FRET-based cyanine probes for monitoring ligation reactions and their applications to mechanistic studies and catalyst screening

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FRET-based Cyanine Probes for Monitoring Ligation Reactions and

their Applications to Mechanistic Studies and Catalyst Screening†

E. Herbst*‡^aand D.Shabat^a

Received 00th January 20xx,

Accepted 00th January 20xx

There is an ever-increasing need to find better methods to selectively connect two molecules under mild aqueous conditions

on small scale. The process of finding such methods significantly relies upon the employment of an appropriate assay. We

report here a modular FRET-based assay to monitor such reactions and exemplify how the assay is used to monitor two

particular reactions: native chemical ligation (NCL) and the oxime ligation. For both reactions we show that by employing

appropriately designed probes FRET measurements could be used to monitor the reaction’s progress. We additionally

demonstrate the usefulness of the developed probe system to study the mechanism of the ligation reactions. For example

in monitoring the formation of a trimeric intermediate in the NCL reaction. Finally, we demonstrate that FRET measurements

conducted in our system allow the quantification of the reaction yield and we show the application of our FRET-based assay

to catalyst screening for the oxime ligation.

DOI: 10.1039/x0xx00000x

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cycloaddition,^7,8the Staudinger ligation,⁹and the oxime

ligation.¹⁰However, these commonly employed synthetic

ligation methods are of second order with a rate constant in the

range of 10^-3-10³M^-1S^-1, whereas biological linking methods,

such as the biotin streptavidin interaction have rate constants

as fast as 10⁶M^-1S^-1.^11,12The importance of ligation reactions in

chemical biology research demands improved kinetics of

current methods.⁴

Introduction

Reactions used to selectively connect two molecules in aqueous

solutions under mild conditions are sought after for various

applications in the continuously expanding field of chemical

biology. For example, in order to monitor a biomolecule, such

as a protein, a lipid or a nucleic acid in a living cell, a site-specific

reaction is needed to connect the reporter molecule and the

biomolecule.^1,2Other common applications include conjugating

two biomolecules, such as a protein and an oligonucleotide,³or

connecting together two peptides,⁴with the promise of

obtaining fully functioning synthetic enzymes.⁵However, site-

specific and high yielding chemistry in biomolecules is very

often challenging. For one reason, biomolecules frequently

contain a rich variety of functional groups. Additionally, many

biomolecules are unstable at high temperatures and harsh

reaction conditions and often have limited solubility in both

organic solvents and water. Also, biomolecules are often only

available in very small quantities due to associated substantial

cost and synthetic challenges. This limits the scope of

transformations to connect such molecules together to

reactions that could be carried out in low concentrations in

aqueous conditions. The required low concentration therefore

implies that 2^nd-order reactions between such molecules have

to be extremely rapid.

The discovery of novel catalysts or alternative conditions for

existing methods has the potential of improving the current

ligations reactions. In order to access the vast majority of

possible combinations of different catalysts and reaction

conditions a high-throughput assay is needed. In the optimal

case the assay would be modular, so that it could be easily

adapted to improve many different ligation reactions. We

therefore turned to develop such an assay and decided to

exemplify its modularity and usefulness on two ligation

reactions, native chemical ligation and the oxime ligation.

Native chemical ligation (NCL), the most commonly used

peptide ligation method, is an efficient method to join together

two unprotected polypeptides by an amide bond.¹³This

reaction takes place between a peptide that contains a

thioester fragment at its C-terminus and another peptide that

bears a cysteine residue at its N-terminus. The oxime ligation

presents another powerful way to link two molecules under

physiological conditions in the μΜ range.^14,15The oxime bond is

formed by reacting a carbonyl, such as a ketone or an aldehyde,

with an aminooxy nucleophile. Contrary to its imine

counterpart, the oxime bond formation is irreversible under

physiological conditions.^11,16In order to achieve effective

reaction rates, the oxime ligation requires the use of a catalyst

when performed in physiological pH.¹⁷Since the original report

of catalysis of the ligation of two peptide fragments by aniline

and 4-methoxyaniline,¹⁶various aniline derivatives have been

Existing reactions that are frequently used to selectively

connect molecules in aqueous solutions under mild conditions

are the native chemical ligation,⁶the [3 + 2] azide-alkyne (click)

^a.School of Chemistry, Department of Organic Chemistry, Raymond and Beverly

Sackler Faculty of Exact Sciences, Tel-Aviv University, Tel Aviv, 69978 Israel

‡Current address: Department of Chemistry, Columbia University 550W 120TH St,

MC 4849, New York, NY 10027; E-mail: eh2639@columbia.edu

†Electronic Supplementary Information (ESI) available

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introduced as alternative oxime ligation catalysts.¹⁷Examples

include 4-aminophenylalanine as a biocompatible aniline

derivative,¹⁸and m-phenylenediamine, a highly water-soluble

catalyst that could be introduced in high concentrations.¹⁹

The assay used to discover m-phenylenediamine as a catalyst

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DOI: 10.1039/C5OB02127H

for the oxime reaction was based on

a fluorescence

enhancement of the aminooxy-dansyl conjugate upon oxime

bond formation with a hydrophobic aldehyde. In a different

assay, nitrobenzoxadiazole (NBD) hydrazine, which is known to

undergo a red shift when it forms a hydrazone, was used to

discover catalysts for the hydrazone ligation.^20,21However,

catalysts that were found in this screen to outperform aniline in

the hydrazone ligation did not necessarily outperform aniline in

the oxime ligation. The substrate scope of both assays described

above is inherently limited. The dansyl-based assay is limited to

the use of hydrophobic electrophiles, while the NBD-based

assay is limited to the use of the NBD hydrazine nucleophile.

Thus, both assays do not allow the use of peptides and other

common biomolecules employed in the biological applications

of the oxime ligation.¹⁷A recent screening assay for oxime-

ligation catalysts has a much wider potential substrate scope.

However, the use of HPLC monitoring in this assay would

challenge its use in high-throughput screening.²²

Fig. 1 A schematic illustration of a ligation reaction between two molecular

probes and the resulting acceptor emission as a result of illumination at the donor

absorption wavelength upon bond formation

derivative

activated ester was immediately reacted with compound 7²⁷to

produce the protected amine . The latter was deprotected

with TFA and reacted with N-Boc-S-Trityl-L-cysteine, EDC, HOBt

and Et₃N to afford the protected cysteine derivative , which

was deprotected to afford compound . TCEP was added to all

reactions of in order to maintain its cysteine side chain in its

6 was activated with DCC and NHS and the resulting

8

9

1

reduced form. DMSO was added as a cosolvent to all ligation

reactions to prevent dye aggregation.

Our probe system for monitoring the oxime ligation includes

Cy3 covalently bound to the carbonyl moiety and Cy5 covalently

The excitement about discovering new oxime ligation catalysts

has prompted us to develop a FRET-based assay for monitoring

ligation reactions. We envisioned this assay to be both modular

and high-throughput. Every probe systems we designed

contains two molecules, each of which includes a fluorophore

moiety and an active functional group appropriate to the

reaction we wished to study (Fig. 1). The separation between

the fluorophore reporting moiety and the reacting moiety is the

source of modularity in our system. Upon bond formation, a

quantitative FRET signal can be conveniently measured by a

spectrophotometer, thus providing an easy way of obtaining

kinetic data for ligation reactions in various conditions. The

fluorescence of many dyes, including cyanines, is influenced by

various factors, such as dye concentration, photobleaching, and

hydrophobic interactions.²³We have therefore employed in this

research ratiometric molecular probes. Such probes provide a

built-in correction for these factors by detecting the

fluorescence signal of both reacted and unreacted probes.²⁴The

use of near-IR dyes in the assay we present here would promote

the assay’s usefulness in studying ligation reactions in vivo in the

future.^25,26

bound to the aminooxy moiety (compounds 12 and 11a

,

respectively, Fig. 6). We decided to additionally synthesize

probe 11b to evaluate if the use of a Cy3-Cy7 FRET pair would

allow better signal to noise ratio than the use of the Cy3-Cy5

FRET pair.²⁸We found that for our application the Cy3-Cy7 FRET

pair performs as well as the Cy3-Cy5 FRET pair (Fig. S12†). The

synthesis of compounds 11a-b is presented in Scheme 2.

Compounds 20 and 21 were synthesized in a similar manner to

previously published procedures.²⁹Compound 22 was prepared

from compound 21 in a similar manner to the synthesis of

compound

8 from compound 6. Compound 22 was deprotected

with TFA and reacted with the carboxylic acid derivative 16 ³⁰

,

EDC, HOBt and Et₃N to form compound 23. The latter was

deprotected with TFA to form the trifluoroacetate

hydroxylammonium salt of 11a that was used without further

purification. The aminooxy moiety of compound 11a is

extremely nucleophilic and demands careful handling to

prevent unwanted oxime bond formation reactions with trace

carbonyl compounds, such as acetone and formaldehyde that

could be also found in trace amounts of common laboratory

solvents, or even in common laboratary plastic equipment.³¹

Immediately after deprotection, a stock solution of 10 mM in

DMSO was made for immediate use or for storage of up to 24

Results and discussion

Organic synthesis

o

hours in -20 C. Deprotection using lower concentrations of 23

led to a higher prevalence of side reactions (data not shown).

