Maleimide-based metal-free ligation with dienes: A comparative study

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Renault, L. Bailly, A. Frisby, P. La Nahenec-Martel, P. Renard and C. Sabot, Org. Biomol. Chem., 2020,

DOI: 10.1039/D0OB00403K.

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Maleimide-based metal-free ligation with dienes: a comparative

study

Alexis Lossouarn,^aKévin Renault, ^aLaetitia Bailly, ^aAxel Frisby, ^aPatricia Le Nahenec-Martel, ^aPierre-

Received 00th January 20xx,

Accepted 00th January 20xx

Yves Renard, ^aand Cyrille Sabot*^a

DOI: 10.1039/x0xx00000x

A brief literature survey reveals that metal-free ligation such as the maleimide-based cycloaddition with electron-rich

(hetero)dienes is a widespread tool for the assembly of (bio)molecular systems with applications in biotechnology, materials

science, polymers and bio-organic chemistry. Despite their everyday use, only scattered data about their kinetics as well as

the stabilities of corresponding products under physiological conditions, are accessible. These key parameters are yet, of

paramount importance to ensure the rapid and effective preparation of stable compounds. Herein is reported a systematic

study regarding the different classes of dienes used in chemoselective ligation, including their accessibility and stability, as

well as comparative kinetic experiments and products stability assays. We took advantage of these data to develop a double

labeling strategy from the combined use of cyclopentadiene and oxazole dienes.

reported the production of an antibody-drug conjugate by

reacting the maleimide-based drug-linker AZ1508 with a

cyclopentadiene-based non canonical lysine residue genetically

incorporated into the human IgG1 antibody.¹⁷Finally,

Overkleeft and coworkers demonstrated the applicability of the

maleimide-based D-A strategy for monitoring the activity of

endogenously expressed proteases in cellular extracts.¹⁸

Introduction

Maleimide is indisputably a privileged scaffold for the design of

biomolecular systems and tailor-made materials that are

finding widespread utility in biotechnology, polymer and

materials science.^1-4The constant interest in maleimide

derivatives is partly due to their remarkable reactivity,

chemoselectivity, stability, and to the fact that a wide range of

maleimide-activated labels are now commercially available

(Figure 1). Moreover, maleimide-based chemical reactions

proceed under metal-free and physiological conditions suitable

for biomolecules modification.

O

N

HO₂C

O

N

O

N

HO₂C

N

H

O

On the one hand, maleimide readily undergoes Michael

addition with thiols or biothiols and prominent examples in this

context include the design of marketed antibody-drug

conjugate (ADCs), like Kadcyla and Adcetris used for cancer

treatment.⁵On the other hand, this cyclic electron-poor system

is a particularly convenient dienophile partner for Diels-Alder

Maleimide-TEMPO

(spin labeling)

Maleimide-NOTA

(radiolabeling)

N

O

H

N

H

O

NH

O

H

N

HN

N

S

N

5

3

S

O

O₂

(D-A)

cycloaddition

reactions

with

diene-modified

O

(bio)molecules, which have been exploited for the site-specific

derivatization of peptides and proteins,^6-8monoclonal

antibodies,^9,10oligonucleotides,^11-14polysaccharides, as well as

for their immobilization on surface.^15,16In this context, recent

examples include the preparation of carbohydrate-based

Maleimide-dansylamide

(fluorescence labeling)

Maleimide-biotine

(affinity labeling)

O

N

n-Bu

affinity adsorbents by Fort and coworkers through

a

O

furan/maleimide strategy for immobilizing oligosaccharides on

solid matrices.¹⁶The resulting glycoconjugates were used for

the purification of lectins and the capture of anti-blood-group A

and –B antibodies. In addition, Read de Alaniz and Christie

N₂

Maleimide-aryl diazonium salts

(surface functionalization)

N-Arachidonyl maleimide

(monoacylglycerol lipase inhibitor)

Figure 1. Representative examples of commercial maleimide-activated labels.

^a.Normandie Univ, CNRS, UNIROUEN, INSA Rouen,COBRA (UMR 6014), 76000

Rouen (France). E-mail: cyrille.sabot@univ-rouen.fr

Electronic Supplementary Information (ESI) available: [details of any supplementary

information available should be included here]. See DOI: 10.1039/x0xx00000x

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We have recently reviewed the current state-of-the-art in the Additionally, a fast dimerization of 2 was observed after 36 h at

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use of maleimides for bioconjugate reactions,⁴and noticed, room temperature (Figures S34-S35). ^DN^Oe^I:v¹e⁰r^.1t⁰h³e⁹l^/e^Ds⁰s^O, ^Bi⁰t⁰⁴w⁰³a^Ks

however, that only a few scattered comparative studies relatively stable when stored at -25 °C, as less than 10%

between the different dienes were reported. Thus selecting the dimerization was observed after 9 months of storage. It is also

appropriate D-A partner according to the targeted applications worth noting that Read de Alaniz and Christie observed that

is not straightforward, although important theoretical studies cyclopentadiene-modified lysine was stable over the 11-day

have recently been reported on the prediction of rate antibody expression process.¹⁷Despite a modest isolated yield

constants¹⁹and factors governing D-A reactions.^20,21To address of 39 %, fulvene 3 requires only one synthetic step from

this gap, we report, herein, a systematic comparative study of

commercially available starting materials.²⁵Furthermore, no

maleimide-based D-A strategies in terms of dienes accessibility isomeric mixture nor chiral centre is present in this structure. In

and stability, reaction rates, as well as cycloadducts isolation contrast to cyclopentadiene 2, fulvene 3 was fully stable for

and stability. Large differences in reaction rate of D-A suggest more than 16 days at room temperature (Figure S36). Next, the

that dienes could be used concomitantly in sequential labeling racemic cyclohexadiene 4 was prepared from commercial

strategies, which should open a new route for the design of cyclohex-3-en-1-ylmethanol in a total of 5 synthetic steps and

multiple conjugate systems.

15% overall yield (Scheme 1).^12,14This cyclic diene was,

however, isolated along with 6 % of its aromatized analog,

benzyl carbamate (Figure S37), as an inseparable mixture,

whatever the purification method used (normal-phase flash

chromatography, RP-HPLC or recrystallization in water). These

past years, our team reported the preparation of 5-

ethoxyoxazole dienes from commercial α-hydroxy- or α-amino

esters for the total synthesis of natural compounds,^27-30and for

bioconjugation purposes.^8,31,32Importantly, the azaphthalimide

linkers generated from corresponding 5-ethoxyoxazoles

revealed fluorescence behaviors, especially in aqueous media

(Figure S2). In the context of modification of peptides, 5-

ethoxyoxazole 8 bearing a carboxylic acid function was

prepared on the gram scale in a total of six steps and 29%

overall yield.⁸Interestingly, this compound proved stable for

several years at - 25 °C. We postulated that protonation of the

nitrogen atom of the oxazole ring by the carboxylic acid moiety

preserved 8 against oxidative degradation, such as the

formation of the N-oxide derivative which could be observed by

HRMS analysis in a sample of degraded 9 (Figure S39). Thus, to

support this hypothesis and to find a reliable procedure for

long-term storage of 5-alkoxyoxazoles, the model compound 9

containing 1 equiv. of acetic acid was also stored at - 25 °C for

nine months. After that time, only 16% of degradation was

observed, while at the same time the model oxazole 9

contained more than 80% of oxidation product (Figure S38). As

a result, a new bioconjugatable analogue 10 of compound 9,

bearing a carboxylic acid, was prepared from commercially

available ethyl N-acetyl-L-tyrosinate in only three steps and 42%

overall yield. As expected, this diene proved stable since less

than 10% of oxidation was observed after nine months of

storage at -25 °C (Figure S40).

Results and discussions

Diene synthesis/accessibility

Dienes of different natures (linear, cyclic, polycyclic,

heterocyclic), which were already reported to react specifically

with maleimides in chemoselective ligation, were investigated

in this study. Position of the substituent on the diene moiety

was selected according to their previous or potential use in

ligation experiments (Figure 2).

a) Linear

b) Cyclic

N₃

OH

CO₂H

O

3

2

O

1

2

3

O

N

OH

H

6

c) Polycyclic

OH

4

d) Heterocyclic

OH

HO₂C

O

5

6

7

OEt

O

Ph

HO₂C

5

O

HO₂C

N

O

N

8

9

10

Figure 2. Examples of functionalized dienes used in chemoselective ligations with

maleimides.

Dienes 1^6,7,11,18,22, and 5-7^11,23are commercially available, while

2,^15,24, 3,²⁵4^12,14and 8-9⁸were prepared according to known

procedures. In addition, we also report here a novel tyrosine-

based oxazole derivative 10 readily obtained from commercially

available tyrosine ethyl ester. First, the cyclopentadiene

derivative 2 was synthesized in a total of three steps through

freshly distilled cyclopentadiene, and tosylate 11,²⁶which was

obtained in two steps from commercially available 2-[2-(2-

chloroethoxy)ethoxy]ethanol (Scheme 1). Monosubstituted

cyclopentadiene 2 was isolated as a mixture of two inseparable

position isomers, 1- and 2-cyclopentadiene (ratio ≈ 1:1),

resulting from a well-established fast [1,5]-hydride shift.²⁷

2 | J. Name., 2012, 00, 1-3

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Scheme 2. D-A cycloaddition reactions between dienes 1 – 10 and probe 16 for

DOI: 10.1039/D0OB00403K

N₃

kinetic comparisons study.

