A modular approach for the construction and modification of glyco-SAMs utilizing 1,3-dipolar cycloaddition

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A modular approach for the construction and modiﬁcation of glyco-SAMs

utilizing 1,3-dipolar cycloaddition

Mike Kleinert,^aTobias Winkler,^bAndreas Terfort*^band Thisbe K. Lindhorst*^a

Received 29th January 2008, Accepted 12th March 2008

First published as an Advance Article on the web 18th April 2008

DOI: 10.1039/b801595c

We report the synthesis of a broad variety of functionalized molecules for assembly on gold, allowing

the formation of biologically relevant SAMs by a modular approach: either utilizing 1,3-dipolar

cycloaddition of alkynes and azides in solution or by ‘click on SAM’. Extensive studies into the various

parameters of SAM formation and stability have been carried out, leading us to deduce reliable

conditions under which glyco-decorated self-assembled monolayers can be formed and studied such as

in SPR-supported binding assays.

density and orientation of the carbohydrate ligands, in addition

Introduction

to being amenable to many spectroscopic techniques like surface

Eukaryotic cell surfaces are covered by a highly complex sugar

plasmon resonance (SPR), ellipsometry or AFM (atomic force

coat, called the glycocalyx. It comprises of glycoproteins, gly-

microscopy).

colipids, complex oligosaccharides as well as proteoglycans and

For the formation of glyco-SAMs on gold, carbohydrate-

other glycoconjugates.¹It may be considered as a cell organelle,²

terminated long-chain thiols or thioacetates have to be prepared,

whose function is essential in cell biology, because processes

as well as their non-carbohydrate analogs, in order to allow

like cellular recognition, cell development and differentiation,

‘dilution’ of the monolayer to avoid steric hindrance at the surface,

fertilisation and immune response are afﬁliated with molecular

which continuously increases during assembly. Furthermore, it has

recognition within the glycocalyx. In addition, states of disease

been our goal to include labelled molecules in SAM formation

can depend on molecular interaction with the glycocalyx, such as

to facilitate characterization of the monolayer once it is formed.

in the case of cancer and metastasis.³Interaction of cell surface

Finally, as SAM formation often is hard to control, we sought

saccharides with specialized proteins called lectins and selectins⁴is

a method to allow modiﬁcation of a preformed monolayer. This

of crucial biological importance. However, in spite of this, lectin–

has led us to a modular approach for the controlled construction

carbohydrate interactions are typically weak in in vitro testing,

of glyco-functionalized SAMs involving a sequence of formation

of a initial monolayer and its modiﬁcation by the attachment of

with K_Dvalues in the millimolar or high micromolar range.⁵It

has been assumed that the multivalency of carbohydrate–protein

biological relevant headgroups ‘on SAM’. We chose the copper(I)-

interactions⁶is fundamental to their biological effect,⁷but so

catalysed modiﬁcation of Huisgen’s 1,3-dipolar cycloaddition of

alkynes to azides,¹³introduced more-or-less simultaneously in

2001 by Meldal¹⁴and Sharpless.¹⁵This reaction allows modiﬁ-

cation of a preformed SAM without the requirement for classical

workup or puriﬁcation procedures.

far, multivalency effects occurring in carbohydrate recognition

have not been conclusively understood.⁸Therefore, molecular

mimetics are needed to study carbohydrate–protein interactions in

detail; such designer molecules should preferably try to mimic the

complexity and heterogeneity of the cell surface. There is a broad

range of approaches, stretching from carbohydrate libraries to

distinct multivalent glycoconjugates (such as glycodendrimers and

glycopolymers, for example),⁹all of which can be regarded as ‘one-

dimensional’ setups; however, a nano-size molecular array such

as the glycocalyx rather requires a ‘two-dimensional’, or ideally

‘three-dimensional’, molecular mimicry. Consequently, surface-

based glycomimetics have recently gained much interest including

glyconanoparticles,¹⁰glycoarray technology,¹¹and in particular

self-assembled monolayers (SAMs).

This so-called ‘click’ chemistry¹⁶has recently become a popular

method in organic and biological chemistry, including the ﬁeld

of self-assembled monolayers. Modiﬁcation of alkyne-terminated

SAMs with azido-functionalized aromatic molecules, nucleotides

and carbohydrates has been shown,¹⁷as well as attachment

of functionalized alkynes to azido-terminated SAMs on silicon

dioxide,¹⁸and binding of biologically and electrochemically rel-

evant molecules like oligonucleotides and ferrocenes to SAMs.¹⁹

Recently, 1,3-dipolar cycloaddition has been used for construction

of carbohydrate SAMs in order to measure carbohydrate–protein

interactions.²⁰

Here we report an extended study on the synthesis of func-

tionalized molecules for the formation of glyco-SAMs, their

modiﬁcation in a modular approach utilizing 1,3-dipolar cy-

cloaddition of alkynes and azides, and biophysical investigation

of a collection of different SAMs. An essential requirement for

the molecules synthesized for self-assembly is that they have to

contain oligoethylene glycol (OEG) portions, to suppress non-

speciﬁc adhesion of proteins to the SAM,²¹and to allow accurate

We became interested in the synthesis of sugar-modiﬁed SAMs,

the so-called ‘glyco-SAMs’,¹²for the study of molecular interac-

tions of carbohydrates, because glyco-SAMs have the potential to

serve as tailor-made carbohydrate arrays, allowing control over

^aOtto Diels Institute of Organic Chemistry, Christiana Albertina University

of Kiel, Otto-Hahn-Platz, 4, 24098, Kiel, Germany. E-mail: tklind@oc.uni-

kiel.de; Fax: +49 431 880 7410

^bDepartment of Chemistry, Philipps-University Marburg, Hans-Meerwein-

Str., 35032, Marburg, Germany. E-mail: aterfort@chemie.uni-marburg.de

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measurement of speciﬁc ligand–receptor interactions.²²To com-

bine ‘click’ chemistry with our rather complex oligoethylene glycol

spacers, ﬁrst in solution and eventually ‘on SAM’, was one of the

main challenges of this project.

are unreactive under cycloaddition conditions and serve to ‘dilute’

the alkyne-functionalized monolayer.

Thus, the commercially available 10-undecenic acid (1,

Scheme 1) was quantitatively converted into the methyl amide

2 via its mixed anhydride using isobutyl chloroformate (IBCF)

under basic conditions. Thioacetic acid was then added to the

terminal double bond of 2 in a photoinduced radical reaction

initiated with AIBN, providing 3. An alkyne-functionalized analog

of 3, the amide 5, was obtained from well known acetyl-

protected mercaptoundecanoic acid 4²³by peptide coupling with

propargylamine, again according to the mixed anhydride method

using IBCF.

With the ﬁrst two basic spacer types 3 and 5 in hand,

oligoethylene glycol-containing spacers had to be prepared next,

to allow the construction of monolayers resistant to non-speciﬁc

binding of proteins. An oligoethylene unit can very easily be

introduced using the monomethyl ether of hexaethylene glycol.

Esteriﬁcation of 4 with this alcohol under standard conditions

employing DCC and DMAP led to the thio-functionalized

Results and discussion

Our modular approach for formation of glyco-SAMs included

introduction of both biorepulsive OEG moieties as well as

the carbohydrate headgroups ‘on SAM’ (Fig. 1), after initial

monolayer formation using a basic thio-functionalized type of

spacer. The latter molecules were synthesized ﬁrst.

Synthesis of basic spacer molecules

Two types of spacer molecules were synthesized, those with a

terminal alkyne group to allow ‘click on SAM’ by 1,3-dipolar

cycloaddition, and a second group of non-alkyne analogs, which

Fig. 1 Surface modiﬁcation of a mixed SAM on gold by coupling of azides. The two possible concepts of generating mixed SAMs containing one

functional compound generated by ‘click’ chemistry are shown. Right: direct assembly of the preformed molecules onto the surface. Left: SAM formation

followed by ‘click’ reaction of the reactive groups. The latter approach permits more ﬂexibility in the surface modiﬁcation, both chemically and spatially.

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Scheme 1 Synthesis of basic spacer molecules. Reagents and conditions: a) NH₂Me, NPr₃, IBCF, DMF, 0 ^◦C → RT, 1.5 h, quant.; b) AcSH, AIBN,

THF, hm, RT, 3 h, 86% for 3, 95% for 8, 83% for 14, 97% for 15; c) propargyl amine, NPr₃, IBCF, DMF, RT, 2 h, 78%; d) DCC, DMAP, DCM, −20 ^◦C →

RT, 15 h, 75%; e) TosCl, DABCO, ethyl acetate, 4 A molecular sieves, 0 ^◦C → RT, 1.5 h, 82%; f) NaN₃, TBAI, DMF, 70 ^◦C, 2 h, 97%; g) PPh₃, 1 : 1

˚

THF–H₂O, RT, 3 d, 63%; h) propiolic acid, DCC, DCM, 0 ^◦C → RT, 3 h, 70%; i) propiolic acid, DCC, DMAP, DCM, −26 ^◦C → RT, 15 h, 73%.

alkyloxy spacer 6 in 75% yield. On the other hand, esteriﬁcation

could be avoided when the literature-known alkene 7²⁴was used

as the starting material, which can be obtained from bromoundec-

11-en and monomethyl hexaethylene glycol under Williamson

etheriﬁcation conditions in over 90% yield. Radical addition

of thioacetic acid under UV irradiation yielded the methoxy-

terminated spacer 8 in excellent 95%. To achieve the synthesis

of an alkyne-functionalized analog of 8, the alkyl oligoethylene

alcohol 9 was subjected to a sequence of OH-activation to

yield the tosylate 10, followed by nucleophilic substitution and

Staudinger reduction of the resulting azide 11 to the amine 12.

It is noteworthy that the use of DABCO, according to Hu¨nig’s

procedure,²⁵allowed us to omit pyridine in the tosylation step.

Next, amide coupling of the amine 12 with propiolic acid had to

be addressed. In this case, however, the use of IBCF led to decom-

position, whereas DCC-mediated peptide coupling was successful

and delivered alkyne 13 in 70%. This was in turn converted into the

terminal acetyl thioate 14 in a photoaddition reaction, as described

earlier. A byproduct carrying thioacetyl units at both termini

of the molecule was formed in this step; it was easily removed

during chromatographic puriﬁcation. The amount of byproduct

formation was diminished when irradiation was performed at

wavelengths >295 nm using a cut-off ﬁlter. This allowed the

preparation of the target alkyne 14 in 30% yield over ﬁve steps

starting with 9.

As molecules with internal amide linkages tend to form rather

stable monolayers due to intermolecular hydrogen bridging,²⁶an

ester analog of 14 was sought for comparison reasons. This is

easily accessible in two steps from 9, through radical addition of

thioacetic acid followed by DCC-mediated esteriﬁcation of the

resulting alcohol 15²⁷with propiolic acid, delivering 16 in 71%

overall yield.

Optimization of 1,3-dipolar cycloaddition conditions for the

convergent synthesis of spacer molecules

According to our synthetic plan, the convergent synthesis of more-

or-less complex spacer molecules using 1,3-dipolar cycloaddition

had to be accomplished next. The literature provides a vast

number of different reaction conditions for the copper(I)-mediated

1,3-dipolar cycloadition, with a variety of solvents, bases and

catalysts involved. Our ﬁrst investigation of reaction conditions

was performed with commercially available undecynic acid (17)

and the acetylthio-functionalized alkyne 5. Cycloaddition of the

alkyne 17 to dodecyl azide delivered triazole 18 in 50% yield,

whereas the reaction of 5 with the tosyl-functionalized triethylene

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Extension of reaction time up to 63 h had no effect on the yields.