We employed a more convergent route to the synthesis of

compound 11b by first introducing the protected amine

functionality to the indolenine moiety in compound 24 and only

then reacting it with compound 25. The synthetic route to

compounds 12 and 18a is presented in Scheme 2. The synthesis

of 28 was based on previously published procedures.²⁹

Compound 29 was deprotected with TFA and reacted with 17

The probe system to monitor the native chemical ligation

reaction includes Cy3 covalently bound to the amino acid

cysteine (compound

activated glycine (compound 2a, Fig. 2). The synthesis of

compounds and 2a is presented in Scheme 1. Activated ester

was reacted with glycine in the presence of Et₃N to form

1), and Cy5 covalently bound to a thioester

1

4

compound 5a. The latter was further reacted with DCC and

thiophenol to afford the desired product 2a. Carboxylic acid

and Et₃N to afford the desired compound 12

.

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Fig. 2 (a) The chemical structure of the FRET-based probe system for monitoring the native chemical ligation. (b) A schematic illustration of a native chemical ligation

reaction between two probes, such as and 2a

1

Scheme 1 The chemical synthesis of compounds

1 and 2a.

Evaluation of the FRET-based probe system for monitoring the Using an independent measurement, we wished to confirm the

NCL reaction

The reaction of

hypothesis that trimer 10 is the intermediate formed during the

and 2a in PBS 7.4 leads to a fluorescent signal course of the NCL reaction between and 2a. Trimer 10

1

in the Cy5 emission spectrum upon excitation in the Cy3

absorbance spectrum when our NCL probes are used but not

when unfunctionalized Cy3-COOH and Cy5-COOH probes are

used in the same conditions (Fig. 3). This confirms our

hypothesis that our probes could be used to monitor the NCL

reaction by FRET.

Isolation and identification of trimer formation in the NCL reaction

Monitoring the progress of the reaction between

1 and 2a by

HPLC (Fig. 4 and Fig. S2†) shows that an intermediate is formed

in considerable quantity before the formation of the final

product in the reaction. We conducted LCMS experiments and

identified two compounds that have both Cy3 and Cy5

absorption (550 nm and 650 nm, respectively). The

experimental m/z values for the two compounds were 562.8

and 549.5 respectively (Fig. S3†). The 562.8 value corresponds

to the calculated m/z value of 562.33 for compound 3a ([M]²⁺)

– the ligation product of compounds

1 and 2a. The 549.5 value

corresponds to the m/z calculated value of 549.0 for trimer 10

([M]³⁺). The formation of this trimer could be reasoned by a

transthioesterification reaction between compound 3a and

Fig. 3 Fluorescence (λex= 490 nm). Reaction conditions between compound

compound 2a: 50 μM of , 50 μM of 2a, and 500 μM of TCEP in PBS (pH 7.4) with

7.5% DMSO (v/v) at r.t. for 120 minutes. Negative control reaction conditions

1 and

1

compound 2a following the reaction of compound

compound 2a to form compound 3a (Fig. 5).

1 and

between Cy3-COOH and Cy5-COOH: 50 μM of Cy3-COOH (6), 50 μM of Cy5-

COOH,³⁸and 500 μM of TCEP in PBS (pH 7.4) with 7.5% DMSO (v/v), at r.t. for 120

minutes.

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Scheme 2 The chemical synthesis of compounds 11a-b, 12, 18a and 30

contains two Cy5 moieties and one Cy3 moiety, whereas

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compound

However, as expected, the reaction between compound

was significantly slower than in the cases of 2b and 2a. Trimer

10 formed in the reaction of 2b and (Fig. S7-S9†). In

accordance with our hypothesis, no trimer formation was

identified in the reaction of 2c and in various reaction

1 in a comparable rate to the case of compound 2a .

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^{DOI: 10.1039/C5OB}2⁰c²¹a²n^7Hd

1

conditions (Fig. S10, S11†). Unproductive internal thioester

formation was previously reported to form between an internal

cysteine residue and a C-terminal thioester and to slow down

reaction rates.^34,35However, to the best of our knowledge, we

show here the first characterization of such an internal thioester

formation in the NCL reaction.

Evaluation of the FRET-based probe system for monitoring the

oxime ligation

The oxime ligation was performed between compound 11a and

compound 12 (Fig. 6, Table 1) and the change over time in the

fluorescence spectrum of the reaction was measured (Fig. 7).

We observed a gradual quenching of the fluorescence of Cy3

molecules, and a gradual increase in the Cy5 fluorescence, as

could be expected with gradual formation of product 18a. This

pattern of change in fluorescence allowed the probes to be used

as ratiometric probes, and thus provided the desired built-in

correction for factors that affect the fluorescence. The structure

of the oxime conjugate 18a was ascertained by a larger scale

synthesis (Scheme 2), followed by NMR and mass spectroscopy.

This shows that the oxime ligation could be monitored on small-

scale by conducting FRET measurements.

Fig. 4 Quantification of relative Cy5 absorption across the HPLC chromatograms of the

NCL reaction between compound 1 and compound 2a. Conditions: 50 μM of compound

1, 50 μM of compound 2a and 500 μM of tris(2-carboxyethyl)phosphine (TCEP) in PBS

(pH 7.4) with 7.5% DMSO (v/v), at room temperature. The percent conversion was

measured by integrating the 650 nm absorption over the HPLC chromatogram. The

separation was performed on a C-18 RP-column (gradient: 10% to 90% ACN in 0.1% (v/v)

TFA aqueous solution over 20 min).

contains two Cy5 moieties and one Cy3 moiety, whereas the

ligation product 3a has one Cy3 moiety and one Cy5 moiety.

Thus, the ratio of 650 nm absorption to 550 nm absorption is

expected to be significantly higher in trimer 10 than in the

ligation product 3a. Indeed, we observed the expected

difference at the 2D HPLC chromatogram (Fig. S4†). The ratio

between the absorption peak area of Cy5 (650 nm) to the

absorption peak area of Cy3 (550 nm) was 35% higher in trimer

Insights from the correlation between the FRET- and the HPLC-

monitoring of the oxime ligation

10 than in the final NCL compound 3a

.

In order to estimate percent conversion to product based upon

the easily conducted FRET-measurements, efforts to correlate

the FRET measurements of the reaction progress with HPLC

measurements of the reaction progress followed. In initial HPLC

measurements with 0.1 % TFA as the aqueous mobile phase, the

conversion of 11b to product was shown to exceed 90% after 5

minutes only (Fig. 8). We suspected that the seemingly rapid

reaction progress might result from acid catalysis of oxime bond

formation by the 0.1% TFA aqueous phase (pH 2) as the oxime

ligation reaction rate is known to be enhanced in acidic

conditions.¹⁶Indeed, replacing the 0.1% TFA aqueous mobile

phase in the HPLC measurements of Reaction 3 to PBS 7.4 has

led to a much slower measured rate of conversion to product

According to previously published studies,^32,33changing the

phenolate leaving group to an aliphatic thiolate leaving group is

expected to slow down the transthioesterification stage of NCL.

A rate reduction of greater magnitude is expected for changing

the amino acid side chain of the C-terminal amino acid to a

bulkier one. We hypothesized that slowing down the

transthioesterification stage would reduce the prevalence of a

trimer such as 10. We therefore synthesized compounds 2b

with an aliphatic thiolate leaving group, and compound 2c with

an aliphatic thiolate leaving group as well as an isoleucine as the

C-terminus amino acid (Fig. S5†). We measured the reaction

progress of compounds 2b and 2c with compound

1 by HPLC

(Fig. S6-S9†). Surprisingly, compound 2b reacted with

Fig. 5 The suggested reaction sequence leading to the NCL product 3a through trimer 10

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Fig. 6 The oxime ligation reaction between compounds 12 and 11a or 11b to afford compound 18a or 18b, respectively. Conditions: see Table 1.

Table 1 Reaction conditions for oxime ligation reactions between compound 11a or 11b and compound 12. All the reactions were performed at room

temperature with 5 mM aniline and 7.5% DMSO (v/v).