O

2

NaH

O

2

- Three steps (62% overal yield)

- Regioisomeric mixture (1:1)

- Relatively good shelf-life at -25 °C

(<10% decomposition after nine months)

TsO

N₃

The comparative efficiency of dienes for cycloaddition with

maleimides was thus easily assayed by determining the time

course of fluorescence intensity enhancement over a 60-minute

time scale. Results are summarized in Figure 3. Dienes from

each class of compounds were also subject to RP-HPLC analysis

at t = 10 minutes for cyclopentadiene 2, and t = 60 minutes for

other dienes to confirm fluorescence recovery assays (Table 1).

The cyclopentadiene derivative 2 exhibited by far the highest

rate of fluorescence enhancement. After 10 min of reaction,

maleimide 16 was entirely consumed, which was also confirmed

by RP-HPLC analysis (Figure S10). A calibration curve that

correlates the fluorescence intensity to the cycloadduct

concentration enabled us to determine the second order kinetic

rate constant k₂= 6.50 ± 0.02 M^-1.s^-1in MeCN/PBS pH 7.4 (1:2,

v:v) at T = 20 °C (Figures S19-S21). While results obtained by

fluorescence experiments matched well with the ones

determined by RP-HPLC, the cycloaddition with fulvene and

anthracene are the exception. In fact, no fluorescence intensity

change could be detected after 60 minutes while cycloadducts

formation was observed by RP-HPLC. This result from the fact

that fulvene and anthracene absorb the incident UV light, which

is confirmed by their absorption profiles that exhibit a band at

330 nm, preventing thus the excitation of the dansyl dye (Figure

Freshly

distilled

2

11

(2 steps)

67%

O

CO₂H

3

- One step (39% after 2 purifications)

- No isomeric mixture or chiral center

- Good shelf-life at room temperature

for 16 days

pyrolidine (cat.)

Et₃N

Freshly

distilled

39%

TBSCl

imidazole

Br

Br₂

OTBS

OH

Br

95%

32%

13

12

O

1) tBuOK

O

N

OH

H

4

2) AcCl, MeOH

3) CDI

13

4

- Five steps (15% overall yield)

- Racemic mixture (1:1)

- Good shelf-life at - 25 °C

H₂N

OH

4

49%

OEt

O

I

CO₂Me

HO₂C

5

EtO₂C

CO₂Et

5

(2 steps)

N

HN

8

steps

- Six steps (29% overall yield)

- Good shelf-life at - 25 °C

(several years)

O

I

CO₂Me

OEt

POCl₃

97%

OEt

HN

MeO₂C

O

61%

HO

14

OEt

O

LiOH

then AcOH

O

HO₂C

N

O

MeO₂C

10

N

O

72%

- Three steps (42% overall yield)

15

- Relatively good shelf-life at -25 °C

(<10% decomposition after nine months)

Scheme 1. Syntheses of conjugatable non-commercially available dienes 2-4 and

5-ethoxyoxazoles 8 and 10.

Comparative kinetics study in D-A reactions of dienes with

maleimide: towards double labeling strategies

With dienes 1-10 in hand, their ability to react with maleimides

was investigated through the use of a maleimide-based probe

16 whose fluorescence is quenched by a photoinduced

electron-transfer (PeT) process, which occurs between the

maleimide and the dansyl dye.^34,35Then, upon reaction with

dienes, the maleimide scaffold is transformed to the

corresponding cycloadduct, which leads to a full dansyl

fluorescence recovery (Scheme 2).

O

H

N

R

N

SO₂

N

SO₂

O

Dienes 1-10

O

x

R

MeCN / PBS pH 7.4 (1:2, v:v)

20 °C

PeT ON

PeT OFF

NMe₂

Fluorescence OFF

Fluorescence ON

S2).

17

Probe 16

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Figure 3. A) Time-dependent fluorescence intensity increase of dansyl-based

probe 16 (1 mM, 1 equiv.) in the presence of dienes 1-10 (5 mM, 5 equiv.) in

MeCN/PBS pH 7.4 (1:2, v:v) recorded at λ_em560 nm upon excitation at λ_ex330 nm.

B) Enlarged view of Figure 3A).

The large difference in reaction rates observed between the

cyclopentadiene and other dienes, such as 5-ethoxyoxazoles,

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DOI: 10.1039/D0OB00403K

prompted us to investigate their combined use in the prospect,

for example, of developing one-pot double labeling strategies

potentially useful for biological applications, or for the design of

(hetero)multifunctional cross-linking reagents.^3,36-39Such an

approach generally relies on the use of mutually orthogonal

chemoselective ligations,⁴⁰or the catalytic activation of one of

the two ligations when they share a common chemical function

such as copper-catalyzed alkyne azide cycloadditions (CuAAC)

used in the combination of strain-promoted alkyne azide

cycloaddition (SPAAC).⁴¹In the present study, since one of the

maleimide-based reagent was common to both chemical

ligations, we questioned whether the selectivity for one of the

two dienes could be controlled by their large kinetic difference.

To verify this hypothesis, a stoichiometric mixture of the two

sufficiently kinetically different dienes, namely cyclopentadiene

2/oxazole 8 (1 equiv. each), was treated with maleimide 16 (1

equiv.) at 25 °C and stirred for 1 h (STEP 1, Figure 4A), before

adding the coumarin-based maleimide MC⁴²(5 equiv.) (STEP 2).

This one-pot 2 steps protocol was followed by RP-HPLC analysis.

Figure 4B shows relevant enlarged views of RP-HPLC

chromatograms recorded at 330 nm (λ_absof dansyl) in order to

simplify chromatograms and focus on the selectivity outcome

of the first step. Full-view data also determined at 254 nm are

also available in the ESI. The first step led exclusively to

cycloadduct 17b, observed as a mixture of 4 distinguishable

isomers. Indeed, the formation of the competitive cycloadduct

or azaphthalimide resulting from the reaction of the oxazole 8

with maleimide 16 was not observed (STEP 1 versus CONTROL,

Figure 4B). Then, the addition of the second maleimide (MC) to

the reaction mixture provided mostly the corresponding

azaphthalimide 18 after 24 h (STEP 2). The aromatization of the

oxabicycle intermediate under “neutral conditions”, we

previously observed in acidic conditions,⁸and ending up in an

irreversible rearomatization reaction process, is presumably

promoted by the presence of an internal proton source thanks

to the carboxylic acid function, combined with a long reaction

time.

Of note their corresponding cycloadducts do not absorb at 330

nm (Figures S5-S8). Moderate to low conversions were

observed with the other dienes. A conversion of 39 % was

reached after 60 minutes of reaction with fulvene 3, the second

fastest diene. Then, 5-ethoxyxazoles and anthracene have

shown to react more slowly, with only 17, 13 and 11% of

conversion for compounds 8, 5 and 9, respectively, after 60

minutes. Finally, cyclohexadiene 4, trans-trans-2,4-hexadiene 1,

and furans 6-7 gave even lower kinetics, with less than 5%

conversion after 60 minutes. The same trend was observed with

a lower probe concentration (50 M, Figure S3). Following these

results, k₂was determined for several of these dienes. Due to

their apparent slow kinetics, these values were determined by

RP-HPLC analysis from calibration curves (Figures S22-S25), and

results are summarized in Table 1.

Table 1 Kinetic Data for the D−A Reaction in aqueous media. ^a

% conv. (min)

1 (60)

k₂(M^-1.s^-1)

(8.9 ± 0.7)*10^-4

6.50 ± 0.02

0.15 ± 0.01

n.d. ^b

Diene

1

2

>99 (10)

39 (60)

4 (60)

3

4

13 (60)

<1 (60)

17 (60)

11 (60)

n.d.

5

(8.9 ± 0.1)*10^-4

n.d.

6

7

(1.2 ± 0.4) *10^-2

(5.1 ± 0.3) *10^-3

n.d.

8

9

10

^aReaction in MeCN/PBS pH 7.4 (1:2, v:v) at 20 °C monitored by RP-HPLC (λ = 330

nm), except for diene 2 where fluorescence spectroscopy was used. ^bn.d.: not

determined.

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Figure 4. (A) Sequential labeling experiment from a stoichiometric mixture of cyclopentadiene 2 and oxazole 8 followed by the successive addition of maleimide 16 (1

equiv.) and MC (5 equiv.). (B) RP-HPLC monitoring of the one-pot two step procedure (STEP 1, STEP 2) and references of possible by-products resulting from the reaction

of 8 with 16 (CONTROL).

Next, an enantiomerically pure maleimide partner 19, namely

This experiment clearly illustrates that the kinetics difference (R)-1-(1-phenylethyl)-1H-pyrrole-2,5-dione, was selected as

between the D-A reactions could be exploited to achieve dienophile partner to study first how its chirality could impact

sequential bioconjugate reactions and to offset the need for HPLC analyses of isolated cycloadducts in terms of number of

chemoselectivity between the reactive functions. Moreover, a isomers, while avoiding issues related to the short half-life of

perfect chemical compatibility between 2 and 8 was observed chiral biomolecules for the stability study in blood plasma. In

in our study, which made very convenient this one-pot fact, the formation of uncontrolled stereogenic centers during

procedure.

the cycloaddition process combined to those already present in

biomolecules, may lead to the formation of multiple isomers.

