Bifunctional Reagents for Formylglycine Conjugation: Pitfalls and Breakthroughs

Combining Chemistry and Biology

Accepted Article

Title: Bifunctional Reagents for Formylglycine Conjugation: Pitfalls and

Breakthroughs

Authors: Nils Janson, Tobias Krüger, Lennard Karsten, Mareile

Boschanski, Thomas Dierks, Kristian M. Müller, and Norbert

Sewald

This manuscript has been accepted after peer review and appears as an

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of the final Version of Record (VoR). This work is currently citable by

using the Digital Object Identifier (DOI) given below. The VoR will be

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the VoR from the journal website shown below when it is published

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content of this Accepted Article.

To be cited as: ChemBioChem 10.1002/cbic.202000416

Link to VoR: https://doi.org/10.1002/cbic.202000416

01/2020

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Bifunctional Reagents for Formylglycine Conjugation: Pitfalls and

Breakthroughs

Nils Janson,^[a]Tobias Krüger,^[a]Lennard Karsten,^[b]Mareile Boschanski,^[c]Thomas Dierks*,^[c]Kristian M.

Müller*,^[b]and Norbert Sewald*^[a]

[a]

[b]

[c]

N. Janson, Dr. T. Krüger, Prof. Dr. N. Sewald

Faculty of Chemistry, Organic and Bioorganic Chemistry

Bielefeld University

Universitätsstraße 25, 33615 Bielefeld

E-mail: norbert.sewald@uni-bielefeld.de

L. Karsten, Prof. Dr. K. M. Müller

Cellular and Molecular Biotechnology

Bielefeld University

Universitätsstraße 25, 33615 Bielefeld

E-mail: kristian.mueller@uni-bielefeld.de

M. Boschanski, Prof. Dr. T. Dierks

Faculty of Chemistry, Biochemistry

Bielefeld University

Universitätsstraße 25, 33615 Bielefeld

E-mail: thomas.dierks@uni-bielefeld.de

Dedicated to the memory of Thomas Dierks

Abstract: Formylglycine-generating enzymes specifically oxidize cys- Introduction

teine within the consensus sequence CxPxR to C^α-formylglycine

(FGly). This non-canonical electrophilic amino acid can subsequently

be addressed selectively via bioorthogonal Hydrazino-iso-Pictet-

Spengler (HIPS) or Knoevenagel ligation to attach payloads like fluor-

ophores or drugs to proteins, while obtaining a defined payload-to-

protein ratio. However, disadvantages of these conjugation tech-

niques include the need of a large excess of conjugation building

block, comparably low reaction rates and limited stability of FGly-con-

taining proteins. Therefore, functionalized clickable HIPS and tan-

dem Knoevenagel building blocks were synthesized, conjugated to

small proteins (DARPins) and subsequently linked to strained alkyne-

containing payloads for protein labeling. This procedure allowed the

selective bioconjugation of one or two DBCO-carrying payloads with

nearly stoichiometric amounts at low concentrations. Furthermore, an

azide-modified tandem Knoevenagel building block enabled the syn-

thesis of branched PEG-linkers and the conjugation of two fluoro-

phores, resulting in an improved signal-to-noise ratio in live cell fluo-

rescence imaging experiments targeting the EGF-receptor.

In recent years site-specific bioconjugation chemistry has been

used as a powerful tool to modify proteins and study their biolog-

ical function.^[1-4]Furthermore, it enables selective protein fusion,

enzyme immobilization or the production of antibody-drug conju-

gates (ADC).^[5-8]In order to retain the protein functions, these

methods are to be biocompatible, highly selective, fast and non-

invasive for living cells.

Non-canonical amino acids like p-azidophenylalanine can be in-

troduced via complex genetic engineering of living cells and an

expanded genetic code.^[9,10]Alternatively, enzymatic modifica-

tions with short amino acid sequences, as catalyzed by sortase^[11-

^13], transglutaminase^[14-17]or protein farnesyl transferase^[18], can

be used to avoid these complex manipulations. Additionally,

unique electrophilic aldehyde moieties can be introduced into pro-

teins. On one hand, an N-terminal α-oxo aldehyde can be ob-

tained by oxidation of serine or threonine^[19]or by a transamination

reaction of N-terminal glycine with pyridoxal-5’-phosphate

Scheme 1. Development of a highly efficient modular conjugation system for mono- and double-conjugation using stoichiometric amounts of DBCO-derivatives.

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(PLP).^[20]On the other hand, the oxygen- and copper-dependent Results and Discussion

formylglycine-generating enzyme (FGE) can be used. It catalyzes

the site-specific oxidation of cysteine within the consensus se-

quence CxPxR (aldehyde tag) to form the non-canonical amino

acid C^α-formylglycine (FGly).^[21-28]This enzyme has already been

used for efficient in vitro and in vivo FGly-formation.^{[6-8, 28]}Com-

pared to the α-oxo aldehyde, which can only be generated N-ter-

minally, the aldehyde tag can be inserted at almost any desired

location within recombinant proteins.

Synthesis of HIPS and Knoevenagel conjugation reagents. In

order to address formylglycine residues with modular HIPS and

Knoevenagel ligation reagents combined with either strain-pro-

moted azide alkyne cycloadditions (SPAAC) or inverse electron-

demand Diels-Alder reaction (IEDDA), we synthesized an array of

bioorthogonally reactive HIPS and Knoevenagel building blocks

(Schemes 2A, 3A and 3B).

Several aldehyde-specific reactions with strong nucleophiles

were developed in the past. First attempts used hydroxylamines

and hydrazines, which form oximes or hydrazones under acidic

conditions, but are sensitive to hydrolysis.^[29-36]Further research

resulted in the development of the (Hydrazino-iso-)Pictet-Speng-

ler (HIPS) reaction using modified tryptamines that undergo a

Mannich-type reaction with respective aldehydes. After proton-

catalyzed condensation (pH 5.5-6.5), the iminium ion formed is

converted by an intramolecular nucleophilic attack of the indole

moiety.^[37-39]Moreover, Pomplun et al. reported a similar reaction

using N-substituted pyrrolyl alanine derivatives.^[40]

Alternatively, carbon nucleophiles may be used for aldehyde-la-

belling, like in Wittig ^[41], Mukaiyama aldol^[42]and aldol reactions

with 2,4-thiazolidinediones^[43]or N-aliphatic substituted pyra-

zolones. In the latter case, a double bond is formed at neutral pH,

which can either be attacked by an intramolecular thiol

(trapped Knoevenagel)^[8]or a second pyrazolone building block

(tandem Knoevenagel).^[44]N-aryl substitution of those moieties

further increases reactivity.^[45]HIPS and Knoevenagel ligation

have already been used in combination with the aldehyde-tag to

produce site-specific ADCs with defined drug-to-antibody ratios

(DAR). However, a large excess of the expensive drug-containing

building block and long reaction times were necessary.^[7,8,44]

To avoid these aforementioned disadvantages, we have estab-

lished an efficient and straightforward synthesis of inexpensive bi-

functionalized HIPS and tandem Knoevenagel building blocks,

which can be used in combination with highly efficient strain-pro-

moted azide alkyne cycloadditions (SPAAC) for selective mono-

or double-derivatization with stoichiometric amounts of DBCO-

modified payloads.

The Fmoc protected hydrazine 1 (Schema 2A) and the pyrazo-

lone-type Knoevenagel core segments 8 and 11 (Scheme 3A and

3B) were synthesized similar to literature protocols.^{[7-8,28,44-45]}Sub-

sequently, bi-functionalized HIPS ligation reagents were obtained

containing the N,N’-dimethylhydrazine group for FGly conjugation

together with an azide, a bicyclononyne (BCN), a dibenzocy-

clooctyne (DBCO) or a 6-methyl-tetrazine (mTet) moiety for

SPAAC or IEDDA, respectively. Amide couplings were accom-

plished using DCC/EDC and pentafluorophenol/HOBt to form the

corresponding PFP or HOBt ester^[28], which reacts with amine

functionalized azide-, BCN-, DBCO- or 6-methyl-tetrazine-deriva-

tives. Finally, Fmoc cleavage was performed by treatment with

sodium hydroxide or 20% piperidine.

However, during the syntheses of different envisaged building

blocks we encountered problems because of incompatibilities of

reagents and/or functional groups. The purification of the alkyne-

containing HIPS-BCN (5) could be performed by RP-HPLC omit-

ting acidic additives, since we observed two major side products

with mass signals at m/z 586.37 (Fig. S8) and m/z 1135.72 (Fig.

S9) during purification in the presence of 0.1% TFA. These sig-

nals could be related to a water adduct of the desired product

(m/z_calc= 586.36 [M+H₂O+H]⁺) and a dimer (m/z_calc= 1135.69),

indicating acid-mediated addition of nucleophiles, like hydrazine

or water, to the strained alkyne. As a result, either an enamine or

an enol is formed that tautomerizes to the corresponding ketone

(Scheme 2B.1). The water addition was also observed and fully

characterized by Spangler et al.^[46]Instead of the nucleophilic ad-

dition of the hydrazine to the vinyl cation formed upon protonation

of the strained alkyne, it can also be considered that the hydrazine

Scheme 2. Bifunctional HIPS reagents and observed side reactions. A) Two step synthesis (coupling and deprotection) of HIPS-CF (2), -mTet (3), -DBCO (4), -BCN

(5), and -azide (6). B) Postulated side reactions during synthesis, purification or conjugation. 1) Postulated acid-mediated decomposition of BCN. 2) Possible

mechanism for proton catalyzed decomposition of DBCO. 3) Observed inter- or intramolecular hydrazine catalyzed decomposition of HIPS-mTet (3).

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Scheme 3. Bifunctional Knoevenagel reagents and observed side reactions during synthesis, purification or conjugation. A) Synthesis of trapped Knoevenagel-CF

(9) starting from 3-mercaptopropionic acid (7). B) Synthesis of tandem Knoevenagel-azide (12), fluorine substituted aryl azide (13) and -mTet (14). C) Postulated

inter- and intramolecular side reaction of pyrazolone with methyl substituted tetrazine.

moiety reacts with the aforementioned formed ketone, which

would lead to the same enamine side product (Scheme 2B.1).