Also, varying the temperature had little effect on conversion.

As the reaction conditions were intended to be applicable ‘on

SAM’, temperatures over 45 ^◦C were not examined thoroughly.

As ligation of the copper ion is of importance for the reaction,

it can be understood that both the nitrogen base applied as well

as the solvent have strong effects on the outcome of the reaction.

Common nitrogen bases such as 2,6-lutidine and diisopropylethyl

amine (DIPEA) were tested. Interestingly, the combination of

methanol with DIPEA as the base (Table 1, entry 8) is a low

yielding solvent–base system, as is lutidine in acetonitrile (entry 6).

On the other hand, DIPEA in acetonitrile or lutidine in methanol

provide good yields, with the desired 1,4-triazoles being the only

cycloaddition products. The reaction conditions with elevated

temperatures applied in entries 9 and 11 led to regioisomeric

mixtures of the 1,4- and 1,5-cycloaddition products.

Scheme 2 Convergent synthesis of spacer molecules employing 1,3-dipo-

lar cycloaddition. Reagents and conditions: a) 1-azidododecane, CuI,

2,6-lutidine, MeCN, 0 ^◦C → RT, 15 h, 50%; b) TosEG₄N₃, CuI, DIPEA,

MeOH, RT, 15 h, 62%.

Finally, a 1 : 1 mixture of DMF and methanol with 0.2

equivalents of cuprous iodide and without any nitrogen base was

identiﬁed to be the best system in homogeneous solution, with a

96% yield at 45 ^◦C.

glycolylazide TosOEG₃N₃gave the cycloaddition product 19 in a

reasonable 64% yield (Scheme 2).

To further optimize the reaction conditions of the copper(I)-

catalysed Huisgen reaction, to allow ‘click on SAM’ with the

molecules prepared here, we performed a systematic study in a

homogeneous solution employing alkyne 5 and hexaethylene gly-

colylazide 20 leading to triazole 21 after 1,3-dipolar cycloaddition

(Scheme 3). Published reaction conditions, using water as solvent

and a Cu(II) ascorbate-system,²⁸were unsuccessful in our case.

This might be due to the extremely polar hexaethylene glycol

moiety causing problems during workup and puriﬁcation in an

aqueous system.

Synthesis of functionalized spacers under optimized 1,3-dipolar

cycloaddition conditions

The results of our study on advantageous reaction conditions for

the 1,3-dipolar cycloaddition of ethylene glycol-type spacers (cf.

Table 1) paved the way for the synthesis of diversely functionalized

spacers, suited for self-assembly of monolayers. Thus, the impor-

tant dendritic building block 22²⁹was converted into the alkyne

23 using propiolic acid in a HATU-mediated peptide coupling

reaction³⁰(Scheme 4). Then, 1,3-dipolar cycloaddition with the

azide 24³¹proceeded in very good yield (91%). Deprotection of

the resulting dendritic triester 25 using TFA in 1,2-dichloroethane

As indicated in Table 1, the solvent, base, temperature, reaction

time and catalyst were varied in the synthesis of 21. Starting

materials were employed as 0.1 mM solutions. Reactions were

ﬁnished after about 8 h, regardless of solvent or temperature.

Scheme 3 Optimization of 1,3-dipolar cycloaddition conditions employing azide 20. Reagents and conditions: a) TosCl, DABCO, ethyl acetate, 0 ^◦C →

RT, 1.5 h, 80%; b) NaN₃, TBABr, DMF, 90 ^◦C, 3.5 h, 72%; c) see Table 1.

Table 1 Reaction conditions for the 1,3-dipolar cycloaddition of 5 and 20 leading to 21

Entry

Base

Solvent

Temp./^◦C

Catalyst

Time/h

Yield (%)^a

1

2

3

4

5

6

7

8

9

1.3 eq. DIPEA

1.3 eq. 2,6-lutidine

1.3 eq. DIPEA

—

MeCN

MeOH

RT

45

1 eq. CuI

5 eq. CuI

1 eq. CuI

2

8

<10

23

62

65

86

33

74

45

8

RT

100

45

15

43

toluene

1 eq. CuI

80^b

50

10

11

12

H₂O–^tBuOH

DMF–MeOH (1 : 1)

CuSO₄+ Na ascorbate

0.2 eq. CuI

—

70

45

91^b

96

^aIsolated yield. ^bBoth regioisomeric triazoles were detected.

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Scheme 4 Synthesis of dendritic triacidic spacer 26, employing 1,3-dipolar cycloaddition. Reagents and conditions: a) propiolic acid, HATU, DIPEA,

DMF, 0 ^◦C → RT, 18 h, 56%; b) CuI, DIPEA, MeCN, RT, 20 h, 91%; c) TFA, DCE, RT, 1 h, 98%.

(DCE) provided the target compound 26 in almost quantitative

yield. This molecule is of interest to modify polarity and acidity,

respectively, of SAMs, which is of importance with regard to the

high sialic acid content of many cells.

We then applied these reaction conditions to the synthesis of

carbohydrate-decorated spacers. The known mannoside 27³²was

employed as the azido component, whereas spacer 13 (Scheme 1)

was used as the alkyne. Cycloaddition with CuI in DMF–MeOH

(1 : 1) at 45 ^◦C yielded 1,4-triazole 28 in 73%, which was

subsequently turned into the thioacetate 29 following the usual

UV irradiation protocol in THF at room temperature (Scheme 5).

ﬂuorescence labels can be used to investigate the properties

of an actual SAM.³³Commercially available dansylcadaverin

(30, Scheme 6) was used as ﬂuorescence dye and had to be

converted into an azido-functionalized derivative for 1,3-dipolar

cycloaddition to a spacer alkyne. Conversion of the terminal

amino group in 30 into the respective azide via a metal-catalysed

diazo transfer with triﬂyl azide³⁴gave the desired product in only

63% yield. Therefore, we decided to employ azidoacetic acid, which

is easily accessible from chloroacetic acid in a quantitative reaction

with sodium azide in an aqueous medium. DCC-mediated peptide

coupling with 30 then led to the azide 31 in 92% yield. 1,3-Dipolar

cycloaddition to the spacer alkynes 5 and 14 delivered triazoles 32

and 33, respectively. Unfortunately, cycloaddition to 33 proceeded

in poor yields (around 60%).

SAM formation and ‘click on SAM’

With this array of thio-functionalized molecules in hand, SAM

formation and ‘click on SAM’ chemistry were evaluated next.

Ellipsometry was used to estimate the success of different reaction

conditions by determination of the layer thickness ex situ. Initial

experiments showed that the acetonitrile–CuI–DIPEA system

developed for the homogeneous solutions only works well for cy-

cloaddition of azides to alkyne-terminated monolayers, resulting

in an increase of layer thickness. In contrast, azido-terminated

Scheme

5

Synthesis of D-mannose-decorated thioacetate spacers.

Reagents and conditions: a) alkyne 13, CuI, DMF–MeOH, 45 ^◦C, 15 h,

73%; b) AcSH, AIBN, THF, hm, RT, 5 h, 40%.

Finally we set out to apply 1,3-dipolar cycloaddition to the

synthesis of ﬂuorescence-labeled spacers, suited for SAM forma-

tion, as labelling is an important tool in bioassays. In addition,

Scheme 6 Synthesis of ﬂuorescence-labelled spacers. Reagents and conditions: a) azidoacetic acid, DCC, HOBt, DCM, 0 ^◦C → RT, 15 h, 92%; b) 5, CuI,

DIPEA, MeCN, 45 ^◦C, 15 h, 84%; c) 14, CuI, DMF–MeOH, 45 ^◦C, 15 h, 59%.

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Table 2 Thicknesses and BSA adsorptions of SAMs of triazole-bridged 21, formed from pure 21 or from a homogeneous mixture of reaction partners

(d: optical thickness)

a

b

˚

Entry

Deposition conditions

Deposition time/h

d

_SAM/A

Dd_BSA/A

1

2

3

Pure 21 and EtOH

Mixture of 5 and 20^c

15

72

19.9 2.7

10.6 1.4

8.8 3.0

1.8 3.2

8.8 2.1

5.0 3.8

a

calc

SAM

= 37.4 A. ^bDd_BSA(Au) = 19.6 1.0 A. ^cReaction mixture: 5.0 mM thioacetate, 5.0 mM azide, 5.0 mM CuI, 6.3 mM DIPEA, MeCN.

˚

d

Table 3 1,3-Dipolar cycloaddition of the spacer azide 20 (MeO-EG₆-N₃)

to SAMs formed from a mixture of the amide 3 and alkyne-terminated 5

(d: optical thickness)

monolayers did not react readily with alkynes. Instead of a smooth

reaction, the formation of a precipitate (presumably a copper(I)–

alkyne complex) was observed, whose formation removes the

catalyst from the reaction solution.

Entry Ratio 5:3^a

d

_SAM/A

d^c_S^a_A^lc_M/A Dd_click/A

˚ ˚

Dd^c_c^a_li^l_c^c_k/A

˚

Therefore, our interest focused ﬁrst on the acetylenic molecule

5, which due to its simple structure should form densely packed

monolayers. In fact, deposition of this molecule from ethanolic

solution reproducibly yielded monolayers with a thickness of

1

2

3

4

1 : 0

1 : 3

1 : 4

0 : 1

20.3 0.8 18.8

20.3 0.6 17.2

18.9 0.9 17.0

16.0 2.4 16.6

11.4 1.0

9.2 0.7

7.0 1.1

−0.6 1.0

18.6

4.7

3.7

0

˚

about 19 1 A, in excellent agreement with a theoretical value

^aRatio in the deposition solution.

˚

of 18.8 A for molecules that have lost their acetyl group, being

packed in the monolayer with a tilt angle of 30^◦.

Reaction of a monolayer formed from 5 with the ethylene glycol

derivative 20 is of interest since attachment of this molecule should

not only increase the layer thickness, but also render the monolayer

resistant to the adhesion of proteins. Two approaches were tested

(Table 2); ﬁrstly, pure triazole 21, the reaction product of 5 and 20,

was used for monolayer formation, and secondly a homogeneous

reaction mixture of alkyne 5 and azide 20 was employed under

‘click’ conditions. The latter option is attractive, as isolation and

puriﬁcation of 21 could be avoided by selectively depositing it from

a reaction mixture.

The layer thickness was in all cases much smaller than

anticipated, hinting on incomplete monolayer formation. This

is a commonly observed phenomenon for longer molecules, in

particular with OEG headgroups, since unfolding of the coiled-

up chain costs too much entropy.³⁵Nevertheless, the resulting

monolayer almost completely suppresses the adsorption of bovine

serum albumin (BSA), a ‘sticky’ protein commonly used to test

not only in layers approaching the expected thickness (column

4 in Table 3) but exceeding it. Since any reactivity of the

methyl derivative 3 could be excluded (as shown in entry 4 in

Table 3), this excess thickness can only be interpreted as a result

of a higher ratio of 5:3 in the SAM than in the deposition

on.