Reaction Number

Solvent

Conc. of 11a

Conc. of 11b

Conc. of 12

1

2

3

PBS (pH 7.4)

0.1% (v/v) TFA in H₂O

PBS (pH 7.4)

50 μM

-

50 μM

(Fig. 8). The rate of Reaction 3, k_obs= 3.442 M^-1S^-1, was calculated oxime ligation reaction.^16,11,36,37Therefore, further research

according to the HPLC monitoring of the reaction (Fig. 8, Fig. should be conducted to examine if the oxime ligation reaction

S15†). We confirmed the rapid oxime bond formation in a 0.1% of other reactants is also enhanced in HPLC runs employing

TFA aqueous solution by comparing the change in the Cy5 / Cy3 0.1% (v/v) TFA aqueous mobile phase. It is possible that the

fluorescence ratio over time as measured when the reaction is effect of acid catalysis by the HPLC mobile aqueous phase is

performed in 0.1% TFA with the change in the same more pronounced in faster reactions, such as the one between

fluorescence ratio over time when the reaction is performed in our probes, which has a higher rate constant compared to other

a PBS 7.4 aqueous solution (Fig. S13†). Our measurements oxime ligation reactions (e.g., k_obs= 3.442 M^-1S^-1vs K_obs= 0.061

show that at least for some reactants the HPLC measurement of M^-1S^-1).¹⁶

progress of the oxime ligation reaction is not informative when After establishing an appropriate HPLC assay to monitor the

a 0.1% (v/v) TFA aqueous mobile phase is employed. This acidic reaction we conducted a correlation analysis between the

aqueous solution is commonly employed for studies of the HPLC- and the fluorescence-monitoring of the reaction

Fig. 7 (A) Changes over time in the fluorescence spectrum (λex = 460 nm) of Reaction 1 (see Table 1 for the reaction conditions). (B) Changes over time in the ratio of

the emission at 670 nm (Cy5 emission) to the emission at 570 nm (Cy3 emission) in the fluorescence spectrum of Reaction 1 (see Table 1 for the reaction conditions).

6 | Org. Biomol. Chem., 2015, 00, 1-3

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27% conversion after 120 minutes, respectively)_V._ieW_{w A}e_rticfu_ler_Ot_nh_lie_ner

show that other compounds that were^Dn^Oo^I:t¹⁰t^.e¹s⁰t³e⁹d^/C5b^Oe^Bfo⁰²re¹²⁷a^Hs

oxime ligation catalysts, such as 4-aminophenol and 3-methoxy-

4-phenylaniline, performed even better in the screen we

conducted (47% and 62% conversion after 120 minutes,

respectively). We hypothesized that thiourea derivatives could

serve as catalysts for the oxime ligation and therefore tested

the ability of 1-(3,5-bis(trifluoromethyl)phenyl)thiourea and

1,3-bis(3,5-bis(trifluoromethyl)phenyl)thiourea to catalyze the

reaction between 11b and 12 (Entries 3, 10). We show that the

monosubstituted thiourea increases the donor to acceptor

fluorescence ratio in the oxime ligation reaction between 11b

and 12 and that the addition of aniline does not enhance that

ratio (entries 1-3). No significant enhancement was observed

for the corresponding disubstituted or unsubstituted thiourea

derivatives (Entries 10 and 11, respectively). This could be

explained by the lack of an unsubstituted nitrogen in the

disubstituted thiourea derivative and an unoptimal pKa in the

case of the unsubstituted thiourea derivative.

Fig. 8 The conversion to product over time in the oxime ligation Reaction 3

between compound 12 and compound 11b (see Table 1 for the reaction-

conditions), as measured by HPLC. The percent conversion to product was

measured by performing integration of the absorbance signal of the product 18b

and the reactant 11b at λ=750 nm and dividing the value obtained for the product

by the sum obtained for both the product and the reactant. The separation in the

case of a 0.1% (v/v) TFA – HPLC aqueous mobile phase was performed on a C-18

RP-column (gradient: 10% to 90% ACN in 0.1% (v/v) TFA aqueous solution over 20

min. The separation in the case of a PBS 7.4 – HPLC aqueous mobile phase was

performed on a C-18 RP-column (gradient: 5% to 70% ACN in a 20 mM PBS 7.4

aqueous solution over 20 min).

We verified our assay’s ability to identify new catalysts for the

oxime ligation by testing one of the identified catalysts in the

reaction between alternative starting materials, 4-

formylbenzoic acid

(

32

)

and (aminooxy)acetic acid

progress. To begin with, a Pearson product-moment correlation

coefficient (Pearson’s r) was calculated between time and the

acceptor to donor emission ratio for Reaction 3 (Fig. S12†).

There was a positive correlation between the variables with r =

0.998 (ρ < 0.001). A linear regression equation for predicting

acceptor to donor emission ratios based on given time value

was calculated to be y = 0.011*x + 0.130. Based on this

equation, the acceptor to donor emission ratio was predicted

for 30 minutes interval values, starting at 5 minutes. A second

Pearson product-moment correlation coefficient was calculated

for Reaction 3 between the natural log of the predicted

acceptor to donor emission ratio and the percent conversion to

product that was measured by HPLC with PBS 7.4 as the

aqueous mobile phase (Fig. 8). There was a positive correlation

between the variables with r = 0.997 (ρ < 0.001, Fig. S14†). The

linear regression formula to predict percent conversion to

product based on the natural logarithm of the acceptor to donor

fluorescence ratio was calculated to be y = 24.377 * ln (x) +

45.866. The excellent correlation between the HPLC method

and the FRET-based method to monitor the oxime ligation

reaction has allowed us to use the latter linear equation to

estimate the conversion to product from the spectral

measurements in catalyst screens.

hemihydryochloride (33) to form oxime product 30 (Fig. S17†).

Indeed, 4-aminophenol serves as a more efficient catalysis than

aniline in the reaction between the alternative starting

materials (K_obs=

2.6938 M^-1S^-1, 0.9968 M^-1S^-1, respectively, Fig.

S18, S19†) as measured in kinetic NMR experiments in

deuterated PBS (pH 7.4). A similar measurement with 1-(3,5-

bis(trifluoromethyl)phenyl)thiourea as a catalyst has shown no

rate enhancement over the uncatalyzed reaction (data not

shown).

Conclusions

A FRET-based assay for convenient monitoring, investigating

and improving small-scale reactions under physiological

conditions was introduced. We showed the application of this

assay in the native chemical ligation reaction and in the oxime

ligation. We demonstrated that this assay could shed light on

our understanding of these reactions by studying parameters

such as alternating functional groups, catalysts and conditions.

The modular design of our FRET-based probe system could be

further applied to other reactants and other ligation reactions

by changing the functional groups that are attached to the

fluorophore moieties.

We characterized a trimeric intermediate formed in the NCL

reaction and showed the effect of changing the thiolate leaving

group and the amino acid residue on the formation of this

intermediate. In the catalyst screen we performed for the oxime

ligation we found new catalysts, such as 3-methoxy-4-

phenylaniline and 4-aminophenol, that outperform leading

previously reported oxime ligation catalysts. We hope that this

demonstration of the utility of our assay for the discovery of

new oxime ligation catalysts will inspire the employment of this

assay to search for new catalysts for the oxime ligation and for

Screening for improved oxime ligation catalysts using the cyanine

FRET-based probes

We show here that the probe system introduced in this work

could be used to screen for catalysts of the oxime ligation (Table

2). In order to test our system’s capacity for catalyst screening

we included 2-amino-5-methoxybenzoic acid in our catalyst

sample. This compound was previously reported to outperform

aniline as an oxime ligation catalyst.²⁰We show that our probe

system also detects this catalyst’s ability to outperform aniline

in catalyzing the oxime ligation in physiological pH (42% and

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Table 2 A screen for oxime ligation catalysts based on FRET measurements of the oxime ligation reaction between compound 11b and compound 12.

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Conversion to

product (%)^b

Entry

1

Catalyst

Aniline

Structure

Acceptor to donor

emission ratio^a

1.00

27.1

2

Aniline + 1-(3,5-

bis(trifluoromethyl)phenyl)thiourea

2.15

45.8

47.1

3

1-(3,5-bis(trifluoromethyl)phenyl)thiourea

2.27

4

5

6

anthranilic acid

0.75

1.87

0.61

20.1

42.4

14.9

2-amino-5-methoxybenzoic acid

2-methoxy-5-methylaniline

7

3-methoxy-4-phenylaniline

4.25

62.5

8

2,5-dimethoxyaniline

0.53

2.26

0.62

11.5

47.1

15.5

9

4-aminophenol

10

1,3-bis(3,5-bis(trifluoromethyl)phenyl)thiourea

11

12

Thiourea

0.44

0.46

6.8

8.1

No Catalyst

-

^aMeasurements were taken after 120 min of reaction and the results were normalized to entry 1. Acceptor emission was measured at 780 nm and donor emission

was measured at 570 nm. ^bPercent conversion to product was calculated according to the acceptor to donor emission ratio of each entry after 120 min of reaction

plugged into the linear regression equation y = 24.377 * ln (x) + 45.866 described above. Reaction conditions: 50 μM of compound 11b, 50 μM of compound 12 and 1

mM of the catalyst at room temperature in PBS (pH 7.4) containing 7.5% DMSO (v/v).

other ligation reactions. Lastly, as the assay we developed is DCM. High-pressure liquid chromatography (HPLC): C-18 5 μ,

based on near-IR cyanine dyes, the direct continuation of this

250x4.6 mm, eluent given in parentheses. Unless stated

research should be the use of this assay to study ligation otherwise, the gradient used for the HPLC separations was 10%

reactions in vivo when applicable.

to 90% ACN in 0.1% (v/v) TFA aqueous solution over 20 minutes.