First attempts to purify the literature known HIPS-DBCO (4)^[47]on

silica gel failed due to complete decomposition during flash chro-

matography. Instead, purification by RP-HPLC led to low yield en-

countered together with unsatisfactory purity. This can be ex-

plained by decomposition reactions, caused by oxidation of the

hydrazine moiety, which was observed for all HIPS reagents

(Fig. S6). Additionally, an intramolecular attachment to the vinyl

cation of the amide nitrogen accompanied by amide hydrolysis

presumably forms 6H-isoindolo[2,1-a]indole (Scheme 2B.2 and

Fig. S6), which would fit to the literature known decomposition of

Cbz-protected DBCO under acidic or basic conditions.^[48]

Improved reactivities towards BCN or cyclooctenes have been

observed for tetrazines as reaction partners.^[49]However, purifica-

tion of the bifunctional building block HIPS-mTet (3) was not pos-

sible due to a fast decomposition upon Fmoc cleavage. LC-MS

analysis of the mixture revealed a major side product with a mass

shift of -22 Da that might correspond to a 5-methyl-1,3,4-oxadia-

zole derivative (Scheme 2B.3 and Fig. S7) formed by hydrazine-

catalyzed decomposition, which proceeds similarly to previ-

ously described reactions.^[50-51]

As observed for 4 and 5, purification of HIPS-azide (6) with RP-

HPLC led to product decomposition. Interestingly, this was even

observed in the absence of TFA and might have been caused due

to an increase inherent susceptibility of 6 towards oxidation at the

hydrazine moiety. However, 6 could be isolated in sufficient puri-

ties upon precipitation of the dibenzofulvene-piperidine side-pro-

duct formed upon Fmoc cleavage by dilution of the reaction mix-

ture with water. In contrast, no side reactions involving the azide

group were observed, indicating sufficient orthogonality between

both functionalities.

fore, HIPS building block 6, showing sufficient stability and orthog-

onality of the functional groups, appeared to be the most promis-

ing candidate for following bioconjugation reactions.

Besides the HIPS conjugation reagents, a similar modular system

for mono-conjugation with the trapped Knoevenagel building

block containing an intramolecular thiol would be conceivable.

However, DBCO, BCN, azides, and tetrazines had previously

been shown to be incompatible with the required reaction condi-

tions or high concentrations of thiols.^[52-55]

Therefore, bi-functionalized tandem Knoevenagel ligation rea-

gents with a pyrazolone for FGly conjugation and an alkyl azide

(12), an aryl azide (13), or a tetrazine (14) were synthesized in

moderate to good yields and in excellent purities (Scheme 3B). A

mixture of tautomers was obtained in each case with varying hy-

droxypyrazole/4H-pyrazolone ratios depending on solvent and pH

(Fig. S1 and S2). DBCO and BCN had to be avoided because of

decomposition (vide supra). Purification of 14 was particularly

challenging because of a rapid inter- or/and intramolecular attack

of the pyrazolone moiety to the electrophilic methyltetrazine

(Scheme 3C), slowly leading to a cyclization at low concentrations

(1 mM; Fig. S5). However, at higher concentrations cyclization as

well as a dimerization of two molecules of 14 was observed

(Fig. S4). Interestingly, in contrast to previous observations^[56]and

the hydrazine-catalyzed decomposition of 3, no nitrogen release

and no further modifications were observed (Scheme

2B.3 and 3C).

In summary, the commercially available clickable groups tetrazine

and strained alkynes show unsatisfactory compatibility with the

reactive groups of the HIPS and Knoevenagel building blocks

(N,N’-dimethylhydrazine and pyrazolone). Hence, the azides 6,

12, and 13 remained as the most promising candidates.

In summary, bifunctional HIPS reagents containing azide, BCN or

DBCO could be prepared, albeit limited compatibility of the hydra-

zine and these functional groups was observed. An acidic envi-

ronment leads to the addition of water to the strained alkynes or

amide hydrolysis, complicating purification procedures. Introduc-

tion of a tetrazine group causes immediate decomposition. There-

FGE mediated formylglycine formation in proteins. We used

Designed Ankyrin Repeat protein (DARPin) E01 (Fig. S10), which

is directed against the epidermal growth factor receptor

(EFGR),^[57]as a model protein for conjugation. The enzymatic

conversion of the CTPSR target sequence in a C-terminally

CTPSR-His₆-tagged DARPin was performed in a bicine buffer

containing 200 mM arginine with 10 mol% of CTPSR-tagged FGE

from Mycobacterium tuberculosis (MtFGE), CuSO₄and CaCl₂

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Figure 1. Stoichiometry- and time-dependent conjugation assay with HIPS-CF (2) and trapped Knoevenagel-CF (9). A) Bioconjugation experiments: DARPin-

CTPSR-His₆(100 µM) was enzymatically oxidized in a bicine buffer containing 10 mol% CuSO₄and 10 mol% CTPSR-MtFGE. The FGly-containing DARPin was

re-buffered (HIPS: 100 mM phosphate, 30 mM NaCl, pH 6.0; Knoevenagel: 50 mM citrate, 50 mM NaCl, pH 7.2) and subsequently fluorescence labeled using 2

and 9 (30 µM FGly-DARPin). B) MALDI-ToF analysis of relevant Cys-(m/z = 2736.6) and FGly-peptide (m/z = 2718.6 and 2700.6). C) SDS-PAGE analysis (15%)

of stoichiometry- (2.5, 5, 7.5 and 10 eq.) and time-dependent conjugation assays. Samples (15 µg) were taken at 2, 6, 10 and 24 hours and analyzed on gel by

fluorescence (upper panels) before Coomassie staining (lower panels). Whole SDS gels and fluorescence images can be found in the SI (Fig. S12 and S13).

(each 10 µM) for 16-20 h at 22-25°C (Fig. 1A). Copper and cal-

cium salts were added for in situ FGE reconstitution. The CTPSR-

tag within FGE serves as an internal standard for FGly conversion

and conjugation reactions. Protein mixtures were directly treated

with trypsin and the fragments analyzed by MALDI-ToF-MS to de-

termine the FGly modification of the DARPins (Fig. 1B). Since

FGly formation leads to a mass shift of -18 Da, intramolecular

imine formation between FGly and the N-terminus of peptides pro-

duces a detectable Schiff base in MALDI-ToF-MS. Integration of

the peaks corresponding to the CTPSR-tag containing peptide

(m/z_obs= 2736.7, m/z_calc= 2736.3), FGly-peptide (m/z_obs= 2718.6,

m/z_calc= 2718.3), and the intramolecular imine (m/z_obs= 2700.6,

m/z_calc= 2700.3) revealed a conversion of approx. >95% assum-

ing similar ionization efficiencies for all three species.^[28]The al-

most quantitative conversion could also be shown by subsequent

conjugation with azide 12 and the absence of the starting material

band in SDS-PAGE analysis (see below, Fig. 2D). Subsequently,

the FGly-containing DARPin was rebuffered either into HIPS liga-

tion buffer (100 mM phosphate, 20 mM NaCl, pH 6.0) or

Knoevenagel ligation buffer (50 mM citrate, 50 mM NaCl, pH 7.2).

However, precipitation of DARPin was observed in the HIPS

buffer, which is probably due to the pH (6.0) close to the isoelec-

tric point of the protein (pI_calc: 4.96).

known from published peptide experiments.^[58]The addition of ar-

ginine during FGly-conversion and SDS-PAGE analysis probably

stabilizes the FGly-containing DARPin through imine formation,

and indeed less fragmentation was observed (Fig. S11). There-

fore, this additive was always included preventively during FGly

conversion.

The success of the fluorescence labeling by standard HIPS and

Knoevenagel ligation with the carboxyfluorescein derivatives 2

and 9 was proven by the selective formation of a protein band at

higher molecular masses and increased fluorescence intensity

over the time (Fig. 1C, S12 and S13). In our experiments, 2.5-10

equivalents of 2 or 9 and 24 h were not sufficient to achieve full

conversion at 30 µM, assuming a similar low reactivity for the

HIPS and trapped Knoevenagel ligation reaction. Full conversion

was obtained when 10 equivalents of 9 were used at higher pro-

tein concentrations (100 µM) (Fig. S14). Therefore, high concen-

trations and an excess of the payload-containing building blocks

are needed for the standard HIPS and Knoevenagel ligation at

low concentrations. Due to the complex and usually expensive

multistep synthesis towards drugs, fluorophores or other payloads

a more efficient procedure needs to be developed.

Conjugation with azide-containing bifunctional linkers gives

superior results. In order to demonstrate an alternative route to-

wards FGly-based bioconjugations with higher efficiencies, proof-

of-concept experiments with the bi-functionalized HIPS- and tan-

dem Knoevenagel-azides 6 and 12 were performed in the same

buffers described above. In both cases, the reagent was added to

the target proteins and incubated either for 16 h at 30°C with

2 mM of 6 or 24 h at 22°C with 10 mM of 12 (Scheme 3).

Conjugated DARPins were subsequently rebuffered into phos-

phate buffer (8% DMSO, 100 mM phosphate, 150 mM NaCl,

pH 7.2) to remove excess reagent. DMSO was added for im-

proved solubility of the DBCO- and BCN-modified payloads. Ad-

ditionally, the fractions with excess azide were also collected,

freeze dried, extracted with dichloromethane (DCM) and analyzed

by LC-MS (Fig. 2A). Interestingly, 12 could be recovered with 94%

Direct fluorophore conjugation with HIPS-CF (2) and trapped

Knoevenagel-CF (9) requires large excess of reagent. In order

to compare the conjugation efficiencies of the standard HIPS and

trapped Knoevenagel ligations with our modular approach involv-

ing bifunctional linkers, 30 µM of the FGly-containing DARPins

were incubated with different equivalents of the 5(6)-carboxyfluo-

rescein derivatives 2 and 9. Samples were taken after 2, 6, 10,

and 24 h, which were then analyzed by reducing SDS-PAGE

(15%) and in-gel fluorescence imaging (Figs. 1C, S12 and S13).

Multiple bands at approx. 16 kDa were observed for the non-con-

jugated FGly-DARPin. These could result from conformational

changes (internal imine formation) or fragmentation; the latter is

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Scheme 4. Azide-derivatization of DARPin by HIPS-azide (6) and tandem Knoevenagel-azide (12). FGly-DARPin was first conjugated with HIPS-azide (6,

2 mM, 16 h, 30°C) or tandem Knoevenagel-azide (12, 10 mM, 24 h, 22°C), rebuffered in phosphate buffer (100 mM phosphate, 150 mM NaCl, pH 7.2, 8% DMSO)

and then used in a sequential one-pot capping and click reaction with different strained alkynes.

and good purities, while 6 underwent decomposition (Fig. 2A and

2B). In our postulated decomposition mechanism, a two-electron

oxidation and subsequent deprotonation leads to the formation of

the hydrazone C (m/z_calc= 460.27 ([M+H]⁺)), which could induce

an intramolecular attack of the indole to form hydrazine D

(m/z_calc= 460.27 ([M+H]⁺)). Another two-electron oxidation,

deprotonation and a third two-electron oxidation finally results in

the formation of the diazacarbazol derivative G (m/z_calc= 456.24

([M]⁺)). This aromatic structure cannot undergo further electron

release.^[59]Further signals might occur due to the hydrolytic re-

lease of formaldehyde by intermediate C to I (m/z_calc= 448.27

([M+H]⁺)), which can then incorporated by another molecule of A

to form H (m/z_calc= 474.28 ([M+H]⁺)). These postulated species

are in accordance with results published by T. Linz et al.^[60]More-

over, partial in source-fragmentation and elimination of the hydra-

zine moiety was used to corroborate the postulated species (Fig-

ures S22 – S26). Additionally, the compound corresponding to

m/z 456.24 shows higher ionization efficiency and increased ab-

sorption at higher wavelength (254 and 280 nm) compared to the

other signals, indicating formation of an expanded and positively

charged aromatic structure (Figure S21).

