A generic building block for C- and N-terminal protein-labeling and protein-immobilization

Bioorganic & Medicinal Chemistry 14 (2006) 6288–6306

A generic building block for C- and N-terminal protein-labeling

and protein-immobilization

Anja Watzke,^a,bMarta Gutierrez-Rodriguez,^a,bMaja Ko¨hn,^a,bRon Wacker,^c

Hendrik Schroeder,^c,fRolf Breinbauer,^a,bJurgen Kuhlmann,^dKirill Alexandrov,^e

¨

Christof M. Niemeyer,^c,fRoger S. Goody^e,*and Herbert Waldmann^a,b,

*

^aMax-Planck-Institut fu¨r molekulare Physiologie, Abteilung Chemische Biologie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany

^bUniversita¨t Dortmund, Fachbereich Chemie, Chemische Biologie, Otto-Hahn-Strasse 6, 44227 Dortmund, Germany

^cChimera Biotec GmbH, Emil-Figge-Strasse 76a, 44227 Dortmund, Germany

^dMax-Planck-Institut fu¨r molekulare Physiologie, Abteilung Strukturelle Biologie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany

^eMax-Planck-Institut fu¨r molekulare Physiologie, Abteilung Physikalische Biochemie,

Otto-Hahn-Strasse 11, 44227 Dortmund, Germany

^fUniversita¨t Dortmund, Fachbereich Chemie, Biologisch-Chemische Mikrostrukturtechnik,

Otto-Hahn-Strasse 6, 44227 Dortmund, Germany

Received 8 March 2006; revised 2 May 2006; accepted 2 May 2006

Available online 24 May 2006

Abstract—Expressed protein ligation (EPL) and bioconjugation based on the maleimide group (MIC-conjugation) provide powerful

tools for protein modiﬁcation. In the light of the importance of site-selectively modiﬁed proteins for the study of protein function, a

ﬂexible method for the introduction of tags and reporter groups into the C-terminus of proteins employing EPL and MIC-conju-

gation was developed. We describe the solid-phase synthesis of a generic building block, equipped with ﬂuorescence markers or dif-

ferent functional groups. This generic building block allows for a ﬂexible incorporation of diﬀerent tags into proteins and was used

for the introduction of ﬂuorescence markers into the C-terminus of Rab and Ras GTPases by EPL or MIC-conjugation techniques.

In addition, a building block appropriately modiﬁed for the incorporation of an azide into proteins was synthesized. Azide-func-

tionalized Ras protein was immobilized on a phosphane-modiﬁed surface by means of Staudinger ligation providing a highly

chemoselective ligation method for the immobilization of proteins.

1. Introduction

in this semi-synthetic approach to yield functionalized

proteins.³In the light of the increasing interest in the

use of precisely modiﬁed proteins as probes for the study

of protein function, we have developed a ﬂexible and

general method for the synthesis of functional building

blocks to be used for protein-modiﬁcation.

Proteins equipped with moieties such as ﬂuorophores

are eﬃcient probes for the study of protein function.

In vitro ligation methods¹provide powerful tools for

the introduction of such small organic molecules into

proteins. Expressed protein ligation²(EPL) is a broadly

applicable method for the site-speciﬁc modiﬁcation of

proteins. Small organic molecules made accessible by

organic synthesis can be ligated by EPL to the C-termi-

nus of proteins. Most frequently, modiﬁed peptides cor-

responding to the C-terminus of proteins have been used

As proteins of interest Rab GTPases, which play an

important role in regulating vesicular transport

processes,⁴and Ras GTPases, which are membrane-

bound signal transducers occupying a central position

in the MAP-kinase signal transduction pathway,⁵were

chosen. Chemical labeling of Rab and Ras GTPases is

a invaluable tool for the elucidation of the biological

function of these small GTPases.

Keywords: Chemical biology; Protein modiﬁcation; Solid-phase syn-

thesis; Protein immobilization.

*

Corresponding authors. Tel.: +49 231 133 2400; fax: +49 231 133

2499; e-mail addresses: roger.goody@mpi-dortmund.mpg.de; herbert.

waldmann@mpi-dortmund.mpg.de

Because of the central role of Ras proteins in signal

transduction the identiﬁcation of Ras interaction

doi:10.1016/j.bmc.2006.05.006

A. Watzke et al. / Bioorg. Med. Chem. 14 (2006) 6288–6306

6289

partners or small molecules interfering with Ras-protein

interactions is of utmost importance.⁶For the study of

protein–protein interactions protein-immobilization is

a powerful technique, reﬂected by an increasing interest

in protein-microarray technology.⁷

required cysteine or a MIC-group and a second amino

group for the introduction of reporter groups. An addi-

tional carboxylic acid group may be further transformed

into a thioester which would allow an N-terminal

attachment to proteins containing an N-terminal cys-

teine by means of EPL. Such a functionalized building

block would provide the possibility for protein-modiﬁ-

cation at well-deﬁned sites.

For the application of protein arrays in proteomics

research^7–10site- and chemoselective immobilization of

proteins via regions of the macromolecules that are

not involved in interactions with other molecules is

preferable or even required. Recently, chemo- and

regioselective expressed protein ligation was employed

for this purpose.¹¹

We note that a suitably functionalized amino acid, for

example, lysine or glutamic acid equipped with three

orthogonally stable protecting groups, would be an

alternative to the aromatic building block developed

by us. Since the chemistry for the functionalization of

amino acids with appropriate protecting groups is well

established and since arene chemistry might oﬀer alter-

natives in terms of reactivity, stability and applicable

reaction types and conditions, we decided to explore

an aromatic building block instead of a more established

approach based on a trifunctional amino acid.

We describe the use of the generic building block for the

site-selective immobilization of Ras. Our approach to a

generic building block for protein-modiﬁcation and

immobilization includes the introduction of an azide

into the C-terminus of the Ras-protein by means of

EPL allowing for the immobilization of azide-modiﬁed

Ras on phosphane-functionalized surfaces by means of

Staudinger ligation. The Staudinger ligation has been

applied to protein immobilization by Raines and

co-workers¹²and Bertozzi and co-workers¹³by non-co-

valent binding of a protein to an immobilized peptide

sequence. The use of the Staudinger ligation in living

cells by Bertozzi and co-workers¹⁴and for the prepara-

tion of small molecule microarrays¹⁵demonstrates its

potential as powerful coupling strategy and its tolerance

of a diverse array of functionalities. Here, we report that

the Staudinger ligation provides a new chemoselective

method for immobilization of proteins equipped with

an azide to a phosphane-modiﬁed surface.¹⁶

3. Synthesis of building blocks used for protein-labeling

For the development of a ﬂexible synthesis of diﬀerently

modiﬁed core molecules we aimed at a solid-phase

synthesis. In order to establish a suitable protocol for

the synthesis of the desired building block, we decided

to explore ﬁrst a solution-phase strategy.

Starting from the commercially available dimethyl-5-

nitro-isophthalate 1 one methyl ester was reduced to the

corresponding alcohol 2 which was subsequently protect-

ed and converted to azide 4 (Scheme 2). The azide was re-

duced to amine 5, which was then coupled to an Mmt-

and Trt-protected cysteine derivative. The obtained inter-

mediate 6 could be further modiﬁed. Reduction of the

nitro group with palladium on charcoal under a hydrogen

atmosphere, followed by attachment of the ﬂuorescent 5-

N,N-(dimethylamino)-naphthalene-1-sulfonyl(dansyl)-

group, yielded ﬂuorescently labeled benzene derivative 8

with good to excellent yield.

2. Design of a triply substituted building block

A benzene derivative equipped with a triply orthogonal

set of functional groups was chosen as core structure. It

should contain a linker group for its introduction into

proteins, so that the remaining two functional groups

can be equipped with tags, labels or reporter groups of

interest. Consequently, the desired building block was

equipped with a cysteine or a maleimidocaproyl

(MIC)-group which is required for introduction into

proteins by expressed protein ligation or MIC-conjuga-

tion, respectively. Substituted benzene derivatives as

shown in Scheme 1 represent suitable building blocks

bearing one amino group for the attachment of the

For the development of a solid-phase synthesis of the

building block based on these reaction conditions the

choice of the linker for connecting the core molecule

to the solid phase had to be considered. To make this

approach more broadly applicable, cleavage of the

desired compounds from the resin should be possible

fluorescence-marker or

other functional group

FG

NH

cysteine or MIC-group for

C-terminal attachment

H

N

Linker

thioester for N-terminal attachment

C

O

RS

Scheme 1. Design of a triply orthogonally substituted benzene derivative as generic building block for protein labeling.

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A. Watzke et al. / Bioorg. Med. Chem. 14 (2006) 6288–6306

Scheme 2. Synthesis of ﬂuorescently labeled building block 8 in solution. Reagents and conditions: (a) 2.5 equiv DIBAL-H, THF, ꢀ70 ꢁC; (b)

1.5 equiv MsCl,10 equiv Et₃N, CH₂Cl₂, 30 min, 0 ꢁC; (c) 5 equiv NaN₃, DMF, 50 ꢁC, 6 h; (d) 6 equiv HS–(CH₂)₃–SH, 4 equiv Et₃N, MeOH, 12 h, rt;

(e) Trt-Cys(Mmt)-OH, 1.2 equiv HOBt, 1.1 equiv HBTU, 2 equiv Et₃N, CH₂Cl₂, 12 h, rt; (f) Pd/C, H₂, MeOH; (g) 1.5 equiv dansylCl, 0.5 equiv

DMAP, 3 equiv Py, CH₂Cl₂, 12 h, rt.

under very mild conditions giving the opportunity for

further modiﬁcation. These requirements are fulﬁlled by

the oxidation-sensitive hydrazide linker.¹⁷Therefore, 4-

Fmoc-hydrazinobenzoyl AM NovaGel (Novabiochem)

was employed for the synthesis, drawing from previous

experience on the synthesis of Ras- and Rab-peptides.⁴

This linker allows for release of the immobilized

compounds as carboxylic acids, methyl esters or amides

making this approach desireably ﬂexible.

was released from the resin by oxidation of the hydrazide

to the acyldiazene with copper acetate and oxygen fol-

lowed by treatment with methanol. After cleavage, the

copper salts could be removed by column chromatogra-

phy to give compound 11 in 55% overall yield.

After removal of the Fmoc-group from compound 11,

ﬂuorescently labeled compound 12 was ligated to trun-

cated Ypt1D3-thioester¹⁸(the yeast analogue of human

Rab7) by expressed protein ligation (Scheme 4). For

the ligation reaction thioester-tagged Ypt1D3 was mixed

with 5-fold molar excess of compound 12 and incubated

for 12 h at room temperature. The ligation reaction did

not depend on the presence of a detergent due to the

good solubility of the building block in the buﬀer

solution.

The solid-phase synthesis started with the immobiliza-

tion of the azide to the solid support (Scheme 3). The

4-Fmoc-hydrazinobenzoyl AM NovaGel resin was

deprotected by agitating it twice with a solution of

30% piperidine in DMF for 10 min. After saponiﬁcation

of the methyl ester building block, 4 was attached to the

resin using DIC/HOBt as coupling reagents in dichloro-

methane, yielding resin 9 after 12 h of agitation. The

introduction of the protected cysteine derivative was

achieved by activating the amino acid with DIC/HOBt

for 20 min and treating resin 9 with the activated cys-

teine in the presence of tributylphosphine for amide

bond formation. The resin was further treated with a

1 M solution of SnCl₂Æ2H₂O in DMF for the reduction

of the nitro group. Afterwards, the dansyl group was

attached to the resin by using dansyl chloride in the pres-

ence of DMAP and pyridine. The desired compound 11

The reaction mixtures were resolved on an SDS–PAGE

gel and the obtained gel was examined under UV light.

Figure 1A shows that a ﬂuorescent product could be

detected at the position corresponding to the semi-syn-

thetic Ypt1 protein. Figure 1B shows the mass spectrum

of the semi-synthetic and ﬂuorescently-labeled Ypt1-

protein.