Since in principle all the alkyne sites should be reactive in

these SAMs, we set out to use higher temperatures for the ‘click’

reaction. As trivial as this might sound, it should be kept in

mind that most SAMs are desorbed into solution at elevated

temperature.³⁶We therefore tested up to which temperature diluted

SAMs deposited from 1 : 1 mixtures of 3 and 5 remain stable in

acetonitrile (Table 4).

Surprisingly, even up to 80 ^◦C no complete desorption took

place, although the upper limit for keeping the monolayers

unaltered seemed to be 60 ^◦C. We therefore decided to carry

out ‘click’ reactions using azide 20 up to this temperature. For

this, pure monolayers of 5 were used, (i) to learn if even in a

dense monolayer all reactive groups can be approached under

these conditions, and (ii) to be certain of the maximum attainable

thickness after the ‘click’ reaction, since – as shown above –

the molecular ratio in a diluted monolayer does not need to

be the same as in the deposition solution. As can be seen in

Table 5, the elevated temperature had only a minor inﬂuence on

the completeness of the ‘click’ reaction: at 50 ^◦C and 60 ^◦C a

˚

bioresistance that typically results in layers about 20 A thick

(depending on the deposition conditions). The situation becomes

worse for the layers prepared from the reaction mixtures: even after

prolonged immersion (entry 3) only partial monolayers could be

attained, and these are not bioresistant.

If entropy is really the force suppressing the formation of

denser monolayers, it would be reasonable to expect that the post-

monolayer formation chemistry, such as ‘click on SAM’ should

result in thicker layers. In fact, when the complete monolayer of

5 was treated with 20 using the above-mentioned catalyst solution

Table 4 Temperature stability in acetonitrile of SAMs formed from 1 : 1

mixtures of alkyne 5 and amide 3 (d: optical thickness)

˚

at room temperature for 16 h, a layer thickness of 31.7 1.8 A

˚

(Dd = 11.4 1.0 A; Table 3, entry 1), signiﬁcantly higher than

attained from the preformed molecule 21, resulted.

a

˚

_SAM/A

d

Nevertheless, this monolayer was still thinner than the expected

Temperature/^◦C

Before treatment

After treatment

˚

(37.4 A), showing again the inﬂuence of entropy, meaning that

a number of reaction sites remain inaccessible. As a test for

this hypothesis, we used so-called diluted monolayers consisting

of a mixture of reactive molecule 5 with its unreactive deriva-

tive 3. The presence of the unreactive molecule should render

all the reactive sites accessible for the azide, thus permitting

complete transformation. In fact, the transformation resulted

50

60

70

80

14.1 0.3

12.6 0.3

14.2 0.1

16.1 0.4

13.1 0.2

12.9 0.5

13.1 0.2

13.7 1.0

a

calc

SAM

˚

d

= 17.7 A.

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Table 5 ‘Click’ reaction of MeOEG₆N₃(20) to SAMs of 5 at different

temperatures (d: optical thickness)

Table 6 Bioresistance of EG₆-containing monolayers as a function of the

number of alkyne groups exposed in the SAM (d: optical thickness)

Temperature/^◦C Time/h Dd_click/A

Dd_BSA/A

Entry

Ratio 16:15^a

d^c_S^a_A^lc_M/A

Dd_BSA/A

a

b

c

˚

d_SAM/A

˚

Entry

d

_SAM/A

1

2

3

4

20.3 0.8 25

19.2 0.6 40

17.8 0.7 50

16.3 1.2 60

15

16

15

11.4 1.0

11.5 0.9

14.9 1.6

13.1 1.9 −1.0 2.2

4.9 0.8

2.0 0.9

0.5 2.6

1

2

3

4

5

6

1 : 0

50 : 1

10 : 1

1 : 1

1 : 2

0 : 1

15.8 1.6

15.3 0.9

22.6 1.5

21.3 0.2

20.3 0.6

24.2 0.2

36.8

36.7

36.5

35.3

34.7

33.7

14.8 2.2

3.1 1.7

1.4 2.1

0.9 0.4

−0.8 0.7

a

calc

SAM

calc

= 18.8 A. ^bDd_click= 18.6 A. ^cDd_BSA(Au) = 20.0 0.4 A.

−2.2 0.4

˚

d

^aIn solution.

It should be mentioned at this point that the de facto proportion

of 15 in the SAMs may exceed the one in solution – the reverse of

the process previously observed for the 5:3 system. Experiments

to clarify this point by determination of the number of reactive

acetylenic groups by ‘click on SAM’ using the azide 20 again

resulted in a surprise: instead of a gain in thickness, we observed

a loss of matter as well as a loss of bioresistance. For example,

a 1 : 1 diluted monolayer of 16 and 15, being completely

˚

bioresistant beforehand, became thinner by almost 12 A instead

˚

of thickening by 18.8 A, accompanied by adherence of 3 A

˚

of BSA compared to 0 A before the cycloaddition. Extensive

experiments (data not shown) showed that OEG-terminated

thiolate SAMs in general are not compatible with Cu⁺ions in

hot acetonitrile. We assume that under these conditions a Cu⁺–

thiolate complex is formed which becomes soluble due to the very

polar OEG part of the molecule. Unfortunately no conditions

could be found to achieve a ‘click on SAM’ reaction without

desorption.

Fig. 2 Time-dependence of the added layer thickness (‘click’ reaction of

20 to SAM of 5 according to entry 3 in Table 5) and the BSA resistance.

˚

couple of A in thickness were gained (compared to Table 3, entry

1), but no complete layer was formed in either case.

Because of this, we returned to the system based on the

monolayers generated by 5 on gold to study carbohydrate–lectin

interactions. Using the optimised conditions for ‘click on SAM’,

the azidoethyl mannoside 27 was attached to a SAM of thioacetate

5 on gold. Surprisingly, the resulting increase of the layer thickness

Protein resistance of the monolayers on the other hand became

signiﬁcantly better with increasing reaction temperature (Table 5,

column 5), an important criterion for future applications. In

continuation of the optimization of the reaction conditions, the

time dependence of the layer properties was studied after reaction

at 50 ^◦C. In a series of time-dependent experiments it was

again observed that the increase in thickness never reached the

˚

(10.8 0.6 A) is signiﬁcantly larger than the expected value (6.5

˚

A). We attribute this either to physisorbed material or to some

kind of side reaction attaching additional material. The water

contact angles of the modiﬁed surfaces were 34 1^◦(advancing)

and 17 2^◦(receding), corresponding to a hydrophilic surface.

However, for a pure hydroxy-terminated surface the value of

the advancing contact angle is too high. This suggests that the

surface exposes not only OH groups, but also methylene groups.

This assumption is supported by the relatively large contact

˚

theoretical value of 18.6 A, but levelled off at about 13 A after

4 h (Fig. 2). Despite this, complete bioresistance was not achieved

before 8 h reaction time, a situation mirroring the temperature-

dependent experiments. We therefore decided to perform all

further ‘click’ reactions for 16 h at 50 ^◦C.

Since the introduction of bioresistance was not the primary goal

of this work, but to use bioresistant molecules as a background

to attach recognition sites (in particular carbohydrate sites), we

switched to the use of the OEG-containing alkyne 16 as SAM-

forming material. The monolayers exclusively formed from 16 are

not only thinner than calculated, but also are not bioresistant

(Table 6, entry 1).

This comes to no surprise, since – as mentioned before –

molecules this long hardly form perfect monolayers. In addition,

these layers should be terminated with a signiﬁcant number of

hydrophobic alkyne groups, thus exposing adsorption sites for

BSA. We hoped that dilution with the molecule 15 of proven

bioresistance (entry 6) would render the monolayers bioresistant

at some point. This expectation was more than fulﬁlled, since even

a proportion as low as 9% of 15 (entry 3) induces bioresistance in

the system.

˚

angle hysteresis of ca. 17 A, indicating an imperfectly ordered

surface.

Since disorder should not hinder the recognition of the man-

nosyl groups (since, for example, the glycocalyx is not a laterally

ordered system) we decided to perform SPR experiments to see

if some speciﬁcity for concanavalin A (ConA), a strongly binding

plant lectin, exists. In fact, the pure monolayer of 5 turned out not

to be very adhesive for ConA, thus permitting a clear distinction

from the very strong response obtained from the mannosyl-

terminated monolayer (Fig. 3).

Since these data were obtained on a home-built SPR system,

the quality of the data does not permit a quantitative assessment

yet, but the data suggest that the speciﬁc recognition is at least

ﬁve times stronger than the non-speciﬁc adhesion, thus proving

our modular system suitable for further investigation.

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without further puriﬁcation. TLC was performed on GF254 silica

gel plates (Merck), detection was effected by the use of UV light

(254 nm and 366 nm) and with mixtures of either 10% sulfuric acid

in ethanol or cerium(IV) sulfate and phosphomolybdate in 10%

sulfuric acid followed by heat treatment. Flash chromatography

was performed on silica gel 60 (230–400 mesh, particle size 0.040–

0.063 mm, Merck). NMR spectra were recorded on Bruker DRX

500 (500 MHz for ¹H, 125.47 MHz for ¹³C) and ARX 300

instruments (300 MHz for ¹H, 75.47 MHz for ¹³C). Spectra were

calibrated with respect to the solvent peak (CDCl₃7.24 ppm for

1

¹H and 77.0 ppm for ¹³C; [D₄]methanol 3.35 ppm for H and

49.30 ppm for ¹³C.) Assignment of the peaks was achieved with the

1

aid of 2D NMR techniques (¹H–¹H-COSY and H–¹³C-HSQC).

Peak values that could not be unequivocally assigned to one atom,

and may therefore be interchangeable, are marked with an asterisk.

In some cases, amide isomers delivered a double set of signals, as

indicated by ‘‡’.

Fig. 3 The adsorption of concanavalin A onto a native monolayer

of alkyne 5 (A) as well as onto a monolayer modiﬁed by ‘click on

SAM’ chemistry using the azidoethyl mannoside 27 (B) as followed by

SPR. Although some non-speciﬁc adsorption takes place in case A, the

recognition of the mannosyl substituents is dominant.

Hydrogen and carbon atoms within the scaffold are indexed

as follows: the sugar residue is numbered as usual from 1 to 6

with the anomeric position being number 1, and aglycon atoms

are numbered in ascending order starting with the atom adjacent

to the glycosidic bond being number 7, as exempliﬁed in Fig. 4

for compound 29. For spacer molecules without a sugar moiety,

the acetyl thio-group was deﬁned as the terminus of the molecule,

and carbon and hydrogen atoms were numbered accordingly, as

exempliﬁed for 29, 33 and the dendritic molecule 25 (Fig. 4).

MALDI-TOF mass spectra were recorded on a Bruker Biﬂex III

19 kV instrument, with norharmane (9H-pyrido[3,4-b]indole) in

Conclusion

We have reported the synthesis of a series of differently function-

alized molecules for the formation of SAMs and their utilization

in a modular approach employing 1,3-dipolar cycloaddition

of alkynes and azides. Thus, we have provided a molecular

toolbox, allowing for the preparation of biorelevant monolayers,

including carbohydrate decoration. We have extensively studied

the parameters of SAM formation and reported on drawbacks and

limitations as well as on breakthroughs en route to glyco-SAMs as

glycocalyx models.