To elucidate the source of the oxidant, HIPS-azide (6) was diluted

in HIPS ligation buffer (100 mM phosphate, 30 mM NaCl, pH 6.0)

and incubated for 16 h at 30°C with different additives and ana-

lyzed by LC-MS. Without any additives partial decomposition was

observed, while the compound was completely stable upon addi-

tion of EDTA to the buffer. The opposite was observed upon the

addition of copper salts (e.g. CuSO₄), while exclusion of oxygen

did not lead to slower oxidation.

Accelerated decomposition occurred in the presence of the FGly-

containing DARPin, which is probably due to the coordination of

CuSO₄during the FGly conversion. Preventive treatment of the

FGly-protein with EDTA could be considered to remove coordi-

nated copper and reduce HIPS decomposition (Figure S20).

In summary, these experiments show that the oxidative decom-

position of the hydrazine is caused by metal ions, able to serve as

electron acceptors.

1 mM N-ethylmaleimide to avoid thiol-yne side reactions. SDS-

PAGE analysis of conjugated DARPin (Fig. 2C and S15) revealed

that DARPin-N₃contains a faint fragmentation band at lower mo-

lecular masses, indicating that the protein was not fully converted

to the azide. Qualitative analysis of tryptic peptides using MALDI-

ToF-MS shows only minor amounts of unconjugated FGly-DAR-

Pin (Fig. S27). Nevertheless, treatment with one or two equiva-

lents of 16 for 3 h at 22°C resulted in a prominent fluorescent pro-

tein band at approx. 19 kDa, confirming successful application of

the modular conjugation protocol. Despite longer reaction times

(22 h), the click reaction with BCN-CF (15) resulted in comparably

lower conjugation efficiencies. At least 5 equivalents were neces-

sary for complete conversion at low concentrations (Fig. 2C and

S15).

Tandem Knoevenagel-azide (12) allows for efficient double

conjugation. Compared to DARPin-N₃, the diazido substituted

DARPin (DARPin-(N₃)₂), generated using the tandem

Knoevenagel building block 12, did not show any protein bands

possibly originating from fragmentations or residual starting ma-

terial, indicating almost full conversion to the conjugate (Fig. 2D

and S16). Additionally, MALDI-ToF-MS analysis detected only

traces of the FGly-DARPin. (Fig. S28). The following click reaction

of DARPin-(N₃)₂with the reactive dye DBCO-CF (16) was per-

formed under similar conditions (22°C for 3 h) and almost full con-

version to the double-conjugate was observed upon addition of

one equivalent per azide. A faint fluorescent protein band below

the double-conjugate is assigned to the mono-conjugate (Fig. 2D

and S16). These results and efficiencies are in agreement to pre-

vious reports on diazido substituted linkers, which were attached

to antibodies by transglutaminase.^[61]Due to the lower reactivity

of BCN^[62], the conjugation tests with DARPin-(N₃)₂were carried

out at higher concentrations (50 µM) and 5 or 10 equivalents per

azide of BCN-CF (15) were applied. Despite the utilization of

higher concentrations, longer reaction times (16 h) and modified

stoichiometry, no complete conversion to the double-conjugate

was observed. Here, approx. 50% mono- and 50% double-conju-

gate were obtained (Fig. 2D and S16). Even an increase to 50 or

100 equivalents did not lead to complete double-conjugation (Fig.

S17-S19). A reason for incomplete conversion to the double-con-

jugate could be the relatively lower reactivity towards aliphatic az-

ides and also the inherent instability of the alkyne moiety (see

above).

Efficient mono-conjugation with HIPS-azide (6). In order to

identify the most suitable and commercially available strained al-

kyne for the conjugation with aliphatic azides, 10 µM of DARPin-

N₃, generated using 6, were treated with different equivalents of

BCN-CF (15) or DBCO-CF (16) (Fig. 2C and S15). Prior to the

addition of any conjugation reagent, residual free thiols were

treated in a sequential one-pot capping and click reaction with

Further click reactions with the less reactive and commercially

available DBCO-PEG_20k(17) were performed to demonstrate the

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Figure 2. Recovery of the bifunctional reagents 6 and 12 after bioconjugation and subsequent SPAAC experiments. A) LC-MS analysis of recovered tandem

Knoevenagel-azide (12) and HIPS-azide (6) after PD10 and extraction. B) Possible mechanism for oxidative decomposition and modifications of 6. C-D) SDS-PAGE

analysis (15%) (lower panels) and in-gel fluorescence (upper panels) of DARPin-N₃, DARPin-(N₃)₂and subsequent click-reactions with different equivalents of BCN-

CF (15) and DBCO-CF (16). On the right part of panel D, MW markers and samples are from the same SDS gel. Only relevant samples are shown. Full SDS gels

and fluorescence images of C and D can be found in the SI (Fig. S15 and S16). E-F) SDS-PAGE analysis (15%) of DARPin-N₃, DARPin-(N₃)₂and subsequent click-

reaction with different equivalents of DBCO-PEG_20k(17). PEGylation was further analyzed with a PEG-specific BaCl₂/I₂staining protocol.

efficiency of the modular system (Fig. 2E and 2F). In general,

higher concentrations, temperatures, and longer reaction times

are recommended for this reactive group.

limited solubilities of both reagents. The mixture was incubated at

22°C overnight and directly analyzed after tryptic digest by

MALDI-ToF-MS (Fig. 3A). It could be shown that both HIPS rea-

gents were successfully conjugated to the protein, but starting

material could still be detected, corresponding to incomplete liga-

tion. Furthermore, in both reactions a signal with a relative mass

shift of +18 Da was detected, indicating acid-mediated alkyne hy-

dration forming a ketone (Scheme 2B.2). Larger amounts of the

water adduct can be observed for BCN, which probably is more

prone to protonation due to the comparatively electron rich char-

acter of the strained alkyne.^[62]Prolonged incubation times during

the HIPS ligation (pH 5.5-6.5) could be a reason for this side prod-

uct. Further incubation with an azide functionalized payload

yielded only traces of the desired conjugate. As previously as-

sumed, strained alkynes seem to be not compatible under these

mild HIPS ligation conditions.

For example, DARPin-N₃(10 µM) was treated for 16 h at 37°C

with one or two equivalents of 17. In addition to Coomassie stain-

ing, SDS-PAGE analysis was also performed using a PEG-spe-

cific BaCl₂/I₂staining protocol (Fig. 2E).^[63,64]In both reactions, a

major PEGylated protein band was observed with an apparent

molecular mass of approx. 50 kDa (MW_calc= 38.6 kDa). The devi-

ation of the predicted molecular mass of a conjugated DARPin

can be explained by the larger hydrodynamic radius of the bound

PEG.^[65]Additionally, DARPin-(N₃)₂was treated with 1-3 equiva-

lents per azide of 16 (Fig 2F). With one equivalent, the double-

PEGylated protein can be observed as a major protein band at

200 kDa. However, the mono-conjugate can still be detected at

50 kDa. By increasing the equivalents to 3 per azide, almost full

conversion to the double-pegylated protein was obtained (Fig. 2F). Compared to aliphatic azides, aryl azides and tetrazines show

In conclusion, our modular approach allows the efficient synthesis

of linear or branched PEG-linkers.

higher reactivities towards BCN.^[62]Hence, the tandem Knoeve-

nagel constructs 13 and 14 (final concentration of 2.5 mM) were

used for DARPin conjugation. Due to their limited solubility, 10%

of DMSO was added. After incubation for 24 h at 22°C and rebuff-

ering into the above described phosphate buffer, 50 µM of the

conjugated DARPins were treated with 2.5, 5, and 10 equivalents

Strained alkynes and tetrazines gave insufficient results. Fur-

ther conjugation tests were also performed with HIPS-DBCO (4)

and HIPS-BCN (5) by incubating the FGly-DARPin (56 µM) with

590 µM of 4 or 5. Higher concentrations were not possible due to

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per azide of BCN-CF (15). Both DARPin-(aryl azide)₂and DAR-

Pin-(mTet)₂did not show any protein bands corresponding to

starting material, indicating full conversion (Fig. 3B). Despite the

large excess of 15 used in the subsequent click reactions, incom-

plete conversion to the double-conjugate was revealed by SDS-

PAGE analysis (Fig. 3B). Here, approx. 50% mono- and 50% dou-

ble-conjugate were obtained, indicating that BCN cannot be used

for the conjugation of two azides or tetrazine, which are in

close proximity.

(generated using 9) or DARPin-(CF)₂(generated using 12 and 16)

and analyzed by confocal fluorescence microscopy.^[66]Both con-

jugates bound to A431 cells (Fig. 4A and D), but not to MCF7 cells

(Fig. 4C and F), which indicates that specific EGFR targeting of

the aldehyde-tagged DARPin is maintained after the modification

via trapped or tandem Knoevenagel reactions.^[57]Internalization

and colocalization with lysosomes have been observed for both

DARPin conjugates at 37 °C (Fig. 4A and D) but not at 4 °C (Fig.

4B and E) suggesting that selective EGFR-mediated endocytosis

occurs.^[67]Analysis of the mean fluorescence intensity of live cell

images taken at the same temperature revealed about a twofold

increased value for DARPin-(CF)₂compared to DARPin-CF

(Fig. 4). Hence, conjugating the DARPin with two carboxyfluores-

cein units via the tandem Knoevenagel reaction significantly im-

proves the signal-to-noise ratio for fluorescence-based studies

(Fig. S29). In contrast to the standard Knoevenagel ligation

(100 µM, 10 eq.), this modular approach allows protein labeling

with stoichiometric amounts of the fluorophore. Live cell imaging

experiments demonstrated that both DARPin conjugates retain

the ability of EGFR binding and successive EGFR-mediated in-

ternalization.

Figure 3. Analysis of the conjugation with HIPS reagents 4 and 5 and tan-

dem Knoevenagel reagents 13 and 14. A) MALDI-ToF-MS analysis of DARPin

conjugated to 4 and 5 showing tryptic peptides, indicating incomplete ligation

and water addition to the strained alkyne. B) Coomassie stained SDS-PAGE

(15%) analysis of DARPin conjugated to 13 and 14 and subsequent click reac-

tion with BCN-CF (15). 1) DARPin-CTPSR-His₆, 2) FGly-DARPin, 3.1) DARPin-

(aryl azide)₂, 3.2) DARPin-(mTet)₂, 4) 2.5 eq. of 15 per azide, 5) 5 eq./azide, 6)

10 eq./azide.

We were able to establish a highly efficient modular conjugation

system for mono- and double-conjugation, which requires a sim-

ple and inexpensive azide modified HIPS or tandem Knoevenagel

building block and stoichiometric amounts of DBCO-derivatives,

while BCN-derivatives are less effective. Another advantage of

this modular system is that these azides and the azide-labeled

proteins can be stored in solution for several months at -80°C.