Additionally, the solid-phase synthesis of a ﬂuorescently

labeled MIC-modiﬁed building block allowing chemical

A. Watzke et al. / Bioorg. Med. Chem. 14 (2006) 6288–6306

6291

Cys(StBu)-NHFmoc

NH

N₃

c, d

a, b

H

N

H

N

O₂N

CO₂Me

N

H

9

O₂N

4

N

O

10

H

O

e, f, g

S StBu

H

N

NH-Fmoc

O

CO₂Me

Dansyl-HN

11

overall yield: 55%

DIC =

N C N

Scheme 3. Synthesis of the ﬂuorescently labeled building block 11 on the solid phase. Reagents and conditions: (a) 1.5 equiv NaOH, H₂O/dioxane;

(b) 3 equiv DIC, HOBt, Et₃N, CH₂Cl₂, hydrazide resin, 12 h, rt; (c) 3 equiv Fmoc-Cys(S-t-Bu)-OH, DIC, HOBt, THF, 20 min, rt; (d) 3 equiv Bu₃P,

12 h, rt, (e) SnCl₂Æ2H₂O, DMF, 2 h, rt; (f) 3 equiv dansylCl, 3 equiv pyridine, 0.5 equiv DMAP, 2 h, rt; (g) 0.005 M Cu(OAc)₂, pyridine, MeOH,

CH₂Cl₂, O₂, 3 h, rt.

Scheme 4. Ligation of the ﬂuorescently labeled building blocks 11 after Fmoc-deprotection to Ypt1D3-thioester. Reagents and conditions: (a)

EtNH₂/CH₂Cl₂(1:4); (b) a—50 mM Na₂HPO₄/NaH₂PO₄, pH 7.5, 0.1 mM MgCl₂, 2 lM GDP, 150 mM 2-mercaptoethanesulfonic acid (MESNA),

12 h, rt.

labeling of proteins via MIC-conjugation and of a ﬂuo-

rescently labeled thioester allowing an N-terminal intro-

duction of a ﬂuorescence marker into the protein was

carried out. To this end, amine 4 was protected with

the Boc-group and after saponiﬁcation compound 14

was immobilized on the solid support (Scheme 5). The

immobilized building block 15 could be further modiﬁed

on the solid phase. After removal of the Boc group, the

amine was coupled to the dansyl group. The second ami-

no group was generated by reduction of the nitro group

and used for the introduction of the MIC-group. The

desired compound 17 was cleaved from the resin and

after puriﬁcation by column chromatography obtained

with a yield of 63%. The analogous solution-phase syn-

thesis of desired ﬂuorescent-labeled compound 17 pro-

vided a lower overall yield of 23%.

and ﬂexibility the solid-phase technique is superior. Thi-

oester 18 was generated in a similar way (Scheme 5).

Due to problems encountered using thiols as nucleo-

philes for the ﬁnal cleavage step we decided to apply a

strategy used for the synthesis of peptide thioesters on

the hydrazine support.¹⁹After oxidation of the hydra-

zide linker with NBS, glycine-S-ethyl-thioester was used

as a nucleophile for the ﬁnal cleavage of thioester 18

from the solid phase. The nitro group was reduced to

the amine before cleavage from the resin to increase

the polarity of the compound required for the ligation

reaction in ligation buﬀer. Ligation of 18 to Rab6A-

protein was achieved with a yield of ca. 10–20% of ﬂuo-

rescently labeled, semi-synthetic protein. Non-ligated

Rab6A-protein was found to be diﬃcult to seperate

from the ligation product.

Apart from the higher yield of the solid-phase synthesis,

a direct comparison of the solution phase and solid-

phase routes revealed that in terms of eﬃciency, speed

MIC-conjugation of 17 to truncated ^1–181N-Ras²⁰was

carried out successfully using only a very small excess

of compound 17 (Fig. 2A). The ligation was complete

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Figure 1. (A) SDS–PAGE gel of the ﬂuorescently labeled semi-synthetic ^1–203Ypt1-protein. The gel was photographed either in UV-light or visible

light after Coomassie blue staining. (B) Deconvoluted MS-spectrum of the semi-synthetic protein. Calculated mass: 23,454 Da [M+H]⁺.

Scheme 5. Synthesis of the ﬂuorescently labeled building block 17 for MIC-conjugation and thioester 18 for N-terminal EPL. Reagents and

conditions: (a) Boc₂O, Et₃N, THF, 0 ꢁC; (b) 1.5 equiv NaOH, H₂O/dioxane; (c) hydrazide resin, 3 equiv HOBt, DIC, Et₃N, CH₂Cl₂, 12 h, rt; (d) 50%

TFA/CH₂Cl₂; (e) 3 equiv dansyl-Cl, pyridine, 0.5 equiv DMAP, 3 h, rt; (f) 1 M SnCl₂Æ2H₂O, DMF, 2 h, rt; (g) NBS, pyridine; (h) 10 equiv H₂N-Gly-

SEt, Et₃N, DMF; (i) 2 equiv MIC, HOBt, HBTU, DIPEA, DMF, 12 h, rt; (k) 0.005 M Cu(OAc)₂, MeOH, pyridine, O₂.

A. Watzke et al. / Bioorg. Med. Chem. 14 (2006) 6288–6306

6293

Figure 2. (A) Ligation of the ﬂuorescently labeled derivative 17 with truncated ^1–181N-Ras. Reagents and conditions: (a) 50 mM Tris-buﬀer, pH 7.4,

0.1 mM NaCl, 2 lM GDP, 12 h, rt. (B) SDS–PAGE gel of the crude ligation mixture. (C) MALDI-TOF spectra of the puriﬁed semi-synthetic

protein. Calculated mass: 21,005 Da [M+H]⁺. The deviation of the observed molecular mass from the calculated values is within the error range of

the mass spectrometer. Matrix: sinapinic acid.

after 12 h and the semi-synthetic product was puriﬁed

by gel ﬁltration. Figure 2C shows the MALDI-TOF

spectrum of the ﬂuorescently labeled N-Ras protein.

lieved to primarily mediate protein-membrane interac-

tions and to be less important for Ras-eﬀector

interactionsthanotherregionsoftheprotein.^5cTherefore,

immobilization of Ras via the C-terminus is desirable.

4. Immobilization of N-Ras-protein

For visualization of proteins on the surface Cy5 was also

coupled to the building block as suitable ﬂuorescence

marker.

Encouraged by the successful ligation of the ﬂuorescently

labeled building block to the C-terminus of truncated

Rab- and Ras-proteins, we aimed at the synthesis of an

azide-tagged Ras-protein suitable for immobilization on

a phosphane-functionalized surface by means of Stau-

dinger ligation (Fig. 3). The hypervariable C-terminal

region of the signal-transducing Ras oncoproteins is be-

For immobilisation experiments, benzene derivative 15

was appropriately functionalized (Scheme 6). The mod-

iﬁcation of the building block with Cy5 was achieved on

the solid support. Due to its high polarity, Cy5 adhered

to the resin and could hardly be removed from the solid

Figure 3. Concept for immobilization of functionalized proteins on phosphine-modiﬁed glass-slides via Staudinger ligation.

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A. Watzke et al. / Bioorg. Med. Chem. 14 (2006) 6288–6306

Scheme 6. Synthesis of diﬀerent functionalized benzene derivatives on the solid phase. Reagents and conditions: (a) SnCl₂Æ2H₂O, DMF, 2 h, rt; (b)

3 equiv MIC, HOBt, DIC, 12 h, rt; (c) 50% TFA/CH₂Cl₂, 30 min; (d) 6 equiv Cy5, HOBt, HBTU, Et₃N, 12 h, rt; (e) NBS, pyridine; (f) MeOH/

CH₂Cl₂; (g) 3 equiv N₃–(CH₂)₅–CO₂H, HOBt, DIC, DIPEA, CH₂Cl₂, 12 h, rt; (h) 3 equiv Fmoc-Cys(S-t-Bu)-OH, 3 equiv HOBT, HBTU, DIPEA,

4 h, rt; (i) H₂O/THF; (m) 4 equiv Cy5-Et-NH₂, 4 equiv HOBt, HBTU, DIPEA, 12 h, rt; (j) 4 equiv Trt-Cys(Mmt)-OH, 4 equiv HOBT, HBTU,

DIPEA, 4 h, rt; (k) 4 equiv H₂N–(CH₂)₆–N₃, THF; (l) 4 equiv Cy5-Et-NH₂, 4 equiv HOBt, HBTU, DIPEA, 12 h, rt; (m) 3 equiv Cy5, 8 equiv HOBt,

HBTU, 4 equiv DIPEA; (n) 10% piperidine/DMF, 5 min.

support. To solubilize the ﬂuorophore, methanol had to

be used as solvent which, however, leads to insuﬃcient

swelling of the resin. Therefore, compound 19 was

obtained with only 5% yield. To overcome this problem,

we decided to synthesize triply substituted building

block 21. To this end, azide- and cysteine-functionalized

carboxylic acid 20 was cleaved from the solid support by

using a mixture of water and THF. Cy5 could then be

coupled to 20 as amide to yield the desired triply func-

tionalized compound 21, which was puriﬁed by prepara-

tive HPLC. The amino derivative of Cy5 employed in

this sequence was synthesized as shown in Scheme 6.

Ethylenediamine derivative 23 was deprotected and then

coupled to Cy5 yielding 24 in moderate yield. In the ﬁnal

step the Fmoc-group was removed under basic condi-

tions, which are in general tolerated by Cy5.

A. Watzke et al. / Bioorg. Med. Chem. 14 (2006) 6288–6306

6295

Figure 4. (A) Ligation of 19 to truncated ^1–181N-Ras-protein. Reagents and conditions: (a) 50 mM Tris-buﬀer, pH 7.4, 0.1 mM NaCl, 2 lM GDP,

12 h, rt. (B) Deconvoluted MS-spectrum of the semi-synthetic protein (calculated mass: 21,411 Da [M+H]⁺) and SDS–PAGE gel of the puriﬁed semi-

synthetic protein. The gel was photographed either in UV-light or visible light after Coomassie blue staining.

The Cy5-labeled compound 19 was ligated to truncated

^1–181N-Ras by MIC-conjugation (Fig. 4A). Only a slight

excess of compound 19 was required for a successful

ligation reaction yielding the semi-synthetic and Cy5-

labeled Ras-protein quantitatively. Figure 4B shows

the mass spectrum of the labeled N-Ras-protein after

puriﬁcation and the SDS–PAGE gel of the puriﬁed liga-

tion product.

shows the LC/MS trace of the semi-synthetic N-Ras. It

is important to note that the ligation reaction with

azide-carrying building block is eﬃcient in the presence

of excess thiol, that is, the azide is not reduced to the

amine under these conditions.

The reduction of alkyl azides by mono- and dithiols has

been investigated before. Thus, the monothiol mercap-

toethanol reduces alkyl azides much slower than, for

example, 1,4-dithiothreitol.²⁰Accordingly, azide 4 is eﬃ-

ciently reduced by propane-1,3-dithiol (Scheme 4)

whereas the monothiol mercaptoethanesulfonic acid

does not reduce the alkyl azide under the conditions

described above.

The azide-modiﬁed building block 22 was generated on

solid phase by using one amino group of the building

block 15 for the introduction of the cysteine, whereas

the desired azide was introduced in the ﬁnal cleavage

step (Scheme 6). To avoid the formation of undesired

complexes formed by copper-salts and azides, NBS

was used to oxidize the hydrazide linker. By proper

selection of the cleavage nucleophile, a further function-

alization could be achieved. For synthesis of compound

22, 6-azido-hexylamine was used as nucleophile. The

cleavage proceeded smoothly, although in contrast to

methanol, the excess of amine could not be removed

by straightforward evaporation or extraction. However,

simple column chromatography suﬃced to purify the

desired compound 22.