Preparative HPLC: C-18 5 μ, 250x21 mm, eluent given in

parentheses. ¹H-NMR spectra were measured using Bruker

Avance operated at 400 MHz or Bruker Ascend operated at 500

MHz, as mentioned. ¹³C-NMR spectra were measured using

Bruker Avance operated at 400 MHz as mentioned. Mass

spectroscopy measurements were performed on Waters Xevo

TQD mass spectrometer equipped with Acqcuity UPLC and an

electronspray ion source. Unless stated otherwise, all reagents,

salts and solvents were purchased from commercial sources

and used without further purification. Absorption and

fluorescence spectra were recorded on Infinite-M200

fluorescent spectrometer. FRET measurements were taken

Experimental Section

General methods

All reactions requiring anhydrous conditions were performed

under Argon atmosphere. Thin layer chromatography (TLC):

silica gel plates Merck 60 F₂₅₄: compounds were visualized by

irradiation with UV light. Flash chromatography (FC): silica gel

Merck 60 (particle size 0.040-0.063 mm), eluent given in

parentheses. For all column chromatography separations that

involved more than 20% MeOH in the eluent mixture, the silica

gel was previously mixed with MeOH, dried and eluted with

8 | Org. Biomol. Chem., 2015, 00, 1-3

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Page 9 of 14

Organic & Biomolecular Chemistry

ARTICLE

after five times dilution (Fig. 3) or ten times dilution (Fig. 7, Fig. was left O.N. at R.T. The reaction was monitored_Vb_iey_wd_Ari_ts_ics_lo_{e O}lv_ni_ln_ing_e

DOI: 10.1039/C5OB02127H

S12, Fig. S13, Fig. S16†, Table 2) of the reaction mixture into ACN one drop of the reaction mixture in (TFA/DCM, 1:1) for 5

to a final volume of 100 μL at the indicated time points. minutes at R.T. and then analyzing the deprotection product by

Deuterated PBS 7.4 was prepared from 126.1 mM NaCl, 10.3 RP-HPLC (10-90% ACN in H₂O, 20 min). After completion, the

mM Na₃PO₄, 1.5 mM K₃PO₄and 13.6 mM DCl in D₂O. The pH reaction mixture was diluted with DCM and the 1,3-

was adjusted to 7.4 with a DCl solution in D₂O.

dicyclohexylurea was filtrated. The solvent was removed under

reduced pressure and the crude product was purified by

column chromatography on silica gel (DCM/MeOH 95:5 →

Chemical Synthesis

DCM/MeOH 93:7) to afford compound

solid.

8 (144 mg, 75%) as a red

2-((1E,3E)-5-((E)-1-(6-((carboxymethyl)amino)-6-oxohexyl)-

3,3-dimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-

trimethyl-3H-indol-1-ium (5a). Compound 4³⁸(111 mg, 0.18

mmol) and glycine (36.5 mg, 0.49 mmol) were dissolved in 2 mL

of DMF. Et₃N (220 μL, 1.6 mmol) was added and the reaction

¹H NMR (400 MHz, CDCl₃+ 1 drop CD₃OD): δ = 8.37 (1 H, t, J =

13.4 Hz ), 7.44-7.30 (4 H, m), 7.21 (2 H, t, J = 7.2 Hz), 7.11 (2 H,

d, J = 7.6 Hz), 6.84 (1 H, d, J = 13.4 Hz), 6.74 (1 H, d, J = 13.1 Hz),

4.05 (2 H, br. t), 3.69 (3 H, s), 3.41 (2 H, m), 3.24 (2 H, m), 2.78-

2.25 (6 H, m), 1.42-1.69 (16 H, m), 1.36 (9 H, s). MS (ESI+): m/z

o

mixture was heated to 30 C for two hours. The reaction was

monitored by TLC (DCM:MeOH:AcOH, 89:10:1). After

completion the solvent was removed under reduced pressure

and the crude product was purified by column chromatography

on silica gel (DCM/MeOH/AcOH 97:2:1 → DCM/MeOH/AcOH

89:10:1) to afford compound 5a (49 mg, 50%) as a blue solid.

¹H NMR (400 MHz, CDCl₃): δ = 7.76 (2 H, m), 7.35 (4 H, m), 7.20-

7.00 (5 H, m), 6.78 (1 H, t, J = 12.4 Hz), 6.63 (1 H, d, J = 13.7 Hz),

6.22 (1 H, d, J = 13.4 Hz), 3.97 (2 H, t, J = 7.2 Hz), 3.86 (2 H, d, J =

3.8 Hz), 3.80 (3 H, s), 2.25 (2 H, t, 6.7 Hz), 1.77 (4 H, m), 1.67 (12

+

calc. for C₃₇H₅₁N₄O₃: 599.40; found: 599.6 [M]⁺. RP-HPLC

product retention time = 17.45 min.

2-((E)-3-((E)-1-((R)-2,2-dimethyl-4,7,12-trioxo-6-

((tritylthio)methyl)-3-oxa-5,8,11-triazaheptadecan-17-yl)-3,3-

dimethylindolin-2-ylidene)prop-1-en-1-yl)-1,3,3-trimethyl-3H-

indol-1-ium (9). Compound

8 (100 mg, 0.15 mmol) was

dissolved in a 1:1 mixture of TFA/DCM and the mixture was

stirred at R.T. for 5 minutes. The reaction was monitored by RP-

HPLC (10-90% ACN in H₂O, 20 min). After completion the

solvents were removed under reduced pressure and 3 mL of

DCM were added, followed by Et₃N (62 μL, 0.45 mmol), EDC (84

mg, 0.45 mmol), HOBt (68 mg, 0.45 mmol) and N-Boc-S-Trityl-L-

cysteine (206 mg, 0.45 mmol) and the reaction mixture was

stirred at room temperature overnight. The reaction was

monitored by RP-HPLC (10-90% ACN in H₂O, 20 min; product

retention time = 22.14 min). After completion, the solvent was

removed under reduced pressure and the crude product was

purified by column chromatography on silica gel (DCM/MeOH

95:5 → DCM/MeOH 93:7) to afford compound

as a red solid.

¹H NMR (400 MHz, CDCl₃): δ = 8.39 (1 H, t, J = 13.3 Hz ), 7.90 (1

H, br. t), 7.73 (1 H, m), 7.59 (1 H, m), 7.38 (18 H, m), 7.18-7.03

(4 H, m), 6.84 (2 H, m), 4.05 (2 H, t, J = 7.7 Hz), 3.89 (1 H, m),

3.73 (3 H, s), 3.46-3.21 (8 H, m), 1.97 (2 H, m), 1.71 (12 H, s),

+

H, s), 1.50 (2 H, m). MS (ESI+): m/z calc. for C₃₄H₄₂N₃O₃: 540.32

; found: 540.5 [M]⁺. RP-HPLC product retention time = 16.67

min.

2-((1E,3E)-5-((E)-3,3-dimethyl-1-(6-oxo-6-((2-oxo-2-

(phenylthio)ethyl)amino)hexyl)indolin-2-ylidene)penta-1,3-

dien-1-yl)-1,3,3-trimethyl-3H-indol-1-ium (2a). Compound 5a

(49 mg, 0.091 mmol) and N,N'-Dicyclohexylcarbodiimide (DCC,

46 mg, 0.23 mmol) were dissolved in 2 mL of DCM. Thiophenol

(20 μL, 0.196 mmol) was added and the reaction mixture was

o

heated to 30 C for two hours. The reaction was monitored by

9 (49 mg, 29%)

RP-HPLC (10-90% ACN in H₂O, 20 min; product retention time =

21.66 min). After completion the solvent was removed under

reduced pressure and the crude product was purified by

preparative RP-HPLC (30-100% ACN in H₂O, 20 min) to afford

compound 2a (18.6 mg, 30%) as a blue solid.

¹H NMR (400 MHz, CDCl₃): δ = 8.29 (1 H, br. s.), 7.81 (2 H, dt, J =

4.1, 12.5 Hz), 7.41-7.28 (9 H, m), 7.23-7.16 (2 H, m), 7.08 (2 H,

m), 6.69 (1 H, t, J = 12.5 Hz), 6.31 (1 H, d, J = 13.7 Hz), 6.19 (1 H,

d, J = 13.5 Hz), 4.30 (2 H, d, J = 6.0 Hz), 4.03 (2 H, t, J = 7.4 Hz),

3.54 (3 H, s), 2.47 (2 H, t, 7.1 Hz), 1.73 (4 H, m), 1.67 (12 H, s),

1.56 (2 H, m). MS (ESI+): m/z calc. for C₄₀H₄₆N₃O₂S⁺: 632.33;

found: 632.3 [M]⁺.