Figure 4. Live cell imaging of DARPin conjugates. A/D) A431 cells at 37 °C,

B/C) A431 cells at 4 °C, and C/F) MCF7 cells at 37 °C were incubated with

100 nM DARPin-CF (A-C) or DARPin-CF₂(D-E) for 6-8 h. Fluorescence of

5(6)-carboxyfluorescein and LysoTracker DND-22 are shown in green and blue,

respectively. For images with merged channels colocalization is pseudo-colored

in magenta and indicated by arrows. Scale bars in the lower right corner repre-

sent 15 µm.

Live cell fluorescence imaging. Finally, to test an appropriate

application for the modular conjugation strategy and to analyze

the impact of this artificial modification, live cell imaging experi-

ments with fluorescently labeled DARPins were conducted

(Fig. 4). A431 and MCF7 cells, which present either high or low

levels of EGFR, respectively, were incubated with DARPin-CF

Conclusion

Fluorescence labeling of C-terminal-His₆-tagged DARPin E01 by

the aldehyde-tag strategy combined with standard HIPS or

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trapped Knoevenagel reactions is compromised by slow kinetics

and the need of a large excess of the expensive conjugation build-

ing block at low concentrations. In our experiments, 2.5 to 10

equivalents of HIPS-CF (2) and trapped Knoevenagel-CF (9) and

24 h were not sufficient for complete conjugation of protein as de-

tected by SDS-PAGE. However, similar efficiencies for HIPS- and

Knoevenagel ligations were obtained.

Therefore, we established a straightforward synthesis of different

bifunctional HIPS and tandem Knoevenagel building blocks capa-

ble of being used in bioorthogonal click reactions. In contrast to

the azide derivatives, tetrazine-, BCN- and DBCO-derivatization

led to several side reactions involving the hydrazine or pyrazolone.

However, azide derivatives showed sufficient chemical orthogo-

nality. They can be stored at -80°C in solution for several months,

and efficiently conjugated to target proteins.

Experimental Section

General information:

Reactions sensitive to oxygen and moisture were performed us-

ing Schlenk technique and an argon atmosphere. Chemicals and

solvents were bought from commercial sources and used without

further purification. Anhydrous solvents were obtained through

distillation over desiccant. For extraction and column chromatog-

raphy, distilled EtOAc and petroleum ether were used. Column

chromatography was performed with silica gel with a diameter of

0.040-0.063 mm (Merck). For substance detection, aluminum-

plates with silica gel 60 and fluorescence-indicator F254 by Merck

and UV radiation (254 nM) and/or staining solutions (bromocresol,

potassium permanganate or ninhydrin) were used. HPLC analysis

were conducted on an Agilent 1200 series LC-MS system with a

C18 column Luna (100 x 2 mm, particle size 3 µm, Phenomenex)

using a linear acetonitrile gradient from 5-95% in 12 min (+ 0.1%

formic acid). HPLCs were monitored at 220 nm. For preparative

RP-HPLC, a LaChrom HPLC system (Merck Hitachi) with Inter-

face D-7000, UV detector L-7520 and pump L-7150 was used.

Purifications were performed with a C18 Jupiter column (250 x

21.2 mm, particle size 10 µm, flowrate 10 mL/min, Phenomenex).

¹H- and ¹³C-NMR spectra were obtained with a BRUKER DRX

500 or BRUKER Avance 500. The measurements were carried

out at 298 K and processed using MestReNova (MestreLab).

Chemical shifts are given relative to residual solvent signals. The

multiplicities are given as s (singlet), d (doublet), t (triplet), q (quar-

tet) and m (multiplet) with coupling constants given in Hertz [Hz].

Azide-modified DARPins were reacted with DBCO-PEG_20k(17)

and DBCO-5(6)-carboxyfluorescein (16) and full conversion was

attained with almost equimolar amounts of the DBCO-containing

building blocks. Additionally, a branched PEG-linker using linear

DBCO-PEG was obtained.

Table 1. Compatibility aspects of the bifunctional building blocks utilized in this

study. Classification is based on the assessment of the obtained results.

Additional re-

active group

Synthetic

Conjugation

efficiency

Solubility

Stability

accessibility

HIPS

aliphatic

azide (6)

DBCO (4)

+

o

-

+

o

-

+/o

-

BCN (5)

mTet (3)

Chemical synthesis:

The chemical synthesis of the Knoevenagel core segment 8,

HIPS-CF (2), HIPS-DBCO (4), clickable precursors, fluorescein

modifications and PEG-linkers were performed similar to proce-

dures describe in the literature and can be found in the supporting

information. The synthesis of the HIPS core segment 1 is already

described in our previous publication.^[27]

-

tandem

Knoevena-

gel

aliphatic

azide (12)

aromatic

azide (13)

mTet

+

o

+

o

+

o

+

o/-

(14)

Fmoc-HIPS-mTet

Fmoc-HIPS (1, 45 mg, 0.09 mmol) was dissolved in DCM/DMF

(9:1, v/v, 4 mL) and HOBt (16 mg, 0.12 mmol) was added. Then,

the obtained solution was combined with (4-(6-methyl-1,2,4,5-te-

Finally, the impact of one- and two-fold fluorescence labeling on

binding behavior was tested by live cell fluorescence imaging.

Treatment of A431 cells (high EGFR expression) and MCF7 cells

(low EGFR expression) with DARPin-CF and DARPin-CF₂re-

sulted in selective receptor mediated endocytosis. Furthermore, a

better signal-to-noise ratio was obtained with DARPins modified

with two molecules of carboxyfluorescein.

All in all, the presented combination of HIPS and Knoevenagel

ligation with click chemistry shows high efficiencies and opens up

new synthetic strategies to complex protein conjugates. This

method could be well suited to generate PEGylated therapeutic

proteins with improved pharmacokinetics or to produce homoge-

nous antibody-drug conjugates with a drug-to-antibody ratio of 2

or 4 for one aldehyde tag at e.g. the heavy chain.

trazin-3-yl)phenyl)methanamine

hydrochloride

(45 mg,

0.19 mmol) and triethylamine (50 µL, 0.36 mmol) and the start of

the reaction was induced by the addition of EDC·HCl (56 mg,

0.29 mmol). The mixture was stirred overnight at rt until a mixture

of petroleum ether/ethyl acetate (1:1, v/v, 30 mL) was added. The

red suspension was washed three times with KHSO₄solution

(0.5 M, 30 mL each) and three times with saturated NaHCO₃so-

lution (30 mL each). The organic layer was dried over MgSO₄and

the solvent was removed in vacuo. The desired product was iso-

lated as a red solid (25 mg, 0.04 mmol, 44%). LC-MS (ESI+): m/z

calc for C₃₉H₃₉N₈O₃[M+H]⁺: 667.31, found: 667.31 (t_R= 11.5 min).

Fmoc-HIPS-BCN

The modular system can be used for the labeling of living cells or

proteins in the lysate. For such experiments, FGly can be intro-

duced by co-expression with FGE. However, a limiting factor in

the lysate are small molecules containing aldehyde moieties.

Such in vivo experiments are part of our current research.

1 (65 mg, 0.13 mmol) and pentafluorophenol (33 mg, 0.18 mmol)

were dissolved in ethyl acetate (abs., 3 mL) and cooled down to

0 °C. Next, DIPEA (30 µL, 0.18 mmol) and solid DCC (29 mg,

0.14 mmol) were added and the reaction mixture was stirred for

2.5 h at rt. After stirring, the obtained suspension was again

cooled to 0 °C, the precipitate was filtered off and the filter cake

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was washed with cold ethyl acetate (10 mL). Subsequently, the

filtrate was washed two times with dH₂O (10 mL each) and two

times with saturated sodium chloride solution (10 mL each). The

organic layer was dried over Na₂SO₄and concentrated in vacuo.

The crude HIPS-PFP ester (116 mg) was then used without fur-

ther purification.

In parallel, BCN-NH₂(25 mg, 0.08 mmol) and HIPS-PFP were dis-

solved in DMF (0.8 mL each), combined under argon and stirred

for 20 h at rt. Afterwards, the solvent was removed in vacuo lead-

ing to the formation of a brownish, solid residue. The residue was

dissolved in a mixture of water and acetonitrile (1:1, v/v, 2 mL)

and directly purified by RP-HPLC with a linear acetonitrile gradi-

ent (5-95% in 50 min, 0.1% TFA). Lyophilization of product-con-

taining fractions enabled the isolation of Fmoc-HIPS-BCN as a

colorless solid (40 mg, 0.05 mmol, 63%). LC-MS (ESI+): m/z calc

for C₄₆H₅₆N₅O₇[M+H]⁺: 790.42, found: 790.46.

197.5 µmol) in DMF (0.5 mL) was stirred for 5 min before a solu-

tion of H₂N-PEG₂-5(6)-CF (118 mg, 217.7 µmol) and DIPEA

(70.5 µL, 414.75 µmol) in DMF (0.5 mL) was slowly added. After

stirring for 4 h, the reaction mixture was directly purified via pre-

parative RP-HPLC. The product (110 mg, 55%) was obtained as

a yellow solid. LC-MS: m/z calc for C₅₈H₅₁N₄O₁₀S [M+H]⁺: 995.33,

found: 995.32 (t_R= 10.1 min). HRMS: m/z calc for C₅₈H₅₁N₄O₁₀S

[M+H]⁺: 995.33204, found: 995.3301.

Trapped Knoevenagel-CF (9)

Trt-trapped Knoevenagel-CF (20 mg, 20.1 µmol) was suspended

in DCM (670 µL) with DMF (3 drops), H₂O (50 µL) and TIPS

(51.4 µL). The reaction mixture was cooled to 0°C und TFA

(410 µL) was slowly added. After 2.75 h of stirring at 0°C, DMF

(3 mL) was added, DCM removed under vacuum and then directly

purified via RP-HPLC. The product (12.6 mg, 83%) was obtained

as a yellow solid. LC-MS: m/z calc for C₃₉H₃₆N₄O₁₀S [M+H]⁺:

753.22, found: 753.26 (t_R= 7.9 min). HRMS: m/z calc for

C₃₉H₃₆N₄O₁₀S [M+H]⁺: 753.22249, found: 753.2219.

HIPS-BCN (5)

Fmoc-HIPS-BCN (11.0 mg, 13.9 µmol) was dissolved in a solu-

tion of dioxane/methanol/2 M NaOH (10:9:1, v/v/v, 1.5 mL) and

stirred for 30 min at rt. The solution was directly purified by RP-

HPLC using a linear acetonitrile gradient (5-95% in 50 min). Ly-

ophilization of product-containing fractions enabled the isolation

of 5 as a pale-yellow solid (4.6 mg, 8.1 µmol, 58%). LC-MS

(ESI+): m/z calc for C₃₁H₄₆N₅O₅[M+H]⁺: 568.34, found: 568.35

(t_R= 7.2 min). HRMS (ESI+): m/z calc for C₃₁H₄₆N₅O₅[M+H]⁺:

568.3494, found: 568.3497.