For the immobilization of the azide-functionalized Ras-

protein glass-slides initially modiﬁed with an intermedi-

ate layer of generation 4 PAMAM dendrimers were

chosen as carriers to achieve maximum surface cover-

age.¹⁵For covalent attachment of the protein by means

of Staudinger ligation the surface of the slides had to be

decorated with an appropriately functionalized phos-

phine.^14aThis phosphane has been used for in vivo stud-

ies applying the Staudinger ligation.^14cPhosphane

groups on surfaces in general are prone to oxidation,

the phosphine used here was developed by Bertozzi

et al. to limit air oxidation.²¹For the spotting experi-

ments, the azide-modiﬁed protein (positive control;

PC) as well as the non-modiﬁed Ras-protein as a nega-

tive control (NC) were dissolved in phosphate buﬀer at

200–100 lM by means of a piezo-driven spotting

robot.²²Subsequent to the spotting process the slides

were washed with buﬀer and blocked with bovine serum

To introduce an azide into the C-terminus of N-Ras,

intermediate 22 was deprotected and afterwards ligated

to truncated ^1–180N-Ras-thioester by expressed protein

ligation (Fig. 5A). After incubation of the thioester-

tagged N-Ras-protein with the azide-modiﬁed building

block for 12 h at room temperature, the obtained

semi-synthetic N-Ras-protein was puriﬁed by gel ﬁltra-

tion using DEAE-column chromatography. Figure 5B

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A. Watzke et al. / Bioorg. Med. Chem. 14 (2006) 6288–6306

Figure 5. (A) Ligation of compound 22 after removal of all protection groups to ^1–180N-Ras-thioester. (B) Deconvoluted LC–MS spectra of the

puriﬁed semi-synthetic protein. Calculated mass: 20,747 Da [M+H]⁺.

albumin (BSA)-containing blocking solution to prevent

non-speciﬁc binding of protein reagents. The slides were

then treated with Cy5-labeled Ras-antibody (50 nM, 1 h

at room temperature). The ﬂuorescent signals were

recorded and quantiﬁed after removal of excess reagent.

This preliminary result for the immobilization of

Figure 6. Preliminary results of immobilization of azide-functionalized N-Ras-protein (2, 4 and 6) as positive control (PC) and non-modiﬁed N-Ras-

protein (1, 3 and 5) as negative control (NC) with various concentrations.

Figure 7. Inﬂuence of diﬀerent buﬀers on the immobilization of azide-modiﬁed N-Ras-protein (PC). Reagents and conditions: Buﬀer 1: 20 mM Tris/

Cl, 5 mM MgCl₂, 150 mM NaCl, pH 7.4 + 0.05% Tween 20. Buﬀer 2: 20 mM Tris, 5 mM MgCl₂, pH 7.4 + 0.05% Tween 20. Buﬀer 3: 10 mM Hepes,

150 mM NaCl, 5 mM MgCl₂, pH 7.4 + 0.05% Tween 20. Buﬀer 4: 20 mM Tris, 5 mM MgCl₂, 2 mM DTE, pH 7.6 + 0.05% Tween 20. Buﬀer 5:

50 mM Na₂HPO₄, 2 mM NaCl, 2 mM MgCl₂, pH 7.5 + 0.05% Tween 20. NC: non-modiﬁed N-Ras-protein.

A. Watzke et al. / Bioorg. Med. Chem. 14 (2006) 6288–6306

6297

Figure 8. Time course and concentration dependence of immobilization by Staudinger ligation.

azide-modiﬁed Ras-protein is shown in Figure 6. The

immobilization of azide-modiﬁed protein (PC) gave

clear ﬂuorescent signals, whereas no signiﬁcant signals

were obtained at any concentration for the negative con-

trol (NC). Encouraged by these ﬁndings we aimed at the

study of the inﬂuence of diﬀerent buﬀers, reaction time

and concentration dependence of immobilization of

azide-modiﬁed Ras-protein by Staudinger Ligation.

The inﬂuence of diﬀerent buﬀers on the immobilization

of azide-modiﬁed N-Ras-protein (PC) is shown in

Figure 7. No signiﬁcant diﬀerences were observed

between buﬀer 1, 2, 3 and 5. Buﬀer 4 gave inferior

results, that is, the use of DTE leads to a decrease of

immobilized protein. The immobilization was carried

out at pH 7.4–7.6 due to the pH-dependent stability of

the Ras-protein. We note, however, that the Staudinger

ligation proceeds well over a relatively wide pH

range (5–8.5).²³

ily modiﬁed on the solid phase in a controlled way and

was successfully applied to the introduction of ﬂuores-

cence markers into Rab and Ras GTPases.

Additionally, the building block was used for the cova-

lent attachment of an azide into the C-terminus of the

Ras-protein yielding azide-functionalized Ras, which

could be further immobilized on phosphane-modiﬁed

glass-slides via highly chemoselective Staudinger liga-

tion. This new strategy for the site-speciﬁc immobiliza-

tion of proteins onto a glass surface may be used for

the creation of protein microarrays.

6. Experimental

6.1. General

Chemicals were obtained from Acros, Advanced Chem-

tech, Aldrich, Biosolve, Fluka or Novabiochem and

used without further puriﬁcation. Analytical chroma-

tography was performed on Merck silica gel 60F₂₅₄alu-

minium sheets and Merck aluminium oxide 60 F₂₅₄

aluminium sheets. Chromatography was performed

using Merck Silica gel 60 and Fluka aluminium oxide.

Dichloromethane, triethylamine, diisopropylethylamine

and piperidine were reﬂuxed under argon over CaH₂,

THF over Na/K, methanol over magnesium and freshly

The time course and concentration dependence of immo-

bilization by Staudinger ligation was studied using diﬀer-

ent concentrations of azide-modiﬁed N-Ras-protein (PC)

and non-modiﬁed N-Ras-protein (NC) in phosphate buﬀ-

er (Buﬀer 5: 50 mM Na₂HPO₄, 2 mM NaCl, 2 mM

MgCl₂, pH 7.5 + 0.05% Tween 20) for 1–16 h as shown

in Figure 8. After incubation with Cy5-labeled Ras-anti-

body (50 nM, 1 h), the ﬂuorescence signals were recorded

and quantiﬁed. The minimum concentration for accept-

able levels of immobilization was found to be ca.

50 lM. An immobilization time of 4 h was suﬃcient for

obtaining clear and reproducible signals.

1

distilled prior to usage. H and ¹³C NMR data were

recorded on a Bruker DRX 500 spectrometer at room

temperature. NMR spectra were calibrated to the sol-

vent signals of CDCl₃(7.26 and 77.00 ppm). ESI-MS

was performed on a Agilent 1100 series binary pump

together with a reversed-phase HPLC column (Mache-

rey-Nagel) and a Finnigan Thermoquest LCQ. Prepara-

tive HPLC was preformed on Agilent 1100 series with a

VP125/21 Nucleosil 120-7C4 column (Macherey-Nagel)

run at a ﬂowrate of 20.0 mL/min, eluant was monitored

at 215 nm. FAB measurements were taken with a Jeol

SX 102A spectrometer using 3-nitrobenzyl alcohol

(3-NBA) as matrix. MALDI-TOF spectra were recorded

with a Voyager-DE Pro BioSpectrometer from PerSep-

tive Biosystems using 2,5-dihydroxybenzoic acid

(DHB) as matrix. Optical rotations were measured using

a Schmidt+Haensch Polartronic HH8. Yield and scale

These results demonstrate that the immobilization of the

azide-modiﬁed Ras-protein onto the phosphane-modi-

ﬁed glass-slides was successful.

5. Conclusion

In summary, a generic building block for protein modi-

ﬁcation was developed. It provides a ﬂexible and gener-

ally applicable tool for the introduction of diﬀerent

functional groups of interest (e.g., ﬂuorescent groups)

into proteins. The benzene derivative equipped with a

triply orthogonal set of functional groups could be eas-

6298

A. Watzke et al. / Bioorg. Med. Chem. 14 (2006) 6288–6306

of the solid-phase reactions are given with respect to the

Fmoc loading as supplied by Novabiochem. All reac-

tions were carried out under an argon atmosphere in

dry solvents unless otherwise noted.

ture. The resin was ﬁltered oﬀ and the solvent was evap-

orated under reduced pressure.

Method B. The resin was treated with a solution of 0.02 M

NBS (5 equiv), and 0.02 M pyridine (5 equiv) in dichloro-

methane for 45 min. The resin was ﬁltered oﬀ and washed

ﬁve times with dichloromethane. Cleavage was achieved

by agitating the resin with a solution of methanol/dichlo-

romethane (1:100) for 2 h. The resin was ﬁltered oﬀ and

the solvent was evaporated under reduced pressure.

6.2. General procedures for the solid-phase synthesis of

building blocks using the phenylhydrazide linker

For all building blocks commercially available Fmoc-4-

hydrazinobenzoyl AM NovaGel resin from Novabio-

chem was used, (Fmoc loading of 0.56 mM/g). All

reactions were carried out under an argon atmosphere

in polyethylene syringe reactors equipped with a fritted

disc. Agitation was achieved using an orbital shaker.

6.3. 3-Hydroxymethyl-5-nitro-benzoic acid methyl ester (2)

To a solution of 7 g (0.029 mmol) 5-nitro-isophthalic

acid dimethyl ester (1) in 200 mL THF, 2.5 equiv

DIBAL-H (72.5 mL of 1 M-solution in toluene) was add-

ed slowly under an argon-atmosphere at ꢀ78 ꢁC. The

reaction mixture was allowed to warm to room tempera-

ture and 50 mL methanol were added. The solvent was

evaporated under reduced pressure and the remaining

residue was dissolved in ethyl acetate. The organic phase

was washed once with each 1 N HCl and a saturated solu-

tion of NaCl and was dried over Na₂SO₄. The solvent was

evaporated under reduced pressure and the crude product

was puriﬁed by column chromatography (silica gel, cyclo-

hexane/ethyl acetate 9:1, 7:3 (v/v)) yielding 2 as an oﬀ-

white solid (3.06 g, 1.45 mmol, 50%).

6.2.1. Loading of the resin (general procedure GP1).

Swelling of the Fmoc-4-hydrazinobenzoyl NovaGel res-

in was carried out in dichloromethane. For Fmoc cleav-

age the resin was treated with a degassed solution of

35% piperidine in DMF for 7 min twice. The resin was

subsequently washed six times with DMF and three

times with dichloromethane. Afterwards 3 equiv of the

acid to be coupled to the resin was preactivated for

5 min with 3 equiv HOBt, 3 equiv DIC and 3 equiv of

Et₃N in dichloromethane. The solution was added to

the resin and the mixture was agitated for 12 h at room

temperature. The resin was subsequently washed six

times with dichloromethane and three times with DMF.

TLC: 0.36 (cyclohexane/ethyl acetate 1:1 (v/v));

mp = 75–76 ꢁC.

6.2.2. Cleavage of the Boc-group (GP2). The Boc-pro-

tecting group was cleaved by agitating the resin once

for 5 min with a solution of 50% TFA in dichlorometh-

ane. The resin was subsequently washed ﬁve times with

dichloromethane and ﬁve times with DMF.

¹H NMR (500 MHz, CDCl₃): d = 8.74 (s, 1H, arom

CH-4), 8.43 (s, 1H, arom CH-6), 8.34 (s, 1H, arom

CH-2), 4.88 (s, 2H, CH₂OH), 3.88 (s, 3H, CO₂CH₃),

2.14 (s, 1H, OH).

6.2.3. Reduction of the nitro group (GP3). The resin was

treated for 2 h at room temperature with a solution of

1M SnCl₂Æ2H₂O in DMF and subsequently washed ﬁve

times with dichloromethane, DMF and ethyl acetate.

¹³C NMR (125 MHz, CDCl₃): d = 164.99 (CO₂CH₃),

148.60 (C-5), 143.60 (C-3), 133.05 (C-2), 131.88 (C-1),

125.24 (C-4), 123.44 (C-6), 63.49 (CH₂OH), 52.83

(CO₂CH₃).

6.2.4. Couplings (GP4). Method A. Amino acids were

coupled using HBTU/HOBt as activating reagents.

Three equivalents of amino acid was preactivated for

5 min with 3 equiv HBTU, 3 equiv HOBt and 3 equiv

i-PrNEt₂in dichloromethane. The solution was added

to the resin and agitated for 4 h at room temperature.

In order to suppress racemisation, cysteine derivatives

were coupled using DIC/HOBt for activation. The resin

was subsequently washed six times with dichlorometh-

ane and three times with DMF.

HR-FAB-MS (m/z): calcd for [C₉H₁₀NO₅]⁺: 212.0559

[M+H]⁺, found: 212.0576.

6.4. 3-Methanesulfonyloxymethyl-5-nitro-benzoic acid

methyl ester (3)

To a solution of 1.50 g (7.11 mmol) 3-hydroxymethyl-5-

nitro-benzoic acid methyl ester (2) in 20 mL dichloro-

methane 10 equiv Et₃N (9.85 mL, 71.1 mmol) and

2 equiv mesyl chloride (1.10 mL, 14.22 mmol) was add-

ed slowly under argon at 0 ꢁC. The reaction mixture

was stirred for 15 min at 0 ꢁC and subsequently treated

with 10 mL of a saturated solution of NaCl. The organic

phase was washed with 1 N KHSO₄and a sat. solution

of Na₂CO₃and dried over Na₂SO₄. The solvent was

evaporated under reduced pressure and the crude prod-

uct was puriﬁed by column chromatography (silica gel,

cyclohexane/ethyl acetate 9:1, 8:2 (v/v)) yielding 3 as a

pale yellow solid (1.95 g, 6.75 mmol, 95%).