We have demonstrated that ‘click on SAM’ chemistry permits

the modiﬁcation of surfaces with biologically relevant headgroups

such as bioinert OEG chains or substrate groups recognized

by lectins. It seems generally advisable to have the acetylenic

compound for the Huisgen reaction in the SAM and the azide

in solution to avoid precipitation of presumably inactive Cu

acetylides rendering them unsuitable for a surface reaction. For

the ligation with long OEG azides, the ‘click on SAM’ approach

permits the synthesis of denser SAMs with a reliable bioresistance

against non-speciﬁc protein adhesion as opposed to the layers

obtained from the pre-formed, complete molecules. Although the

optimizations of the click reactions conditions were performed

on a single and stable short-chain SAM using OEG as a ligand,

we have shown that the reaction conditions were also useful

for the attachment of bio-entities such as mannose, resulting in

surfaces recognized by lectins. Investigations are now under way

to understand how surface libraries made from this building-block

toolbox inﬂuence the recognition process by, for example, proteins.

Experimental

General remarks

Reactions were carried out in dried glassware under argon or nitro-

gen and using distilled solvents unless otherwise indicated. THF

was dried by distillation from sodium/potassium ketyl, methanol

by distillation from magnesium turnings, and dichloromethane by

distillation from calcium hydride, each under argon. Commercially

available starting materials, reagents and pure DMF were used

Fig. 4 Numbering of hydrogen and carbon atoms for assignment of NMR

data.

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THF being used as a matrix. For sample preparation, a drop of

matrix solution was ﬁrst placed on the target and left to evaporate.

Afterwards, a solution of the sample in THF or in methanol was

placed on the pre-crystallized matrix.

Even though NMR spectra showed no contamination and

analytical HPLC showed no impurities, correct elemental analyses

could not be obtained for most of the reported substances.

ESI-MS measurements were consulted to prove purity. High-

resolution mass spectra were recorded on a Mariner (Part-No.

V800600) instrument. For analytical HPLC chromatography a

Merck-Hitachi machine with a diode array detector L-7455 was

Protein adsorption

The samples were immersed for 2 h in a solution of 1 mg

mL⁻¹bovine serum albumin (BSA) in aqueous KH₂PO₄/K₂HPO₄

buffer (pH = 7.0). After immersion each sample was rinsed with

100 cm³of demineralised water and dried in a stream of nitrogen.

The amount of adsorbed BSA was determined by ellipsometric

measurements.

Ellipsometry

R

used with either LiChrosorb^ꢀRP-8 7 lm silica or a Chromolith^ꢀ

performance RP-18 100 × 4.6 mm column. Preparative HPLC

chromatography was carried out on a Shimadzu system with a

Optical ﬁlm thicknesses were determined assuming a complex

refractive index N = n − ik with a real part n = 1.45 and an

imaginary coefﬁcient k = 0 for both the SAMs as well as the

BSA adlayers.³⁸The parameters n and k of the gold substrates

were obtained by ellipsometric measurements of the plasma-

cleaned ﬁlms before monolayer formation. For each experiment

six readings were recorded to calculate an average value as well as

the standard deviation.

R

Merck Hibar^ꢀRT250–25 mm column with LiChrosorb^ꢀRP-8

7 lm silica.

Ellipsometric contact angle measurements and protein adsorp-

tion experiments were performed under ambient conditions. Ellip-

sometry was performed on a SE 400 (Sentech Instruments GmbH)

ellipsometer under an incident angle of 70^◦at a wavelength of

633 nm. Water contact angles were measured by the sessile drop

method using a Multiskop (Optrel GBR, Germany) instrument.

SPR measurements were also carried out on the Multiskop

instrument using the Kretschmann geometry with prism coupling

(BK7), p-polarized laser radiation (785 nm) being used for the

excitation of the plasmon. The prism and the gold-covered glass

substrate were optically connected using a matching ﬂuid with a

refractive index of n = 1.51. Home-made PDMS chambers with

a volume of 36 lL were used as ﬂow cells. The solutions were

pumped through the cell using syringe pumps (Braun Perfusor

Secura, Germany).

Contact angle

Contact angles were measured for water as contact ﬂuid. The

droplets were imaged by a CCD camera and the contact angle was

calculated from the shape of the recorded image using an included

software. In all cases the advancing and the receding contact angle

was determined. The value of each contact angle (as well as its

standard deviation) was obtained by averaging six readings.

SPR measurements

The binding of concanavalin A (ConA) to mannosyl-terminated

monolayers was monitored using surface plasmon resonance spec-

troscopy. The angle shift was calculated from the resonance angles

determined before and after lectin adsorption. The measurements

were carried out in HEPES-buffered saline (150 mM NaCl,

10 mM NaHEPES, 0.005% Tween 20) containing Ca²⁺(1 mM

CaCl₂·2H₂O) and Mn²⁺(1 mM MnCl₂·4H₂O) ions for ConA

activation. Brieﬂy, adsorption was performed by ﬁrst running

buffer until a stable signal was observed, followed by 10 lM of

ConA dimer in buffer solution (10 min) and then again buffer

(10 min). The resonance angles were determined after the ﬁrst and

the second buffer purge, respectively. For all experiments a ﬂow

rate of 30 mL h⁻¹was chosen.

Au(111)-covered substrates for the SAM formation were pre-

pared by electron beam deposition of 1.5 nm of chromium as

adhesion promotor followed by the deposition of 200 nm of

gold onto Si(100) wafers (Wacker Siltronic AG). For the SPR

experiments, gold-covered microscope slides (2 nm Cr, 50 nm Au)

were used.

A Harrick Plasma Cleaner/Sterilizer PDG-32G was used to

generate a microwave-induced hydrogen plasma for the cleaning

of the gold surfaces.³⁷

Monolayer formation

Before use, the gold-covered substrates were cleaned by the treat-

ment with hydrogen plasma for 60 s. Thiolate SAMs were formed

by immersion of the cleaned gold substrates in 5 mM solutions

of the corresponding thioacetate in dry ethanol overnight under

nitrogen. After immersion, substrates were rinsed thoroughly with

ethanol and dried in a stream of nitrogen.

=

H₂C C₉C(O)NHMe (2). 10-Undecenic acid (0.91 g,

10.85 mmol) was dissolved in dried DMF (15 ml) and cooled to

0 ^◦C. Tripropylamine (4.13 ml, 21.70 mmol) and IBCF (1.48 ml,

11.40 mmol) were added and the reaction mixture was stirred

for 30 min. Then 2 M methylamine solution in THF (5.43 ml,

10.85 mmol) and more tripropylamine (2 ml, 10.85 mmol) were

added, the reaction mixture was warmed to ambient temperature

and kept stirring overnight. Subsequently the solvents were

removed under reduced pressure and the colourless crude product

was puriﬁed by column chromatography on silica using ethyl

acetate and cyclohexane (1 : 1 → 2 : 1) as the solvent system.

The alkene 2 was obtained as a colourless amorphous solid

(2.11 g, 99%). The spectroscopic data were in accordance with the

literature.³⁹

Monolayer modiﬁcation by ‘click on SAM’

Substrates covered with alkyne-terminated monolayers were

treated with a solution of CuI (5.0 mM), DIPEA (6.5 mM)

and the corresponding azide (5.0 mM) in oxygen-free acetonitrile

overnight at a temperature of 50 ^◦C. Then the substrates were

thoroughly rinsed with acetonitrile, immersed in demineralised

water for 2 h to remove physisorbed material and dried in a stream

of nitrogen.

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AcSC₁₀C(O)NHMe (3). To a stirred solution of alkene amide

2 (1.00 g, 5.07 mmol) in abs. THF (30 cm³) were added thioacetic

acid (0.90 cm³, 12.67 mmol) and AIBN (832.2 mg, 0.51 mmol) at

room temperature and the UV irradiation (k ≥ 295 nm) was started

and maintained for 2 h. For workup the solvents were removed

under reduced pressure and the residual pale yellow crude oil was

puriﬁed by ﬂash chromatography on silica using an ethyl acetate–

cyclohexane gradient (2 : 1 → 4 : 1). The product 3 was obtained

as a colourless amorphous solid (1.19 g, 86%). m_max(ﬁlm/cm⁻¹)

3308s, 3088w, 2920s, 2849s, 1690s, 1643s, 1560s, 1459w, 1411m,

1237w, 1142m, 707w, 631m; d_H(500 MHz, CDCl₃, Me₄Si) 5.82

(1H, br, NH), 2.86 (2H, m≈t, J 7.26, 2 × 12-H), 2.81 (3H, d, J

4.85, 3 × 1-H), 2.33 (3H, s, 3 × 14-H), 2.16 (m≈t, J 7.33, 2 × 3-H),

1.58 (4H, m, 2 × 4-H, 2 × 11-H), 1.27 (12H, m, 2 × (5–10-H));

d_C(125 MHz, CDCl₃, Me₄Si) 196.18 (13-C), 173.81 (2-C), 36.71

(3-C), 30.64 (14-C), 29.45 (12-C), 29.32–28.74 (5–11-C), 26.25 (1-

C), 25.75 (4-C); m/z (ESI-MS) 296.1302 (M⁺+ Na. C₁₄H₂₇NO₂S

requires 296.1655).

34.96 (16-C), 32.41-29.76 (17–24-C), 30.54 (26-C), 26.00*; m/z

(MALDI-TOF-MS) 561.5 (M⁺+ Na. C₂₆H₅₀O₉S + Na requires

561.5), (ESI-MS) 434.3474 (M⁺+ H. C₂₃H₄₇NO₆+ H requires

434.3476).

AcSC₁₁EG₆OMe (8). To a stirred solution of alkene 7 (2.92 g,

6.50 mmol) in abs. THF (30 cm³), thioacetic acid (1.16 cm³,

16.27 mmol) and AIBN (840 mg, 5.21 mmol) were added and

the mixture was irradiated with UV light for approx. 3 h at room

temperature. The solvents were then evaporated and the yellow

crude product was puriﬁed by chromatography with a methanol–

DCM gradient (1 : 20 → 1 : 18) to yield a colourless oil (3.245 g,

95%). m_max(ﬁlm/cm⁻¹) 2925s, 2855s, 1692s, 1461m, 1352m, 1299w,

1249w, 1116s, 952m, 884w, 627 m; d_H(300 MHz, CD₃OD) 3.73–

3.68 and 3.66–3.54 (22H, m, 2 × (2-13-H)), 3.44 (2H, t, J 6.81,

2 × 14-H), 3.38 (3H, s, 3 × H-1), 2.85 (2H, t, J 7.36, 2 × 24-

H), 2.31 (3H, s, 3 × 26-H), 1.59–1.49 (4H, m, 2 × 15-H, 2 ×

23-H), 1.30–1.24 [14H, m, 2 × (16–22-H)]; d_C(75 MHz, CD₃OD)

196.12 (25-C), 71.92–70.39 (3–13-C), 59.05 (1-C), 30.66 (26-C),

29.62–28.81 (15–22-C), 26.08 (24-C); m/z (CI-MS) 511 (M⁺+ H),

100.0%), 496 (13.5), 435 (1.3), 361 (1.4), 325 (2.5), 317 (1.7), 283

(2.8), 229 (2.0), 187 (2.6), 177 (2.8) 133 (3.9), (ESI-MS) 547.3267

(M⁺+ Na. C₂₅H₅₂O₈S +Na requires 547.3275).