Tandem Knoevenagel-methyl ester

Ethyl 3-oxobutanoate (587 mg, 4.51 mmol), methyl 4-hydrazino-

benzoate (750 mg, 4.51 mmol), and sodium acetate (740 mg,

9 mmol) were suspended in AcOH (9 mL) and stirred for 3 h at

75°C. After cooling to rt, the solvent was removed under reduced

pressure and a brown solid was obtained. The crude was sus-

pended in H₂O (25 mL) and extracted with DCM (2 x 75 mL). The

combined organic phases were washed with sat. NaHCO₃

(25 mL), brine (25 mL) and dried over MgSO₄. The solvent was

removed under reduced pressure and the product (594 mg,

2.6 mmol, 57%) was obtained after column chromatography (Elu-

ent: EtOAc:PE, 1:4) as a yellow solid. R_f= 0.2 (EtOAc:PE, 1:3).

¹H-NMR (500 MHz, CDCl₃, 25°C) = 8.05 (d, ³J = 8.5 Hz, 2H, Ar-

Fmoc-HIPS-azide

1 (50 mg, 103 µM), HOAt (17.6 mg, 129 µmol), 1-amino-11-az-

ido-3,6,9-trioxaundecane (45.1 mg, 207 µmol) and DIPEA (72 µL,

413 µmol) were dissolved in DCM/DMF (9:1, 1 mL) and after-

wards EDC·HCl (59.5 mg, 310 µmol) was added portionswise.

The mixture was stirred for 5 h at rt, concentrated in vacuo and

finally diluted with DMF. After RP-HPLC and lyophilization, Fmoc-

HIPS-azide (52 mg, 74%) was isolated as a brown oil. LC-MS

(ESI+): m/z calc for C₃₇H₄₆N₇O₆[M+H]⁺: 684.35, found: 684.35

(t_R= 11 min). HRMS (ESI+): m/z calc for C₃₇H₄₅N₇O₆Na [M+Na]⁺:

706.33235, found: 706.3321.

3

H), 7.99 (d, J = 8.6 Hz, 2H,Ar-H), 3.90 (s, 3H, -OCH₃), 3.45 (s,

2H, -CH₂-), 2.20 (s, 3H, -CH₃). ¹³C-NMR (126 MHz, CDCl₃) =

170.89 (N-C=O), 166.74 (-CO-OCH₃), 157.02 (N-C-CH₃), 141.90

(N-C_Ar), 130.69 (C_Ar-C_Ar-C=O), 126.17 (C_Ar-C=O), 117.74 (N-C_Ar-

C_Ar), 52.16 (-CO-OCH₃), 43.27 (CH3-CN-CH₂), 17.19 (-CH₃). LC-

MS (ESI+) = m/z calc for C₁₂H₁₃N₂O₃[M+H]⁺: 233.09, found

233.12 (t_R= 7.5 min). HRMS (ESI+): m/z calc for C₁₂H₁₂N₂O₃Na

[M+Na]⁺: 255.07401, found 255.0739.

HIPS-azide (6)

Fmoc-HIPS-azide (50 mg) was dissolved in a solution of 20% pi-

peridine in THF (2.3 mL) and stirred for 1 h at rt. Water was added

to the reaction mixture until a colorless precipitate formed. The

solid was filtered off and the filtrate was concentrated in vacuo

and the residue was freeze-dried. Finally, the lyophilizate

(25.9 mg, 77%) could be directly employed for bioconjugations,

since LC-MS analysis revealed >95% purity and significant deg-

radation during purification by RP-HPLC were observed. Moreo-

ver, due to rapid oxidative decomposition of purified 6, the ob-

tained oil was immediately dissolved in a mixture of water and

acetonitrile (1:1, v/v) to a final concentration of 1 M and aliquots

of that solution were stored at -80°C until further use in bioconju-

gation reactions. LC-MS (ESI+): m/z calc for C₂₂H₃₆N₇O₄[M+H]⁺:

462.28, found: 462.32 (t_R= 5.2 min). HRMS (ESI+): m/z calc for

C₂₂H₃₆N₇O₄[M+H]⁺: 462.28233, found: 462.2839.

Tandem Knoevenagel-acid (11)

Tandem Knoevenagel-methyl ester (500 mg, 2 mmol) was dis-

solved in 1 M NaOH (15 mL) and stirred for 3.5 h at rt. Afterwards,

the reactions mixture was diluted with aq. HCl until pH of 4 was

reached. The product (414 mg, 93%) was obtained through filtra-

tion as a beige-colored solid. R_f= 0.6 (DCM:MeOH:AcOH,

9:1:0.01). ¹H-NMR (500 MHz, DMSO-d₆, 25°C, Iminol): 7.98 (d, ³J

= 8.7 Hz, 2H, Ar-H), 7.88 (d, ³J = 8.6 Hz, 2H, Ar-H), 5.39 (s, 1H,

CN-CH-C-OH), 2.13 (s, 3H, -CH₃). ¹³C-NMR (126 MHz, DMSO-

d₆): 171.71(-N-C=O), 166.87 (-COOH), 166.82 (-COOH) 159.37

(-C-CH₃), 153.92 (-N-C-OH), 149.08 (-C-CH₃), 142.49 (-C_Ar),

141.63 (-C_Ar), 130.48 (-C_Ar), 130.36 (-C_Ar), 126.49 (-C_Ar), 126.04

(-C_Ar), 119.23 (-C_Ar), 116.92 (-C_Ar), 88.20 (-CH₃-CN-CH=), 43.16

(-CH₃-CN-CH₂-), 16.71 (-CH₃), 14.26 (-CH₃). LC-MS (ESI+): m/z

calc for C₁₁H₁₁N₂O₃[M+H]⁺: 219.07, found 219.09 (t_R= 5.6 min).

HRMS (ESI+): m/z calc for C₁₁H₁₂N₂O₃Na [M+Na]⁺: 241.05836,

found 241.0578.

Trt-trapped Knoevenagel-CF

A

solution of Trt-trapped Knoevenagel-acid (8, 100 mg,

197.7 µmol), HATU (75 mg, 197.2 µmol) and DIPEA (33.5 µL,

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Tandem Knoevenagel-azide (12)

78.9 µmol) were dissolved in DMF (1 mL) under argon atmos-

phere and then EDC·HCl (65.9 mg, 34.4 µmol) was added in por-

tions over 5 min. The reaction mixture was stirred overnight at rt

in the dark. The product (30 mg, 66%) was directly purified via

preparative RP-HPLC and obtained as a pale orange-colored

H₂N-PEG₃-N₃(75 mg, 34.4 µmol), HOAt (46.8 mg, 34.4 µmol),

tandem Knoevenagel-acid (11, 50 mg, 22.9 µmol) and DIPEA

(60 µL, 34.4 µmol) were dissolved in DMF (1 mL) under argon at-

mosphere and EDC·HCl (65.9 mg, 34.4 µmol) was added in por-

tions over 10 min. The reaction mixture was stirred overnight at rt

and then directly purified via preparative RP-HPLC. After freeze

drying, azide 12 (106 mg, 74%) was obtained as a yellowish solid.

¹H-NMR (500 MHz, CDCl₃, 25°C): 7.96 (d, ³J = 8.4 Hz, 2H, Ar-H),

7.82 (d, ³J = 8.5 Hz, 2H, Ar-H), 6.80 (s, 1H, -NH-), 3.63 (m, 14H,

1

solid. Amide-iminol-ratio: 8:2. H-NMR (600 MHz, CDCl₃, 25°C):

3

7.95 (d, J = 8.8 Hz, 2H, Ar-H, Amide), 7.80 (d, J = 8.8 Hz, 2H,

Ar-H, Amide), 7.69 (m, 2H, Ar-H, Iminol), 7.50 (m, 2H, Ar-H, Imi-

3

nol), 7.42 (m, 2H, N₃-Ar-H, Amide), 7.36 (d, 2H, J = 8.6 Hz, N₃-

Ar-H, Iminol), 7.20 (s, 1H, N-H), 7.00 (s, 1H, N-H), 5.64 (s, 2H,

-CN-CH=C-OH, Iminol), 3.63 (m, 16H, PEG), 3.46 (s, 2H,Pz-H,

Amide), 2.32 (s, 3H, CH₃, Iminol), 2.21 (s, 3H, CH₃, Amide). ¹³C-

NMR (151 MHz, CDCl₃, 25°C): 171.07 (-N-CO-CH₂-), 167.71 (-

CO-NH-PEG), 164.75 (-NH-CO-C_Ar-C_ArH-C_ArF-), 160.64 (-N-C-

OH), 157.27 (-N=C-CH₃), 156.10 (-C_Ar), 154.43 (-C_Ar), 150.01 (-

N-C-CH₃), 140.85 (-CO-N-C_Ar), 136.75 (HO-C-N-C_Ar), 133.09 (-

C_Ar-CO-NH-PEG), 131.13 (-C-F), 130.07 (-C_Ar-CO-NH-PEG),

128.46 (-C_Ar-C_Ar-CO-NH-PEG), 128.08 (-C_Ar-C_Ar-CO-NH-PEG),

121.67 (-N-C_Ar-C_Ar) 118.07 (-N-C_Ar-C_Ar), 111.43 (-NH-CO-C_Ar-C_Ar-

C_Ar-F), 93.27 (-CH-C-OH), 70.52 (PEG), 70.48 (PEG), 70.29

(PEG), 70.24 (PEG), 69.83 (PEG), 69.68 (PEG), 43.31 (-CH₂-

CO-), 40.19 (PEG), 40.04 (PEG), 17.16 (-CH₃), 12.33 (-CH₃). LC-

MS (ESI+): m/z calc for C₂₆H₃₀F₂N₇O₆[M+H]⁺: 574.22, found

574.24 (t_R= 8.1 min). HRMS (ESI+): m/z calc for C₂₆H₂₉F₂N₇O₆Na

[M+Na]⁺: 596.20396, found 596.2042.

3

PEG), 3.44 (s, 2H, -CN-CH₂-C=O), 3.33 (t, J = 5.1 Hz, 2H, N₃-

CH₂-), 2.19 (s, 3H, CH₃). ¹³C-NMR (126 MHz, CDCl₃, 25°C):

166.90 (-C_Ar-C=O), 156.91 (CH₃-C-N), 140.68 (-C_Ar-N), 130.58 (-

C_ar-C=O), 128.00 (-C_Ar-C_Ar-C=O), 117.97 (-C_Ar-C_Ar-N), 70.77

(PEG), 70.74 (PEG), 70.67 (PEG), 70.35 (PEG), 70.12 (PEG),

69.91 (PEG), 50.73 (-CH₂-N₃), 43.25 (-CH₂-C=O), 39.86 (PEG),

17.16 (-CH₃). LC-MS (ESI+): m/z calc for C₁₉H₂₇N₆O₅[M+H]⁺:

419.20, found 419.22 (t_R= 6.3 min). HRMS (ESI+): m/z calc for

C₁₉H₂₆N₆O₅Na [M+Na]⁺: 441.18569, found 441.1849.