Method B. A solution of 3 equiv dansylchloride, 3 equiv

pyridine and 0.5 equiv DMAP in DMF was added to the

resin, and the mixture was left standing for 3 h at room

temperature. The resin was afterwards subsequently

washed six times with dichloromethane and three times

with DMF.

6.2.5. Cleavage from the solid support (GP5). Method A.

The resin was treated with a solution of Cu(OAc)₂

(5.4 mg, 29 lmol), pyridine (48 lL, 588 lmol) and meth-

anol (240 lL, 5.9 mmol) in dichloromethane (7 mL)

under an oxygen atmosphere for 3 h at room tempera-

TLC: 0.32 (cyclohexane/ethyl acetate 1:1 (v/v));

mp = 82 ꢁC.

A. Watzke et al. / Bioorg. Med. Chem. 14 (2006) 6288–6306

6299

¹H NMR (500 MHz, CDCl₃): d = 8.84 (s, 1H, arom

CH-6), 8.44 (s, 1H, arom CH-4), 8.37 (s, 1H, arom

CH-2), 5.33 (s, 2H, CH₂O), 3.98 (s, 3H, CO₂CH₃),

3.08 (s, 3H, O₂SCH₃).

¹³C NMR (125 MHz, CDCl₃): d = 164.97 (CO₂CH₃),

148.31 (C-5), 144.60 (C-3), 133.80 (C-2), 131.72 (C-1),

125.87 (C-4), 122.88 (C-6), 52.55 (CH₂NH₂), 44.57

(CO₂CH₃).

¹³C NMR (125 MHz, CDCl₃): d = 164.35 (CO₂CH₃),

136.38 (C-3), 134.61 (C-2), 132.59 (C-1), 126.84 (C-4),

125.01 (C-6), 68.17 (CH₂O), 53.04 (CO₂CH₃), 38.19

(O₂SCH₃).

HR-FAB-MS (m/z): calcd for [C₉H₁₁N₂O₄]⁺: 311.0719

[M+H]⁺, found: 211.0730 (in 3-NBA-Matrix).

6.7. 3-{Amino-[^L-cysteine-(S- monomethoxytrityl)-N-tri-

tyl]-methyl}-5-nitro-benzoic acid methyl ester (6)

HR-FAB-MS (m/z): calcd for [C₁₀H₁₁NO₇SNa]⁺:

312.0154 [M+Na]⁺, found: 312.0142.

To a solution of 1.57 g (2.54 mmol) Trt-Cys(Mmt)-OH in

5 mL dichloromethane 389 mg (2.54 mmol) HOBt and

320 lL (2.54 mmol) DIC were added. Activation of the

amino acid to the active ester was controlled by TLC.

Afterwards, a solution of 500 mg (2.12 mmol) 3-azido-

methyl-5-nitro-benzoic acid methyl ester (5) in 1 mL

dichloromethane was added to the reaction mixture.

The reaction mixture was stirred for 12 h at room temper-

ature. The solvent was evaporated under reduced pressure

and the crude product was puriﬁed by column chroma-

tography (silica gel, cyclohexane/ethyl acetate 8:2, 7:3

(v/v)) yielding 6 as a white solid (1.67 g, 2.01 mmol, 95%).

6.5. 3-Azidomethyl-5-nitro-benzoic acid methyl ester (4)

To a solution of 1.30 g (4.50 mmol) 3-methanesulfonyl-

oxymethyl-5-nitro-benzoic acid methyl ester (3) in

20 mL DMF 5 equiv of sodium azide (1.46 g, 22.49 mmol)

were added. The reaction mixture was stirred for 2 h at

50 ꢁC. The solvent was evaporated under reduced pres-

sure and the remaining residue was dissolved in ethyl ace-

tate. The organic phase was washed once with water and a

saturated solution of NaCl and was dried over Na₂SO₄.

The solvent was evaporated under reduced pressure and

the crude product was puriﬁed by column chromatogra-

phy (silica gel, cyclohexane/ethyl acetate 9:1, 7:3 (v/v))

yielding 4 as a pale yellow solid (1.06 g, 4.5 mmol, quant.).

TLC: 0.5 (cyclohexane/ethyl acetate 7:3 (v/v)); mp = 118–

20

D

119 ꢁC; ½aꢁ +55.7 (c 1.0, CHCl₃).

¹H NMR (500 MHz, CDCl₃): d = 8.83 (s, 1H, arom CH-

6), 8.39 (s, 1H, arom CH-4), 8.33 (s, 1H, arom CH-2),

7.98 (m, 1H, NH), 7.45–7.51 (m, 6H, 6· arom CH Trt,

Mmt), 7.36–7.21 (m, 21H, 21· arom CH Trt, Mmt),

6.81 (d, J = 5.0 Hz, 2H, 2· arom CH Mmt), 4.57

(s, 2H, CH₂NH), 4.01 (s, 3H, CO₂CH₃), 3.90 (m, 1H,

a-CH), 3.78 (s, 3H, OCH₃Mmt), 2.81–2.85 (m, 2H,

b-CH₂), 2.65 (m, 1H, NH).

TLC: 0.83 (cyclohexane/ethyl acetate 1:1 (v/v));

mp = 69–72 ꢁC.

¹H NMR (500 MHz, CDCl₃): d = 8.75 (s, 1H, arom

CH-6), 8.35 (s, 1H, arom CH-4), 8.29 (s, 1H, arom

CH-2), 4.57 (s, 2H, CH₂N₃), 3.97 (s, 3H, CO₂CH₃).

¹³C NMR (125 MHz, CDCl₃): d = 164.47 (CO₂CH₃),

148.47 (C-5), 138.38 (C-3), 134.23 (C-2), 132.31 (C-1),

126.37 (C-4), 124.00 (C-6), 53.25 (CH₂N₃), 52.83

(CO₂CH₃).

¹³C NMR (125 MHz, CDCl₃): d = 173.75 (CONH), 158.16,

148.43, 144.95, 144.59, 141.22, 136.41, 134.34, 131.76,

130.94, 129.49, 129.44, 128.83, 128.70, 128.01, 127.99,

127.92, 127.81, 127.78, 126.93, 126.77, 126.43, 123.3 (22·

arom CH), 113.23 (2· arom CH Mmt), 71.44 (quart C

Trt), 66.75 (quart C Mmt), 56.24 (OCH₃Mmt), 55.12

(a-CH), 52.75 (CO₂CH₃), 42.26 (CH₂NH), 36.79 (b-CH₂).

FAB/LR-MS (m/z): calcd for [C₉H₈NO₄]⁺: 194.05

[MꢀN₃]⁺, found: 194.00.

6.6. 3-Aminomethyl-5-nitro-benzoic acid methyl ester (5)

ESI-MS+ m/z: calcd for C₅₁H₄₅N₃O₆SNa [M+Na]⁺:

850.3, found: 850.4.

To a solution of 200 mg (0.85 mmol) 3-azidomethyl-

5-nitro-benzoic acid methyl ester (4) in 5 mL metha-

nol 4 equiv Et₃N (470 lL, 3.39 mmol) and 6 equiv

of propanedithiol (511 lL, 5.08 mmol) were added

under an argon atmosphere. The reaction mixture

was stirred for 12 h at room temperature. The sol-

vent was evaporated under reduced pressure and

the crude product was puriﬁed by column chroma-

tography (silica gel, dichloromethane/methanol 10:1

(v/v)) yielding 5 as a pale yellow solid (151.7 mg,

0.723 mmol, 85%).

MALDI-TOF (DHB) calcd for C₅₁H₄₅N₃O₆SNa

[M+Na]⁺: 850.3, found: 850.4.

FAB/LR-MS (m/z): calcd 828.30 [M+H]⁺, found: 828.00.

6.8. 3-{Amino-[^L-cysteine-(S-monomethoxytrityl)-N-tri-

tyl]-methyl}-5-amino-benzoic acid methyl ester (7)

To a solution of 50 mg (0.06 mmol) 3-{amino-[^L-cys-

teine-(SMmt)-N-trityl]-methyl}-5-nitro-benzoic

acid

TLC: 0.3 (dichloromethane/methanol 10:1 (v/v));

mp = 84 ꢁC.

methyl ester (6) in 3 mL methanol 10 mg palladium on

charcoal was added. The reaction mixture was stirred

for 4 h under an hydrogen atmosphere at room temper-

ature. The catalyst was ﬁltered oﬀ and the solvent was

evaporated under reduced pressure. The crude product

was puriﬁed by column chromatography (silica, cyclo-

¹H NMR (500 MHz, CDCl₃): d = 8.61 (s, 1H, arom

CH-6), 8.30 (s, 1H, arom CH-4), 8.21 (s, 1H, arom

CH-2), 3.91 (s, 2H, CH₂NH₂), 3.86 (s, 3H, CO₂CH₃).

6300

A. Watzke et al. / Bioorg. Med. Chem. 14 (2006) 6288–6306

hexane/ethyl acetate 1:1 (v/v)) yielding 7 as a white solid

(47.8 mg, 0.06 mmol, quant.).

124.49, 123.94, 122.76, 119.69, 118.17, 115.02 (26· arom

CH), 112.94 (2· arom CH Mmt), 69.63 (quart C Trt),

66.13 (quart C Mmt), 56.12 (OCH₃Mmt), 54.85 (a-CH),

51.94 (CO₂CH₃), 45.07 (CH₂NH), 42.47 (N(CH₃)₂), 36.45

(b-CH₂).

TLC: 0.44 (cyclohexane/ethyl acetate 1:1 (v/v));

20

D

mp = 124–126 ꢁC; ½aꢁ +18.0 (c 1.0, CHCl₃).

¹H NMR (500 MHz, CDCl₃): d = 7.95 (s, 1H, arom CH),

7.42–7.35 (m, 5H, 5· arom CH), 7.27–7.24 (m, 3H, 3· arom

CH), 7.23–7.10 (m, 20H, 20· arom CH), 7.03 (s, 1H, arom

CH), 6.65 (d, ³J = 10.8 Hz, 2H, 2· arom CH Mmt), 4.00–

3.98 (dd, J₁= 15.0 Hz, J₂= 7.1 Hz, 2H, CH₂NH), 3.82

(s, 3H, CO₂CH₃), 3.68 (s, 3H, OCH₃Mmt), 3.66 (m, 1H,

a-CH), 3.08 (m, 1H, b-CH_2a), 2.99 (m, 1H, b-CH_2b), 2.86

(br s, 2H, NH₂), 2.41–2.36 (m, 1H, NH).

ESI-MS+ m/z: calcd for C₆₃H₅₈N₄O₆S₂[M]: 1030.3,

found: 850.4 [MꢀMmt+2Na+H].

MALDI-TOF (DHB) calcd for C₆₃H₅₈N₄O₆S₂[M]:

1030.3, found: 811.31 [MꢀMmt+Na+H].

HR-FAB-MS (m/z): calcd: 1031.3876 [M+H]⁺, found:

1031.3936.

3

¹³C NMR (125 MHz, CDCl₃): d = 173.24 (CONH), 158.15,

145.31, 144.92, 144.87, 136.58, 130.95, 129.49, 129.46,

128.86, 127.98, 127.92, 127.86, 126.93, 126.78, 126.74 (15·

arom CH), 113.44 (2· arom CH Mmt), 71.67 (quart C

Trt), 66.58 (quart C Mmt), 56.79 (OCH₃Mmt), 55.27

(a-CH), 52.29 (CO₂CH₃), 43.21 (CH₂NH), 36.97 (b-CH₂).

6.10. 3-{Amino-[^L-cysteine-(S-tert-butyl)-N-ﬂuorenyl-

methoxy-carbonyl]-methyl}-5-(5-dimethylamino-naphtha-

lene-1-sulfonyl-amino)-benzoic acid methyl ester (11)

The loaded resin (9) (50 mg, 0.05 mmol) was agitated in

2 mL dichloromethane. To a solution of 126.2 mg

(0.2 mmol) Fmoc-Cys(S-t-Bu)-OH in 2 mL THF 30.6 mg

(0.2 mmol) HOBt and 25.2 lL DIC (0.2 mmol) were added.

