) β-D-Glucopyranoside [Ac(M-AMB)Glc]: The

aminooxy linker M-AMB (30 mg, 0.09 mmol) was treated with glu-

cose in the same two-step procedure as described for Ac(B-AE)Glc.

Purification with vacuum-column chromatography (MeOH 0 to

20% in EtOAc) of the reaction crude after treatment with acetic

anhydride gave a single peracetylated glucose conjugate, as charac-

terized by HPLC-MS (eluent condition 1, 5 to 100% of B). MS

(ESI): Calcd. C₂₇H₃₇N₃O₁₂[M + H]⁺, 596.2; found 596.2. Follow-

ing deacetylation, the pure product Ac-MAMB-Glc (14 mg, 49%)

was obtained as a white solid. ¹H NMR (300 MHz, D₂O): δ = 7.75

(d, J = 8.4 Hz, 2 H, ArH), 7.55 (d, J = 8.4 Hz, 2 H, ArH), 4.88 (d,

J = 10.8 Hz, 1 H, OCH_2aAr), 4.84 (d, J = 10.8 Hz, 1 H, OCH_2bAr),

4.16 (d, J = 9.0 Hz, 1 H, 1-H), 3.91 (dd, J = 12.3, 2.0 Hz, 1 H, 6a-

H), 3.74 (dd, J = 12.3, 5.0 Hz, 1 H, 6b-H), 3.58 (t, J = 9.0 Hz, 1

H, 2-H), 3.55–3.50 (m, 3 H), 3.47–3.37 (m, 4 H), 2.80 (s, 3 H,

NCH₃), 1.99 (NCOCH₃) ppm. ¹³C NMR (75 MHz, D₂O): δ =

174.4, 170.7, 139.9, 133.6, 129.4, 127.2, 93.2, 77.2, 77.0, 74.5, 69.9,

N-[N-Benzyl-O-(Acetamidoethyl)hydroxylamino] β-D Glucopyran-

oside, Ac(B-AE)Glc: The aminooxy linker B-AE (40 mg, 0.17 mmol)

and glucose (153 mg, 0.85 mmol) were placed in a reaction vial

provided with stirrer and dissolved in a 5:1 mixture of methanol

and acetic acid (5 mL), whereupon the vial was sealed and allowed

to stir at room temp. for 72 h. The reaction mixture was then re-

duced to dryness in vacuo and the residue taken up in pyridine

(4 mL) and treated with acetic anhydride (1 mL). The vial was se-

aled and the reaction stirred at room temp. for 24 h. The solvents

were removed and the residue azeotroped several times in toluene

in order to thoroughly remove pyridine. Purification with vacuum-

column chromatography (EtOAc 0 to 100% in heptane) of the reac-

tion crude after treatment with acetic anhydride gave a single per-

acetylated glucose conjugate, as characterized by HPLC-MS [elu-

Eur. J. Org. Chem. 2010, 540–554

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551

E. Cló, O. Blixt, K. J. Jensen

FULL PAPER

69.4, 60.7, 39.2, 38.7, 38.7, 21.8 ppm. HR-MS (ESI): Calcd. for

C₁₉H₂₉N₃O₈[M + Na]⁺, 450.1852; found 450.1832.

gram) were hand-collected and freeze-dried. When a conjugate was

present in more than one isomer, no effort was done to separate

them from one anther; hence the different isomers were pooled to-

gether. The freeze-dried pellets were taken up in print buffer

(200 µL) and the conjugate concentration for each sample was esti-

mated via calibration curves obtained with stock solutions of the

relevant free linker. Purification procedure by extraction: reaction

mixtures of N-acetyllactosamine and mannobiose conjugates of B-

AMB were freeze-dried and the resulting pellets taken up with

0.1  NH₄OAc buffer (200 µL), pH 9.0 in an Eppendorf vial. Then

DCM (800 µL) was added to each vial and the resulting two-layer

mixtures were vigorously shaken for 5 min. After centrifugation,

the DCM layer was removed with a pipette. A total of 8 extraction

cycles with DCM were performed, the last which was kept whilst

stirring overnight. After the extraction series, the water layer was

analyzed by HPLC-MS (elution condition 1), showing only negligi-

ble traces of free linker left. The extracted samples were freeze-

dried and the resulting pellets taken up in print buffer (200 µL) and

their concentrations estimated via a calibration curve as mentioned

above. (AMB)Lac, HR-MS (ESI): calcd. for C₂₂H₃₅N₃O₁₂[M +

H]⁺, 534.2294; found 534.2317; (AMB)LacNAc, HR-MS (ESI):

calcd. for C₂₄H₃₈N₄O₁₂[M + H]⁺, 575.2559; found 575.2519;

(AMB)Man₂, HR-MS (ESI): calcd. for C₂₂H₃₅N₃O₁₂[M + Na]⁺,

556.2113; found 556.2771; (AMB)GlcNAc₅, HR-MS (ESI): calcd.