≡

AcSC₁₀C(O)NHCH₂C CH (5). A solution of the carboxylic

acid 4 (1.60 g, 6.14 mmol), IBCF (956.4 ll, 7.37 mmol) and

tripropylamine (2.34 cm³, 12.29 mmol) in abs. DMF (25 cm³)

was cooled to 0 ^◦C and stirred for 50 min before a cooled

solution of propargylamine (505.8 ll, 7.37 mmol) in abs. DMF

(5 cm³) and tripropylamine (1.17 cm³, 6.15 mmol) were added

dropwise. The clear reaction mixture was slowly warmed to

room temperature and stirring was maintained for 2 h. Then

the solvents were removed under reduced pressure and the crude

product was puriﬁed by column chromatography on silica using

ethyl acetate and cyclohexane (1 : 1.5) as solvent system. The

product 5 was obtained as a colourless amorphous solid (1.43 g,

78%). m_max(ﬁlm/cm⁻¹) 3311s, 3287s, 2919s, 2849s, 1691s, 1634s,

1534s, 1534s, 1471m, 1454w, 1418m, 1357w, 1284w, 1232m, 1209w,

1141m, 959w, 643m, 579w, 549w; d_H(500 MHz, CDCl₃, Me₄Si)

4.05 (2H, dd, J 2.56 and 5.24, 2 × 3-H), 2.86 (2H, t, J 7.37, 2 ×

14-H), 2.32 (3H, s, 3 × 16-H), 2.23 (1H, t, J 2.55, 1-H), 2.20

(2H, m, 2 × 5-H), 1.63 (2H, m, 2 × 13-H), 1.56 (2H, m, 2 ×

6-H), 1.27 (12H, m, 12H, 2 × (7–12-H)); d_C(125 MHz, CDCl₃,

Me₄Si) 196.10 (15-C), 172.78 (4-C), 79.69 (2-C), 71.38 (1-C), 36.37

(5-C), 30.59 (16-C), 29.48-28.70 (3-C, 6–13-C), 25.48 (4-C); m/z

(ESI-MS) 298.1828 (M⁺+ H. C₁₆H₂₇NO₂S requires 298.1835).

=

TosOEG₆C₉CH CH₂(10). To a stirred solution of the alcohol

9 (4.0 g, 9.20 mmol) in ethyl acetate (13 cm³) were added DABCO

˚

(2.07 g, 18.4 mmol) and 4 A molecular sieves (100 mg). The

reaction mixture was cooled to 0 ^◦C and tosyl chloride (2.63 g,

13.80 mmol) was added in portions. Upon addition of TosCl a

highly viscous suspension formed immediately. The mixture was

warmed to room temperature and stirred for 1.5 h before the

slurry was ﬁltered through ﬁlter paper. The ﬁltrate was acidiﬁed

by dilute hydrochloric acid (5 cm³) and washed with sat. NaHCO₃

(5 cm³) in demineralised water prior to drying over Na₂SO₄. After

ﬁltration, solvents were evaporated and the crude product was

puriﬁed by column chromatography using silica and an ethyl

acetate–cyclohexane gradient (2 : 1 → 4 : 1). The product 10

was obtained as a pale yellow oil (4.43 g, 82%). m_max(ﬁlm/cm⁻¹)

3509w, 3072w, 2925s, 2856s, 1736w, 1640m, 1598m, 1454m, 1359s,

1292m, 1248m, 1189s, 1178s, 1113br, 1019m, 923s, 817m, 775m,

664s, 555s; d_H(300 MHz, CDCl₃, Me₄Si) = 7.80 (2H, dt, J 1.88

and 8.3, 2 × Ar-H), 7.48 (2H, m, 2 × Ar-H), 5.81 (1H, ddt, J 6.63,

10.29, 17.1; 21-H), 5.03–5.01 and 4.97–4.90 (2H, m, 2 × 23-H),

4.16 (2H, t, J 4.81, 2 × 1-H), 3.70–3.56 (22H, m, 2 × (2–12-H)),

3.44 (2H, t, J 6.8, 2 × 15-H), 2.45 (3H, s, 3 × Ar-CH₃), 2.07–

2.00 (2H, m, 2 × 21-H), 1.59–1.55 (2H, m, 2 × 14-H), 1.30-1.28

(12H, m, 2 × (15–20-H)); d_C(75 MHz, CDCl₃, Me₄Si) 144.74

(C-Ar), 139.19 (22-C), 132.90 (C-Ar), 129.78 (C-Ar), 127.94 (C-

Ar), 114.07 (23-C), 71.49 (2-C), 70.53 (3–11-C), 69.99 (1-C), 69.20

(12-C), 68.62 (13-C), 33.77 (21-C), 29.10 (14–19-C), 26.03*, 21.61

(CH₃-Ar); m/z (CI-MS) 598 (M⁺+ H, 100%), 545 (1.5), 501 (2.6),

437 (41.4), 375 (9.6), 331 (29.6), 287 (27.7), 241 (37.5), 199 (59.8),

133 (18.9), 89 (39.5).

AcSC₁₀C(O)OEG₆OMe (6). A solution of carboxylic acid 4

(430 mg, 1.65 mmol) and hexaethylene glycol monomethyl ether

(538.3mg, 1.82 mmol) in dry DCM (3 cm³) was cooled to −20 ^◦C

and a mixture of DCC (357.8 mg, 1.73 mmol) and DMAP

(20.2 mg, 0.17 mmol) in DCM (3 cm³) were added slowly. The

reaction mixture was then allowed to warm to room temperature

and was stirred overnight. The solvents were removed under

reduced pressure and the viscous crude product was puriﬁed by

column chromatography (MeOH–DCM, 1 : 20) to yield the title

compound as a pale yellow oil (667.2 mg, 75%). m_max(ﬁlm/cm⁻¹)

2924s, 2855s, 1735s, 1691s, 1456m, 1352m, 1298w, 1246m, 1111s,

1045w, 954m, 852m, 628m; d_H(500 MHz, CD₃OD) 4.20 (2H, m,

2 × 13-H), 3.69 (2H, m, 2 × 12-H), 3.63 (18 H, m, 2 × (3–11-H)),

3.53 (2H, m, 2 × 2-H), 3.35 (3H, s, CH₃), 2.86 (2H, t, J 7.30, 2 ×

24-H), 2.33 (2H, t, J 7.41, 15-H), 2.30 (3H, s, 3 × 26-H), 1.61 (2H,

m, 2 × 16-H), 1.55 (2H, m, 23-H), 1.30 (12H, m, 2 × (17–22-H));

d_C(125 MHz, CD₃OD) 197.50 (25-C), 175.35 (14-C), 72.96 (15-

C), 71.59-71.35 (3-12-C), 70.14 (2-C), 64.55 (13-C), 59.10 (1-C),

=

N₃EG₆C₉CH CH₂(11). The tosylate 10 (4.39 g, 7.46 mmol),

sodium azide (2.43 g, 37.28 mmol) and tetrabutylammonium

iodide (1.54 g, 4.20 mmol) were dissolved in abs. DMF (50 cm³)

and warmed to 70 ^◦C for 2 h with stirring. Then the solvents

were removed under reduced pressure and the crude material

was puriﬁed by ﬂash chromatography on silica using ethyl

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acetate–cyclohexane (2 : 1). The title compound was obtained

as a colourless oil (3.33 g, 97%). m_max(ﬁlm/cm⁻¹) 3590w, 3074w,

2924s, 2854s, 2103s, 1737w, 1340m, 1455s, 1348s, 1300s, 1249m,

1118s, 1039m, 994m, 910s, 853m, 722w, 644w, 556w; d_H(500 MHz,

CDCl₃, Me₄Si) 5.80 (1H, ddt, J 6.75, 10.28, 17.14; 22-H), 4.98 (1H,

ddt, J 1.65, 2.20, 17.06; 23-H), 4.94 (1H, ddt, J 1.19, 2.02, 10.18;

23-H), 3.68–3.62 (20H, m, 2 × (3–12-H)), 3.58–3.56 (2H, m, 2 ×

2-H), 3.43 (2H, t, J 6.79, 2 × 13-H), 3.38 (2H, t, J 5.14, 2 × 1-H),

2.05–2.00 (2H, m, 2 × 12-H), 1.56 (2H, q, J 14.67, 2 × 14-H),

1.36 (2H, t, J 7.25, 2 × 15-H), 1.32-1.28 (10H, m, 2 × (16-20-H);

d_C(125 MHz, CDCl₃, Me₄Si) 139.23 (22-C), 114.09 (23-C), 71.53

(13-C), 70.63 (3–12-C), 70.03 (2-C), 50.65 (1-C), 33.80 (21-C),

29.61-28.90 (14–20-C), 26.00*; m/z (CI-MS) 460 (M⁺+ H, 9.61%),

432 (100.0), 388 (1.2), 292 (2.1), 241 (2.5), 177 (1.4), 147 (2.1), 133

(2.1), 97 (3.0), (ESI-MS) 482.3270 (M⁺+ Na. C₂₃H₄₅O₆N₃requires

482.3201).

(1-C), 70.58–69.24 (5–16-C), 39.58 (4-C), 33.76 (24-C), 29.58–

28.88 (17–23-C), 26.04*; m/z (CI-MS) 485 (M⁺, 0.5%), 391 (7.4),

280 (2.5), (ESI-MS) 508.3319 (M⁺+ Na. C₂₆H₄₇NO₇+Na requires

508.3245).

≡

AcSC₁₁EG₆NHC(O)C CH (14). The alkyne 13 (200 mg,

0.42 mmol) were dissolved in abs. THF (3 cm³), and thioacetic

acid (0.06 cm³) and AIBN (66.4 mg, 0.42 mmol) were added at

room temperature. The reaction mixture was irradiated with UV

light (k ≥ 295 nm) for 3 h. Then dichloroethane (2 cm³) was added

and the solvents were removed under reduced pressure to yield

a brownish crude product. This was subjected to ﬂash column

chromatography (MeOH–DCM, 1 : 20) to yield a pale yellow oil

(0.19 g, 83%). m_max(ﬁlm/cm⁻¹) 3324m, 2925s, 2852s, 2106m, 1692s,

1656m, 1573w, 1538w, 1437w, 1352m, 1245m, 1118s, 954s, 627m,

542w; d_H(300 MHz, CDCl₃, Me₄Si) 3.66 (24 H, m, 2 × (4–15-H)),

3.44 (2H, t, J 6.83, 2 × 16-H), 2.86 (2H, t, J 7.29, 2 × 26-H),

2.85 (1H, s, 1-H), 2.33 (3H, s, 3H, 3 × 28-H), 1.56 (4H, m, 2 ×

17-H, 2 × 25-H), 1.26 (14H, m, 2 × (18–24-H)); d_C(75 MHz,

CDCl₃, Me₄Si) 196.74 (27-C), 152.53 (3-C), 73.41 (2-C), 71.54

(1-C), 70.59–69.26 (4–16-C), 30.74 (28-C), 29.60-26.06 (17-26-C);

m/z (ESI-MS) 584.3260 (M⁺+ Na. C₂₈H₅₁NO₈S + Na requires

584.3228).