+

1.53 (4 H, m), 1.37 (9 H, s). MS (ESI+): m/z calc. for C₅₉H₇₀N₅O₄:

944.51; found: 944.8 [M]⁺.

2-((E)-3-((E)-1-(6-((2-((R)-2-ammonio-3-

mercaptopropanamido)ethyl)amino)-6-oxohexyl)-3,3-

dimethylindolin-2-ylidene)prop-1-en-1-yl)-1,3,3-trimethyl-3H-

indol-1-ium (1). Compound

9 (45 mg, 0.045 mmol) was

dissolved in 1 mL of DCM. 0.5 mL of TFA and 0.1 mL of Et₃SiH

were added and the mixture was stirred at R.T. for 5 minutes.

The reaction was monitored by TLC (DCM:MeOH, 90:10). After

completion the solvents were removed under reduced pressure

and the crude product was purified by preparative RP-HPLC (10-

2-((E)-3-((E)-1-(6-((2-((tert-

butoxycarbonyl)amino)ethyl)amino)-6-oxohexyl)-3,3-

dimethylindolin-2-ylidene)prop-1-en-1-yl)-1,3,3-trimethyl-3H-

indol-1-ium (8). Compound 6²⁹(150 mg, 0.28 mmol), DCC (87

mg, 0.42 mmol) and NHS (48.3 mg, 0.42 mmol) were dissolved

in 3 mL of DCM and the reaction mixture was stirred at room

temperature for 45 minutes. The reaction was monitored by RP-

HPLC (10-90% ACN in H₂O, 20 min). After completion,

90% ACN in H₂O, 20 min) to afford compound

as a red solid.

1 (4.75 mg, 13%)

¹H NMR (400 MHz, CDCl₃+ 1 drop CD₃OD): δ = 8.38 (1 H, t, J =

13.5 Hz ), 7.5-7.15 (9 H, m), 6.33 (2 H, m), 4.13 (1 H, br. t), 4.05

(2 H, br. t), 3.62 (3 H, s), 3.48 (2 H, m), 3.39 (2 H, m), 3.29 (2 H,

compound 7²⁷

(134 mg, 0.84 mmol) was added and the reaction

This journal is © The Royal Society of Chemistry 2015

Org. Biomol. Chem., 2015, 00, 1-3 | 9

Organic & Biomolecular Chemistry

Page 10 of 14

ARTICLE

Organic & Biomolecular Chemistry

m), 3.03 (2 H, m), 1.71 (14 H, m), 1.46 (2 H, m). MS (ESI+): m/z 3-(2-((1E,3E)-5-((E)-1-(5-carboxypentyl)-3,3-dimethylindolin-

View Article Online

calc. for C₇₀H₉₄N₁₀O₄S₂²⁺: 601.34; found: 601.3 [M]⁺. RP-HPLC 2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium-

DOI: 10.1039/C5OB02127H

product retention time = 13.72 min.

1-yl)propane-1-sulfonate (21). Compound 19⁴⁰(200 mg, 0.71

mmol) and compound 13⁴¹(219 mg, 0.85 mmol) were dissolved

in a 1:1 solvent mixture of AcOH and Ac₂O (2 mL of each). The

mixture was refluxed for 20 minutes and monitored by RP-HPLC

(10-90% ACN in H₂O, 20 min; product retention time = 11.80

min, product λ_max= 437 nm). After cooling down to room

temperature, the solvents were removed under reduced

2-((1E,3E)-5-((E)-1-(6-((2-((2-ethoxy-2-oxoethyl)thio)-2-

oxoethyl)amino)-6-oxohexyl)-3,3-dimethylindolin-2-

ylidene)penta-1,3-dien-1-yl)-1,3,3-trimethyl-3H-indol-1-ium

(2b). Compound 5a (33 mg, 0.061 mmol) and DCC (46 mg, 0.17 pressure and the crude product was purified by column

mmol) were dissolved in 1 mL of DMF. Ethyl-2-mercaptoacetate chromatography on silica gel (DCM/MeOH 90:10

→

(20 μL, 0.18 mmol) was added and the reaction mixture and was DCM/MeOH 80:20) to afford compound 20 (197 mg, 61%) as a

stirred at room temperature overnight, protected from light by dark yellow solid, that was directly used without further

aluminum foil.³⁹The reaction was monitored by RP-HPLC (10- characterization. Compound 20 (197 mg, 0.435 mmol) and

90% ACN in H₂O, 20 min; product retention time = 18.51 min). compound 14³⁸(154 mg, 0.435 mmol) were dissolved in 20 mL

After completion the solvent was removed under reduced of EtOH and NaOAc (43 mg, 0.52 mmol) was added. The reaction

pressure and the crude product was purified by preparative RP- mixture was refluxed for 20 minutes and monitored by RP-HPLC

HPLC (30-100% ACN in H₂O, 20 min) to afford compound 2b (34 (10-90% ACN in H₂O, 20 min; product retention time = 20.86

mg, 82%) as a blue solid.

min). After cooling down to room temperature the solvent was

¹H NMR (400 MHz, CDCl₃): δ = 8.42 (1 H, br. t), 7.81 (2 H, dt, J = removed under reduced pressure and the crude product was

5.1, 13.2 Hz), 7.38 (4 H, m), 7.24 (2 H, m), 7.12 (1 H, d, 7.6 Hz), purified by column chromatography on silica gel (DCM/MeOH

7.07 (1 H, d, 8 Hz), 6.73 (1 H, t, J = 12.1, 15.2 Hz), 6.35 (1 H, d, J 90:10 → DCM/MeOH 80:20) to afford compound 21 (192 mg,

= 13.6 Hz), 6.25 (1 H, d, J = 13.2 Hz), 4.22 (2 H, d, J = 6.8 Hz), 4.05 75%) as a blue solid.

(2 H, t, J = 7.2 Hz), 3.66 (2 H, s), 3.59 (3 H, s), 3.46 (2 H, m), 2.43 ¹H NMR (400 MHz, CDCl₃): δ = 7.86 (2 H, m), 7.38-7.28 (4 H, m),

(2 H, t, 6.74 Hz), 1.79 (4 H, m), 1.69 (12 H, s), 1.57 (2 H, m), 1.35 7.22-7.12 (4 H, m), 7.03 (1 H, d, J = 6.8 Hz), 6.31 (2 H, m), 4.49 (2

(3 H, m). MS (ESI+): m/z calc. for C₃₈H₄₈N₃O₄S⁺: 642.34; found: H, br. t), 4.01 (2 H, br. t), 3.10 (2 H, m), 2.39 (2 H, m), 2.32(2 H,

642.5 [M]⁺.

m), 2.22 (2 H, t, J = 8.0 Hz), 2.01 (2 H, m), 1.79 (6 H, m), 1.69 (6

H, s), 1.65 (6 H, s), 1.53 (2 H, m). MS (ESI+): m/z calc. for

C₃₄H₄₂N₂NaO₅S⁺: 613.27; found: 613.5 [M + Na]⁺.

2-((1E,3E)-5-((E)-1-(6-(((2S,3S)-1-((2-ethoxy-2-oxoethyl)thio)-

3-methyl-1-oxopentan-2-yl)amino)-6-oxohexyl)-3,3-

dimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-

3-(2-((1E,3E)-5-((E)-1-(6-((2-((tert-

trimethyl-3H-indol-1-ium (2c). Compound

4 (40 mg, 0.065 butoxycarbonyl)amino)ethyl)amino)-6-oxohexyl)-3,3-

mmol) and L-isoleucine (26 mg, 0.19 mmol) were dissolved in 1 dimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-

mL of DMF. Et₃N (90 μL, 0.65 mmol) was added and the reaction 3H-indol-1-ium-1-yl)propane-1-sulfonate (22). Compound 21

o

mixture was heated to 40 C for three hours. The reaction was (192 mg, 0.32 mmol), DCC (72 mg, 0.35 mmol) and NHS (40.2

monitored by HPLC (10-90% ACN in H₂O, 20 min; product mg, 0.35 mmol) were dissolved in 3 mL of DMF and the reaction

retention time = 20.10 min). After completion the solvent was mixture was stirred at room temperature for 2 hours. The

removed under reduced pressure and the crude product was reaction was monitored by RP-HPLC (10-90% ACN in H₂O, 20

purified by column chromatography on silica gel min). After completion, compound 7 (160 mg, 1 mmol) was

(DCM/MeOH/AcOH 97:2:1 → DCM/MeOH/AcOH 91:8:1) to added and the reaction was stirred overnight at room

afford compound 5b (11.8 mg, 30%) as a blue solid. The latter temperature. The reaction was monitored by dissolving one

(11.8 mg, 0.02 mmol) and DCC (12.4 mg, 0.06 mmol) were drop of the reaction mixture in (TFA/DCM, 1:1) for 5 minutes at

dissolved in 1 mL of DMF. Ethyl-2-mercaptoacetate (6.5 μL, 0.06 R.T. and then analyzing the deprotection product by RP-HPLC

mmol) was added and the reaction mixture and was stirred at (10-90% ACN in H₂O, 20 min; product retention time = 16.60

room temperature overnight, protected from light by aluminum min). Upon completion, the solvent was removed under

foil.³⁹The reaction was monitored by RP-HPLC (10-90% ACN in reduced pressure and the crude product was purified by column

H₂O, 20 min; product retention time = 20.09 min). After chromatography on silica gel (DCM/MeOH 90:10

→

completion the solvent was removed under reduced pressure DCM/MeOH 80:20) to afford compound 22 (166 mg, 71%) as a

and the crude product was purified by preparative RP-HPLC (30- red solid.