for C₅₀H₈₀N₈O₂₇[M + H]⁺, 1225.5206; found 1225.5292; (M-

AMB)Lac, HR-MS (ESI): calcd. for C₂₃H₃₇N₃O₁₂[M + H]⁺,

548.2450; found 548.2480; (M-AMB)LacNAc, HR-MS (ESI):

calcd. for C₂₅H₄₀N₄O₁₂[M + H]⁺, 589.2715; found 589.2720; (M-

AMB)Man₂, HR-MS (ESI): calcd. for C₂₃H₃₇N₃O₁₂[M + H]⁺,

548.2450; found 548.2447; (B-AMB)Lac, HR-MS (ESI): calcd. for

C₂₉H₄₁N₃O₁₂[M + H]⁺, 624.2763; found 624.2778; (B-AMB)-

LacNAc, HR-MS (ESI): calcd. for C₃₁H₄₄N4O₁₂[M + H]⁺,

665.3028; HPLC purified; found 665.3032; extraction purified;

found 665.3041; (B-AMB)Man₂, HR-MS (ESI): calcd. for

C₂₉H₄₁N₃O₁₂[M + H]⁺, 624.2763; HPLC purifed; found 624.2732;

extraction purified; found 624.2741.

N-(N-Benzyl-O-{4-[(2-acetamidoethyl)aminocarbonyl]benzyl}-hydrox-

ylamino) β-D-Glucopyranoside [Ac-BAMB-Glc]: The aminooxy

linker B-AMB (35 mg, 0.095 mmol) was treated with glucose in the

same two-step procedure as described for Ac(B-AE)Glc. Purifica-

tion with vacuum-column chromatography (MeOH 0 to 40% in

EtOAc) of the reaction crude after treatment with acetic anhydride

gave a single glucose conjugate, as characterized by HPLC-MS

(eluent condition 1,

5 to 100% of B). MS (ESI): calcd.

C₃₃H₄₁N₃O₁₂[M + H]⁺, 672.3; found 672.2.2. Following deacety-

lation, the pure product Ac-BAMB-Glc (16 mg, 35%) was obtained

as a white solid. ¹H NMR (300 MHz, D₂O): δ = 7.55 (d, J = 8.3 Hz,

2 H, ArH), 7.47–7.39 (m, 5 H, ArH), 7.28 (d, J = 8.3 Hz, 2 H,

ArH), 4.64 (d, J = 10.3 Hz, 1 H, OCH_2aAr), 4.47 (d, J = 10.3 Hz,

1 H, OCH_2bAr), 4.22 (d, J = 12.8 Hz, 1 H, NCH_2aAr), 4.18 (d, J

= 9.0 Hz, 1 H, 1-H), 4.08 (d, J = 10.3 Hz, 1 H, NCH_2bAr), 3.93

(dd, J = 12.3, 2.0 Hz, 1 H, 6a-H), 3.76 (dd, J = 12.3, 5.0 Hz, 1 H,

6b-H), 3.69 (t, J = 9.0 Hz, 1 H, 2-H), 3.52–3.46 (m, 3 H), 3.43–

3.37 (m, 4 H), 1.98 (s, 3 H, NCOCH₃) ppm. ¹³C NMR (75 MHz,

D₂O): δ = 174.4, 170.7, 139.4, 136.3, 133.6, 130.2, 129.6, 128.5,

127.9, 127.1, 91.6, 77.2, 77.2, 76.0, 70.0, 69.4, 60.8, 56.8, 39.2, 38.7,

21.8 ppm. HR-MS (ESI): Calcd. for C₂₅H₃₃N₃O₈[M + Na]⁺,

526.2165; found 526.2196.

Hydrolytic Stability of Glucose-Linker Conjugates: Stock solutions

of each glucose conjugate (20 m in DMSO) were prepared and

50 µL of these were diluted to 1 mL with each of the relevant buffer

solutions (50 m, sodium phosphate pH 3; sodium acetate pH 4

and 5) in order to obtain 1 m final concentration of conjugate

(time of dilution set to be t₀of the hydrolysis reaction course). The

solutions were incubated at room temp. inside the HPLC autosam-

pler in the dark and injected into HPLC at intervals during 4 d.

All hydrolysis were monitored with either Phenomenex Hydro

150ϫ3.0 mm, 3 Å or Dionex Acclaim 150ϫ5 mm, 5 Å columns

with flow rate 1.0 mL/min and linear gradient flow of B 5 to 50%

in A over 15 min (A = NH₄OAc 10 m, pH 5.8; B = 9:1 v/v MeCN/

NH₄OAc 100 m pH 5.8). Peaks area at 230 nm [except for Ac(B-

AE)Glc where 215 nm was used instead] were used to estimate the

conjugate/linker ratio, which is reported as fraction of conjugate

remaining according to the formula [A_conjugate/(A_conjugate+ A_linker)]

and plotted vs. time for each of the three buffer systems. The points

thus collected fitted well an exponential decay function, which al-

lowed calculating the pseudo first-order rate constant and half-lives

of the conjugates.