Activation of the amino acid was controlled by TLC. The

reaction mixture was stirred for 10 min and then added to

the resin. To the resin 6 equiv Bu₃P (74 lL, 0.3 mmol) was

added subsequently. After 12 h the resin was ﬁltered oﬀ

and washed ﬁve times with dichloromethane and ﬁve times

with DMF. The reduction of the nitro group was carried out

according to GP3. Afterwards, the resin was treated with

27 mg (0.1 mmol) dansyl chloride, 3 mg (2.5 · 10^ꢀ5mol)

DMAP and 5 lL (0.06 mmol) pyridine as described in

GP4 (method B). The cleavage from the resin was carried

out according to GP5 (method A). The crude product was

puriﬁed by column chromatography (silica gel, cyclohex-

ane/ethyl acetate 6:4 (v/v)) yielding 11 as a yellow solid

(22.7 mg, 0.027 mmol, 55%).

MALDI-TOF (DHB) calcd for C₅₁H₄₇N₃O₄SNa

[M+Na]⁺: 820.3, found: 820.6; calcd for C₅₁H₄₇N₃O₄SK

[M+K]⁺: 836.3, found: 836.6.

FAB/LR-MS (m/z): calcd: 798.33 [M+H]⁺, found:

799.15.

6.9. 3-{Amino-[^L-cysteine-(S-monomethoxytrityl)-N-tri-

tyl]-methyl}-5-(5-dimethylamino-naphthalen-1-sulfonyla-

mino)-benzoic acid methylester (8)

To a solution of 770 mg (0.966 mmol) 3-{amino-[^L-cys-

teine-(SMmt)-N-trityl]-methyl}-5-amino-benzoic acid

methyl ester (7) in 3 mL dichloromethane 1.5 equiv dan-

syl chloride (7.6 mg, 0.028 mmol), 3 equiv pyridine

(4.46 mg, 0.056 mmol) and 0.5 equiv dimethylaminopyri-

dine (1.7 mg, 0.015 mmol) were added. The reaction mix-

ture was stirred for 4 h at room temperature. The solvent

was evaporated under reduced pressure and the crude

product was puriﬁed by column chromatography (silica,

cyclohexane/ethyl acetate 8:2, 1:1 (v/v)) yielding 8 as a

pale yellow solid (786.0 mg, 0. 763 mmol, 79%).

TLC: 0.64 (cyclohexane/ethyl acetate 1:1 (v/v));

20

D

mp = 112–113 ꢁC; ½aꢁ ꢀ85.1 (c 1.0, CHCl₃).

3

¹H NMR (500 MHz, CDCl₃): d = 8.46 (d, J = 8.5 Hz,

1H, H-2 dansyl), 8.20 (d, J = 8.8 Hz, 1H, H-4 dansyl),

3

7.75–7.72 (m, 4H, H-8 dansyl, H-1, H-8 Fmoc, arom

CH-4), 7.58–7.50 (m, 4H, H-4, H-5 Fmoc, H-3 dansyl,

arom CH-6), 7.40–7.26 (m, H, H-6, H-7 dansyl, H-2,

H-3, H-6, H-7 Fmoc), 7.12 (s, 1H, arom CH-2), 4.42–

4.18 (m, 5H, H-9, H-10 Fmoc, CH₂NH), 4.01 (t,

³J = 8.8 Hz, 1H, a-CH); 3.78 (s, 3H, OCH₃), 3.15–3.03

(m, 8H, b-CH₂Cys, N(CH₃)₃), 1.33 (s, 9H, S-t-Bu).

TLC: 0.58 (cyclohexane/ethyl acetate 1:1 (v/v));

20

D

mp = 120–121 ꢁC; ½aꢁ ꢀ46.1 (c 1.0, CHCl₃).

3

¹H NMR (500 MHz, CDCl₃): d = 8.46 (d, J = 8.5 Hz,

1H, H-2 dansyl), 8.28 (d, J = 9.2 Hz, 1H, H-4 dansyl),

3

8.20 (d, ³J = 6.8 Hz, 1H, H-8 dansyl), 7.55–7.44 (m, 4H,

4· arom CH), 7.40–7.35 (m, 7H, 7· arom CH), 7.26–

ESI-MS+ m/z: calcd for C₄₃H₄₇N₄O₅S₃[M+H]⁺: 827.2,

found: 827.5, calcd for C₄₃H₄₆N₄O₅S₃Na [M+Na]⁺:

859.2, found: 859.5.

3

7.04 (m, 22H, 22· arom CH), 6.65 (d, J = 8.0 Hz, 2H,

2· arom CH Mmt), 6.30 (br s, 1H, NH), 3.88–3.92 (dd,

3

³J₁= 14.9 Hz, J₂= 6.0 Hz, 2H, CH₂NH), 3.72 (s, 3H,

CO₂CH₃), 3.66 (s, 3H, OCH₃Mmt), 3.64 (m, 1H, a-

CH), 3.12–3.05 (m, 2H, b-CH₂), 2.84 (s, 6H, N(CH₃)₂),

2.32 (dd, ³J₁= 12.3 Hz, ³J₂= 6.3 Hz, 1H, NH).

HR-FAB-MS (m/z): calcd: 827.2606 [M+H]⁺, found:

827.2552.

6.11. 3-[Amino-(tert-butyloxycarbonyl)-methyl]-5-nitro-

benzoic acid methyl ester (13)

¹³C NMR (125 MHz, CDCl₃): d = 172.98 (CONH), 165.85

(CO₂CH₃), 151.72, 144.89, 144.50, 139.42, 137.10, 136.27,

133.87, 130.98, 130.66, 130.61, 129.93, 129.54, 129.25,

129.16, 128.55, 128.36, 127.70, 127.55, 126.51, 126.69,

To a solution of 340 mg (1.61 mmol) 3-aminomethyl-5-

nitro-benzoic acid methyl ester (4) in 15 mL THF a solu-

A. Watzke et al. / Bioorg. Med. Chem. 14 (2006) 6288–6306

6301

tion of 2 equiv Et₃N (446 lL, 3.22 mmol) and 1.5 equiv

Boc₂O (457 mg, 2.09 mmol) in 5 mL THF was added at

0 ꢁC. The reaction mixture was stirred for 12 h and the

solvent was removed under reduced pressure. The crude

product was puriﬁed by column chromatography (silica,

cyclohexane/ethyl acetate 7:3 (v/v)) yielding 13 as a col-

ourless oil (499 mg, 1.61 mmol, quant.).

with 61.3 mg (0.294 mol) maleimidocaproic acid, 91 mg

(0.588 mol) HOBt, 336 mg (0.882 mol) HBTU and

52 lL (0.294 mol) i-PrNEt₂according to GP4 (method

A). Cleavage from the resin was carried out according

to GP5 (method A). The crude product was puriﬁed

by column chromatography (silica gel, cyclohexane/eth-

yl acetate 1:1, 4:6 (v/v)) yielding 17 as a colourless oil

(49 mg, 0.081 mol, 63%).

TLC: 0.67 (cyclohexane/ethyl acetate 1:1 (v/v)).

TLC: 0.22 (cyclohexane/ethyl acetate 1:1 (v/v)).

¹H NMR (500 MHz, CDCl₃): d = 8.73 (s, 1H, arom CH-

6), 8.32 (s, 1H, arom CH-4), 8.27 (s, 1H, arom CH-2),

5.13 (br s, 1H, NH), 4.45 (s, 2H, CH₂NH), 3.97 (s,

3H, CO₂CH₃), 1.46 (s, 9H, O-t-Bu).

¹H NMR (500 MHz, CDCl₃): d = 8.40 (d, ³J =

10.5 Hz, 1H, H-2 dansyl), 8.22 (d, ³J = 10.8 Hz, 1H,

H-4 dansyl), 8.13 (d, ³J = 9.1 Hz, 1H, H-8 dansyl),

7.80 (s, 1H, arom CH-6), 7.53 (s, 1H, arom CH-4),

7.45 (t, ³J = 9.5 Hz, 1H, H-3 dansyl), 7.40 (t, ³J =

10.3 Hz, 1H, H-7 dansyl), 7.30 (s, 1H, arom CH-2),

7.09 (d, ³J = 10.0 Hz, 1H, H-6 dansyl), 6.62 (s, 2H,

CH@CH Mic), 5.27 (s, 1H, NH), 4.00 (s, 2H,

¹³C NMR (125 MHz, CDCl₃): d = 164.87 (CO₂CH₃),

148.55 (C-5), 142.18 (C-3), 133.77 (C-2), 132.03 (C-1),

125.81 (C-4), 123.33 (C-6), 52.78 (CO₂CH₃), 43.67

(CH₂NH), 28.29 (O-t-Bu).

3

CH₂NH), 3.76 (s, 3H, CO₂CH₃), 3.44 (t, J = 5.0 Hz,

HR-FAB/MS (m/z): calcd for [C₁₄H₁₈N₂O₆]⁺: 311.1243

2H, e-CH₂), 2.82 (s, 6H, N(CH₃)₂), 2.25 (t,

³J = 9.5 Hz, 2H, a-CH₂), 1.64 (m, 2H, b-CH₂), 1.55

(m, 2H, d-CH₂), 1.28 (m, 2H, c-CH₂).

[M+H]⁺, found: 311.1241.

6.12. 3-[Amino-(tert-butyloxycarbonyl)-methyl]-5-nitro-

benzoic acid (14)

¹³C NMR (125 MHz, CDCl₃): d = 172.02 (CONH),

170.91 (CO₂CH₃), 166.55 (OCNCO), 157.70, 138.39,

137.61, 134.88 (4· arom CH), 133.97 (C@C), 130.15,

130.01, 129.47, 129.39, 129.34, 128.01, 123.88, 123.49,

123.12, 119.77, 119.66, 115.09 (12· arom CH), 51.98

(CO₂CH₃), 46.37 (CH₂NH), 45.26 (N(CH₃)₂), 41.57

(e-CH₂), 37.40 (a-CH₂), 28.50 (d-CH₂), 26.07 (b-CH₂),

24.71 (c-CH₂).

To a solution of 140 mg (0.45 mmol) 3-[amino-(tert-butyl-

oxycarbonyl)-methyl]-5-nitro-benzoic acid methyl ester

(13) in 2 mL water/dioxane (1:1) 1.5 equiv NaOH (675 lL

of a 1 N solution, 0.675 mmol) was added over 1 h. The

pH of the solution was adjusted to pH 2 with 1 N HCl.

The reaction mixture was extracted three times with ethyl

acetate. The combined organic phases were dried over

Na₂SO₄. The solvent was removed under reduced pressure

yielding pure 14 (133 mg, 0.45 mmol, quant.) as a colourless

oil.

ESI-MS+ m/z: calcd for C₁₃H₃₅N₄O₇S [M+H]⁺: 607.2,

found: 607.5.

MALDI-TOF (DHB) calcd for C₁₃H₃₄N₄O₇SNa

[M+Na]⁺: 629.2, found: 629.6.

TLC: 0.2 (cyclohexane/ethyl acetate 1:1 (v/v)).

¹H NMR (500 MHz, CDCl₃): d = 8.56 (s, 1H, arom CH-

6), 8.25 (s, 1H, arom CH-4), 8.20 (s, 1H, arom CH-2),

4.29 (s, 2H, CH₂NH), 3.21 (s, 3H, CO₂H), 1.37 (s, 9H,

O-t-Bu).

FAB/LR-MS (m/z): calcd: 607.21 [M+H]⁺, found: 607.84.

6.14. 3-[Amino-(5-dimethylaminonaphthalene-1-sulfonyl)-

methyl]-5-amino-benzoic acid-(glycine-S-ethylthio ester)

amide (18)

¹³C NMR (125 MHz, CDCl₃): d = 164.18 (CO₂CH₃),

148.68 (C-5), 143.18 (C-3), 133.71 (C-2), 132.82 (C-1),

125.46 (C-4), 122.37 (C-6), 43.05 (CH₂NH), 27.61 (O-t-Bu).