This may complicate the reaction monitoring, as well as the

bioconjugate isolation and characterization in particular with

low-molecular weight conjugates (a few kDa).^5,13,43Besides,

Isolation and stability of conjugates, evaluation of isomer

amounts.

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isomeric products may display significant difference in leads directly to the aromatized azaphthalimide scaffold under

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biological, or physicochemical properties.⁴⁴Accordingly, acidic conditions (the oxabicyclic interm^De^Od^Ii^:a¹t⁰e^.1b⁰³e⁹in^/Dg⁰g^Oe^Bn⁰e⁰⁴ra⁰l³l^Ky

products 20-26 were prepared in moderate to good yields from unstable during chromatographic purification, but prone to

corresponding dienes 1-6 and 8 in a solution of THF/water aromatization), and therefore could not be subjected to a retro-

mixture, whereas one equivalent of trifluoroacetic acid was also D-A process. The reversible behaviour of cycloadducts 20-25

added to the reaction mixture for compound 8 to furnish the was investigated at 37 °C, in a iPrOH/PBS pH 7.4 (1:1) solution

corresponding the fluorescent azaphthalimide 26 (Scheme 3). containing 10 equiv. of N-acetylcysteine (NAC) as a fast

Multiple isomers were observed by ¹H NMR analysis for all maleimide-trapping agent (Figure S49).⁴⁹Formation of the thiol

cycloadducts 20-25, with the exception of azaphthalimide 26 Michael adduct 27, resulting from thio-Michael addition

whose stereogenic centers were suppressed by the reaction between NAC and 19, will be observed only if rD-A

aromatization step. Moreover, a mixture of two isomers was reactions occurred (Scheme 4). Thioether 27 was synthesized,

observed by RP-HPLC only for cycloadducts 21 and 25 generated characterized and used as reference for this study. This

from the cyclopentadiene and furan derivatives, respectively. compound was found to be fully stable after 24 h in iPrOH/PBS

This set of experiments clearly illustrates the advantage of the pH 7.4 (1:1), 37 °C. Stability experiments revealed that all

oxazole- based bioconjugate reaction for low molecular weight cycloadducts were stable for 24

h under physiological

bioconjugates, since, provided the occurring further conditions, except oxabicycle 25, for which thiol Michael adduct

aromatization process, this is the only reaction yielding only one was observed in noticeable amount (30%, Figure S55) which

isomer (either regio or stereoisomer). Consequently, this is the was consistent with recent studies.⁵⁰Of note, the

only diene avoiding the formation of heterogeneous conjugates azaphthalimide 26 was stable under these reaction conditions.

likely to have different properties.

Finally, the chemical and enzymatic stability of conjugates were

investigated by incubating compounds 20-26 in human blood

plasma (HBP) at 37 °C for 24 h.⁵¹HBP is composed of proteins

(8%), organic compounds (1%), and mineral salts (1%). After 24

O

THF / H₂

(2:1, v:v)

O

N

R

+

Conjugate

Ph

20°C

O

h

incubation, HPLC-MS analyses showed that most of

20-26

1-6 and 8

(1.0 equiv.)

19

(1.0 equiv.)

cycloadducts appeared to be stable, except compounds 20 and

26 for which monohydrolysis of the corresponding imide was

observed in about 35 and 4%, respectively, along with

presumably epimerization for 20 and traces of N-oxidation for

26. A plausible explanation for this is that bridged ring systems

in 21-25 provide additional steric hindrances in the vicinity of

the imide function protecting this imide against hydrolysis,

compared to simply fused ring scaffolds 20 and 26. Of note,

degradation products of 20 and 26 maintain nevertheless a

covalent linkage between the two conjugation partners through

the remaining amide bond.

O

N₃

O

HN

O

N

O

N

Ph

N

4

N

O

Ph

CO₂

H

OH

Ph

HO

O

20

(72 h, 79% yield)

22

23

21

(4.5 h, 76% yield)

(2 h, 90% yield)

(72 h, 52% yield)

RP-HPLC : 1

¹H NMR : 2

Isomers: RP-HPLC : 1

¹H NMR : 2

RP-HPLC : 2

RP-HPLC : 1

¹H NMR : 2

¹H NMR : multiple

Ph

N

HO

OH

O

HO₂

C

NHAc

5

N

Ph

O

OH

O

N

HS

N

HO₂C

Ph

O

S

i-PrOH / PBS 7.4

(1:1, v:v)

AcHN

(NAC)

CO₂H

24

26^a

25

R

+

N

O

N

(120 h, 54% yield)

(24 h, 48% yield)

RP-HPLC : 1

¹H NMR : 1

(48 h, 80% yield)

Ph

37°C

RP-HPLC : 1

¹H NMR : 2

RP-HPLC : 2

¹H NMR : 3

O

Ph

27

19

20 - 26

Scheme 3. Conjugates 20-26 obtained from Diels-Alder reactions of dienes 1-6 and

a

8 with maleimide 19, and experimentally identified isomers. Prepared in the

presence of TFA (1 equiv.).

Scheme 4. rD-A study in the presence of an excess of NAC as fast maleimide-

trapping agent.

The stability of the conjugate adduct is important to ensure that

the two ligated units will not prematurely dissociate in

biological media. In fact, the reversible character of the D-A

reaction is closely associated with the nature of the diene-

dienophile pair. Moreover, imide-containing cycloadducts may

also be subject to hydrolysis under physiological conditions, as

reported for example for some thiosuccinimides resulting from

the addition of biothiols to maleimides.^4,45-48Accordingly, the

reversibility, and more generally the stability of imide-based

cycloadducts obtained from different classes of dienes was next

investigated. It should be stressed that the oxazole strategy

Experimental

General

All chemicals were used as received from commercial sources

without further purification, except cyclopentadiene which was

freshly distilled prior to use. Solvents, unless otherwise stated,

were purchased in reagent grade or HPLC grade and used as

received. PBS (pH 7.4, 0.1 M) and aq. mobile phases for HPLC

were prepared with water that was purified by means of a

6 | J. Name., 2012, 00, 1-3

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MilliQ system (purified to 18.2 MΩ cm). For Diels-Alder reaction

ARTICLE

¹H, ¹³C spectra were recorded on 300 MHz Bruker FT-NMR

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DOI: 10.1039/D0OB00403K

in aqueous media, MilliQ H₂O was used in the solvents system. machine operating at ambient probe temperature. The solvent

Human blood plasma (HBP) was furnished by the Centre resonance was used as the internal standard for ¹H-NMR

Hospitalier Universitaire de Rouen and was collected under (chloroform-d at 7.26 ppm ; acetonitrile-d3 at 1.94 ppm ;

medical conditions and supervisions from unknown donors. acetone-d6 at 2.05 ppm ; methanol-d4 at 3.31 ppm ; DMSO-d6

(CAUTION! HBP is a biological substance that must be handled at 2.50) and ¹³C-NMR (chloroform-d at 77.0 ppm ; acetonitrile-

with care by trained personnel. Vials, needles, pipettes, etc. d3 at 1.3 ppm ; acetone-d6 at 29.8 ppm ; methanol-d4 at 49.0

must be considered and treated as biological wastes). All ppm ; DMSO-d6 at 39.5). Chemical shift (δ) were quoted in parts

reactions were monitored by thin layer chromatography (TLC) per million (ppm). Coupling constants (J) were quoted in Hertz

and/or RP-HPLC. TLC were carried out on Merck DC Kieselgel 60 (Hz). The following abbreviations were used to give the

F-254 aluminum sheets. Visualization of spots was performed multiplicity of the NMR signals: s: singlet, bs: broad singlet, d:

under a UV lamp at λ = 254 or 365 nm, and/or staining with a doublet, t: triplet, dd: doublet of doublet…

KMnO₄solution/K₂CO₃+ 5% NaOH, developed with heat. Flash

Fluorescence spectroscopic studies (emission/excitation

column chromatography purifications were performed spectra and time course) were performed on a Varian Cary

manually on silica gel (40–63 µM) under pressurized air flow.

Eclipse® spectrophotometer using a quartz fluorescence cell

(Hellma, 104F-QS, 10 × 4 mm, pathlength 10 mm, chamber

volume 3.5 mL or 1.4 mL). Excitation/emission spectra were

recorded at 20 °C (330/560 nm, excitation and emission filters:

auto, excitation and emission slit: 5 nm or 10 nm). UV-Vis

spectroscopy was performed on a Agilent Cary 60 UV-Vis® at 20

°C.

For conjugates stabilities in HBP, centrifugations were

performed with a VWR MicroStar 12® at 11.000 rotations/min

for 30 min.