Printing of Arrays: The glycan arrays were fabricated by robotic

contact printing of approx. 6 nL of glycan-linker conjugates in

print buffer (150 m phosphare, 0.005% tween-20, pH 8.5) onto

NHS-activated glass slide (Nexterion Slide H, Schott AG, Mainz,

Germany). The conjugates were distributed in 384-well source

plates (20 µL per well) and structure was printed in 4 different con-

centrations in fivefold dilutions (250–2 µ) and each dilution de-

posited 4 times, thus creating a 4ϫ4 subgrid with a 0.21 mm pitch

between spots for each conjugate. The needles dwell time in the

wells was of 4 seconds and the pins underwent 3 wash cycles in

between visits. The complete array pattern was printed in 16 repli-

cas, distributed in two columns and 8 rows. Immediately after print-

ing the slides were incubated at 80% humidity for 60 min. The

NHS groups in the unprinted areas of the slides were blocked by

immersion in the blocking buffer (50 m ethanolamine in 50 m

borate buffer, pH 9.2) for 1 h. Slides were rinsed in Millipore water,

dried by centrifuging and probed. The slide that were not to be

probed immediately were stored at –18 °C before the blocking step.

General Preparation of Glycoconjugate Probes for Microarray Fabri-

cation: Stock solutions (40 m) of each of the free reducing glycans,

stock solutions (120 m) of linkers M-AMB and B-AMB and a

60 m stock solution of linker AMB were prepared in 0.1 

NH₄OAc buffer, pH 4.5 (to the chitopentaose stock solution 20%

DMSO was added for solubility). Aliquots from the relevant glycan

and the linker stock solution were mixed 1:1 (v/v, typically 50 or

100 µL) in a tight seal Eppendorf tube and incubated at 37 °C un-

der gentle shaking. At regular intervals the reaction mixtures were

injected into HPLC (elution condition 2). After 24 to 72 h all the

reactions reached equilibration and no more product formation

was observed. The above procedure was implemented for the prep-

aration of the batch of (B-AMB)LacNAc where 100 µL of reaction

mixture contained 50 m B-AMB (41.6 µL) and 1 m N-acetyllac-

tosamine (2.5 µL) and purified by extraction. Purification procedure

by HPLC: 20 to 40 µL aliquots of the reaction mixtures at equili-

bration were injected directly into HPLC (elution condition 2) and

the fractions containing the product (as assessed by UV chromato-

On-Slide Hydrolysis of Glucose-Linker Conjugates: A printed and

blocked slide was equipped with the 16-well superstructure in order

to create incubation wells for each of the 16 replica subarrays pres-

ent on a slide. Nine wells were filled with 90 µL of phosphate buffer

50 m, pH 3 each and the remaining wells filled with print buffer,

whereupon an adhesive cover tape was applied over the whole slide.

After 6 h the eight wells holding pH 3 solution were emptied and

backfilled with print buffer. After 24 h all wells were emptied and

backfilled with print buffer. Finally, after 36 h all wells were emp-

552

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Eur. J. Org. Chem. 2010, 540–554

Glycans in Microarrays

Wilson, R. Cummings, N. Bovin, C.-H. Wong, J. C. Paulson,

Proc. Natl. Acad. Sci. USA 2004, 101, 17033–17038; b) J. L.

De Paz, C. Noti, P. H. Seeberger, J. Am. Chem. Soc. 2006, 128,

2766–2767.

tied and the slide washed by immersion in PBST (phosphate-buff-

ered saline, 0.05% Tween-20), PBS and Millipore water whereupon

lectin staining with RCA I (10 µg/mL) followed as described below.

Lectin Staining: Blocked glycan-linker microarrays not coming

from the hydrolysis study were equipped with the 16-well super-

structure and immersed in PBS for 5 min. Incubation followed a

two-step procedure, in which at first solutions of biotin-tagged lec-

tins RCA I, ConA, ECA and WGA in PBST (all applied at 10 µg/

mL, 90 µL per well) were placed in the relevant wells. Then Alexa

Fluor 488-conjugate streptavidin in PBST (0.4 µg/mL, 90 µL per

well) was placed in all wells for detection. The slides were incubated

for 1 h at 80% humidity for each incubation step. In between incu-

bations the slides were washed by immersing them 2 min in each i)

PBST, ii) PBS, and iii) Millipore water. Finally, the superstructure

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1

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Received: October 29, 2009

Published Online: December 9, 2009

554

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Chemoselective reagents for covalent capture and display of glycans in microarrays

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