The loading of 50 mg Fmoc-4-hydrazinobenzoyl resin

(0.56 mmol/g, 0.05 mmol) with compound 14 was car-

ried out according to GP1. Removal of the Boc-group

was carried out according to GP2. The resin was treated

with 40 mg (0.15 mmol) dansyl chloride, 3.4 mg

(0.03 mmol) DMAP and 7.5 lL (0.15 mmol) pyridine

according to GP4 (method B). The reduction of the ni-

tro group was carried out according to GP3. The cleav-

age from the resin was carried out according to GP5

(method B). In the ﬁnal cleavage step the resin was treat-

ed with a solution of 10 equiv glycine ethyl thioester

(0.5 mmol, 250 mg) and 10 equiv i-PrNEt₂(0.5 mmol,

86 lL) in 2 mL DMF for 3 h. The resin was ﬁltered oﬀ

and the solvent was removed under reduced pressure.

The crude product was puriﬁed by column chromatog-

raphy (silica gel, dichloromethane/methanol 50:1, 20:1

(v/v)) yielding 18 as a yellow solid (10.8 mg, 0.021 mol,

43%).

HR-FAB/MS (m/z): calcd for [C₁₃H₁₆N₂O₅Na]⁺:

319.0906 [M+Na]⁺, found: 319.0901.

6.13. 3-[Amino-(5-dimethylaminonaphthalene-1-sulfonyl)-

methyl]-5- (maleimidocaproyl-amino)-benzoic acid methyl

ester (17)

The loading of 150 mg Fmoc-4-hydrazinobenzoyl resin

(0.56 mmol/g, 0.147 mmol) with compound 14 was car-

ried out according to GP1. Removal of the Boc-group

was carried out according to GP2. The resin was treated

with 94 mg (0.348 mmol) dansyl chloride, 10.4 mg

(0.085 mmol) DMAP and 17.4 lL (0.348 mmol) pyri-

dine as described in GP4 (method B). The nitro group

was reduced according to GP3 and the resin was treated

6302

A. Watzke et al. / Bioorg. Med. Chem. 14 (2006) 6288–6306

TLC: 0.50 (dichloromethane/methanol 20:1 (v/v)).

¹H NMR

6.16. 3-{Amino-[^L-cysteine-(S-tert-butyl)-N-ﬂuorenyl-

methoxy-carbonyl]-methyl}-5-(6-azido-hexoyl-amino)-

benzoic acid (20)

(500 MHz, CDCl₃): d = 8.43

(d,

³J = 10.3 Hz, 1H, H-2 dansyl), 8.21 (d, ³J = 11.5 Hz,

1H, H-4 dansyl), 8.14 (d, ³J = 9.0 Hz, 1H, H-8 dan-

syl), 7.48 (t, ³J = 10.0 Hz, 1H, H-3, dansyl), 7.43 (t,

³J = 10.5 Hz, 1H, H-3, dansyl), 7.10 (d, ³J = 8.8 Hz,

1H, H-6 dansyl), 6.80 (s, 2H, arom CH-2), 6.72 (s,

2H, arom CH-6), 6.41 (s, 1H, arom CH-4), 6.30 (br

s, 1H, NH), 4.17 (br s, 1H, NH), 3.91-3.87 (m, 2H,

CH₂NH), 3.62–3.61 (m, 2H, CH₂Gly), 3.41 (s, 2H,

NH₂), 3.09–3.08 (m, 2H, CH₂CH₃), 2.81 (s, 6H,

N(CH₃)₂), 1.18 (s, 3H, CH₂CH₃).

Resin 15 (50 mg, 0.05 mmol) was agitated in 2 mL

dichloromethane. The reduction of the nitro group

was carried out according to GP3. According to GP4

(method A) a solution of 21.3 mg (0.15 mmol) 6-azido-

hexanoic acid, 22.9 mg (0.15 mmol) HOBt, 56.3 mg

(0.15 mmol) HBTU and 20.2 lL (0.15 mmol) Et₃N in

2 mL dichloromethane was added to the resin. Removal

of the Boc-protecting group was carried out according

to GP2. According to PG4 (method A) the resin was

treated with 126.2 mg (0.2 mmol) Fmoc-Cys(S-t-Bu)-

OH, 30.6 mg (0.2 mmol) HOBt, 74.4 mg (0.2 mmol)

HBTU and 34.5 lL (0.2 mmol) i-PrNEt₂in 2 mL dichlo-

romethane. The cleavage from the resin was carried out

according to GP5 (method B). In the ﬁnal cleavage step

the resin was treated with 100 lL H₂O in 900 lL THF

for 3 h. The resin was ﬁltered oﬀ and the solvent was re-

moved under reduced pressure. The crude product was

puriﬁed by column chromatography (silica gel, dichloro-

methane/methanol 50:1, 20:1 (v/v)) yielding 20 as a white

solid (8.9 mg, 0.021 mol, 25%).

ESI-MS+ m/z: calcd for C₂₄H₂₈N₄O₄S₂[M+H]⁺: 501.1,

found: 501.4.

HR-FAB-MS (m/z): calcd: 501.1630 [M+H]⁺, found:

501.1644.

6.15. 3-(Cy5-aminomethyl)-5-(maleimidocaproyl-amino)-

benzoic acid methyl ester (19)

Resin 15 (150 mg, 0.147 mmol) was agitated in 3 mL

dichloromethane. Reduction of the nitro group was car-

ried out according to GP3. The resin was afterwards

treated with 93.0 mg (0.441 mmol) maleimidocaproic

acid (MIC), 68.3 mg (0.441 mmol) HOBt and 69.1 lL

(0.441 mmol) DIC according to PG4 (method A).

Removal of the Boc-group was carried out according

to GP2. Afterwards, the resin was treated with

576.8 mg (0.882 mmol) Cy5, 134.9 mg (0.882 mmol)

HOBt, 330.8 mg (0.882 mmol) HBTU and 132.0 lL

(0.882 mmol) Et₃N in 2 mL DMF for 12 h. Cleavage

from the resin was carried out according to GP5 (meth-

od B). The crude product was puriﬁed by HPLC (linear

gradient of water (solvent A) and CH₃CN (solvent B);

20–100 B% 15 min) yielding 19 as a blue solid (7.4 mg,

7.35 · 10^ꢀ5mol, 5%).

TLC: 0.30 (dichloromethane/methanol 20:1 (v/v)); mp:

20

D

92–93 ꢁC; ½aꢁ ꢀ54.9 (c 1.0, CHCl₃).

¹H NMR (500 MHz, CDCl₃): d = 7.91 (br s, 1H, NH),

7.73 (d, ³J = 9.6 Hz, 2H, H-1, H-8 Fmoc), 7.69–7.63

(m, 3H, H-4, H-5 Fmoc, arom CH-6), 7.52 (m, 2H, H-

2, H-7 Fmoc), 7.39–7.30 (m, 4H, H-3, H-6 Fmoc, 2·

arom CH-4, CH-2), 4.49 (m, 1H, H-9 Fmoc), 4.39–

4.29 (m, 4H, H-10 Fmoc, CH₂NH), 4.12 (m, 1H, a-

3

CH Cys), 3.17 (t, J = 8.0 Hz, 2H, CH₂N₃), 3.11–3.00

(m, 2H, b-CH₂Cys), 2.26 (m, 2H, a-CH₂), 1.61 (m,

2H, b-CH₂), 1.51 (t, ³J = 8.8 Hz, 2H, d-CH₂), 1.33–

1.28 (m, 2H, c-CH₂), 1.24 (s, 9H, S-t-Bu).

¹³C NMR (125 MHz, CDCl₃): d = 172.29 (CONH),

143.54, 141.08, 140.71, 128.16, 127.58, 126.96, 126.51,

126.01, 124.96, 119.78, 117.12, 110.78 (12· arom C),

66.34 (OCH₂Fmoc), 51.07 (a-CH Cys), 48.09

(CH₂NH), 46.98 (CH₂N₃), 43.45 (b-CH₂Cys), 42.64

(CH Fmoc), 36.71 (a-CH₂), 29.61 (S-t-Bu), 28.41(d-

CH₂), 26.15 (c-CH₂), 24.82 (b-CH₂).

¹H NMR (500 MHz, CDCl₃): d = 8.32–8.26 (m, 2H,

arom. CH Cy5), 8.05 (s, 1H, arom CH-6), 7.98–7.84

(m, 3H, H-4, 2· arom CH Cy5), 7.67 (s, 1H, arom

CH-2), 7.34–7.29 (dd, ³J₁= 10.3 Hz, ³J₂= 6.4 Hz,

2H, 2· arom CH Cy5), 6.75 (s, 2H, HC@CH Mic),

6.67–6.60 (m, 1H, C@CHCy5), 6.34–6.27 (t,

³J = 16.6 Hz, 2H, HC@CHCy5), 4.38 (s, 2H,

CH₂NH), 4.14–4.12 (m, 4H, e-CH₂, N⁺@C–CH@,

C_quart@CH Cy5), 3.86 (s, 3H, CO₂CH₃), 3.47 (t,

³J = 8.3 Hz, 2H, e-CH₂Mic), 2.35 (t, ³J = 8.5 Hz,

2H, a-CH₂Cy5), 2.29 (t, ³J = 8.2 Hz, 2H, a-CH₂

Mic), 1.85–1.60 (m, 6H, b-CH₂, d-CH₂Mic, N–

CH₂–CH₃Cy5), 1.70 (s, 12H, 4· CH₃Cy5), 1.60 (t,

³J = 8.5 Hz, 2H, d-CH₂Cy5), 1.50 (m, 2H, c-CH₂

Cy5), 1.37 (t, ³J = 9.0 Hz, 2H, c-CH₂Mic), 1.33–

1.28 (m, 3H, NCH₂–CH₃Cy5).

ESI-MS+ m/z: calcd for C₃₆H₄₃N₆O₆S₂[M+H]⁺: 719.3,

found: 719.2, calcd for C₃₆H₄₂N₆O₆S₂Na [M+Na]⁺:

741.3, found: 741.3.

HR-FAB-MS (m/z): calcd: 719.2675 [M+H]⁺, found:

719.2669.

6.17. 3-{Amino-[^L-cysteine-(S-tert-butyl)-N-ﬂuorenyl-

methoxy-carbonyl]-methyl}-5-(6-azido-hexoyl-amino)-

benzoic acid-(Cy5-ethyl) amide (21)

ESI-MS+ m/z: calcd for C₅₂H₆₂N₅O₁₂S⁺[M]⁺1012.4,

found: 1012.4.

To a solution of 8.2 mg (1.15 · 10^ꢀ5mol) 3-{amino-[L-

cysteine-(S-tert-butyl)-N-ﬂuorenylmethoxycarbonyl]-

methyl}-5-(6-azido-hexoyl-amino)-benzoic acid (25) in

1 mL DMF a solution of 8 mg (1.15 · 10^ꢀ5mol) Cy5-

FAB/LR-MS (m/z): calcd: 1013.38 [M+H]⁺, found:

1013.52.

A. Watzke et al. / Bioorg. Med. Chem. 14 (2006) 6288–6306

6303

ethylamine (29), 7.0 mg (4.58 · 10^ꢀ5mol) HOBt,

17.4 mg HBTU and

s, 1H, NH), 4.14–4.11 (m, 2H, CH₂NH), 3.79–3.68 (m,

1H, a-CH Cys), 3.66 (s, 3H, OCH₃), 3.34–3.22 (m, 6H,

CH₂N₃, b-CH₂Cys, CH₂NH), 2.33–2.22 (m, 2H,

CH₂), 1.55 (t, ³J = 9.2 Hz, 2H, CH₂), 1.33–1.26 (m,

4H, 2· CH₂).

(4.58 · 10^ꢀ5mol)

4 lL

(2.29 · 10^ꢀ5mol) i-PrNEt₂was added. The reaction

mixture was stirred for 12 h at room temperature. the

solvent was removed under reduced pressure and the

crude product was puriﬁed by HPLC (linear gradient

of water (solvent A) and CH₃CN (solvent B); 20–100

B% 15 min) yielding 21 as a blue solid (7.2 mg,

5.18 · 10^ꢀ6mol, 45%).