=

H₂NEG₆C₉CH CH₂(12). The azide 11 (2.20 g, 4.79 mmol)

was dissolved in a 1 : 1 mixture (24 cm³) of THF and water at

room temperature, triphenylphosphine (1.30 g, 4.96 mmol) was

added and the solution was stirred for 3 d vigorously. Then the

solution was extracted three times with a 1 : 1 mixture (200 cm³)

of diethyl ether and cyclohexane followed by three subsequent

extractions with ethyl acetate, diethyl ether and cyclohexane

(50 cm³each). The combined extracts were washed with brine

and dried over Na₂SO₄prior to column chromatography on silica

using a methanol–dichloromethane gradient (1 : 20 → 1 : 10) as

solvent system. Solvent evaporation provided the puriﬁed product

12 as a colourless amorphous solid (1.30 g, 63%). m_max(ﬁlm/cm⁻¹)

3372br, 2925s, 2856s, 1640w, 1590w, 1438s, 1350w, 1300w, 1182m,

1119s, 1037s, 996w, 750w, 722s, 696s, 542s; d_H(500 MHz, CDCl₃,

Me₄Si) 5.81 (1H, ddt, J 6.66, 10.16, 16.88; 22-H), 4.97 (2H, m,

23-H), 3.66 (18H, m, 2 × (3–11-H)), 3.58 (2H, m, 2 × 12-H), 3.51

(2H, t, J 5.12, 2 × 13-H), 3.43 (2H, t, J 6.81, 2 × 2-H), 2.86 (2H,

t, J 5.23, 2 × 1-H), 2.03 (2H, dd, J 6.7, 14.36; 2 × 21-H), 1.52

(4H, m, 2 × 14-H, 2 × 20-H), 1.28 (10 H, m, 2 × (15–19-H));

d_C(125 MHz, CDCl₃, Me₄Si) 139.00 (22-C), 113.96 (23-C), 71.34

(13-C), 70.45–69.88 (2–12-C), 33.62 (1-C), 29.51-28.74 (14–21-C),

25.91*; m/z (MALDI-TOF-MS) 456.5 (M⁺+ Na. C₂₃H₄₇NO₆+

Na requires 456.32), (ESI-MS) 434.3474 (M⁺+ H. C₂₃H₄₇NO₆+

H requires 434.3476).

AcSC₁₁EG₆OC(O)C CH (16). The alcohol 15²⁷(1.29 g,

≡

2.53 mmol) and propiolic acid (0.16 cm³, 2.53 mmol) were

dissolved in dry DCM (10 cm³) and cooled to −26 ^◦C. Then DCC

(547.6 mg, 2.65 mmol) and DMAP (30.9 mg, 0.25 mmol) were

added, the reaction mixture was warmed to room temperature and

stirred overnight. For workup the solvents were removed under

reduced pressure and the crude product was puriﬁed by column

chromatography using an ethyl acetate–cyclohexane gradient (2 :

1 → 4 : 1) providing the title compound as a yellow oil (1.04 g,

73%). m_max(ﬁlm/cm⁻¹) 3217m, 2925s, 2855s, 2113s, 1716s, 1692s,

1462m, 1352m, 1228s, 1111s, 956s, 756m, 628m; d_H(500 MHz,

CDCl₃, Me₄Si) 4.35 (2H, m, 2 × 4-H), 3.74 (2H, m, 2 × 5-H),

3.66–3.58 (20 H, m, 2 × (6–15-H)), 3.44 (2H, t, J 6.81, 2 × 16-H),

2.99 (1H, s, 1-H), 2.86 (2H, t, J 7.26, 2 × 26-H), 2.32 (3H, s, 3 ×

28-H), 1.56 (4H, m, 2 × 17-H, 2 × 25-H), 1.26 (14H, m, 2 ×

(18–24-H)); d_C(125 MHz, CDCl₃, Me₄Si) 196.05 (27-C), 152.58

(3-C), 75.28 (2-C), 74.45 (1-C), 71.46–69.95 (5–15-C), 68.47 (16-

C), 65.17 (4-C), 30.59 (28-C), 29.54 (26-C), 29.48–28.73 (17–25-C),

26.00*; m/z (MALDI-TOF-MS) 585.0 (M⁺+ Na. C₂₈H₅₀O₉S +

Na requires 585.3), (ESI-MS) 585.3063 (M⁺+ Na. C₂₈H₅₀O₉S +

Na requires 585.3068).

=

≡

H₂C CHC₉EG₆NHC(O)C CH (13). To a stirred solution of

DCC (740 mg, 3.59 mmol) in dry DCM (19.5 cm³), propiolic

acid (0.18 cm³, 2.92 mmol) was slowly added at 0 ^◦C. After

10 min stirring the amine 12 (1.267 g, 2.924 mmol), dissolved

in dry DCM (7 cm³), was added dropwise. The reaction mixture

was stirred for 1 h at 0 ^◦C, warmed to room temperature and

then stirred for another 2 h before the solvents were removed

in vacuo. The crude product was puriﬁed by chromatography on

silica gel (MeOH–DCM, 1 : 20) to yield the title compound as a

colourless amorphous solid (1.20 g, 70%). m_max(ﬁlm/cm⁻¹) 3324m,

2926s, 2850s, 2108m, 1226s, 1573w, 1467w, 1436w, 1346w, 1242m,

1116s, 962w, 893w, 843w, 640m, 542m, 417w; d_H(500 MHz, CDCl₃,

Me₄Si) 6.99 (1H, br, NH), 5.81(1H, ddt, J 6.69, 10.18, 16.94; 25-

H), 4.96 (2H, m, 2 × 26-H), 3.66 (22H, m, 2 × (4–14-H)), 3.50 (2H,

m, 2 × 15-H), 3.44 (2H, t, J 6.82, 2 × 16-H), 2.85 (1H, s, 1-H),

2.04 (2H, m, 2 × H-24), 1.57 (2H, m, 2 × 17-H), 1.37 (2H, m, 2 ×

23-H), 1.28 (10H, m, 2 × (18–22-H)); d_C(125 MHz, CDCl₃, Me₄Si)

152.23 (3-C), 139.19 (25-C), 114.07 (26-C), 73.21 (2-C), 71.51

9-(1-Dodecyl-1H-[1,2,3]triazol-4-yl)nonanic acid (18). Un-

decynic acid (17, 50.0 mg, 0.27 mmol) and 1-azidododecane

(60.9 mg, 0.29 mmol) were dissolved in acetonitrile (1.5 cm³),

the mixture was degassed and ﬂushed thoroughly with nitrogen

and cooled to 0 ^◦C before CuI (104.5 mg, 0.55 mmol) and 2,6-

lutidine (0.06 cm³, (0.55 mmol) were added. The clear solution

was warmed to room temperature and then stirred overnight. The

reaction was quenched by addition of dilute hydrochloric acid

(0.5 cm³), the phases were separated and the aqueous phase was

extracted three times with DCM (1 cm³). The combined organic

extracts were washed with brine, dried over Na₂SO₄, and then it

was ﬁltered and the ﬁltrate evaporated under reduced pressure to

provide a brownish crude product, which was puriﬁed by ﬂash

chromatography using a gradient of ethyl acetate and cyclohexane

2128 | Org. Biomol. Chem., 2008, 6, 2118–2132

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(2 : 1 → 4 : 1). The title triazole was obtained as a colourless

amorphous solid (29.6 mg, 50%). d_H(500 MHz, CDCl₃, Me₄Si)

7.25 (1H, s, 11-H), 4.30 (2H, t, J 7.29 Hz, 2 × 12-H), 2.70 (2H,

m≈t, 2 × 9-H), 2.34 (2H, t, J 7.50, 2 × 2-H), 1.88 (2H, m, 2 ×

13-H, 1.64 (4H, m, 2 × 3-H, 2 × 8-H), 1.38 and 1.25 (26H, m, 2 ×

(4–7-H, 14–22-H)), 0.88 (3H, t, J 6.98, 3 × 23-H); d_H(125 MHz,

CDCl₃, Me₄Si) 178.87 (1-C), 148.30 (10-C), 120.43 (11-C), 50.26

(12-C), 34.08 (2-C), 31.91 (21-C), 30.34-25.56 (4–9-C, 13–20-C),

24.71 (3-C), 22.69 (22-C), 14.12 (23-C); m/z (MALDI-TOF-MS)

394.3 (M⁺+ H. C₂₃H₄₃N₃O₂+ H requires 394.6).

AcSC₁₀C(O)NHCH₂-triazole-EG₄Tos (19) The alkyne 5 (30 mg,

0.10 mmol) and tosylated triethylene glycol azide (TosEG₄N₃,

34.3 mg, 0.10 mmol) were dissolved in MeOH (1 cm³), the solution

was degassed and rinsed with nitrogen prior to addition of CuI

(96.0 mg, 0.50 mmol) and DIPEA (0.09 cm³, 0.50 mmol). The

mixture was stirred at room temperature overnight, then the

suspension was ﬁltered through a 0.45 lm HPLC ﬁlter. Analytical

HPLC (250/4 LiChrosorp 7 lm C8, A = H₂O, B = MeCN,

0% B → 90% B, 60 min, 1 cm³min⁻¹, R_t= 26.49 min) proved

the high purity of product 19 (43.3 mg, 62%) without further

chromatography. d_H(500 MHz, CDCl₃, Me₄Si) 7.78 (2H, d, J

8.34, 2 × Ar-H), 7.73 (1H, s, 9-H), 7.35 (2H, d, J 8.10, 2 × Ar-H),

6.42 (1H, br s, NH), 4.51 (4H, m, 2 × 8-H, 2 × 11-H), 4.15 (2H,

m, 2 × 1-H), 3.87 (2H, dd, J 4.63 and 9.83, 2 × 7-H), 3.69 (2H, m,

2 × 2-H), 3.60 (8H, m, 2 × (3–6-H)), 2.86 (2H, dd, J 7.20, 14.56;

2 × 22-H), 2.45 (3H, s, 3 × Ar-CH₃), 2.32 (3H, s, 3 × 24-H), 2.19

(2H, m, 2 × 13-H), 1.56 (4H, m, 2 × 14-H, 2 × 21-H), 1.49 (12

H, m, 2 × (15–20-H)); d_C(125 MHz, CDCl₃, Me₄Si) 196.00 (23-

C), 173.13 (12-C), 144.83 (Ar-C), 144.45 (10-C), 132.82 (Ar-C),

129.89 (Ar-C), 127.86 (Ar-C), 123.20 (9-C), 70.66–68.60 (1–7-C),

54.35 (Ar-CH₃), 50.21 (8-C), 42.67 (11-C), 36.47 (13-C), 30.57 (24-

C), 29.39–28.69 (14–21-C), 25.54 (22-C); m/z (ESI-MS) 693.3025

(M⁺+ Na. C₃₁H₅₀N₄O₈S requires 693.2962).

2-H), 3.39 (2H, m≈t, J 5.23, 2 × 1-H), 3.38 (3H, s, 3 × 13-H);

d_C(125 MHz, CDCl₃, Me₄Si) 71.87 (12-C), 70.64–70.45 (3–11-C),

69.96 (2-C), 58.96 (13-C), 50.62 (1-C); m/z (ESI-MS) 344.1767

(M⁺+ Na. C₁₃H₂₇N₃O₆+ Na requires 344.1792).