Instruments and methods

System A: RP-HPLC analyses were performed with a Thermo

Scientific Ultimate® 3000 RS instrument, equipped with a diode

array detector (DAD-3000RS) and temperature of the column

compartment was fixed at 25 °C. A Thermo Fisher Hypersyl

GOLD® column (1.9 µm, 2.1 × 50 mm) was used with a binary

solvent system composed of MeCN and 0.1% aq. formic acid

(aq. FA, pH 2) as eluents (linear gradient from 5 to 100% MeCN

over 6 min; 100% MeCN for 1.5 min; linear gradient from 100 to

5% MeCN over 1.5 min; 5% MeCN for 2 min) at a flow rate of

0.600 mL/min. System B: RP-HPLC analyses were performed

with a Thermo Scientific Ultimate® 3000 RS instrument,

equipped with a diode array detector (DAD-3000RS) and

temperature of the column compartment was fixed at 25 °C. An

Agilent SB-C18® column (1.8 µm, 2.1 × 50 mm), equipped with

a Phenomenex AJO-9000® C18 pre-column, was used with a

binary solvent system composed of MeCN and 0.1% aq. formic

acid (aq. FA, pH 2) as eluents (linear gradient from 5 to 100%

MeCN over 6 min; 100% MeCN for 1.5 min; linear gradient from

100 to 5% MeCN over 1.5 min; 5% MeCN for 2 min) at a flow

rate of 0.600 mL/min. System C: RP-HPLC-MS analysis were

performed on a Agilent 1200 Series® and Bruker HCT ultra®

instruments, equipped with a Thermo Fisher Accucore® C18

column (2.6 µM, 2.1 × 150 mm). Temperature was maintained

to 22 °C. Injection volume was 4 µL. UV detector (set at 254 nm)

was coupled with a low-resolution MS detector (ESI/ion trap).

Solvent system was composed of MeCN and 0.1% aq. formic

acid (aq. FA, pH 2) as eluents (5% MeCN for 1 min, linear

gradient from 5 to 100% MeCN over 14 min; 100% MeCN for 3

min; linear gradient from 100 to 5% MeCN over 1 min; 5% MeCN

for 11 min) at a flow rate of 0.300 mL/min. System D: Semi-

Synthesis

Diene synthesis

Synthesis of the cyclopentadiene (2)

Tosylate-azide (11). To

a

solution of 2-(2-(2-

azidoethoxy)ethoxy)ethanol⁵⁴(2.5 g, 14.3 mmol, 2 equiv.) in dry

acetonitrile (20 mL), were successively added Et₃N (8.47 mL,

62.7 mmol, 4.4 equiv.) and TsCl (1.36 g, 7.1 mmol, 1.0 equiv.).

The reaction mixture was stirred at room temperature for 2 h.

After completion, a solution of saturated aq. NH₄OH (50 mL)

was poured onto the reaction mixture, followed by brine (25

mL). The aqueous layer was extracted with EtOAc (3 × 50 mL).

Combined organic layers were washed with brine (50 mL), dried

over MgSO₄, and evaporated under vacuum. The crude product

was purified by flash-column chromatography (silica gel,

cyclohexane/EtOAc as gradient from 100:0 to 90:10, v/v) to

provide the product (2.32 g, 7.03 mmol, 99 % yield) as a yellow

oil. The following ¹H NMR analysis was in accordance with

literature data.^{25 1}H NMR (300 MHz, CDCl₃): δ 7.82 – 7.76 (m,

2H), 7.37 – 7.30 (m, 2H), 4.19 – 4.13 (m, 2H), 3.73 – 3.66 (m, 2H),

3.66 – 3.61 (m, 2H), 3.60 (s, 4H), 3.40 – 3.33 (m, 2H), 2.44 (s,

3H).

preparative RP-HPLC were performed with

a Interchim

Cyclopentadiene (2). To a solution of freshly distilled

cyclopentadiene (380 µL, 4.56 mmol, 3 equiv.) in dry THF (5mL)

cooled at 0 °C, under argon atmosphere, was added sodium

hydride (60% w/w in mineral oil, 182 mg, 4.56 mmol, 3 equiv.)

in one portion into the reaction mixture, which became rapidly

dark red. After 15 min, the homogenous mixture was cooled at

-78 °C, and a solution of 2-(2-(2-azidoethoxy)ethoxy)ethyl 4-

methylbenzenesulfonate 11 (500 mg, 1.52 mmol, 1 equiv.) in

PuriFlash® 4250 instument equipped with a UV-visible detector,

and a Hypersil Gold® column (5 µm, 250 × 20 mm) with MeCN

and 0.1% aq. FA as eluents (5% MeCN for 5 min, 5–100% MeCN

for 48 min) at a flow rate of 20 mL/min.

High Resolution Mass spectrometry (HRMS) was performed

using a Waters Micromass LCT Premier XE® equipped with an

orthogonal acceleration time-of-flight (oa-TOF) and an

electrospray source in positive or negative mode.

This journal is © The Royal Society of Chemistry 20xx

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dry THF (5 mL) was introduced dropwise to the mixture. After as gradient from 100:0 to 95:5, v/v)) to pro v_Vid_iee_{w A}t_rth_ice_{le O}t_nit_linle_e

stirring at – 78 °C for 1 h, the reaction mixture was allowed to compound (270 mg, 0.70 mmol, 32 % yie^Dld^O)^I:a¹s⁰a^.10li³g⁹h^/Dt ⁰y^Oel^Blo⁰⁰w⁴⁰o³i^Kl.

warm to 0 °C over 2 h. After completion, the mixture was This procedure was applied several times in order to get

1

filtered on a Celite® plug and the precipitate was washed four sufficient amount of product. The following H NMR analysis

times with EtOAc and DCM. The filtrate was concentrated under was in accordance with literature data.^{14 1}H (300 MHz, CDCl₃): δ

vacuum to give a brown solid, which was purified by flash- 4.73 – 4.64 (m, 2H), 3.48 (d, J = 5.7 Hz, 2H), 2.54 – 2.40 (m, 1H),

column chromatography (silica gel, cyclohexane/EtOAc as 2.26 – 2.14 (m, 1H), 2.14 – 2.02 (m, 1H), 2.02 – 1.90 (m, 2H),

gradient from 100:0 to 90:10, v/v) to afford 1-(2-(2-(2- 1.67 – 1.54 (m, 2H), 0.90 (s, 9H), 0.05 (s, 6H).

azidoethoxy)ethoxy)ethyl)cyclopenta-1,3-diene 2 (227 mg, 0.95

mmol, 67 % yield) as a light yellow oil. IR (neat): 2924, 2865,

2099, 1745, 1709, 1442, 1346, 1284, 1110, 928, 811. Two

Cyclohexadiene (4). To a solution of tert-butyl((3,4-

dibromocyclohexyl)methoxy)dimethylsilane 13 (685 mg, 1.77

mmol, 1 equiv.) in dry THF (25 mL), at room temperature and

inseparable isomers were observed by NMR in a 1:1 ratio and

under argon atmosphere, was added Aliquat 336 (0.02 mL,

are reported below: ¹H NMR (300 MHz, CDCl₃): δ 6.44 (ddq, J =

0.035 mmol, 0.02 equiv.). tBuOK (436 mg, 3.89 mmol, 2.2

equiv.) was rapidly added in one portion to the mixture, which

was stirred for 3 h. After, completion, the reaction was

quenched with water (5 mL) and sat. aq. NH₄Cl (20 mL). The

resulting mixture was extracted with EtOAc (2 × 30 mL).

Combined organic layer dried over MgSO₄, and evaporated

under vacuum. The crude product was partially purified by a

12.0, 3.1, 1.5 Hz, 3H), 6.27 (ddd, J = 5.3, 2.7, 1.4 Hz, 1H), 6.24 –

6.19 (m, 1H), 6.11 – 6.05 (m, 1H), 3.71 – 3.59 (m, 16H), 3.38 (t, J

= 5.0 Hz, 4H), 2.94 (dtd, J = 4.2, 2.9, 1.5 Hz, 4H), 2.94 (dtd, J =

4.2, 2.9, 1.5 Hz, 4H), 2.75 – 2.63 (m, 4H), 1.66 (s, 1H), 1.34 – 1.18

(m, 1H). ¹³C NMR (75 MHz, CDCl₃): δ 146.0, 143.6, 134.7, 133.8,

132.4, 131.1, 127.7, 127.4, 77.5, 77.1, 76.7, 71.3, 70.8, 70.7,

70.3, 70.2, 70.1, 50.7, 43.8, 41.4, 31.0, 30.3. HRMS (ESI+)

rapid flash-column chromatography (silica gel, cyclohexane)

Calculated for C₁₁H₂₁N₄O₂[M +H]⁺: 241.1665 ; found : 241.1664.

and a light yellow oil was obtained, which was directly engaged

in the next step. To a solution of tert-butyl(cyclohexa-2,4-dien-

HPLC System A (λ = 254 nm) t_R= 4.24 min.

1-ylmethoxy)dimethylsilane (≈ 1.77 mmol, 1 equiv.) in dry

methanol (10 mL), at 0 °C and under argon atmosphere, was

added acetyl chloride (0.02 mL, 0.27 mmol, 0.15 equiv.). After

30 min, the solvent was evaporated under vacuum. The crude

Synthesis of the cyclohexadiene (3).

tert-Butyl(cyclohex-3-en-1-ylmethoxy)dimethylsilane (12).