¹³C NMR (125 MHz, CDCl₃): d = 174.85 (CONH),

164.93 (CONH), 158.24, 148.45, 146.78, 144.93,

145.81, 144.61, 141.25, 136.39, 136.36, 131.92, 130.84,

129.43, 129.40, 129.19, 128.80, 128.73, 128.65, 128.50,

128.38, 128.13, 128.03, 128.00, 127.98, 127.90, 127.84,

127.82, 127.71, 127.55, 127.41, 127.26, 127.16, 127.08,

126.94, 126.85, 126.82, 124.75, 120.83, 113.28, 113.14

(39· arom C), 71.47 (quart C Trt), 66.80 (quart C

Mmt), 56.30 (OCH₃), 55.12 (a-CH Cys), 51.28

(CH₂NH), 42.35 (CH₂N₃), 40.14 (CH₂NH), 36.79

(b-CH₂Cys), 28.66 (CH₂), 26.88 (CH₂), 26.41 (CH₂),

26.32 (CH₂).

20

½aꢁ +32.6 (c 1.0, CHCl₃).

D

¹H NMR (500 MHz, CDCl₃): d = 8.97 (s, 1H, arom CH-

4), 8.29–8.27 (m, 2H, 2· arom. CH Cy5), 7.87 (d,

³J = 6.8 Hz, 2H, H-1, H-8 Fmoc), 7.82 (d,

3

³J = 10.4 Hz, 1H, NH), 7.75 (d, J = 9.3 Hz, 2H, H-4,

H-5 Fmoc), 7.65–7.60 (m, 4H, 4· arom. CH Cy5),

7.42 (s, 1H arom. CH-2), 7.34–7.20 (m, 5H, H-2, H-3,

H-6, H-7 Fmoc, arom CH-6), 6.63 (t, ³J = 15.4 Hz,

1H, C@CHCy5), 6.27 (t, ³J = 21.0 Hz, 2H,

HC@CHCy5), 4.46–4.43 (m, 3H, H-9, H-10 Fmoc),

ESI-MS+ m/z: calcd for C₅₆H₅₅N₇O₅SNa [M+Na]⁺:

960.4, found: 960.3.

3

4.22 (t, J = 5.8 Hz, 1H, a-CH Cys), 4.13–4.10 (m, 2H,

CH₂NH), 3.26–3.19 (m, 12H, b-CH₂Cys, N⁺@C–

CH@ Cy5, C_quart@CHCy5, HN–CH₂–CH₂–NH, e-

CH₂Cy5, CH₂N₃), 2.31–2.27 (m, 2H, d-CH₂Cy5), 2.17

(t, ³J = 19.5 Hz, 2H, a-CH₂Cy5), 1.70 (s, 12H, 4·

CH₃Cy5), 1.67–1.59 (m, 6H, b-CH₂Cy5, 2· CH₂),

1.40–1.28 (m, 9H, NCH₂–CH₃Cy5, c-CH₂Cy5, 2·

CH₂), 1.32 (s, 9H, S-t-Bu).

MALDI-TOF (DHB) calcd for C₅₆H₅₅N₇O₅S [M]:

937.40, found: 704.1 [MꢀMmt+H+K]⁺.

FAB/LR-MS (m/z): calcd: 937.40 [M+H]⁺, found:

937.26.

6.19. 1-N-Fluorenylmethoxycarbonyl-2-N-(tert-butyloxy-

carbonyl)-ethane-1,2-diamine (23)

ESI-MS+ m/z: calcd for C₇₁H₈₇N₁₀O₁₂S₄[M]⁺: 1399.5,

found: 1399.5 [M]⁺.

To a solution of N-(tert-butyloxycarbonyl)-ethane-1,2-

diamine (100 mg, 0.625 mmol) in 5 mL dichlorometh-

ane/methanol (1:1) 316.2 mg (0.938 mmol) Fmoc-OSu

and 173.2 lL (1.25 mmol) Et₃N were added at 0 ꢁC.

The reaction mixture was stirred for 12 h at room tem-

perature and the organic phase was washed with a solu-

tion of 1 N NaHSO₄and NaHCO₃. The combined

organic phases were dried over MgSO₄. The solvent

was removed under reduced pressure yielding pure 23

(186 mg, 0.487 mmol, 78%) as a white solid.

MALDI-TOF (DHB) calcd for C₇₁H₈₇N₁₀O₁₂S₄[M]⁺:

1399.5, found: 1399.3 [M]⁺.

6.18. 3-{Amino-[^L-cysteine-(S-monomethoxytrityl)-N-tri-

tyl]-methyl}-5-nitro-benzoic acid-(6-azido-hexyl) amide

(22)

The loaded resin (15) (150 mg, 0. 147 mmol) was agitat-

ed in 2 mL dichloromethane. Removal of the Boc-pro-

tecting group was carried out according to GP2.

According to GP4 (method A) the resin was treated with

a solution of 283.5 mg (0.441 mmol) Trt-Cys(Mmt)-OH,

68.3 mg (0.441 mmol) HOBt, 69.1 lL (0.441 mmol) DIC

and 66.0 lL (0.441 mmol) Et₃N in 2 mL dichlorometh-

ane. Cleavage from the resin was carried out according

to GP5 (method B). In the ﬁnal cleavage step the resin

was treated with a solution of 4 equiv 6-azido-hexyl-

amine (0.588 mmol, 83.5 mg) in 3 mL THF for 3 h.

The resin was ﬁltered oﬀ and the solvent was removed

under reduced pressure. The crude product was puriﬁed

by column chromatography (silica gel, cylohexane/ethyl

acetate 6:4 (v/v)) yielding 22 as a white solid (79.5 mg,

0.085 mol, 58%).

TLC: 0.50 (cyclohexane/ethyl acetate 1:1 (v/v)); mp:

158–159 ꢁC.

3

¹H NMR (500 MHz, CDCl₃): d = 7.76 (d, J = 9.5 Hz,

3

2H, H-1, H-8 Fmoc), 7.58 (d, J = 9.0 Hz, 2H, H-4, H-

5 Fmoc), 7.40 (t, ³J = 9.5 Hz, 2H, H-2, H-7 Fmoc),

7.31 (t, ³J = 9.0 Hz, 2H, H-3, H-6 Fmoc), 4.40 (d,

³J = 8.0 Hz, 2H, OCH₂Fmoc), 4.21 (t, ³J = 8.0 Hz,

1H, CH Fmoc), 3.27 (m, 4H, 2· NHCH₂), 1.45 (s, 9H,

C(CH₃)₃).

¹³C NMR (125 MHz, CDCl₃): d = 156.71 (CO₂NH),

143.90, 141.31, 127.76, 127.02, 119.94 (4· CH Fmoc),

66.68 (OCH₂Fmoc), 47.26 (CH Fmoc), 41.60 (NCH₂),

40.56 (NCH₂), 28.35 (C(CH₃)₂).

TLC: 0.60 (cylohexane/ethyl acetate 1:1 (v/v)); mp: 115–

116 ꢁC; ½aꢁ +47.9 (c 1.0, CHCl₃).

¹H NMR (500 MHz, CDCl₃): d = 8.47 (s, 1H, arom CH-

6), 8.15 (s, 1H, arom CH-4), 7.94 (s, 1H, arom CH-2),

7.35–7.10 (m, 27H, 27· arom CH Trt, Mmt), 6.45 (br

20

D

ESI-MS+ m/z: calcd for C₂₂H₂₆N₂O₄Na [M+Na]⁺:

405.2, found: 405.2.

MALDI-TOF (DHB) calcd for C₂₂H₂₆N₂O₄Na

[M+Na]⁺: 405.2, found: 405.5.

6304

A. Watzke et al. / Bioorg. Med. Chem. 14 (2006) 6288–6306

HR-FAB-MS (m/z): calcd: 383.1893 [M+H]⁺, found:

383.1998.

6.34 (t, ³J = 18.8 Hz, 2H, HC@CH), 4.58 (br s, 1H, NH),

4.18–4.14 (m, 2H, N⁺@C–CH@, C_quart@CH), 3.35 (m,

4H, HN–CH₂–CH₂–NH), 2.90 (m, 2H, e-CH₂), 2.21 (t,

³J = 8.5 Hz, 2H, a-CH₂), 1.83 (m, 2H, d-CH₂), 1.73 (s,

12H, 4· CH₃), 1.65 (t, ³J = 9.0 Hz, 2H, b-CH₂), 1.39–1.36

(m, 5H, NCH₂–CH₃, c-CH₂).

6.20. 1-N-Fluorenylmethoxycarbonyl-2-N-(Cy5)-ethane-

1,2-diamine (24)

For removal of the Boc-group 43.5 mg (0.114 mmol)

1-N-ﬂuorenylmethoxycarbonyl-2-N-(tert-butyloxycarbon-

yl)-ethane-1,2-diamine (23) was dissolved in 1 mL of a

solution of 50% TFA in dichloromethane. The reaction

mixture was stirred for 10 min at room temperature and

the solvent was coevaporated with toluene (10 mL). The

crude product was used without further puriﬁcation. To

a solution of 50 mg (7.615 · 10^ꢀ5mol) Cy5 in 2 mL

DMF 8 equiv HOBt (93.2 mg, 0.5 mmol), 8 equiv DIC

(94.4 lL, 0.610 mmol) and 4 equiv Et₃N (42.0 lL,

0.304 mmol) were added. The reaction mixture was stir-

red for 20 min at room temperature. The crude 1-N-ﬂu-

ESI-MS+ m/z: calcd for C₃₅H₄₇N₄O₇S₂[M]⁺: 699.3,

found: 699.7.

MALDI-TOF (DHB) calcd for C₃₅H₄₇N₄O₇S₂[M]⁺:

699.3, found: 699.7; calcd for C₃₅H₄₇N₄O₇S₂Na

[M+Na]⁺: 721.3, found: 721.6.

FAB/LR-MS (m/z): calcd: 700.29 [M+H]⁺, found:

700.07.

6.22. Protein expressions and ligation reactions

orenylmethoxycarbonyl-ethane-1,2-diamine

(32 mg,

1–203

0.114 mmol) was added and the reaction mixture was

stirred for 48 h. The solvent was evaporated under

reduced pressure and the crude product puriﬁed by

HPLC (linear gradient of water (solvent A) and CH₃CN

(solvent B); 20–100 B% 15 min) yielding 24 as a blue

solid (20 mg, 0.217 mol, 29%).

6.22.1. Cloning, expression and puriﬁcation of

Ypt1D3-MESNA. Ypt1 truncated by three-amino-acid

residues was C-terminally fused to an intein/chitin-bind-

ing domain assembly as implemented in the pTWIN-1

vector (New England Biolabs). Protein expression in

Escherichia coli and puriﬁcation of thioester-tagged pro-

tein were performed as described earlier.^3cThe resulting

Ypt1D3-MESNA thioester protein was desalted on a

PD-10 column (Amersham) equilibrated with 10 mM

Na-phosphate, pH 7.5, 0.1 mM MgCl₂, 2 mM GDP

and concentrated.

¹H NMR (500 MHz, CDCl₃): d = 8.30–8.23 (m, 2H, 2·

arom. CH), 7.97 (m, 4H, 4· arom. CH), 7.74 (d,

³J = 9.1 Hz, 2H, H-1, H-8 Fmoc), 7.60 (d, J = 9.3 Hz,

2H, H-4, H-5 Fmoc), 7.36–7.26 (m, 4H, H-2, 3, 6, 9

3

Fmoc), 6.63 (m, 1H, C@CH), 6.28 (t, ³J = 17.0 Hz,

3

2H, HC@CH), 4.30 (d, J = 8.5 Hz, 2H, N⁺@C–CH@,

6.22.1.1. Ligation of compound 12 to ^1–203Ypt1D3-

MESNA. In the ligation reaction 10 lL ^1–203Ypt1-MES-

NA (12 mg/mL) was mixed with 5 equiv of 30 mM com-

pound 12 (dissolved in H₂O/acetonitrile 1:4) in 50 lL of

50 mM Na₂HPO₄/NaH₂PO₄, pH 7.5, 0.1 mM MgCl₂,

2 lM GDP and 10 lL of 150 mM 2-mercaptoethane-

sulfonic acid (MESNA) and allowed to react overnight

at room temperature with vigorous agitation. The reac-

tion mixture was centrifuged (1 min at 13,000 rpm) to

remove protein and peptide precipitate. The pellet was

washed with methanol, methylene chloride (4 times),

methanol (4 times) and distilled water (4 times). Semi-

synthetic protein obtained was quantitatively deter-

mined by HPLC–MS (calculated mass: 23454 [M+H]⁺,

found: 23455).