AcSC₁₀C(O)NHCH₂-triazole-EG₆Me

(21). The

alkyne

thioate 5 (100.0 mg, 0.54 mmol) and the azide 20 (103.1 mg,

0.32 mmol) were dissolved in acetonitrile (2 cm³), the mixture

was degassed and rinsed with nitrogen twice. CuI (61.1 mg,

0.32 mmol) was added to the stirred colourless solution before

DIPEA (72.6 ll) was added slowly. The colour changed

immediately to yellow. The reaction mixture was stirred overnight

at room temperature prior to removal of the solvents under

reduced pressure. The crude product was puriﬁed by column

chromatography using a ethyl acetate–cyclohexane gradient (4 :

1 → 10 : 1). This procedure provided the title compound as a

yellow amorphous solid (0.17 g, 86%). For further evaluation of

the reaction conditions according to Table 1, 1 : 1 mixtures of

alkyne 5 and azide 20 (20.0 mg, 0.03 mmol each) were dissolved

in the solvents listed (1.5 cm³) and the resulting solution was

degassed and ﬂushed with nitrogen twice. Catalyst was added as

listed and the reaction mixture was stirred under the condition

provided in Table 1; workup was performed as described above.

m_max(ﬁlm/cm⁻¹) 3292w, 2919s, 2852m, 1694s, 1635s, 1548s, 1471s,

1421w, 1348s, 1287w, 1225w, 1110s, 962s, 848m, 775w, 714m,

664w, 631s, 419w; d_H(500 MHz, CDCl₃, Me₄Si) 7.76 (1H, s,

14-H), 6.63 (1H, m≈t, J 5.38, NH), 4.54 (2H, d, J 4.9, 13-H),

4.52 (2H, d, J 5.39, 16-H), 3.87 (2H, t, J 5.10, 2 × 12-H), 3.63

(18H, m, 2 × (3–10-H)), 3.54 (2H, m, 2 × 2-H), 3.37 (3H, s,

3 × 1-H), 2.86 (2H, t, J 7.36, 2 × 27-H), 2.32 (3H, s, CH₃), 2.19

(2H, m≈t, J 7.50, 2 × 18-H), 1.54 (4H, m, 2 × (19-H, 26-H),

1.25 (12H, m, 2 × (20–25-H)); d_C(125 MHz, CDCl₃, Me₄Si)

196.04 (28-C), 173.22 (17-C), 144.66 (15-C), 123.53 (14-C), 71.88

(2-C), 70.57–70.44 (3–12-C), 69.37 (3-C), 59.00 (1-C), 50.36

(13-C), 36.50 (16-C), 34.71 (18-C), 30.64 (29-C), 29.47–28.76

MeEG₆N₃(20). Hexaethylene glycol monomethyl ether

(19–27-C), 25.60 (28-C); m/z (MALDI-TOF-MS) 657.6 (M⁺

+

(1.85 g, 6.24 mmol) and DABCO (1.40 g, 12.49 mmol) were

K. C₁₉H₅₄N₄O₈S + K requires 657.4), m/z (ESI-MS) 641.3625

3

˚

dissolved in 7 cm ethyl acetate, and dried 4 A molecular sieves

(M⁺+ Na. C₁₉H₅₄N₄O₈S + Na requires 641.3555).

(100 mg) were added. The reaction mixture was cooled to 0 ^◦C

and tosyl chloride (1.79 g, 9.63 mmol) was added to the stirred

reaction mixture, which was then warmed to ambient temperature.

Stirring was maintained for 1.5 h, the suspension was then

ﬁltered and the ﬁltrate washed twice with 2 M hydrochloric

acid. After phase separation the aqueous layer was extracted

with ethyl acetate twice and the recombined organic fractions

were washed with brine and ﬁnally dried over sodium sulfate.

The tosylate was obtained as a colourless oil (2.25 g, 80%) and

used without further puriﬁcation. The spectroscopic data were ac-

cording to the literature.⁴⁰The intermediate MeEG₆Tos (1-tosy1-

1,4,7,10,13,16,19-heptaoxaicosane tosylate, 2.50 g, 5.00 mmol)

was dissolved in dry DMF (50 cm³) and sodium azide (2.03 g,

31.21 mmol) and tetrabutylammonium bromide (50 mg) were

added at room temperature. The stirred reaction mixture was

warmed to 90 ^◦C for 3.5 h prior to stopping the reaction by solvent

removal under reduced pressure. The crude product was puriﬁed

by column chromatography using a gradient of MeOH and DCM

(1 : 20 → 1 : 15) providing the product 20 as a colourless oil (1.16 g,

72%). m_max(ﬁlm/cm⁻¹) 3441br, 2875s, 2107s, 1721w, 1642w, 1454m,

1349m, 1300m, 1250m, 1199w, 1106s, 948m, 850w; d_H(500 MHz,

CDCl₃, Me₄Si) 3.66 (20H, m, 2 × (3–12-H)), 3.55 (2H, m, 2 ×

Propiolic acid tris(2-tert-butoxycarbonylethyl)methylamide (23).

Propargylic acid (100.0 mg, 1.42 mmol) was dissolved in abs. DMF

◦

(1 cm³) and cooled to 0 C. HATU (593.3 mg, 1.560 mmol) was

added and a solution of the amine 22²⁶(589.5 mg, 1.42 mmol)

in DMF (1.5 cm³) was added dropwise to the stirred reaction

mixture. Addition of DIPEA (0.371 cm³, 2.128 mmol) turned

the clear solution yellow. Stirring was maintained for 3 h at

0 ^◦C, followed by stirring at room temperature overnight. For the

workup the solvents were removed under reduced pressure and the

crude product was puriﬁed by column chromatography with ethyl

acetate–cyclohexane (1 : 1) as the eluent to yield the title alkyne as

a colourless oil (373 mg, 56%). d_H(300 MHz, CDCl₃, Me₄Si) 6.40

(1H, s, NH), 2.71 (1H, s, 7-H), 2.56 (6H, m, 6 × 2-H), 1.99 (6H,

m, 6 × 3-H), 1.44 (27H, m, 9 × CH₃); d_C(75 MHz, CDCl₃, Me₄Si)

172.62 (1-C), 151.16 (5-C), 80.86 (CCH₃), 77.80 (7-C), 71.63 (6-C),

58.79 (4-C), 29.82–29.67 (2-C, 3-C), 29.09 (CH₃); m/z (ESI-MS)

490.1831 (M⁺+ Na. C₂₅H₄₁NO₇+ Na requires 490.2775).

4-[Tris(2-tert-butoxycarbonylethyl)methylcarbamoyl]-1-[x-(11-

acetylthioundecyl)hexaethylene glycol]-1H-[1,2,3]triazole (25). In

degassed and nitrogen-saturated acetonitrile (1 cm³) were

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dissolved the azide 24 (20.0 mg, 0.037 mmol) and the alkyne

23 (26.2 mg, 0.056 mmol), and CuI (8.5 mg, 0.044 mmol) and

DIPEA (8.5 × 10⁻³cm) were added at room temperature. The

reaction mixture was stirred for 20 h and was then ﬁltered through

a 0.45 lm HPLC ﬁlter before the solvent was removed in vacuo.

Puriﬁcation was performed by preparative HPLC (A = water, B =

MeCN, 0% B, 10 min, 0% B → 70% B, 30 min, 70% → 100%

B, 30 min, 10 cm³min⁻¹, R_t= 65.1 min) to yield a colourless

lyophilisate (34.1 mg, 91%). d_H(500 MHz, CDCl₃, Me₄Si) 8.19

(1H, s, 7-H), 6.84 (1H, s, NH), 4.58 (2H, m≈t, 2 × 8-H), 3.88 (2H,

m≈t, 2 × 9-H), 3.65–3.63 (18H, m, 2 × (10–18-H)), 3.57 (2H, m,

2 × 19-H), 3.44 (2H, t, J 6.82, 2 × 20-H), 2.86 (2H, t, J 7.34, 2 ×

30-H), 2.32 (3H, s, 3 × 32-H), 2.26 (6H, m, 6 × 2-H), 2.08 (6H, m,

6 × 3-H), 1.56 (4H, m, 2 × 21-H, 2 × 29-H), 1.43 (27H, m, 9 ×

CH₃), 1.26 (14H, m, 2 × (22–28-H)); d_C(125 MHz, CDCl₃, Me₄Si)

196.03 (31-C), 172.33 (1-C), 159.33 (5-C), 143.55 (6-C), 126.32 (7-

C), 80.51 (CCH₃), 71.52–70.02 (9–19-C), 69.21 (7-C), 57.68 (4-C),

57.68 (8-C), 30.62 (20-C), 30.00 (2-C), 29.64 (3-C), 29.61–28.79

(21–30-C), 28.06 (CH₃); m/z (MALDI-TOF-MS) 1026.1 [M⁺+

Na. C₅₀H₉₀N₄O₁₄S + Na requires 1026.3).

162.67 (11-C), 143.87 (10-C), 140.12 (33-C), 127.78 (9-C), 114.70

(34-C), [101.78 + 101.70]‡ (1-C), [75.00 + 74.84]‡ (5-C), [72.44 +

72.41]‡ (3-C), 72.37 (24-C), [72.02 + 71.84]‡ (2-C), 71.37–71.26

(–CH₂OCH₂–), [68.51 + 68.34]‡ (4-C), [62.84 + 62.77]‡ (6-C),

[51.74 + 51.45]‡ (8-C), 40.03*, 34.86*, 30.66–30.09 (CH₂), 27.16*.

m/z (MALDI-TOF-MS) 733.5 (M⁺C₃₄H₆₂N₄O₁₃requires 734.9);

m/z (ESI-MS) 735.4383 (M⁺+ H. C₃₄H₆₂N₄O₁₃+ H requires

735.4386); m/z 757.4234 (M⁺+ Na. C₃₄H₆₂N₄O₁₃+ Na requires

757.4206).

ManC₂-triazole-C(O)NH-EG₆C₁₁SAc

(29). Alkene

28

(70.0 mg, 0.10 mmol) was dissolved in THF (1 cm³) and thioacetic

acid (34.0 ll, 0.48 mmol) and AIBN (20 mg, 0.12 mmol) were

added. The reaction mixture was stirred at room temperature and

then UV irradiation (k ≥ 295 nm) was started and maintained

for 5 h. For workup the solvents were removed under reduced

pressure and the residual pale yellow crude oil was puriﬁed by

ﬂash chromatography on silica using methanol–dichloromethane

(1 : 18) as the solvent system. The product was obtained as a

colourless oil (22.0 mg, 40%). d_H(500 MHz, CD₃OD) 8.29 (1H, s,

9-H), 4.64 (1H, m≈s, 1-H), 4.59 (2H, m, 2 × 8-H), 4.05 (1H, m,

7-H), 3.81 (2H, m, 6-H, 7-H), 3.74 (2H, m, 2 × 12-H), 3.69 (1H,

dd, J 2.43, 11.79, 2-H), 3.64 (2H, m, 3-H, 6-H), 3.54 (20H, m, 2 ×

(14–23-H)), 3.53 (2H, m, 4–5-H), 3.36 (2H, t, J 6.65, 2 × 13-H),

3.32 (2H, t, J 4.95, 2 × 24-H), 2.75 (2H, t, J 7.29, 2 × 34-H),

2.21 (3H, s, 2 × 36-H), 1.45 (4H, m, 2 × (24-H, 33-H), 1.20 (12H,

m, 12H, 2 × (26–32-H)); d_C(125 MHz, CD₃OD) 197.61 (35-C),

162.65 (11-C), 143.85 (10-C), 127.80 (9-C), [101.78 + 101.69]‡

(1-C), [75.03 + 74.88]‡ (5-C), [72.39 + 72.36]‡ (3-C), 71.84 (24-C),

[71.44 + 71.30]‡ (2-C), 71.06–70.60 (–CH₂OCH₂–), 68.31 (4-C),

62.78 (6-C), [51.71 + 51.42]‡ (8-C), 40.05*, 30.76–29.79 (CH₂),

27.19*. m/z (ESI-MS) 811.4307 (M⁺+ H. C₃₆H₆₆N₄O₁₄S + H

requires 811.4375).