100% ACN in H₂O, 20 min) to afford compound 2c (4.5 mg, 32%) ¹H NMR (400 MHz, DMSO-d₆): δ = 8.35 (2 H, t, J = 11.8 Hz), 7.79

as a blue solid.

(1 H, br. t), 7.61 (2 H, d, J = 7.4 Hz), 7.49-7.34 (4 H, m), 7.29-7.19

¹H NMR (400 MHz, CDCl₃): δ = 7.97 (2 H, m), 7.3-7.4 (6 H, m), (2 H, m), 6.77 (1 H, br. t), 6.57 (1 H, t, J = 12.3 Hz), 6.46 (1 H, d,

7.24-7.20 (2 H, m), 7.20-7.00 (5 H, m), 6.53 (1 H, d, J = 13.6 Hz), J = 13.8 Hz), 6.32 (1 H, d, J = 13.8 Hz), 4.58 (2 H, t, J = 5.5 Hz),

6.39 (1 H, d, J = 13.5 Hz), 4.13 (8 H, m), 3.66 (5 H, m), 3.48 (4 H, 4.28 (2 H, t, J = 6.6 Hz), 4.09 (2 H, t, J = 7.3 Hz), 3.51 (2 H, m),

m), 2.51 (2 H, m), 1.77 (12 H, s) 1.67 (2 H, m). MS (ESI+): m/z 3.18 (2 H, m), 3.04 (2 H, m), 2.94 (2 H, m), 2.82 (2 H, t, 6.5 Hz),

+

calc. for C₄₂H₅₆N₃O₄: 698.40; found: 698.6 [M]⁺.

2.58 (2 H, m), 2.04 (2 H, m), 1.69 (6 H, s), 1.68 (6 H, s), 1.36 (9

10 | Org. Biomol. Chem., 2015, 00, 1-3

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Organic & Biomolecular Chemistry

ARTICLE

H, s). MS (ESI+): m/z calc. for C₄₁H₅₆N₄NaO₆S⁺: 755.38; found: dimethylindolin-2-ylidene)hepta-1,3,5-trien-1-yl)-_V3_ie,_w3_A-_{rticle Online}

755.6 [M + Na]⁺.

dimethyl-3H-indol-1-ium-1-yl)propane-1-sulfonate

(26).

DOI: 10.1039/C5OB02127H

Compound 19 (400 mg, 1.4 mmol) and Glutaconaldehydedianil

hydrochloride (483 mg, 1.7 mmol) were dissolved in a 1:1

solvent mixture of AcOH and Ac₂O (4 mL each). The mixture was

refluxed for 30 minutes and the reaction was monitored by RP-

3-(2-((1E,3E)-5-((E)-1-(2,2-dimethyl-4,8,13-trioxo-3,6-dioxa-

5,9,12-triazaoctadecan-18-yl)-3,3-dimethylindolin-2-

ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium-1-

yl)propane-1-sulfonate (23).Compound 22 (152 mg, 0.21 HPLC (10-90% ACN in H₂O, 20 min; product retention time =

mmol) was dissolved in a 1 mL solvent mixture of 1:1 TFA/DCM 13.24 min, product λ_max= 482 nm). After cooling to room

and the reaction mixture was stirred at room temperature for 5 temperature the solvents were removed under reduced

minutes. The reaction was monitored by RP-HPLC (10-90% ACN pressure and the crude product was purified by column

in H₂O, 20 min). After completion the solvents were removed chromatography on silica gel (DCM/MeOH 90:10

→

under reduced pressure and 3 mL of DMF were added, followed DCM/MeOH 85:15) to afford compound 25 (185 mg, 28%) as a

by Et₃N (172 μL, 1.2 mmol), EDC (120 mg, 0.62 mmol), HOBt (64 dark red solid, that was directly used without further

mg, 0.2 mmol) and compound 16³⁰(120 mg, 0.62 mmol) and the characterization. Compound 25 (155 mg, 0.33 mmol) and

o

reaction mixture was stirred at 40 C overnight. The reaction compound 24 (135 mg, 0.33 mmol) were dissolved in 3 mL of

progress was monitored by RP-HPLC (10-90% ACN in H₂O, 20 pyridine. The reaction mixture was heated to 50 ^oC for 30

min; product retention time = 15.84 min), and upon completion, minutes and the reaction progress was monitored by RP-HPLC

the solvent was removed under reduced pressure and the crude (10-90% ACN in H₂O, 20 min; product retention time = 17.80

product was purified by column chromatography on silica gel min). Upon completion the solvent was removed under reduced

(DCM/MeOH 90:10 → DCM/MeOH 85:15) to afford compound pressure and the crude product was purified by column

23 (101 mg, 60%) as a blue solid.

chromatography on silica gel (DCM/MeOH 90:10

→

¹H NMR (400 MHz, DMSO-d₆): δ = 8.33 (2 H, t, J = 12.9 Hz), 7.99 DCM/MeOH 85:15) to afford compound 26 (131 mg, 52%) as a

(2 H, br. s), 7.60 (2 H, m), 7.46 (2 H, d, J = 8.0 Hz), 7.37 (2 H, m), blue solid.

7.21 (2 H, t, J = 6.3 Hz ), 6.54 (1 H, t, J = 12.2 Hz), 6.43 (1 H, d, J = ¹H NMR (400 MHz, DMSO-d₆): δ = 8.35 (2 H, t, J = 13.1 Hz), 7.78

13.9 Hz), 6.29 (1 H, d, J = 13.7 Hz), 4.26 (2 H, br. t), 4.12 (2 H, s), (1 H, t, 5.4 Hz), 7.62 (2 H, d, J = 7.5 Hz), 7.41 (4 H, m), 7.26 (2 H,

4.07 (2 H, br. t), 3.14 (4 H, m), 2.53 (2 H, m), 2.05-1.95 (4 H, m), m), 6.77 (1 H, t, J = 5.4 Hz), 6.57 (1 H, t, J = 12.4 Hz), 6.28 (2 H,

1.66 (12 H, s), 1.56 (4 H, m), 1.37 (9 H, s), 1.32 (2 H, m). MS m), 4.58 (2 H, t, J = 5.5 Hz), 4.09 (2 H, t, J = 7.4 Hz), 3.51 (2 H, m),

(ESI+): m/z calc. for C₄₃H₅₉N₅NaO₈S⁺: 828.40; found: 828.7 [M + 3.42 (2 H, m), 3.31 (2 H, m), 3.02 (2 H, m), 2.95 (2 H, m), 2.06 (2

Na]⁺.

H, m), 1.69 (12 H, s), 1.54 (4 H, m), 1.36 (9 H, s). MS (ESI+): m/z

calc. for C₄₃H₅₈N₄NaO₆S⁺: 781.39; found: 781.6 [M + Na]⁺.