Tandem Knoevenagel PEG-amine trifluoroacetate

Tandem Knoevenagel-azide (12, 50 mg, 119.5 µmol) was dis-

solved in THF (970 µL) and then PPh₃(94 mg, 358.4 µmol) was

added in portions. After stirring for 1 h at rt, H₂O (100 µL) was

added and an orange suspension was obtained. To obtain better

solubility, further THF (4 mL) and H₂O (400 µL) was added. After

17 h, only the iminophopshorane was detected via LC-MS, which

is why the solution was acidified wit 1 M HCl until pH 1 was

reached and then the solvent was removed under reduced pres-

sure. Again, the iminophosphorane was obtained as the major

product. Therefore, the crude was again dissolved in THF:H₂O

(1:1, 5 mL) and heated to 60°C for 3.5 h. Still iminophosphrane

was detected, consequently AcOH was added until pH 4 was

reached and then the reaction mixture was heated to 80°C and

stirred for 20 h. After removal of the solvent and preparative RP-

HPLC, the product (45.5 mg, 75%) was obtained as a colorless

Tandem Knoevenagel-PEG-tert butyl ester

Tandem Knoevenagel-acid (11, 100 mg, 0.46 mmol), HOAt

(93.6 mg, 0.69 mmol) and H₂N-PEG₃-COOtBu (181 mg,

0.68 mmol) were dissolved in DMF (1 mL) under argon atmos-

phere and then DIPEA (117 µL, 0.69 mmol) was added. Finally,

EDC·HCl (131.8 mg, 0.69 mmol) was added in portions over 5

min. The reaction mixture was stirred overnight at rt before the

product (90 mg, 42%) was purified directly via preparative RP-

1

HPLC. Amid-Iminol-ratio: 20:1. H-NMR (600 MHz, CDCl₃, 25°C):

3

7.96 (d, J = 8.8 Hz, 2H, Ar-H), 7.84 (d, J = 8.8 Hz, 2H, Ar-H),

7.04 (br s, 1H, NH) 3.96 (s, 2H, CH₂-COOtBu), 3.66 (m, 12H,

PEG), 3.44 (s, 2H, -CN-CH₂-CO), 2.20 (s, 3H, CH₃), 1.44 (s, 9H,

C(CH₃)₃). ¹³C-NMR (151 MHz, CDCl₃, 25°C): 170.85 (-N-N-CO),

169.77 (-COOtBu), 167.33 (-CO-NH), 156.92 (-N-C-CH₃), 140.75

(-N-N-C_Ar), 130.38 (-C_Ar-CO-NH), 128.12 (-C_Ar-C -CO-NH),

1

oil. Amide-Iminol-ratio: 8:2. H-NMR (500 MHz, ACN-d₃, 25°C):

3

7.95 (d, J = 8.8 Hz, 2H, Ar-H, Amide), 7.88 (d, ³J = 8.9 Hz, 2H,

Ar-H, Iminol), 7.86 (d, ³J = 8.9 Hz, 2H, Ar-H, Amide), 7.77 (d, ³J =

8.7 Hz, 2H, Ar-H, Iminol), 7.6 (s, 1H, N-H, Iminol), 7.54 (s, 1H, N-

H, Amide), 7.34 (s, 3H, NH₂), 5.53 (s, 1H, -CN-CH=C-OH, Iminol),

3.70 (m, PEG), 3.66 (m, PEG), 3.59 (m, PEG), 3.48 (s, 2H, -CN-

CH₂-CO, Amide), 3.13 (m, PEG), 3.09 (m, PEG), 2.24 (s, 3H, -

CH3, Iminol), 2.13 (s, 3H, -CH₃, Amide). ¹³C-NMR: 172.65 (-N-

C=O), 168.36 (-C_Ar-C=O), 168.04 (-C_Ar-C=O), 160.67 (-C-OH),

160.82 (TFA), 159.59 (-C-CH₃), 151.65 (-C-CH₃), 142.18 (-C_Ar-N),

139.95 (-C_Ar-N), 132.62 (-C_Ar-CO-NH-), 130.58 (-C_Ar-CO-NH-),

129.15 (-C_Ar-C_Ar-CO-), 129.07 (-C_Ar-C_Ar-CO-), 121.49 (-N-C_Ar-C_Ar),

118.36 (-N-C_Ar-C_Ar), 117.36 (TFA), 92.70 (-CH-C-OH), 70.90

(PEG), 70.86 (PEG), 70.80 (PEG), 70.73 (PEG), 70.70 (PEG),

70.64 (PEG), 70.51 (PEG), 70.45 (PEG), 67.27 (PEG), 43.90 (-

r

A

118.00 (-C_Ar-C -N-CO), 81.86 (-C(CH ) ), 70.73 (PEG), 70.64

r

A

3 3

(PEG), 70.62 (PEG), 70.32 (PEG), 69.94 (PEG), 69.06 (-CH₂-

COOtBu), 43.28 (-CH₂-CO-N-), 40.03 (PEG), 28.21 (-C(CH₃)₃),

17.16 (-N-C-CH₃). LC-MS (ESI+): m/z calc for C₂₃H₃₄N₃O₇[M+H]⁺:

464.24, found 464.25 (t_R= 7.5 min). HRMS (ESI+) m/z calc for

C₂₃H₃₃N₃O₇Na[M+Na]⁺: 486.22107, found 486.2207.

Tandem Knoevenagel-PEG-acid

Tandem Knoevenagel-PEG-tert butyl ester (61 mg, 132 µmol)

was dissolved in DCM (10 mL) and a 4 M solution of HCl in diox-

CH₂-C-OH), 40.59 (PEG), 40.57 (PEG) 40.51 (PEG), 17.09 (-CH₃), ane (10 mL) was added. The reaction mixture was stirred for 3.5 h

13.03 (-CH₃). LC-MS (ESI+): m/z calc for C₁₉H₂₉N₄O₅[M+H]⁺:

393.21, found 393.25 (t_R= 3.9 min). HRMS (ESI+): m/z calc for

C₁₉H₂₉N₄O₅[M+H]⁺: 393.21325, found 393.2127.

at rt. After removal of the solvent, this procedure was repeated for

2.5 h. The product (42 mg, 78%) was obtained after preparative

RP-HPLC as a yellowish oil. Amid-Iminol-ratio: 7:3. ¹H-NMR (600

MHz, CDCl₃, 25°C): 7.93 (d, ³J = 8.82 Hz, 2H, Ar-H, Iminol), 7.84

(d, ³J = 8.22 Hz, 2H, Ar-H, Amide), 7.81 (d, ³J = 8.67 Hz, 2H, Ar-

Tandem Knoevenagel-aryl azide (13)

3

Tandem Knoevenagel-PEG-amine trifluoroacetate (23.6 mg,

78.9 µmol), HOAt (16.1 mg, 118.5 µmol), 4-Azido-3,5-difluoro-

benzoic acid (23.6 mg, 118.5 µmol) and DIPEA (14 µL,

H, Iminol), 7.64 (d, J = 7.78 Hz, 2H, Ar-H, Amide), 7.49 (s, 1H,

N-H, Amide), 7.34 (s, 1H, N-H, Iminol), 5.63 (s, 2H, -CN-CH=CO-,

Iminol), 4.05 (s, 2H, -CH₂-COOH, Amide), 4.03 (s, 2H, -CH₂-

COOH, Iminol), 3.57 (m, 12H, PEG), 3.47 (s, 2H, -CN-CH₂-CO,

10

10.1002/cbic.202000416

ChemBioChem

FULL PAPER

Amide), 2.28 (s, 3H, -CH₃, Amide) , 2.13 (s, 3H, -CH₃, Iminol). ¹³C-

NMR (151 MHz, CDCl₃, 25°C): 172.66 (-N-N-CO), 172.50 (-

COOH), 172.32 (-COOH), 169.00 (-CO-NH), 167.60 (-CO-NH),

161.21 (-CN-CH=CO-), 159.56 (-CN-CH₃), 151.45 (-CN-CH₃),

141.99 (-N-C_Ar), 138.17 (-N-C_Ar), 133.97 (-C_Ar-CO-NH), 130.82 (-

C_Ar-CO-NH), 129.29 (-C_Ar-C_Ar-CO), 128.99 (-C_Ar-C_Ar-CO), 122.45

(-C_Ar-C_Ar-N) 118.37 (-C_Ar-C_Ar-N), 93.48 (CH=C-OH), 71.50 (PEG),

71.45 (PEG), 70.13 (PEG), 71.06 (PEG), 70.04 (PEG), 68.82 (-

CH₂-COOH), 43.91 (-CH₂-CO), 40.68 (PEG), 40.55 (PEG), 17.09

(-CH₃), 12.59 (-CH₃). LC-MS (ESI+): m/z calc for C₁₉H₂₆N₃O₇

[M+H]⁺: 408.17, found 408.18 (t_R= 5.2min). HRMS (ESI+) m/z

calc for C₁₉H₂₅N₃O₇Na [M+Na]⁺: 430.15847, found 430.1577.

DNaseI were added. Cells lysis was performed mechanically by

French Press (3 x 1000 psi). Cell debris was removed via centrif-

ugation (4°C, 10000 rpm, 30 min) and filtration (pore size 0.22

µm). The proteins were purified via an chromatography system

(ÄKTA Explorer, GE Healthcare) at 4°C and Ni²⁺-NTA affinity

chromatography (HisTrap HP, 1 mL, GE Healthcare). The sample

was loaded and washed with binding buffer (50 mM NaH₂PO₄,

300 mM NaCl, 25 mM Imidazol, pH 8.0) and then eluted using a

linear imidazole gradient (25-250 mM). The isolated enzyme,

tested by SDS-Gel, was pooled and rebuffered in PBS (pH 7.4)

using Vivaspin centrifugal concentrators (MWCO 5000, Sartorius).

For MtFGE and DARPin, an average of 30-40 mg was obtained

per 1 L of cell culture. The enzyme was either used immediately

or stored in the presence of 200 mM arginine and the DARPin with

20 mM arginine at -80°C until further use.

Tandem Knoevenagel-mTet (14)

Tandem Knoevenagel-PEG-acid (40 mg, 98.2 µmol), 6-methyl-

tetrazine-amine (28 mg, 117.8 µmol), HOAt (20 mg, 147.3 µmol)

and DIPEA (25 µL, 147.3 µmol) were dissolved in DMF (1 mL).

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was per-

formed using 10, 12.5 or 15% acrylamide in the running gel and

EDC·HCl (28.2 mg, 147.3 µmol) was added in portions over 5 min. 5% in the stacking gel. Bisacrylamide was used as crosslinker.