4-[Tris(2-carboxyethyl)methylcarbamoyl]-1-[x-(11-acetylthioun-

decyl)hexaethylene glycol]-1H-[1,2,3]triazole (26). Into the

solution of triester 25 (34.0 mg, 0.034 mmol) in DCM (0.7 cm³)

triﬂuoric acid (0.7 cm³) was added at room temperature. The

mixture was stirred over 1 h and then the solvent was removed

under reduced pressure to yield a colourless oil (27.7 mg, 98%),

which was used without further puriﬁcation. m_max(ﬁlm/cm⁻¹)

3398br, 2926s, 2856s, 1723s, 1569m, 1511w, 1460m, 1416m,

1352m, 1301w, 1203w, 1180m, 1102s, 1027m, 952m, 628m; d_H

(300 MHz, CD₃OD) 8.46 (1H, s, 7-H), 4.67 (2H, m, 2 × 8-H),

3.95 (2H,m, 2 × 9-H), 3.66 (20H, m, 2 × (10-19-H)), 3.50 (2H, t,

J 6.61, 2 × 20-H), 2.90 (2H, t, J 7.20, 2 × 30-H), 2.39 (6H, m, 6 ×

2-H), 2.34 (3H, s, 3 × 32-H), 2.21 (6H, m, 6 × 3-H), 1.59 (4H, m,

2 × 21-H, 2 × 29-H), 1.34 (14H, m, 2 × (22-28)); d_C(75 MHz,

CD₃OD) 197.64 (31-C), 176.83 (1-C), 162.10 (5-C), 140.14 (6-C),

128.17 (7-C), 72.36–70.13 (9-19-C), 59.25 (4-C), 51.66 (8-C),

30.86-29.26 (2-C, 3-C, 21–30-C); m/z (ESI-MS) 857.4116 (M⁺+

Na. C₃₈H₆₆N₄O₁₄S + Na requires 857.4188).

N-Acidoacetyl-dansylcadaverine

(31). Azidoacetic

acid

(137 mg, 1.36 mmol) and DCC (280 mg, 1.36 mmol) HOBt

(183.1 mg, 1.36 mmol) were dissolved in abs. DCM (6 cm³) and

cooled to 0 ^◦C. To the stirred solution of dansylcadaverine (30,

500 mg, 1.49 mmol) in abs. DCM (4 cm³) was slowly added. The

reaction mixture was slowly warmed to room temperature over

10 min and stirring was maintained overnight. Distilled water

(10 cm³) was added and the aqueous phase was extracted with

DCM (7 cm³) four times. After drying (Na₂SO₄) the organic

solvents were removed under reduced pressure and the crude

material was puriﬁed by ﬂash chromatography using a ethyl

acetate–cyclohexane gradient (1 : 1 → 2 : 1). Product fractions

were detected using a standard UV lamp. The ﬂuorescent product

31 was obtained as viscous oil (523.0 mg, 92%). m_max(ﬁlm/cm⁻¹)

3310br, 2938s, 2863m, 2789w, 2106s, 1661s, 1575m, 1538m,

1455m, 1355w, 1314s, 1201w, 1160s, 1144s, 1074m, 912w, 792s,

732s, 683w, 626s, 571s; d_H(500 MHz, CDCl₃, Me₄Si) 8.55 (1H,

d, J 8.52, 8-H), 8.29 (1H, d, J 8.63, 6-H), 8.24 (1H, dd, J 1.15, J

7.27, 7-H), 7.55 (2H, m, 3–4-H), 7.45 (1H, m≈t, CONH), 7.20

(1H, d, J 7.50, 2-H), 6.27 (1H, br, SO₂NH), 3.97 (2H, s, 2 ×

16-H), 3.15 (2H, q, J 7.36, 2 × 27-H), 2.90 (8H, m, 3 × 9-H,

3 × 9^ꢀ-H, 2 × 10-H), 1.39 (4H, m, J 7.50, 2 × (11-H, 13-H)),

1.25 (2H, m, 2 × 12-H); d_C(125 MHz, CDCl₃, Me₄Si) 166.65

(15-C), 152.10 (1-C), 134.74 (5-C), 130.45 (8-C), 129.93 (4a-C),

129.68 (8a-C), 129.65 (6-C), 128.43 (3-C), 123.25 (7-C), 118.70

(4-C), 115.23 (2-C), 52.74 (16-C), 45.44 (9-C), 42.93 (10-C), 38.95

=

ManC₂-triazole-C(O)NHEG₆C₉C CH₂(28). Alkyne 13

(70.0 mg, 0.14 mmol) and azide 27 (35.9 mg, 0.14 mmol) were

dissolved in dried MeOH (0.5 cm³) and dried DMC (0.5 cm³),

and the solution was degassed. Copper(I) iodide (5.5 mg) was

then added and the reaction mixture was warmed to 45 ^◦C

and kept stirred at that temperature overnight. After that the

suspension was ﬁltered and the solvents were removed under

reduced pressure. The yellowish crude product was puriﬁed

by column chromatography on silica gel with methanol and

dichloromethane (1 : 20) as the solvent system. The alkene 28

was obtained as a pale yellow amorphous solid (77.5 mg, 73%).

d_H(500 MHz, CD₃OD) 8.44 (1H, s, 9-H), 5.84 (ddt, 1H, J 6.80,

10.18, 16.98, 33-H), 4.97 (2H, m, 2 × 34-H), 4.78 (1H, m≈s,

1-H), 4.73 (2H, m, 2 × 8-H), 4.19 (1H, m, 7-H), 3.95 (2H, m,

6-H, 7-H), 3.88 (2H, m, 2 × 12-H), 3.82 (1H, dd, J 2.30, 11.77,

2-H), 3.78 (2H, m, 3-H, 6-H), 3.68 (20H, m, 2 × (14–23-H)), 3.63

(2H, m, 4-H, 5-H), 3.51 (2H, t, J 6.70, 2 × 13-H), 3.46 (2H, t, J

4.95, 2 × 24-H), 2.01 (2H, m≈q, 2 × 32-H), 1.61 (2H, m, 2 ×

25-H), 1.35 (12H, m, 2 × (26–31-H)); d_C(125 MHz, CD₃OD)

2130 | Org. Biomol. Chem., 2008, 6, 2118–2132

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(14-C), 28.92 (11-C), 28.71 (13-C), 23.34 (12-C); m/z (ESI-MS)

441.1439 (M⁺+ Na. C₁₉H₂₆N₆O₃S + Na requires 441.1683); m/z

(ESI-MS) 418.17871 (M⁺. C₁₉H₂₆N₆O₃S requires 418.17855).

27.19 (13-C), 24.67 (12-C); m/z (ESI-MS) 1002.5463 (M⁺+ Na.

C₄₇H₇₇N₇O₁₁S₂+ Na requires 1002.5015).

Acknowledgements

Dansylcadaverine-NHC(O)CH₂-triazole-CH₂NHC(O)C₁₀SAc

(32). Alkyne 5 (60.0 mg, 0.18 mmol) and azide 31 (100 mg,

0.24 mmol) were dissolved in acetonitrile (1 cm³) and twice

degassed and ﬂushed with nitrogen. CuI (18 mg, 0.10 mmol) and

DIPEA (21 ll, 0.12 mmol) were subsequently added to the stirred

reaction mixture and it was heated to 45 ^◦C overnight before the

mixture was ﬁltered and concentrated in vacuo. The crude product

was puriﬁed by ﬂash chromatography (ethyl acetate–cyclohexane,

4 : 1; then MeOH–DCM, 1 : 20). Product fractions were detected

using a standard UV lamp. The ﬂuorescent product 32 was

obtained as viscous oil (112.8 mg, 84%). m_max(ﬁlm/cm⁻¹) 3297s,

3081w, 2921s, 2849s, 1692s, 1668s, 1634s, 1549s, 1446s, 1418m,

1354w, 1319m, 1261m, 1232m, 1201w, 1184w, 1143s, 1058m, 947w,

785s, 682m, 628s, 570s; d_H(500 MHz, CDCl₃, Me₄Si) 8.51 (1H, d,

J 8.47, 8-H), 8.30 (1H, d, J 8.63, 4-H), 8.18 (1H, d, J 7.26, 6-H),

7.90 (1H, s, 17-H), 7.49 (2H, m, 3-H, 7-H), 7.15 (1H, d, J 7.53,

2-H), 7.11 (1H, br, 15-CNH), 6.99 (1H, br, 20-CNH), 6.21 (1H,

m≈t, J 5.64, SO₂NH), 5.15 (2H, s, 2 × 16-H), 4.54 (2H, m≈t, J

5.43, 2 × 19-H), 3.13 (2H, m, 2 × 14-H), 2.87 (6H, s, 3 × 9-H),

2.84 (4H, m≈t, J 7.40, 2 × (10-H, 30-H)), 2.31 (3H, s, 32-CH₃),

2.13 (2H, m≈t, J 7.50, 2 × 21-H), 1.51 (6H, m, 2 × (11-H, 13-

H, 22-H)), 1.36 (4H, m, 2 × (12-H, 23-H)), 1.18 (12H, m, 2 ×

(24–29-H)); d_C(125 MHz, CDCl₃, Me₄Si) 196.13 (31-C), 173.95

(20-C), 165.47 (15-C), 151.87 (1-C), 145.46 (18-C), 134.81 (5-C),

130.28 (6-C), 129.83 (4a-C), 129.56 (8a-C), 129.27 (8-C), 128.33 (3-

C), 124.80 (17-C), 123.16 (7-C), 118.92 (4-C), 115.25 (2-C), 53.05

(16-C), 45.39 (9, 9^ꢀ-C), 42.73 (10-C), 39.23 (14-C), 36.41 (21-C),

34.73 (19-C), 30.63 (32-C), 29.67–29.12 (13-C, 24–28-C), 29.05

(30-C), 28.76 (23-C), 28.60 (29-C), 28.19 (13-C), 25.57 (22-C),

23.23 (12-C); m/z (ESI-MS) 738.3398 (M⁺+ Na. C₃₅H₅₃N₇O₅S₂+

Na requires 738.3443).

This work was partly supported by the DFG in the frame of the

collaborative network SFB 470 and by the Fonds of the German

Chemical Industry. We would like to thank Mrs Dipl.-Chem. Nina

Schwalb for her valuable preparative assistance in optimisation of

the cycloaddition in solution. We thank the Wacker Siltronic AG

for a generous gift of Si(100) wafers.

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A modular approach for the construction and modification of glyco-SAMs utilizing 1,3-dipolar cycloaddition

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