1-(6-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-6-

oxohexyl)-2,3,3-trimethyl-3H-indol-1-ium (24). Compound 14 3-(2-((1E,3E,5E)-7-((E)-1-(2,2-dimethyl-4,8,13-trioxo-3,6-

(800 mg, 2.26 mmol), DCC (840 mg, 4.05 mmol) and NHS (390 dioxa-5,9,12-triazaoctadecan-18-yl)-3,3-dimethylindolin-2-

mg, 3.4 mmol) were dissolved in 4 mL of DMF and the reaction ylidene)hepta-1,3,5-trien-1-yl)-3,3-dimethyl-3H-indol-1-ium-

mixture was heated to 40 ^oC for 6 hours and afterwards kept at 1-yl)propane-1-sulfonate (27). Compound 26 (84 mg, 0.11

room temperature overnight. The reaction was monitored by mmol) was dissolved in a 1 mL solvent mixture of 1:1 TFA/DCM

TLC (DCM:MeOH, 90:10). After completion, compound

7 (1084 and the reaction mixture was stirred at room temperature for 5

mg, 6.8 mmol) was added and the reaction was stirred for 2 minutes. The reaction was monitored by RP-HPLC (10-90% ACN

hours at room temperature. The reaction was monitored by in H₂O, 20 min). After completion the solvents were removed

dissolving one drop of the reaction mixture in (TFA/DCM, 1:1) under reduced pressure and 2 mL of DMF were added, followed

for 5 minutes at R.T. and then analyzing the deprotection by Et₃N (46 μL, 0.33 mmol), EDC (43 mg, 0.22 mmol), HOBt (34

product by RP-HPLC (10-90% ACN in H₂O, 20 min; product mg, 0.22 mmol) and compound 16 (43 mg, 0.22 mmol) and the

retention time = 12.45 min). Upon completion, the solvent was reaction mixture was stirred at room temperature for two

removed under reduced pressure and the crude product was hours. The reaction progress was monitored by RP-HPLC (10-

purified by column chromatography on silica gel (DCM/MeOH 90% ACN in H₂O, 20 min; product retention time = 17.07 min),

90:10 → DCM/MeOH 80:20) to afford compound 24 (815 mg, and upon completion, the solvent was removed under reduced

86%) as a beige solid.

pressure and the crude product was purified by preparative RP-

¹H NMR (400 MHz, DMSO-d₆): δ = 7.76 (1 H, t, J = 5.3 Hz), 7.12 HPLC (10-90% ACN in H₂O, 20 min) to afford compound 27 (20

(1 H, d, J = 7.2 Hz), 7.07 (1 H, dt, J = 1.12, 7.6 Hz), 6.75 (1 H, t, mg, 22%) as a blue solid.

5.34), 6.69 (1 H, t, J = 7.47 Hz), 6.62 (1 H, d, J = 7.8 Hz), 5.58 (2 ¹H NMR (400 MHz, DMSO-d₆): δ = 10.27 (1 H, s), 8.02 (1 H, t,

H, d, J = 7.86 Hz), 3.87 (1 H, d, J = 1.4 Hz), 3.83 (1 H, d, J = 1.4 5.14), 7.84 (3 H, m), 7.58 (2 H, t, J = 6.9 Hz), 7.48 (1 H, d, J = 7.8

Hz), 3.48 (2 H, t, J = 7.2 Hz), 3.04 (2 H, m), 2.96 (2 H, m), 2.04 (2 Hz), 7.38 (3 H, m), 7.23 (2 H, m), 6.54 (3 H, t, J = 12.7 Hz), 6.35

H, d, J = 7.4 Hz), 1.52 (4 H, m), 1.38 (9 H, s), 1.26 (6 H, s). MS (1 H, d, J = 13.6 Hz), 4.25 (2 H, t, 7.0 Hz), 4.15 (2 H, s), 4.05 (2 H,

+

(ESI+): m/z calc. for C₂₄H₃₆N₃O₃: 416.29; found: 416.4 [M]⁺.

t, J = 6.5 Hz), 3.13 (4 H, m), 2.54 (2 H, m), 2.07 (2 H, t, J = 7.2 Hz),

2.00 (2 H, m), 1.64 (6 H, s), 1.63 (6 H, s), 1.56 (4 H, m), 1.40 (9 H,

s), 1.35 (2 H, m). MS (ESI+): m/z calc. for C₄₅H₆₁N₅NaO₈S⁺:

854.41; found: 854.7 [M + Na]⁺.

3-(2-((1E,3E,5E)-7-((E)-1-(6-((2-((tert-

butoxycarbonyl)amino)ethyl)amino)-6-oxohexyl)-3,3-

Org. Biomol. Chem., 2015, 00, 1-3 | 11

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Page 12 of 14

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Organic & Biomolecular Chemistry

solution in DMSO that was kept at -20 ^oC for immediate use. RP-

3-(2-((E)-3-((E)-1-(5-carboxypentyl)-3,3-dimethylindolin-2-

ylidene)prop-1-en-1-yl)-3,3-dimethyl-3H-indol-1-ium-1-

View Article Online

HPLC retention times for products 11a,

1^D1^Ob^I:=¹1^0.3¹⁰.8³3^9/,^C1⁵4^O.4^B01²m¹²i⁷n^H,

yl)propane-1-sulfonate (28). Compound 19 (400 mg, 1.41 respectively.

mmol) and compound 15⁴²(277 mg, 1.41 mmol) were dissolved

o

in a 3 mL of Ac₂O. The mixture was heated to 120 C for 30 3-(2-((E)-3-((E)-1-(6-((2-(4-formylbenzamido)ethyl)amino)-6-

minutes and the reaction was monitored by RP-HPLC (10-90% oxohexyl)-3,3-dimethylindolin-2-ylidene)prop-1-en-1-yl)-3,3-

ACN in H₂O, 20 min; product retention time = 14.88 min). After dimethyl-3H-indol-1-ium-1-yl)propane-1-sulfonate (12). 4-

cooling down to room temperature compound 14 in 3 mL of carboxybenzaldehyde (156 mg, 1.04 mmol), DCC (247 mg, 1.2

pyridine was added and the reaction mixture was stirred at mmol) and NHS (138 mg, 1.2 mmol) were dissolved in 4 mL of

room temperature for 40 minutes and at 40 ^oC for 40 additional DCM and the reaction mixture was heated to 40 ^oC for 3 hours.

minutes. Upon completion the solvents were removed under The reaction was monitored by TLC (EtOAc:Hex, 40:60). Upon

reduced pressure and the crude product was purified by column completion, the reaction mixture was diluted with DCM and the

chromatography on silica gel (DCM/MeOH 95:5 → DCM/MeOH 1,3-dicyclohexylurea was filtrated out. The solvent was

90:10) to afford compound 28 (493 mg, 62%) as a red solid.

removed under reduced pressure and the crude product 17

¹H NMR (400 MHz, DMSO-d₆): δ = 12.04 (1 H, br. s, 8.36 (1 H, t, immediately used without further purification. Compound 29

J = 13.5 Hz ), 7.64 (2 H, dd, J = 2.3, 7.3 Hz ), 7.58 (1 H, d, J = 8.0 (100 mg, 0.14 mmol) was dissolved in a 1 mL solvent mixture of

Hz), 7.45 (3 H, m), 7.30 (2 H, m), 6.62 (1 H, d, J = 13.4 Hz), 6.49 1:1 TFA/DCM and the reaction mixture was stirred at room

(1 H, d, J = 13.4 Hz), 4.29 (2 H, t, J = 7.4 Hz), 4.12 (2 H, t, J = 7.2 temperature for 5 minutes. The reaction was monitored by RP-

Hz), 2.59 (2 H, t, J = 7.2 Hz), 2.23 (2 H, t, J = 7.2 Hz), 2.06 (2 H, HPLC (10-90% ACN in H₂O, 20 min). Upon completion the

quintet, J = 7.2 Hz), 1.78 (2 H, m), 1.71 (6 H, s), 1.70 (6 H, s), 1.58 solvents were removed under reduced pressure and 3 mL of

(2 H, quintet, J = 7.4 Hz), 1.43 (2 H, m). MS (ESI+): m/z calc. for DCM were added, followed by Et₃N (100 μL, 0.71 mmol) and

C₃₂H₄₁N₂NaO₅S⁺: 587.25; found: 587.4 [M + Na]⁺.

compound 17 (0.7 mmol). The reaction mixture was stirred at

room temperature overnight. The reaction progress was

monitored by RP-HPLC (10-90% ACN in H₂O, 20 min; product

retention time = 15.31 min), and upon completion, the solvent

was removed under reduced pressure and the crude product

28 was purified by preparative RP-HPLC (10-90% ACN in H₂O, 20

3-(2-((E)-3-((E)-1-(6-((2-((tert-

butoxycarbonyl)amino)ethyl)amino)-6-oxohexyl)-3,3-

dimethylindolin-2-ylidene)prop-1-en-1-yl)-3,3-dimethyl-3H-

indol-1-ium-1-yl)propane-1-sulfonate (29). Compound

(290 mg, 0.51 mmol), DCC (159 mg, 0.77 mmol) and NHS (89 mg, min) to afford compound 12 (35 mg, 33%) as a red solid

.