To a solution of cyclohex-3-en-1-ylmethanol (2.00 g, 17.8 mmol,

1 equiv.) in dry DMF (8 mL) under argon atmosphere, was added

product was partially purified by

a rapid flash-column

imidazole (2.43 g, 35.7 mmol, 2 equiv.). The reaction mixture

was cooled to 0 °C and then, tert-butyldimethylsilyl chloride

(3.23 g, 21.4 mmol, 1.2 equiv.) was added. After 5 min, the

reaction was allowed to warm to room temperature for and was

stirred for 1 h. After completion, the reaction was quenched

with water (80 mL) and the resulting mixture was extracted with

Et₂O (3 × 40 mL). Combined organic layer was washed with

water (50 mL) and brine (50 mL) and was dried over MgSO₄, and

evaporated under vacuum. The crude product was purified by

flash-column chromatography (silica gel, cyclohexane) to

provide the title compound (3.65 g, 16.1 mmol, 95 % yield) as a

colorless liquid. The following ¹H NMR analysis was in

accordance with literature data.^{14 1}H NMR (300 MHz, CDCl₃): δ

5.67 (d, J = 2.2 Hz, 2H), 3.48 (d, J = 1.9 Hz, 1H), 3.49 (d, J = 1.9

Hz, 1H), 2.12 – 2.00 (m, 3H), 1.85 – 1.63 (m, 3H), 1.34 – 1.15 (m,

1H), 0.90 (s, 9H), 0.04 (s, 6H).

chromatography (silica gel, cyclohexane/EtOAc, 8:2, v/v) and a

light yellow oil was obtained, which was in part directly engaged

in the next step. To a solution of cyclohexa-2,4-dien-1-

ylmethanol (255 mg, 2.32 mmol, 1.0 equiv.) in dry MeCN (8 mL)

under argon atmosphere and room temperature, was added

di(1H-imidazol-1-yl)methanone (451 mg, 2.78 mmol, 1.2

equiv.). The reaction was stirred for 1 h at room temperature.

After that time, 6-aminohexan-1-ol (326 mg, 2.78 mmol, 1.2

equiv.) was added and the reaction was stirred overnight. After

completion, the solvent was removed by vacuum. The crude

mixture was dissolved in DCM (20 mL) and was washed with H₂O

(2 × 20 mL), then brine (20 mL). The organic layer was dried over

MgSO₄and solvents were removed by vacuum. The crude

product was purified by flash-column chromatography (silica

gel, cyclohexane/EtOAc as gradient from 100:0 to 60:40, v/v) to

provide the racemic product 4 (56 mg, 0.22 mmol, 49 % yield)

1

tert-Butyl((3,4-dibromocyclohexyl)methoxy)dimethyl-

as a white solid. NB: Aromatized analog was observed by H

silane (13). To

a solution of tert-butyl(cyclohex-3-en-1- NMR (6 %) but was inseparable from the title compound by

ylmethoxy)dimethylsilane 11 (500 mg, 2.2 mmol, 1 equiv.) in dry flash-column chromatography, RP-HPLC (Systems A and B) or

DCM (5 mL), cooled to 0 °C under argon atmosphere, was added recrystallization in water. The following ¹H NMR analysis was in

NaHCO₃(185 mg, 2.2 mmol, 1.0 equiv.). A solution of bromine accordance with literature data.^{14 1}H NMR (300 MHz, acetone-

(0.12 mL g, 2.42 mmol, 1.2 equiv.) in DCM (3 mL) was added d6): δ 6.19 (bs, 1H), 6.00 – 5.60 (m, 4H), 4.01 – 3.85 (m, 2H),

dropwise at 0 °C. The reaction mixture was stirred at 0 °C for 2 3.58 – 3.47 (m, 2H), 3.11 (dd, J = 13.0, 6.9 Hz, 2H), 2.68 – 2.48

h after the addition of bromine solution. Then, reaction was (m, 1H), 2.30 – 2.16 (m, 2H), 1.60 – 1.25 (m, 8H). HPLC System

quenched with a mixture of sat. aq. Na₂S₂O₃and sat. aq. Na₂CO₃A (λ = 254 nm) t_R= 3.65 min.

(15 mL, 1:1, v/v). The resulting mixture was extracted with

EtOAc (3 × 10 mL). Combined organic layer dried over MgSO₄, Synthesis of oxazoles

and evaporated under vacuum. The crude product was purified

by flash-column chromatography (silica gel, cyclohexane/EtOAc

8 | J. Name., 2012, 00, 1-3

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Oxazole (9). To a solution of (S)-ethyl 2-acetamido-3- 15.11, 14.40. HRMS (ESI+) Calculated for C₁₆H₂₀NO₅[M +H]⁺:

View Article Online

DOI: 10.1039/D0OB00403K

phenylpropanoate (1.00 g, 4.25 mmol, 1 equiv.) in dry toluene 306.1341 ; found :306.1341.

(10 mL), was added POCl₃(0.61 mL, 6.38 mmol, 1.5 equiv.). The

Oxazole (10). To a solution of methyl 2-(4-((5-ethoxy-2-

mixture was heated at 80 °C for 3 h. After completion, the

reaction mixture was cooled to room temperature and poured

onto a saturated solution of aq. NaHCO₃(100 mL) cooled to 0

°C. The aqueous layer was extracted with EtOAc (2 × 100 mL).

The organic layers were combined and washed with brine (50

mL), dried over MgSO₄, and evaporated under vacuum. The

crude product was purified by flash-column chromatography

(silica gel, cyclohexane/EtOAc 70:30, v/v) to furnish the

corresponding product 8 (691 mg, 3.18 mmol, 75 % yield) as a

methyloxazol-4-yl)methyl)phenoxy)acetate 14 (200 mg, 0.644

mmol, 1 equiv.) in THF (6 mL), were successively added

deionized water (0.91 mL) and LiOH (31 mg, 1.31 mmol, 2

equiv.). The resulting reaction mixture was stirred at 70 °C for 3

h. After completion, the reaction mixture was cooled to room

temperature and glacial AcOH was added (150 mL, 2.62 mmol,

4 equiv.). The reaction mixture was stirred at room temperature

for 1 h. The solution was diluted with deionized water (2 mL)

and extracted with EtOAc (3 × 20 mL), and the combined organic

layers dried over MgSO₄, and evaporated under vacuum. The

crude product was co-evaporated several times with toluene (5

× 8mL) to obtained the title compound (137 mg, 0.47 mmol, 72

% yield) as a colorless oil. IR (neat): 2927, 2507, 1739, 1673,

1612, 1583, 1510, 1440, 1375, 1250, 1207, 1177, 1112, 1078,

1013, 976, 909. ¹H (300 MHz, CDCl₃): δ 11.43 (s, 1H), 7.13 (d, J =

8.7 Hz, 2H), 6.85 – 6.73 (d, J = 8.7 Hz, 2H), 4.53 (s, 2H), 4.09 (q, J

= 7.1 Hz, 2H), 3.65 (s, 2H), 2.33 (s, 3H), 1.31 (t, J = 7.1 Hz, 3H).

¹³C NMR (75 MHz, CDCl₃): δ 171.94, 156.59, 153.94, 153.64,

132.15, 129.69, 115.42, 114.69, 70.76, 65.47, 29.32, 15.06,

13.85. HRMS (ESI+) Calculated for C₁₅H₁₆NO₅[M +H]⁺: 290.1028

; found : 290.1033.

1

yellow oil. The following H NMR analysis was in accordance

with literature data.^{27 1}H NMR (300 MHz, CDCl₃): δ 7.32 – 7.13

(m, 5H), 4.07 (q, J = 7.1 Hz, 2H), 3.71 (s, 2H), 2.30 (s, 3H), 1.31 (t,

J = 7.1 Hz, 3H).

Tyrosine ethyl ester (14). To a solution of ethyl acetyl-L-

tyrosinate (1.454 g, 6.2 mmol, 1 equiv.) in dry DMF (7.2 mL), was

added K₂CO₃(1.5 g, 12.4 mmol, 2 equiv.). The resulting reaction

mixture was stirred at room temperature for 30 min, then

methyl iodide (1.17 mL, 12.4 mmol, 2 equiv.) was added. The

reaction mixture was stirred at room temperature for 12 h. The

solution was diluted with EtOAc (30 mL) and the resulting

solution was washed with water (5 × 50 mL), dried over MgSO₄,

and evaporated under vacuum. The crude product was purified

by flash-column chromatography (silica gel, cyclohexane/EtOAc

50:50, v/v) to provide the title compound (1.095 g, 3.39 mmol,

61 % yield) as a white amorphous powder. mp 56 °C. IR (neat):

3312, 1736, 1635, 1514, 1374, 1294, 1225, 1198, 1138, 1077,

1006, 862. ¹H (300 MHz, CDCl₃): δ 7.02 (d, J = 8.7 Hz, 2H), 6.82

(d, J = 8.7 Hz, 2H), 5.94 (d, J = 7.5 Hz, 1H), 4.81 (dt, J = 7.8, 5.8

Hz, 1H), 4.60 (s, 2H), 4.16 (tt, J = 7.2, 3.7 Hz, 2H), 3.80 (s, 3H),

3.06 (dd, J = 5.7, 1.8 Hz, 2H), 1.98 (s, 3H), 1.24 (t, J = 7.2 Hz, 2H).

¹³C NMR (75 MHz, CDCl₃): δ 171.68, 169.73, 169.27, 156.78,

130.32, 129.23, 114.54, 65.19, 61.29, 53.24, 52.09, 36.90, 22.86,

14.00. HRMS (ESI+) Calculated for C₁₆H₂₂NO₆[M +H]⁺: 324.1447

; found : 324.1459.

Oxazole Methyl ester (15). To a solution of ethyl (S)-2-

acetamido-3-(4-(2-methoxy-2-oxoethoxy)phenyl)propanoate

13 (500 g, 1.54 mmol, 1 equiv.) in dry toluene (5 mL), was added

POCl₃(187 mL, 2.01 mmol, 1.3 equiv.). The resulting reaction

mixture was stirred at 80 °C for 4.5 h. After completion, the

solution was added dropwise to a solution of saturated aq.