Finally, the reaction mixture was stirred overnight at rt and then

directly purified via preparative HPLC. 14 (53 mg, 86%) was ob-

tained as a red solid. LC-MS analysis showed a slow formation of

a side product, which is why this product was only analyzed via

LC-MS and HRMS. LC-MS (ESI+): m/z calc for C₂₉H₃₅N₈O₈

[M+H]⁺: 591.27, found 591.20 (t_R= 6.8 min). HRMS (ESI+): m/z

calc for C₂₉H₃₅N₈O₈[M+H]⁺: 591.26741, found 591.2670.

Protein samples were incubated either for 5 min at 95°C or for 1 h

at 37°C with 4x Laemmli buffer plus 20% 2-mercaptoethanol.

Separation was obtained overnight with 4 mA or in 3 h with 30 mA.

After completion, the gel was washed with water for 5 min and

then treated with staining solution (50% MeOH, 10% AcOH, 40%

H₂O, 0.1% Coomassie Brilliant Blue) for 3 h. Destaining was per-

formed after several washing steps with 40% MeOH, 10% AcOH

and 40% H₂O, followed by documentation with a CCD camera

(Fujifilm).

BCN-CF (15)

BCN-NHS (4.9 mg, 16.7 µmol), H₂N-PEG₂-5(6)-CF (10 mg,

18.4 µmol) and DIPEA (6.1 µL, 35 µmol) were dissolved in dry

DMF (0.5 mL) and stirred overnight at rt. The reaction mixture was

directly purified via preparative RP-HPLC. 15 (quant.) was ob-

tained as a yellow solid. LC-MS (ESI+): m/z calc for C₃₈H₃₉N₂O₁₀

[M+H]⁺: 683.26, found: 683.26 (t_R= 8.4 min). HRMS (ESI+): m/z

calc for C₃₈H₃₉N₂O₁₀[M+H]⁺: 683.25992, found: 683.2595.

Protein concentrations were determined via Bradford assay with

Coomassie brilliant blue G-250 (200 µL) and a BSA standard. All

samples were measured in duplicates.

Enzymatic conversion of DARPin E01 with MtFGE

DARPin-E01 with CTPSR-motif (574 µL in PBS buffer containing

20 mM arginine, 7.63 mg/mL) was diluted with bicine buffer

(800 µL, 150 mM bicine, 200 mM NaCl, 600 mM arginine), water

(721 µL) and 100 mM DTT (120 µL) and subsequently incubated

with MtFGE (180 µL in PBS + 20 mM arginine, 5 mg/mL, final con-

centration 10 µM), 10 mM CuSO₄(2.4 µL, final concentration

10 µM) and 10 mM CaCl₂(2.4 µL, final concentration 10 µM) for

16-20 h at 22-25°C. The reaction progress was monitored via

tryptic digest (4 h at 37°C) and MALDI-ToF-MS (UltrafleXtreme,

Bruker Daltonics). Precipitated MtFGE was centrifuged and the

FGly-containing DARPin rebuffered into either HIPS (100 mM

phosphate, 20 mM NaCl, pH 6.0) or Knoevenagel ligation buffer

(50 mM citrate, 50 mM NaCl, pH 7.2) using size exclusion desalt-

ing with a PD10 (GE) column.

DBCO-CF (16)

DBCO-NHS (7.2 mg, 16.7 µmol), H₂N-PEG₂-5(6)-CF (10 mg, 18.4

µmol) and DIPEA (6.1 µL, 35 µmol) were dissolved in dry DMF

(0.5 mL) and stirred overnight at rt. The reaction mixture was di-

rectly purified via preparative RP-HPLC and 16 (4.2 mg, 31%)

was obtained as a yellow solid with a purity of approx. 90%

(HPLC). LC-MS (ESI+): C₄₈H₄₄N₃O₁₀[M+H]⁺: 822.30, found:

822.29 (t_R= 4.6 min). HRMS (ESI+): C₄₈H₄₄N₃O₁₀[M+H]⁺:

822.30212, found: 822.3015.

Biochemical experiments:

Expression and purification of MtFGE and DARPin E01

For MtFGE a 60 mL LB pre-culture containing ampicillin was in-

oculated with E. coli BL21 (DE3) containing the vector pET14b-

mtFGE-His₆. For the DARPin-construct a kanamycin pre-culture

with E. coli BL21 (DE3) containing pET24b-DARPinE01-CTPSR-

His₆(see supplement material; sequences) was generated. After

overnight incubation at 37°C and shaking, a 1 L main culture was

inoculated by the harvested cells, respectively. The cultures were

incubated at 37°C and 160 rpm until an optical density (OD₆₀₀) of

0.6 was reached. Afterwards, the cells were cooled to 18°C, IPTG

(200 µM) was added and the cultures were incubated for 16 h at

18°C and 150 rpm. Finally, cells were harvested and stored at -

20°C until further use. The pellets were resuspended in PBS (pH

7.4) and EDTA-free protease inhibitor (Complete, Roche) and

Time- and equivalent-dependent fluorescent labeling of

FGly-containing DARPin E01

30 µM of FGly-containing DARPin E01 in HIPS or Knoevenagel

buffer was treated for 24 h at 22°C with 2.5, 5, 7.5 and 10 equiv-

alents of HIPS-CF (2) or trapped Knoevenagel-CF (9). Samples

(15 µg of protein) were taken after 2, 6, 10 and 24 h and stored at

-80°C until they were analyzed via SDS-PAGE (15%). Therefore,

the samples were diluted with 4xLaemmli buffer (containing 20%

2-mercaptoethanol) and incubated for 5 min at 95°C.

Preparative fluorescent labeling via trapped Knoevenagel

For complete labeling via standard Knoevenagel chemistry,

100 µM of the previously obtained FGly-containing DARPin was

incubated for 24 h at 22°C with 1 mM of trapped Knoevenagel-

11

10.1002/cbic.202000416

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FULL PAPER

CF (9). Afterwards, DARPin-CF was rebuffered into PBS (pH 7.4)

using PD10 and stored at -80°C until further use.

Acknowledgements

This work was supported by Deutsche Forschungsgemeinschaft

as part of the priority program SPP 1623 (DI 575/9-1, MU 2286/6-

1, SE 609/15-1). The pET14b-mtFGE-His₆plasmid was provided

by David Rabuka (Redwood Bioscience, Emeryville, USA).

Conversion of FGly-DARPin with azides 6 and 12

The obtained FGly-containing DARPins were directly treated ei-

ther with 2 mM of 6 for 16 h at 30°C or with 10 mM of 12 for 24 h

at 22°C. Afterwards, DARPin-N₃(generated using 6) and DAR-

Pin-(N₃)₂(generated using 12) were rebuffered into a phosphate

buffer (8% DMSO, 100 mM phosphate, 150 mM NaCl, pH 7.2)

and stored at -80 until further use.

Conflict of Interest

The authors declare no conflict of interest.

Stability assay with HIPS-azide (6)

HIPS-azide (6) was diluted under aerobic conditions in HIPS liga-

tion buffer (final concentration of 2 mM) and different additives

(1 mM EDTA, 0.1 mM CuI, 0.1 mM CuSO₄) were added. Addition-

ally, HIPS-azide (6) was incubated without additives under aero-

bic and anaerobic conditions. The samples were incubated for

16 h at 30°C and subsequently analyzed via LC-MS.

Keywords: bioconjugation

Knoevenagel ligation • click chemistry • branched PEG-linker

•

formylglycine

•

HIPS and

[1]

[2]

[3]

C.D. Spicer, B.G. Davis, Nat. Commun. 2014, 5, 1-14.

A. Borrmann, J. C. M. Van Hest, Chem. Sci. 2014, 5, 2123–2134.

E. M. Sletten, C. R. Bertozzi, Angew. Chem. Int. Ed. 2009, 48, 6974–

6998.

Click-reaction with different strained alkynes

DARPin-N₃and DARPin-(N₃)₂were treated with different equiva-

lents of DBCO-CF (16) for 4 h at 22°C, BCN-CF (15) for 16 h at

22°C or DBCO-PEG_20k(17) for 16 h at 37°C. Afterwards, the re-

actions were directly analyzed via SDS-PAGE. Samples were di-

luted with 4xLaemmli buffer (containing 20% 2-mercaptoethanol)

and incubated for 1 h at 37°C.

For selective PEG-staining, the colored/non-colored gel was

washed with water (3x5 min), incubated for 10 min with a 5%

BaCl₂solution, washed again with water (3x2 min) and finally in-

cubated for 5 min with 0.1% iodine solution.

[4]

[5]

P. Shieh, C. R. Bertozzi, Org. Biomol. Chem. 2014, 12, 9307–9320.

J. E. Hudak, R. M. Barfield, G. W. Dehart, P. Grob, E. Nogales, C. R.

Bertozzi, D. Rabuka, Angew. Chem. Int. Ed. 2012, 51, 4161–4165.

S. Bloess, T. Beuel, T. Krüger, N. Sewald, T. Dierks, G. Fischer von Mol-

lard, Appl. Microbiol. Biotechnol. 2019, 103, 2229–2241.

P. M. Drake, A. E. Albers, J. Baker, S. Banas, R. M. Barfield, A. S. Bhat,

G. W. De Hart, A. W. Garofalo, P. Holder, L. C. Jones, et al., Bioconjug.

Chem. 2014, 25, 1331–1341.

[6]

[7]

[8]

[9]

R. Kudirka, R. M. Barfield, J. McFarland, A. E. Albers, G. W. De Hart, P.

M. Drake, P. G. Holder, S. Banas, L. C. Jones, A. W. Garofalo, et al.,

Chem. Biol. 2015, 22, 293–298.

Q. Wang, A. R. Parrish, L. Wang, Chem. Biol. 2009, 16, 323–336.

For preparative scale, the DARPin-CF₂, generated by 12 and 16,

was rebuffered into PBS (pH 7.4) using PD10 and stored at -80°C

until further use.

[10] H. Neumann, FEBS Lett. 2012, 586, 2057–2064.

[11] K. Strijbis, E. Spooner, H. L. Ploegh, Traffic 2012, 13, 780–789.

[12] R. R. Beerli, T. Hell, A. S. Merkel, U. Grawunder, PLoS One 2015, 10,

1–17.

Live cell imaging

[13] M. W. L. Popp, H. L. Ploegh, Angew. Chem. Int. Ed. 2011, 50, 5024–

5032.