0.77 mmol) were dissolved in 3 mL of DMF and the reaction ¹H NMR (400 MHz, DMSO-d₆): δ = 10.05 (1 H, s), 8.74 (1 H, t, J =

mixture was stirred overnight at room temperature. This step 5.5 Hz ), 8.34 (1 H, t, J = 13.4 Hz ), 8.10-7.90 (5 H, m), 7.63 (2 H,

was monitored by RP-HPLC (10-90% ACN in H₂O, 20 min). After d, J = 7.3 Hz), 7.55 (1 H, d, J = 8.0 Hz), 7.44 (3 H, m), 7.29 (2 H,

completion, compound 7 (160 mg, 1 mmol) was added and the m), 6.67 (1 H, d, J = 13.4 Hz), 6.42 (1 H, d, J = 13.4 Hz), 4.30 (2 H,

reaction was stirred for 30 minutes at room temperature. This t, J = 7.3 Hz), 4.06 (2 H, t, J = 7.1 Hz), 3.32 (2 H, m), 3.23 (2 H, m),

step was monitored by TLC (DCM:MeOH, 90:10). Upon 2.61 (2 H, t, 7.0 Hz), 2.08 (4 H, m), 1.69 (12 H, s), 1.64 (2 H, m),

complete conversion, the solvent was removed under reduced 1.58 (2 H, m), 1.38 (2 H, m). ¹³C NMR (400 MHz, DMSO-d₆): δ =

pressure and the crude product was purified by column 194.20, 175.32, 175.00, 173.67, 168.81, 151.12, 143.25, 143.20,

chromatography on silica gel (DCM/MeOH 95:5 → DCM/MeOH 141.96, 140.82, 139.03, 131.17, 131.00, 130.68, 130.00, 129.25,

90:10) to afford compound 29 (300 mg, 83%) as a red solid.

126.57, 126.48, 123.83, 113.01, 112.80, 104.41, 103.90, 56.24,

¹H NMR (400 MHz, DMSO-d₆): δ = 8.36 (1 H, t, J = 13.5 Hz ), 7.87 50.25, 50.20, 49.24, 45.04, 44.21, 39.43, 36.49, 28.83, 28.77,

(1 H, t, J = 5.6 Hz ), 7.64 (2 H, t, J = 7.4 Hz), 7.58 (1 H, d, J = 8.0 28.05, 27.07, 26.16, 24.87. MS (ESI+): m/z calc. for

Hz), 7.45 (3 H, m), 7.29 (2 H, m), 6.78 (1 H, t, J = 5.5 Hz ), 6.66 (1 C₄₂H₅₀N₄NaO₆S⁺: 761.34; found: 761.6 [M + Na]⁺.

H, d, J = 13.4 Hz), 6.46 (1 H, d, J = 13.4 Hz), 4.30 (2 H, t, J = 7.1

Hz), 4.11 (2 H, m), 3.17 (4 H, m), 3.02 (2 H, m), 2.93 (2 H, m), Dicyanine oxime conjugate 18a. Compound 23 (28 mg, 0.038

2.84 (2 H, t, J = 6.4 Hz), 2.09 (2 H, m), 1.71 (6 H, s), 1.70 (6 H, s), mmol) was dissolved in a 1 mL of TFA and the reaction mixture

1.58 (2 H, m), 1.38 (2 H, m), 1.36 (9 H, s). MS (ESI+): m/z calc. for was stirred at room temperature for 5 minutes. The reaction

C₃₉H₅₄N₄NaO₆S⁺: 729.36; found: 729.6 [M + Na]⁺. RP-HPLC was monitored by RP-HPLC (10-90% ACN in H₂O, 20 min). Upon

product retention time = 15.95 min.

completion, TFA was removed under reduced pressure and

compound 12 (17 mg, 0.023 mmol) dissolved in 1.5 mL of

General procedure for obtaining compounds 11a-b. pyridine was added. The reaction mixture was stirred at room

Compounds 23 and 27 were used as the Boc-protected temperature for 2 hours. Upon completion, the solvent was

precursors for compounds 11a and 11b, respectively. The removed under reduced pressure and the crude product was

appropriate Boc protected derivative (5 mg, 0.006 mmol) was purified by preparative RP-HPLC (10-90% ACN in H₂O, 20 min)

dissolved in 0.5 mL of TFA and the reaction mixture was stirred to afford compound 18a (11.6 mg, 23%) as a purple solid

.

at R.T. for 5 minutes. The reaction progress was monitored by ¹H NMR (400 MHz, DMSO-d₆): δ = 8.62 (1 H, t, J = 5.5 Hz ), 8.34

RP-HPLC (10-90% ACN in H₂O, 20 min). The solvent was (4 H, m), 8.02 (1 H, J = 5.3 Hz), 7.93 (1 H, t, J = 5.3 Hz), 7.86 (3

removed under reduced pressure and compounds 11a-b were H, m), 7.68-7.54 (8 H, m), 7.50-7.34 (8 H, m), 7.32-7.17 (4 H, m),

used without further purification to prepare a 10 mM stock 6.66 (1 H, d, J = 13.5 Hz), 6.52 (1 H, d, J = 12.5 Hz), 6.45 (2 H, m),

12 | Org. Biomol. Chem., 2015, 00, 1-3

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Organic & Biomolecular Chemistry

ARTICLE

Notes and references

6.28 (1 H, d, J = 13.5 Hz), 4.52 (2 H, s), 4.30 (4 H, m), 4.05 (4 H,

m), 3.30 (4 H, m), 3.23-3.07 (8 H, m), 2.63 (4 H, m), 2.04 (8 H,

m), 1.69 (12 H, s), 1.67 (12 H, s), 1.59 (2 H, m), 1.49 (2 H, m),

1.38 (2 H, m), 1.30 (2 H, m). MS (ESI+): m/z calc. for

C₈₀H₉₉N₉Na₂O₁₁S₂²⁺: 735.83; found: 735.9 [M + 2Na]²⁺. RP-HPLC

product retention time = 16.12 min.

View Article Online

DOI: 10.1039/C5OB02127H

1

2

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4-(((2-methoxy-2-oxoethoxy)imino)methyl)benzoic acid (31).

4-formylbenzoic acid (110 mg, 0.73 mmol) and

(Aminooxy)acetic acid hemihydrochloride (73 mg, 0.67 mmol)

were dissolved in 2 mL methanol. Acetic acid (210 μL, 3.4 mmol)

was added dropwise and the reaction was stirred at room

temperature. After 7 hours a precipitate formed, 2 more mL of

methanol were added and the reaction was left overnight. The

reaction was monitored by TLC (EtOAc:Hex:AcOH, 80:19:1).

Upon completion the solvent was removed under reduced

pressure and the crude product was purified by column

chromatography (EtOAc/Hex/AcOH, 69:30:1) to afford

compound 31 (108 mg, 68%) as a white solid.

5

6

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¹H-NMR (500 MHz, DMSO-d₆): δ = 13.05 (1 H, br. s), 8.46 (1 H,

s), 7.98 (2 H, d, J = 8.3 Hz), 7.74 (2 H, d, J = 8.3 Hz), 4.81 (2 H, s),

+

3.69 (3 H, s). MS (ESI+): m/z calc. for C₁₁H₁₁NNaO₅: 260.05;

-

found: 260.038 [M + Na]+; MS (ESI-): m/z calc. for C₁₁H₁₀NO₅:

236.06; found: 236.06 [M - H]^-.

4-(((carboxymethoxy)imino)methyl)benzoic

acid

(30).

Compound 31 (50 mg, 0.21 mmol) was dissolved in an NaOH

solution in H₂O (1 N) and the reaction was stirred at room

temperature for 90 minutes. The reaction was monitored by TLC

(EtOAc:Hex:AcOH, 80:19:1). Upon completion the reaction was

placed in an ice bath (0^oc) and an HCl solution in H₂O (6 N) was

added dropwise until the solution pH reached 4. Et₂O (25 mL)

was then added, followed by 3 mL of sat. NaCl solution in H₂O

(3 mL). A precipitate formed and was filtered. The filtrate was

placed again at 0^oc and Et₂O (20 mL) was added. A precipitate

formed and was filtered. The organic phase of the filtrate was

extracted and then the aqueous phase was washed with

additional Et₂O (25 mL). The combined organic phase was dried

with MgSO₄and the solvent was removed under reduced

pressure to yield compound 30 (35 mg, 75%) as a white solid.

¹H-NMR (500 MHz, DMSO-d₆): δ = 12.95 (2 H, br. s), 8.43 (1 H,

s), 7.97 (2 H, d, J = 8.4 Hz), 7.73 (2 H, d, J = 8.4 Hz), 4.69, (2 H, s).

-

MS (ESI-): m/z calc. for C₁₀H₈NO₅: 222.04; found: 221.949 [M -

H]^-.

Acknowledgements

D.S. thanks the Israel Science Foundation (ISF), and the German

Israeli Foundation (GIF) for financial support. This work was

partially supported by grants from the Israeli National

Nanotechnology Initiative (INNI), Focal Technology Area (FTA)

program: Nanomedicine for Personalized Theranostics, and by

The Leona M. and Harry B. Helmsley Nanotechnology Research

Fund. The authors would like to thank Dr. Casey J Brown and

Andrew V Anzalone for critically reading the manuscript.

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Org. Biomol. Chem., 2015, 00, 1-3 | 13

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View Article Online

DOI: 10.1039/C5OB02127H

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14 | Org. Biomol. Chem., 2015, 00, 1-3

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FRET-based cyanine probes for monitoring ligation reactions and their applications to mechanistic studies and catalyst screening

DOI: 10.1039/c5ob02127h

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