NaHCO₃(50 mL), and the resulting mixture was extracted with

EtOAc (3 × 25 mL). The organic layers were combined, dried

over MgSO4, and evaporated under vacuum. The crude product

was purified by flash-column chromatography (silica gel,

cyclohexane/EtOAc 50:50, v/v) to furnish the title comound

(454.2 mg, 1.49 mmol, 97% yield) as a pale yellow oil. IR (neat):

2993, 2955, 1760, 1669, 1612, 1586, 1510, 1438, 1375, 1348,

1269, 1204, 1176, 1085, 1022, 953, 909. ¹H (300 MHz, CDCl₃): δ

7.17 (d, J = 8.3 Hz, 2H), 6.87 – 6.78 (d, J = 8.3 Hz, 2H), 4.59 (d, J

= 0.9 Hz, 2H), 4.07 (qd, J = 7.1, 1.2 Hz, 2H), 3.79 (d, J = 1.4 Hz,

3H), 3.63 (s, 2H), 2.29 (d, J = 1.1 Hz, 3H), 1.31 (td, J = 7.1, 1.2 Hz,

3H). ¹³C NMR (75 MHz, CDCl₃): δ 169.61, 156.38, 153.98, 152.55,

132.97, 129.71, 115.96, 114.74, 70.47, 65.59, 52.28, 30.16,

Maleimide synthesis

Maleimide-dansylamide (16). To a solution of 1-(2-amino-

ethyl)-pyrrole-2,5-dione hydrochloride (113 mg, 0.81 mmol, 2.2

equiv.) and dansyl chloride (100 mg, 0.37 mmol, 1.0 equiv.) in

DMF (6 mL) was added DIEA (142 µL, 0.82 mmol, 2.2 equiv.).

The reaction mixture was stirred at room temperature for 2 h.

After completion, the reaction was poured onto water (60 mL)

and the aqueous layer was extracted with DCM (3 × 20 mL).

Combined organic layers were dried over MgSO₄and solvents

were removed by vacuum. The crude product was purified by

flash-column chromatography (silica gel, DCM/EtOAc 90:10,

v/v) to provide the title compound (97 mg, 0.26 mmol, 70 %

yield) as a pale yellow solid. The following ¹H NMR analysis was

in accordance with literature data.^{7 1}H NMR (300 MHz, CDCl₃):

δ 8.51 (d, J = 8.6 Hz, 1H), 8.24 – 8.15 (m, 2H), 7.53 (ddd, J = 10.1,

8.6, 7.5 Hz, 2H), 7.17 (dd, J = 7.6, 0.6 Hz, 1H), 6.34 (s, 2H), 5.07

(t, J = 6.1 Hz, 1H), 3.53 (t, J = 5.7 Hz, 2H), 3.16 (dd, J = 11.5, 6.0

Hz, 2H), 2.88 (s, 6H). HPLC System A (λ = 254 nm) t_R= 3.40 min.

Chiral maleimide (19). To a solution of (R)-1-phenylethanol

(500 mg, 4.09 mmol, 1.0 equiv.) in dry THF (15 mL, 0.41 M) and

under argon atmosphere, cooled to 0 °C, were successively

added PPh₃(1.180 mg, 4.5 mmol, 1.1 equiv.) and diisopropyl

azodicarboxylate (0.88 mL, 4.5 mmol, 1.2 equiv.). The reaction

was stirred for 15 min at 0 °C, then 1H-pyrrole-2,5-dione (597

mg, 6.14 mmol, 1.5 equiv.) was introduced into the mixture,

which was stirred for 30 min at 0 °C, and overnight at room

temperature. After completion, the solvent was removed by

vacuum. The crude product was purified by flash-column

chromatography (silica gel, cyclohexane/EtOAc 90:10, v/v). to

furnish the title compound (278 mg, 1.38 mmol, 34 % yield) as

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1

129.9, 129.8, 129.4, 129.4, 128.4, 128.4, 126.3, 123.3, 119.0,

a yellow oil. The following H NMR analysis was in accordance

with literature data.^{55 1}H NMR (300 MHz, CDCl₃): δ 7.43 – 7.37

(m, 2H), 7.34 – 7.21 (m, 3H), 6.61 (s, 2H), 5.34 (q, J = 7.4 Hz, 1H),

1.81 (d, J = 7.4 Hz, 3H). HPLC System A (λ = 254 nm) t_R= 3.76

min.

View Article Online

118.9, 115.2, 70.7, 70.5, 70.1, 70.0, 70.0, 69.9, 69.0, 68.7, 56.7,

DOI: 10.1039/D0OB00403K

55.7, 52.1, 50.7, 50.7, 48.9, 48.1, 47.3, 46.9, 45.7, 45.4, 45.2,

44.7, 41.9, 41.7, 37.5, 37.2, 31.1, 30.6, 29.7. HRMS (ESI+)

Calculated for C₂₉H₃₆N₆O₆S [M+H]⁺: 597.2495 ; found :

597.2493. HPLC System A (λ = 254 nm) t_R= 4.56 min, 4.61 min.

Cycloadduct fulvene-dansylamide (17c). To a solution of 5-

(cyclopenta-2,4-dien-1-ylidene)hexanoic acid 3 (10 mg, 0.058 mmol,

1.0 equiv.) in a solution of THF/H₂O (0.7 mL, 2:1 v/v) was added a

solution of 16 (24 mg, 0.064 mmol, 1.1 equiv.) in THF/H₂O (0.5 mL,

2:1 v/v). The mixture was stirred at room temperature for 3 h. After

completion, the reaction mixture was diluted with water (10 mL) and

the aqueous layer extracted with EtOAc (3 × 10 mL). The organic

layers were combined, dried over MgSO₄, and solvents were

removed under vacuum. The crude product was purified by flash-

column chromatography (silica gel, DCM/EtOAc/MeOH as gradient

from 100:0:0 to 90:10:0, 80:20:0, 70:30:0 and 50:40:10 v/v) to

provide the title compound (29 mg, 0.053 mmol, 91 % yield) as a pale

yellow oil. IR (neat):3268, 2927, 2858, 2794, 2253, 1770, 1689, 1589,

1575, 1397, 1324, 1143, 909, 728. ¹H NMR (300 MHz, CDCl₃): δ 8.52

(d, J = 8.5 Hz, 1H), 8.30 – 8.13 (m, 2H), 7.65 – 7.45 (m, 2H), 7.19 (d, J

= 7.5 Hz, 1H), 6.12 – 6.01 (m, 2H), 5.22 (bs, 1H), 3.76 – 3.63 (m, 2H),

3.40 (overlaped triplet, J = 5.7 Hz, 2H), 3.03 (dd, J = 11.4, 5.8 Hz, 2H),

2.98 – 2.89 (m, 1H), 2.85 (s, 6H, overlaped with multiplet at 2.84

ppm), 2.84 (m, 1H, overlaped with singlet at 2.85 ppm), 2.29

(overlaped triplet, J = 7.2 Hz, 2H), 1.97 (t, J = 7.3 Hz, 2H), 1.70 (dt, J =

7.2, 7.2 Hz, 2H), 1.54 (s, 3H). ¹³C NMR (75 MHz, CDCl₃): δ 178.4,

177.4, 177.3, 151.9, 148.7, 134.8, 134.7, 134.4, 130.4, 129.8, 129.4,

128.5, 123.3, 119.0, 118.96, 115.3, 113.9, 45.4, 45.1, 44.8, 44.4, 44.2,

41.6, 37.5, 32.9, 32.6, 22.6, 16.9. HRMS (ESI-) Calculated for

C₂₉H₃₄N₃O₆S [M+H]⁺: 552.2168 ; found : 552.2166. HPLC System B

(λ = 330 nm) t_R= 4.16 min, 4.25 min.

Preparation of cycloadducts

Cycloadduct linear diene-dansylamide (17a). To a solution of

(2E,4E)-hexa-2,4-dien-1-ol 1 (10.6 mg, 0.108 mmol, 2.0 equiv.) in a

solution of THF/H₂O (0.5 mL, 2:1 v/v) was added a solution of 16 (20

mg, 0.054 mmol, 1.0 equiv.) in THF/H₂O (0.5 mL, 2:1 v/v). The mixture

was stirred at room temperature for 2 days. After completion, the

reaction mixture was diluted with water (10 mL) and the aqueous

layer extracted with EtOAc (3 × 10 mL). The organic layers were

combined, dried over MgSO₄, and solvents were removed under

vacuum. The crude product was purified by flash-column

chromatography (silica gel, DCM/EtOAc as gradient from 100:0 to

90:10, 80:20 and 50:50 v/v) to provide the title compound (22 mg,

0.047 mmol, 86 % yield) as a pale yellow oil. IR (neat): 3451, 3284,

2926, 2851, 1767, 1684, 1588, 1573, 1403, 1327, 1141, 975, 945, 789.