C_quart@CH), 4.19–4.12 (m, 2H, H-10 Fmoc), 4.02 (t,

³J = 6.8 Hz, 1H, H-9 Fmoc), 3.28–3.20 (m, 6H, HN–

CH₂–CH₂–NH, e-CH₂), 2.18 (t, ³J = 9.0 Hz, 2H, a-

CH₂), 1.73 (m, 2H, d-CH₂), 1.71 (s, 12H, 4· CH₃),

3

1.65 (t, J = 9.5 Hz, 2H, d-CH₂), 1.42–1.39 (m, 2H, c-

3

CH₂), 1.35 (t, J = 8.8 Hz, 2H, NCH₂–CH₃).

ESI-MS+ m/z: calcd for C₅₀H₅₇N₄O₉S₂[M]⁺: 921.36,

found: 921.7.

MALDI-TOF (DHB) calcd for C₅₀H₅₇N₄O₉S₂[M]⁺:

921.36, found: 921.7; calcd for C₅₀H₅₇N₄O₉S₂Na

[M+Na]⁺: 943.4, found: 943.7.

6.21. Cy5-ethylamine (25)

6.22.2. Cloning, expression and puriﬁcation of ^1–181N-

Ras. Truncation of the full-length N-Ras cDNA (Acces-

sion No. X02751) was achieved using high-ﬁdelity Pfu

DNA polymerase (Stratagene). For the truncated ver-

sion of N-Ras two stop codons were introduced at posi-

tions 182 and 183 of the N-Ras cDNA. The resulting

fragment N-RasD181 was puriﬁed and digested with

EcoRI and SmaI, and was subcloned into the ptac

expression vector. The plasmid was sequenced to prove

PCR ﬁdelity and correct mutagenesis, and transformed

into the E. coli strain CK600K (Stratagene).

A solution of 12 mg (1.346 · 10^ꢀ5mol) 1-N-ﬂuorenyl-

methoxycarbonyl-2-N-(Cy5)-ethane-1,2-diamine (24) in

700 lL DMF was cooled to 0 ꢁC with an ice-bath. To

the solution 300 lL piperidine was added at 0 ꢁC and

the reaction mixture was allowed to warm up to room

temperature. The reaction mixture was stirred for 1 h

and the solvent was evaporated under reduced pressure.

The crude product was puriﬁed by HPLC yielding (lin-

ear gradient of water (solvent A) and CH₃CN (solvent

B); 20–100 B% 15 min) 25 as a blue solid (8 mg,

1.144 · 10^ꢀ5mol, 85%).

Escherichia coli carrying the expression plasmid for

N-RasD181 was cultivated in LB-medium containing

100 lg/mL ampicillin and 25 lg/mL canamycin at

37 ꢁC. Expression was induced at a cell density absor-

¹H NMR (500 MHz, CDCl₃): d = 8.29–8.27 (m, 2H, 2·

arom. CH), 7.89–7.85 (m, 4H, 4· arom. CH), 7.33 (t,

³J = 12.5 Hz, 1H, NH), 6.69 (t, ³J = 15.1 Hz, 1H, C@CH),

A. Watzke et al. / Bioorg. Med. Chem. 14 (2006) 6288–6306

6305

bance of A₆₀₀= 0.6 with 500 lM of isopropyl b-D-thiog-

alactoside and incubation was continued at 30 ꢁC over-

night. Pellets were resuspended in 20 mM Tris (pH 7.4),

5 mM MgCl₂, and 2 mM DTE (buﬀer A). Phenylmeth-

ylsulfonyl ﬂuoride (PMSF) to 0.1 mM and 0.5 mg

DNase I were added and the cells were lysed using a

microﬂuidizer (Microﬂuidics). Following centrifugation,

the clear supernatant was applied to a DEAE–Sephar-

ose column equilibrated with buﬀer A. After washing

with 2 column volumes, the protein was eluted over 5

column volumes using a linear gradient of 0–1 M NaCl

in buﬀer A. Fractions containing N-Ras were deter-

mined by SDS–PAGE, pooled and concentrated by pre-

cipitation with 3 M ammonium sulfate (ﬁnal

concentration) for 30 min. After centrifugation, the pre-

cipitate was resuspended in gel ﬁltration buﬀer (buﬀer A

containing 200 mM NaCl, 10 lM GDP) and applied to

a gel ﬁltration column (HiLoad Superdex 200, Amer-

sham Pharmacia Biotech). Fractions containing N-Ras

were pooled and again concentrated by ammonium sul-

fate precipitation. All puriﬁcation steps were carried out

at 4 ꢁC. Puriﬁed protein was stored at ꢀ80 ꢁC.

Tag) vector pTYB2 (New England Biolabs). E. coli

Bl21(DE3) were transformed with the Ras-intein plas-

mid and expression of the protein was performed at

20 ꢁC due to strong aggregation of the fusion protein

at higher temperatures. After harvesting of the bacteria,

the cells were lysed by ultrasoniﬁcation and the soluble

fraction was applied to a chitin-modiﬁed aﬃnity col-

umn. The Ras-MESNA thioester was formed on column

by application of 200 mM 2-mercaptoethanesulfonic

acid for 30 min at 4 ꢁC and concentrated by size-exclu-

sion ﬁltration. After aliquotation, the protein was

shock-frozen and stored at ꢀ80 ꢁC.

The synthesized building block 22 was treated with 40%

TFA/CH₂Cl_2.After complete deprotection (followed by

TLC), the solvent was removed under reduced pressure

and deprotected compound 22 was ligated to ^1–180N-

Ras-MESNA.

In the ligation reaction 10 lL N-Ras-thioester (77.2 mg/

mL) was mixed with 5 equiv of 40 mM deprotected com-

pound 22 (dissolved in H₂O/acetonitrile 1:5) in 50 lL of

50 mM Na₂HPO₄/NaH₂PO₄, pH 7.5, 0.1 mM MgCl₂,

2 lM GDP and 10 lL of 150 mM 2-mercaptoethane-

sulfonic acid and allowed to react overnight at room

temperature with vigorous agitation. The reaction mix-

ture was centrifuged (1 min at 13,000 rpm) to remove

protein and peptide precipitate.

6.22.2.1. Conjugation of MIC-labeled compound 17 to

^1–181N-Ras. In the conjugation reaction 10 lL ^1–181N-

Ras (50 mg/mL) was mixed with 1.2 equiv of 40 mM

compound 17 (dissolved in H₂O/acetonitrile 1:4) in

50 lL of 50 mM Tris-buﬀer, pH 7.4, 0.1 mM NaCl

and 2 lM GDP and allowed to react overnight at room

temperature with vigorous agitation. The reaction mix-

ture was centrifuged (1 min 13,000 at rpm) to remove

protein and peptide precipitate.

The ligation mixture was puriﬁed on a DEAE-gel ﬁltra-

tion column (FPLC-system), eluted with a linear gradi-

ent of 20 mM Tris/HCl, pH 7.4 and 5 mM MgCl₂. The

solution of puriﬁed, semi-synthetic protein was concen-

trated to 13 g/L (yield: 95%, calculated mass: 20747

[M+H]⁺, found: 20747 by HPLC–MS).

The ligation mixture was puriﬁed afterwards on a

DEAE-gel ﬁltration column (FPLC-system), eluted with

a linear gradient of 20 mM Tris/HCl, pH 7.4, and 5 mM

MgCl₂. The solution of puriﬁed, semi-synthetic protein

was concentrated to 40 g/L (yield: 98%, calculated mass:

23,454 [M+H]⁺, found: 23,455 by MALDI-TOF).

6.23. Preparation of the phosphine-functionalized slides

Dry N,N-dimethylformamide (60 mL) (DMF), N,N-

diisopropylethyl amine (4.8 mM) (DIPEA), 1-hydrox-

ybenzotriazole (4.8 mM) (HOBt) and N,N⁰-diisopropyl-

carbodiimide (4.8 mM) (DIC) were added to N-Fmoc-

protected aminocaproic acid (4.8 mM) and stirred for

10 min under argon to activate the acid. Then, the solu-

tion was transferred into a Schlenk ﬂask containing four

6.22.2.2. Conjugation of MIC-labeled compound 19 to

^1–181N-Ras. In the conjugation reaction 10 lL ^1–181N-

Ras (40 mg/mL) was mixed with 1.2 equiv of 30 mM

compound 19 (dissolved in H₂O) in 50 lL of 50 mM

Tris-buﬀer, pH 7.4, 0.1 mM NaCl and 2 lM GDP and

allowed to react overnight at room temperature with

vigorous agitation. The reaction mixture was centri-

fuged (1 min at 13,000 rpm) to remove protein and pep-

tide precipitate.

NH₂-terminated

polyamidoamine-dendrimer-coated

glass slides (Chimera Biotec, Dortmund) mounted on

a Teﬂon rack. The mixture was stirred overnight under

argon, the supernatant solution was removed and the

slides were rinsed twice with DMF, then stirred twice

for 20 min in DMF for washing and rinsed again with

DMF. Afterwards, they were treated twice for 20 min

with 60 mL of a 20% piperidine solution in DMF for

deprotection. The washing procedure was repeated add-

ing a 15 min stirring step in dichloromethane (DCM) as

well as rinsing with DCM. During the whole procedure

the slides were kept in the Schlenk ﬂask under argon.

The slides were then dried in vacuo. In the following

step, the phosphane reagent carrying the free acid func-

tion (5.2 mM), dry DMF (65 mL), HOBt (5.2 mM),

DIPEA (10.4 mM) and DIC (5.2 mM) were added sub-

sequently under argon to the slides. The mixture was

stirred overnight and the supernatant solution was again

The ligation mixture was puriﬁed afterwards on a

DEAE-gelﬁltration column (FPLC-system), eluted with

a linear gradient of 20 mM Tris/HCl, pH 7.4 and 5 mM

MgCl₂. The solution of puriﬁed, semi-synthetic protein

was concentrated to 30 g/L (yield: 98%, calculated mass:

21411 [M+H]⁺, found: 21414 by HPLC–MS).

6.22.3. Cloning and expression of ^1–180H-Ras and gener-

ation of the corresponding MESNA-thioester. To

produce the protein core for native chemical ligation,

the DNA sequence coding for truncated H-Ras (amino

residues 1–180) was cloned into the IMPACT (Intein-

Mediated Puriﬁcation with an Aﬃnity Chitin-binding

6306

A. Watzke et al. / Bioorg. Med. Chem. 14 (2006) 6288–6306

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removed followed by a washing procedure consisting of

rinsing twice with dry DMF, stirring in dry DMF/DCM

(1:1) twice for 20 min and rinsing again twice with dry

DMF. While stirring, the solution was degassed three

times. Afterwards, the slides were treated twice for

10 min with a solution of acetic anhydride (4.8 mM)

and pyridine (43.2 mM) in dry DMF (60 mL). After

removing the supernatant, the slides were rinsed with

dry DMF and dry DCM, stirred twice for 20 min in

dry DCM, rinsed again with dry DCM, dried in vacuo

and stored under argon until usage. Again, the solution

was degassed three times while stirring as mentioned

above.

6.24. Spotting process

The spotting process of 10–200 lM protein solution in

diﬀerent buﬀers (50 mM Na₂HPO₄, 2 mM NaCl and

2 mM MgCl₂, pH 7.5) was carried out using a GeSiM

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0.25 nL droplets of protein solutions (spot size 400 lm

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The slides were incubated with bovine serum albumin

(BSA) blocking-buﬀer (Chimera Biotec, Dortmund)

for 30 min and subsequently washed twice with

phosphate buﬀer (50 mM Na₂HPO₄, 2 mM NaCl,

2 mM MgCl₂and 0.05 vol % Tween 20) for 30 min.

The slides were incubated with 50 nM Ras-antibody

(50 lM TETBS, Tween, EDTA and Tris-buﬀer, pH

7.4) for 45 min and afterwards washed with phosphate

buﬀer (50 mM Na₂HPO₄, 2 mM NaCl and 2 mM

MgCl₂) for 30 min.

Acknowledgments

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This work was supported by the Fonds der Chemischen

Industrie, the Deutsche Forschungsgemeinschaft and

the Volkswagen-stiftung.

17. Stieber, F.; Greter, U.; Waldmann, H. Angew. Chem.

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Article Doi

A generic building block for C- and N-terminal protein-labeling and protein-immobilization

DOI: 10.1016/j.bmc.2006.05.006

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Full text of DOI:10.1016/j.bmc.2006.05.006

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