A431 and MCF7 cell lines were cultivated in µ-Slides 8 Well (ibidi

GmbH) with 2.5·10⁴cells per 1.0 cm²surface area well (37°C, 5 %

CO₂atmosphere). Cells were washed with RPMI medium once

before and twice after incubation with 100 nM DARPin conjugates

(in PBS, pH 7.4) for 6-8 h either at 4°C or 37°C. 5 % CO₂atmos-

phere was not maintained at 4°C. LysoTracker DND-22 was

added to a final concentration of 150 nM to cells incubated at

37°C. Imaging was performed with the confocal fluorescence mi-

croscope Zeiss LSM 780 using the objective LCI Plan-Neofluar

63x/1.3 Imm Corr DIC M27. The line sequential scanning mode

for the green (ex. 488 nm, em. 499-553 nm) and blue (ex. 405 nm,

em. 415-466 nm) channel was used to minimize the effect of ly-

sosomal movement for colocalization analysis. The argon ion la-

ser was run with 2.0% laser power to excite CF. The green fluo-

rescence was detected by the 32-ch GaAsp detector (gain 700,

detector offset 0 digital gain 1.0). 12-bit images were processed

with the ImageJ software: equal brightness adjustment for green

channels (3-345), rescaling to 16-bit images, analysis of mean flu-

orescence intensity, individual thresholding for both channels for

pseudo-coloring of overlaying pixels.

[14] P. R. Spycher, C. A. Amann, J. E. Wehrmüller, D. R. Hurwitz, O. Kreis,

D. Messmer, A. Ritler, A. Küchler, A. Blanc, M. Béhé, et al., ChemBio-

Chem 2017, 18, 1923–1927.

[15] P. Dennler, A. Chiotellis, E. Fischer, D. Brégeon, C. Belmant, L. Gauthier,

F. Lhospice, F. Romagne, R. Schibli, Bioconjug. Chem. 2014, 25, 569–

578.

[16] V. Siegmund, S. Schmelz, S. Dickgiesser, J. Beck, A. Ebenig, H. Fittler,

H. Frauendorf, B. Piater, U. A. K. Betz, O. Avrutina, et al., Angew. Chem.

Int. Ed. 2015, 54, 13420–13424.

[17] H. Schneider, L. Deweid, O. Avrutina, H. Kolmar, Anal. Biochem. 2020,

595, 113615.

[18] M. Rashidian, S. C. Kumarapperuma, K. Gabrielse, A. Fegan, C. R. Wag-

ner, M. D. Distefano, J. Am. Chem. Soc. 2013, 135, 16388–16396.

[19] K. F. Geoghegan, J. G. Stroh, Bioconjug. Chem. 1992, 3, 138–146.

[20] J. M. Gilmore, R. A. Scheck, A. P. Esser-Kahn, N. S. Joshi, M. B. Francis,

Angew. Chem. Int. Ed. 2006, 45, 5307–5311.

[21] J. Landgrebe, T. Dierks, B. Schmidt, K. Von Figura, Gene 2003, 316, 47–

56.

[22] J. Peng, S. Alam, K. Radhakrishnan, M. Mariappan, M. G. Rudolph, C.

May, T. Dierks, K. Von Figura, B. Schmidt, FEBS J. 2015, 282, 3262–

3274.

[23] K. Von Figura, B. Schmidt, T. Selmer, T. Dierks, BioEssays 1998, 20,

505–510.

[24] M. Knop, T. Q. Dang, G. Jeschke, F. P. Seebeck, ChemBioChem 2017,

18, 161–165.

[25] P. G. Holder, L. C. Jones, P. M. Drake, R. M. Barfield, S. Bañas, G. W.

De Hart, J. Baker, D. Rabuka, J. Biol. Chem. 2015, 290, 15730–15745.

12

10.1002/cbic.202000416

ChemBioChem

FULL PAPER

[26] T. Dierks, B. Schmidt, L. V. Borissenko, J. Peng, A. Preusser, M. Mariap-

pan, K. Von Figura, Cell 2003, 113, 435–444.

[64] A. Moosmann, E. Müller, H Böttinger, Methods Mol. Biol. - Protein Down-

str. Process. Des. Dev. Appl. High Low-Resolution Methods 2014, 1129,

572–538.

[27] T. Dierks, EMBO J. 1999, 18, 2084–2091.

[28] T. Krüger, S. Weiland, G. Falck, M. Gerlach, M. Boschanski, S. Alam, K.

M. Müller, T. Dierks, N. Sewald, Angew. Chem. Int. Ed. 2018, 57, 7245–

7249.

[65] F. Selis, G. Focà, A. Sandomenico, C. Marra, C. Di Mauro, G. S. Jotti, S.

Scaramuzza, A. Politano, R. Sanna, M. Ruvo, et al., Int. J. Mol. Sci. 2016,

17, 1–15.

[29] R. J. Spears, M. A. Fascione, Org. Biomol. Chem. 2016, 14, 7622–7638.

[30] P. Agarwal, C. R. Bertozzi, Bioconjug. Chem. 2015, 26, 176–192.

[31] A. Dirksen, P. E. Dawson, Bioconjug. Chem. 2008, 19, 2543–2548.

[32] A. Dirksen, T. M. Hackeng, P. E. Dawson, Angew. Chem. Int. Ed. 2006,

45, 7581–7584.

[66] R. C. Feiner, I. Pennè, B. Müller, K. M. Müller, Biochemistry 2019, 58,

1043–1047.

[67] R. B. Dickson, J. A. Hanover, M. C. Willingham, I. Pastan, Biochemistry

1983, 22, 5667–5674.

[33] B. M. Mueller, W. A. Wrasidlo, R. A. Reisfeld, Bioconjug. Chem. 1990, 1,

325–330.

[34] I. S. Carrico, B. L. Carlson, C. R. Bertozzi, Nat. Chem. Biol. 2007, 3, 321–

322.

[35] M. A. Frese, T. Dierks, ChemBioChem 2009, 10, 425–427.

[36] J. Kalia, R. T. Raines, Angew. Chem. Int. Ed. 2008, 47, 7523–7526.

[37] T. Sasaki, K. Kodama, H. Suzuki, S. Fukuzawa, K. Tachibana, Bioorg.

Med. Chem. Lett. 2008, 18, 4550–4553.

[38] P. Agarwal, J. Van Der Weijden, E. M. Sletten, D. Rabuka, C. R. Bertozzi,

Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 46–51.

[39] P. Agarwal, R. Kudirka, A. E. Albers, R. M. Barfield, G. W. De Hart, P. M.

Drake, L. C. Jones, D. Rabuka, Bioconjug. Chem. 2013, 24, 846–851.

[40] S. Pomplun, M. Y. H. Mohamed, T. Oelschlaegel, C. Wellner, F. Berg-

mann, Angew. Chem. Int. Ed. 2019, 58, 3542-3547.

[41] M. J. Han, D. C. Xiong, X. S. Ye, Chem. Commun. 2012, 48, 11079–

11081.

[42] J. Alam, T. H. Keller, T. P. Loh, J. Am. Chem. Soc. 2010, 132, 9546–

9548.

[43] P. Wang, S. Zhang, Q. Meng, Y. Liu, L. Shang, Z. Yin, Org. Lett. 2015,

17, 1361–1364.

[44] R. A. Kudirka, R. M. Barfield, J. M. McFarland, P. M. Drake, A. Carlson,

S. Banas, W. Zmolek, A. W. Garofalo, D. Rabuka, ACS Med. Chem. Lett.

2016, 7, 994–998.

[45] J. Yu, D. Shen, H. Zhang, Z. Yin, Bioconjug. Chem. 2018, 29, 1016–1020.

[46] B. Spangler, S. Yang, C. M. Baxter Rath, F. Reck, B. Y. Feng, ACS Chem.

Biol. 2019, 14, 725–734.

[47] R. Walder, M. A. Leblanc, W. J. Van Patten, D. T. Edwards, J. A. Green-

berg, A. Adhikari, S. R. Okoniewski, R. M. A. Sullan, D. Rabuka, M. C.

Sousa, et al., J. Am. Chem. Soc. 2017, 139, 9867–9875.

[48] M. F. Debets, S. S. Van Berkel, S. Schoffelen, F. P. J. T. Rutjes, J. C. M.

Van Hest, F. L. Van Delft, Chem. Commun. 2010, 46, 97–99.

[49] B. L. Oliveira, Z. Guo, G. J. L. Bernardes, Chem. Soc. Rev. 2017, 46,

4895–4950.

[50] S. Goswami, H. S. Jena, S. Konar, Inorg. Chem. 2014, 53, 7071–7073.

[51] S. G. Tolshchina, G. L. Rusinov, V. N. Charushin, Chem. Heterocycl.

Compd. 2013, 49, 66–91.

[52] A. L. Handlon, N. J. Oppenheimer, Pharm Res. 1998, 5, 297-299.

[53] R. Van Geel, G. J. M. Pruijn, F. L. Van Delft, W. C. Boelens, Bioconjug.

Chem. 2012, 23, 392–398.

[54] C. Zhang, P. Dai, A. A. Vinogradov, Z. P. Gates, B. L. Pentelute, Angew.

Chem. Int. Ed. 2018, 57, 6459-6463.

[55] S. Samanta, S. Ray, A. B. Ghosh, P. Biswas, RSC Adv. 2016, 6, 39356–

39363.

[56] K. Renault, C. Guillou, P. Y. Renard, C. Sabot, Org. Biomol. Chem. 2019,

17, 388–396.

[57] D. Steiner, P. Forrer, A. Plückthun, J. Mol. Biol. 2008, 382, 1211–1227.

[58] S. Zhang, L. M. De Leon Rodriguez, I. K. H. Leung, G. M. Cook, P. W. R.

Harris, M. A. Brimble, Angew. Chem. Int. Ed. 2018, 57, 3631–3635.

[59] Z. J. Liu, S. Martínez Cuesta, P. van Delft, S. Balasubramanian, Nat.

Chem. 2019, 11, 629–637.

[60] T. Linz, D. Yeo, Q. Hong, W. Zmolek, J. McFarland, R. Barfield, W.

Haskins, D. Rabuka, Antibodies 2018, 7, 40.

[61] Y. Anami, C. M. Yamazaki, W. Xiong, X. Gui, N. Zhang, Z. An, K.

Tsuchikama, Nat. Commun. 2018, 9, 1-9.

[62] J. Dommerholt, O. Van Rooijen, A. Borrmann, C. F. Guerra, F. M. Bickel-

haupt, F. L. Van Delft, Nat. Commun. 2014, 5, 1–7.

[63] M. M. Kurfürst, Anal. Biochem. 1992, 200, 244–248.

13

10.1002/cbic.202000416

ChemBioChem

FULL PAPER

Entry for the Table of Contents

Full Paper

Increased efficiency:

The combination of az-

ide-functionalized HIPS

or tandem

Nils Janson, Tobias

Krüger, Lennard Kars-

ten, Mareile

Boschanski, Thomas

Dierks, Kristian M.

Müller and Norbert Se-

wald*

Knoevenagel ligation

reagents and SPAAC

enables the fast and

efficient mono- and

double-derivatization of

proteins with stoichio-

metric amounts of

DBCO-modified pay-

loads at low concentra-

tion.

Page No. – Page No.

Bifunctional Reagents

for Formylglycine Con-

jugation: Pitfalls and

Breakthroughs

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Bifunctional Reagents for Formylglycine Conjugation: Pitfalls and Breakthroughs

DOI: 10.1002/cbic.202000416

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