¹H NMR (300 MHz, MeOD): δ 8.56 (d, J = 8.5 Hz, 1H), 8.26 (d, J = 8.7

Hz, 1H), 8.14 (dd, J = 7.3, 1.2 Hz, 1H), 7.63 – 7.52 (m, 2H), 7.29 (d, J =

7.6 Hz, 1H), 5.69 (dt, J = 9.0, 3.0 Hz, 1H), 5.61 (dt, J = 9.2, 2.9 Hz, 1H),

3.97 (dd, J = 11.1, 6.9 Hz, 1H), 3.86 (dd, J = 11.0, 8.1 Hz, 1H), 3.39 (t,

J = 6.1 Hz, 2H), 2.97 (t, J = 6.2 Hz, 2H), 2.95 – 2.90 (m, 1H), 2.88 (s,

6H), 2.73 (dd, J = 8.4, 7.0 Hz, 1H), 2.41–2.30 (m, 2H), 1.34 (d, J = 7.4

Hz, 3H), 1.31 – 1.24 (m, 1H). ¹³C NMR (75 MHz, MeOD): δ 179.6,

179.2, 153.2, 136.8, 135.8, 131.2, 130.8, 130.3, 129.2, 124.4, 120.6,

116.5, 63.4, 46.2, 45.8, 43.8, 40.8, 40.1, 38.9, 32.2, 17.1. HRMS

(ESI+) Calculated for C₂₄H₃₀N₃O₅S [M+H]⁺: 472.1906 ; found :

472.1895. HPLC System B (λ = 330 nm) t_R= 3.84 min.

Cycloadduct furan-dansylamide (17d). To a solution of furan-2-

ylmethanol 6 (25 mg, 0.27 mmol, 5.0 equiv.) in a solution of THF/H₂O

(1.0 mL, 2:1 v/v) was added a solution of 16 (20 mg, 0.054 mmol, 1.0

equiv.) in THF/H₂O (0.5 mL, 2:1 v/v). The mixture was stirred at room

temperature for 3 days. After completion, the reaction mixture was

diluted with water (10 mL) and the aqueous layer extracted with

EtOAc (3 × 15 mL). The organic layers were combined, dried over

MgSO₄, and solvents were removed under vacuum. The crude

product was purified by flash-column chromatography (silica gel,

DCM/EtOAc as gradient from 100:0 to 90:10, 80:20, 70:30, 60:40 and

50:50 v/v) to provide the title compound (21 mg, 0.045 mmol, 82 %

yield) as a pale yellow oil. IR (neat): 3483, 3290, 2921, 2828, 1692,

1398, 1323, 1141, 789. ¹H NMR (300 MHz, MeOD): δ 8.61 – 8.51 (m,

1.4H), 8.27 (d, J = 8.7 Hz, 1H), 8.26 (d, J = 8.7 Hz, 0.35H), 8.15 (dd, J =

7.4, 1.0 Hz, 1.34H), 7.64 – 7.51 (m, 2.75H), 7.29 (d, J = 7.3 Hz, 1H),

7.27 (d, J = 7.7 Hz, 0.36H), 6.52 – 6.46 (m, 0.67H), 6.35 (dd, J = 5.8,

Cycloadduct cyclopentadiene-dansylamide (17b). To a

solution of 1-(2-(2-(2-azidoethoxy)ethoxy)ethyl)cyclopenta-1,3-

diene and 2-(2-(2-(2-azidoethoxy)ethoxy)ethyl)cyclopenta-1,3-

diene 2 (47 mg, 0.21 mmol, 5.0 equiv.) in a solution of

MeCN/H₂O (1mL, 1:2 v/v) was added compound 16 (15 mg,

0.042 mmol, 1.0 equiv.). The mixture was stirred at room

temperature for 3 h. After completion, the reaction mixture was

diluted with water (10 mL) and the aqueous layer extracted with

EtOAc (2 × 10 mL). Combined organic layers were dried over

MgSO₄, and solvents were removed under vaccum. The crude

product was purified by flash-column chromatography (silica

gel, DCM/EtOAc as gradient from 100:0 to 90:10, and 80:20, v/v)

to obtain the product (22 mg, 0.037 mmol, 92 % yield) as a dark

yellow foam. At least 2 stereoisomers were observed by NMR in

a ratio of 0.83:1.00. IR (neat): 3276, 2925, 2868, 2100, 1769,

1

1692, 1588, 1574, 1396, 1329, 1143, 909, 790, 727. H NMR

(300 MHz, CDCl₃): δ 8.52 (d, J = 8.6 Hz, 2H), 8.25 (d, J = 8.7 Hz, 1.6 Hz, 1H), 6.22 (d, J = 5.8 Hz, 1H), 5.14 (dd, J = 5.5, 1.6 Hz, 1H), 5.03

2H), 8.20 (ddd, J = 7.3, 3.4, 1.2 Hz, 2H), 7.59 (dd, J = 8.5, 7.7 Hz,

1.9H), 7.55 – 7.46 (m, 2.1H), 7.17 (d, J = 7.6 Hz, 2H), 5.93 (dd, J

= 5.7, 3.0 Hz, 0.8H), 5.80 (d, J = 5.6 Hz, 0.8H), 5.64 –5.50 (m, 1H),

5.38 – 5.30 (t, J = 5.6 Hz, 1H), 5.11 (d, J = 5.6 Hz, 0.9H), 3.70 –

3.57 (m, 11H), 3.57 – 3.42 (m, 8H), 3.41 – 3.33 (m, 8H), 3.25 –

3.18 (m, 2H), 3.16 – 3.11 (m, 1H), 3.09 – 2.96 (m, 5H), 2.94 (dd,

J = 2.8, 1.5 Hz, 2H), 2.87 (s, 11H), 2.36 – 2.02 (m, 6H), 1.74 – 1.64

(m, 2.7H), 1.41 (t, J = 9.4 Hz, 3H), 1.29 – 1.24 (m, 1.8H), 1.21 (t,

J = 7.0 Hz, 1.8H), 1.14 (d, J = 6.4 Hz, 1H), 0.92 – 0.77 (m, 2.9H).

¹³C NMR (75 MHz, CDCl₃): δ 178.3, 177.8, 177.7, 177.4, 152.0,

152.0, 146.9, 137.8, 134.5, 134.4, 134.2, 130.4, 130.4, 129.9,

(d, J = 1.0 Hz, 0.34H), 4.86 (s, 8H), 4.15 – 3.95 (m, 2.48H), 3.80 (d, J =

12.8 Hz, 0.33H), 3.45 (t, J = 6.5 Hz, 0.84H), 3.35 (s, 2.77H), 3.25 (dd, J

= 7.6, 5.5 Hz, 1.35H), 3.06 (d, J = 7.8 Hz, 1H), 3.05 (d, J = 6.1 Hz, 0.66H),

2.96 (t, J = 6.1 Hz, 2.25H), 2.88 (s, 6H), 2.86 (s, 2H), 2.60 (d, J = 6.4 Hz,

0.34H), 2.52 (d, J = 6.5 Hz, 0.34H). ¹³C NMR (75 MHz, MeOD): δ 178.0,

176.9, 176.8, 176.6, 153.3, 138.8, 138.0, 136.7, 136.6, 136.2, 131.3,

131.26, 130.8, 130.77, 130.4, 130.35, 129.2, 124.4, 120.6, 116.6,

93.5, 92.9, 82.1, 80.4, 61.5, 60.8, 51.5, 46.7, 45.8, 40.9, 40.7, 39.4,

38.9. HRMS (ESI+) Calculated for C₂₃H₂₆N₃O₆S [M+H]⁺: 472.1542 ;

10 | J. Name., 2012, 00, 1-3

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ARTICLE

found : 472.1541. HPLC System B (λ = 330 nm) t_R= 3.19 min, 3.49 at room temperature for 2 h. After completion,_Vt_ieh_we_Arr_tie_cla_ec_Ot_ni_lo_inn_e

min.

Azaphtalimide - coumarin (18). To a solution of 6-(5-ethoxy-

2-methyloxazol-4-yl)hexanoic acid 7 (9.4 mg, 0.039 mmol, 1.0

equiv.) in a solution of THF/H₂O (0.5 mL, 2:1 v/v) were added 7-

(diethylamino)-N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-

mixture was diluted with water (10 mL) ^Da^On^Id^{: 1}t⁰h^.1e⁰³a⁹q^/u^De^0Oou^B0s⁰la⁴⁰y³e^Kr

extracted with EtOAc (2 × 15 mL). Combined organic layers were

dried over MgSO₄, and solvents were removed under vacuum.

The crude product was purified by flash-column

chromatography (silica gel, cyclohexane/EtOAc as gradient from

100:0 to 90:10, 80:20 and 70:30, v/v) to provide the title

compound (65 mg, 0.15 mmol, 90 % yield) as a white foam. At

least 4 stereoisomers were observed by NMR in a ratio

1.00:1.00:1.70:0.95. IR (neat): 2941, 2968, 2100, 1694, 1453,

yl)ethyl)-2-oxo-2H-chromene-3-carboxamide MC⁴²(15 mg,

0.039 mmol, 1.0 equiv.) and TFA (3 µL, 0.043 mmol, 1.1 equiv.).

The mixture was stirred at room temperature for 26 h. After

completion, and solvents were removed under vacuum. The

crude product was purified by RP-HPLC (System D) and fractions

were collected and lyophilized to provide the title compound

(12 mg, 0.019 mmol, 49 % yield) as a yellow powder. IR (neat):

3330, 2976, 2927, 2858, 1708, 1617, 1581, 1514, 1402, 1352,

1

1354, 1188, 1113, 913, 728, 698. H NMR (300 MHz, CDCl₃): δ

7.43 – 7.34 (m, 10H), 7.33 – 7.26 (m, 9H), 7.25 – 7.18 (m, 6H),

5.98 (dd, J = 5.6, 2.9 Hz, 1H), 5.89 – 5.81 (m, 2H), 5.70 (d, J = 5.0

Hz, 1H), 5.60 – 5.55 (m, 1.7H), 5.50 (s, 1H), 5.23 (dd, J = 14.5, 7.6

Hz, 4.4H), 3.69 – 3.56 (m, 29.5H), 3.56 – 3.47 (m, 7.4H), 3.41 –

3.33 (m, 11.4H), 3.31 – 3.16 (m, 18.9H), 3.01 (dd, J = 7.3, 6.1 Hz,

2.2H), 2.42 – 2.26 (m,2.5H), 2.26 – 2.10 (m, 4.3H), 1.97 – 1.83

(m, 3.8H), 1.72 – 1.64 (m, 20.5H), 1.49 – 1.42 (m, 5.2H). ¹³C NMR

(75 MHz, CDCl₃): δ 177.8, 177.7, 177.4, 177.4, 177.2, 177.2,

177.1, 177.1, 146.8, 146.7, 139.9, 139.6, 139.5, 137.8, 137.7,

134.1, 134.0, 128.2, 128.1, 128.0, 127.8, 127.6, 127.6, 127.5,

127.5, 126.5, 126.5, 70.6, 70.6, 70.0, 69.9, 69.9, 69.9, 69.0, 68.9,

68.7, 57.0, 56.9, 55.5, 55.5, 52.2, 50.6, 50.1, 49.6, 49.6, 49.5,

48.3, 47.9, 47.8, 46.9, 46.8, 46.6, 46.6, 45.5, 45.3, 45.3, 45.0,

45.0, 31.1, 31.1, 30.8, 30.4, 16.8, 16.3, 16.2, 16.0. HRMS (ESI+)

Calculated for C₂₃H₂₉N₄O₄[M+H]⁺: 425.2189 ; found : 425.2180.

HPLC System A (λ = 254 nm) t_R= 4.55 min, 4.71 min.

1

1227, 1189, 1134, 910, 795. H NMR (300 MHz, DMSO-d6): δ

11.95 (bs, 1H), 8.74 (t, J = 6.1 Hz, 1H), 8.57 (s, 1H), 7.65 (d, J =

9.0 Hz, 1H), 6.79 (dd, J = 8.9, 2.3 Hz, 1H), 6.60 (d, J = 2.0 Hz, 1H),

3.76 – 3.65 (m, 2H), 3.58 – 3.50 (m, 2H), 3.48 (q, J = 7.0 Hz, 4H),

2.81 (t, J = 7.6 Hz, 2H), 2.58 (s, 3H), 2.20 (t, J = 7.3 Hz, 3H), 1.69

– 1.57 (m, 2H), 1.56 – 1.46 (m, 2H), 1.42 – 1.31 (m, 2H), 1.14 (t,

J = 7.0 Hz, 6H). ¹³C NMR (75 MHz, DMSO-d6): δ 174.5, 168.0,

162.8, 161.3, 157.2, 152.4, 147.6, 131.5, 120.4, 110.1, 109.4,

107.6, 95.9, 44.3, 37.6, 37.4, 33.6, 32.1, 28.4, 27.3, 24.3, 19.6,

12.3. HRMS (ESI+) Calculated for C₃₀H₃₅N₄O₈[M+H]⁺: 579.2455

; found : 579.2453. HPLC System A (λ = 254 nm) t_R= 3.96 min.

Cycloadduct linear diene-chiral maleimide (20). To a

solution of (2E,4E)-hexa-2,4-dien-1-ol 1 (25 mg, 0.25 mmol, 1.0

equiv.) in a solution of THF/H₂O (1 mL, 2:1 v/v) was added a

solution of 19 (50 mg, 0.25 mmol, 1.0 equiv.) in THF/H₂O (1 mL,

2:1 v/v). The mixture was stirred at room temperature for 3

days. After completion, the reaction mixture was diluted with

water (10 mL) and the aqueous layer extracted with EtOAc (3 ×

15 mL). The organic layers were combined, dried over MgSO₄,

and solvents were removed under vacuum. The crude product

was purified by flash-column chromatography (silica gel,

cyclohexane/EtOAc as gradient from 100:0 to 90:10, 80:20 and

70:30 v/v) to provide the title compound (59 mg, 0.20 mmol, 79

% yield) as a pale yellow oil. Two stereoisomers were observed

by NMR in a ratio of 1:1. IR (neat): 3437, 3090, 3065, 3034, 2969,

2934, 2878, 2847, 1764, 1687, 1452, 1363, 1225, 1189, 912,

727, 694. ¹H NMR (300 MHz, CDCl₃): δ 7.37 – 7.19 (m, 10H), 5.82

– 5.54 (m, 4H), 5.35 (q, J = 7.2Hz, 1H), 5.35 (q, J = 7.2 Hz, 1H),

4.05 – 3.85 (m, 3H), 3.76 (t, J = 10.5 Hz, 1H), 3.28 (dd, J = 15.9,

7.4 Hz, 1H), 3.16 (bs, 1H), 3.00 (ddd, J = 8.6, 6.2, 5.0 Hz, 2H), 2.62

– 2.49 (m, 2H), 2.49 – 2.32 (m, 2H), 1.72 (d, J = 7.3 Hz, 3H), 1.71

(d, J = 7.3 Hz, 3H), 1.48 (d, J = 7.4 Hz, 3H), 1.43 (d, J = 7.4 Hz, 3H).

¹³C NMR (75 MHz, CDCl₃): δ 178.8, 178.7, 177.3, 177.2, 139.2,

139.2, 135.2, 135.2, 128.4, 128.3, 128.2, 128.2, 127.6, 127.5,

127.1, 127.1, 62.7, 62.7, 49.8, 44.7, 44.6, 42.6, 42.4, 38.2, 31.2,

16.7, 16.7, 16.5, 16.4. HRMS (ESI+) Calculated for C₁₈H₂₂NO₃

[M+H]⁺: 300.1600 ; found : 300.1609. HPLC System A (λ = 254

nm) t_R= 3.88 min.

Cycloadduct fulvene-chiral maleimide (22). To a solution of

5-(cyclopenta-2,4-dien-1-ylidene)hexanoic acid 3 (45 mg, 0.25

mmol, 1.0 equiv.) in a solution of THF/H₂O (0.75 mL, 2:1 v/v)

was added a solution of 19 (30 mg, 0.15 mmol, 1.0 equiv.) in

THF/H₂O (0.5 mL, 2:1 v/v). The mixture was stirred at room

temperature for 3 days. After completion, the reaction mixture

was diluted with water (10 mL) and the aqueous layer extracted

with EtOAc (3 × 15 mL). The organic layers were combined, dried

over MgSO₄, and solvents were removed under vacuum. The

crude product was purified twice by flash-column

chromatography (silica gel, cyclohexane/EtOAc as gradient from

100:0 to 90:10, 80:20, 70:30 and 60:40 v/v) to furnish the title

compound (72 mg, 0.19 mmol, 76 % yield) as a colorless oil. Two

stereoisomers were observed by NMR in a ratio of 1:1. IR (neat):

2939, 1766, 1693, 1378, 1354, 1219, 1188, 911, 835, 768, 729,

698. ¹H NMR (300 MHz, CDCl₃): δ 8.67 (bs, 1H), 7.40 – 7.33 (m,

2H), 7.33 – 7.23 (m, 3H), 6.17 – 6.09 (m, 1H), 6.02 – 5.94 (m, 1H),

5.26 (q, J = 7.3 Hz, 1H), 3.83 – 3.69 (m, 2H), 3.17 – 3.05 (m, 2H),

2.24 (overlaped triplet, J = 7.3 Hz, 2H), 1.97 (overlaped triplet, J

= 7.3 Hz, 2H), 1.69 (d, J = 7.3 Hz, 3H, overlaped with signal at

1.68 ppm), 1.68 (dt, J = 7.3, 7.3 Hz, 2H, overlaped with signal at

1.69 ppm), 1.55 (overlaped singlet, 3H). ¹³C NMR (75 MHz,

CDCl₃): δ 179.1, 177.0, 176.9, 148.7, 139.4, 134.6, 134.6, 134.5,

128.1, 127.6, 113.6, 50.0, 44.76, 44.72, 44.70, 44.66, 44.52,

44.46, 44.41, 44.35, 32.94, 32.60, 22.6, 16.9, 16.4. HRMS (ESI-)

Calculated for C₂₃H₂₄NO₄[M-H]^-: 378.1705 ; found : 378.1703.

HPLC System B (λ = 254 nm) t_R= 4.36 min.

Cycloadduct cyclopentadiene-chiral maleimide (21). To a

solution of 1-(2-(2-(2-azidoethoxy)ethoxy)ethyl)cyclopenta-1,3-

diene and 2-(2-(2-(2-azidoethoxy)ethoxy)ethyl)cyclopenta-1,3-

diene 2 (39 mg, 0.17 mmol, 1.0 equiv.) in a solution of THF/H₂O

(1 mL, 2:1 v/v) was added a solution of 19 (50 mg, 0.25 mmol,

1.0 equiv.) in THF/H₂O (1 mL, 2:1 v/v) . The mixture was stirred

Cycloadduct cyclohexadiene-chiral maleimide (23). To a

solution

of

cyclohexa-2,4-dien-1-ylmethyl

(6-

J. Name., 2013, 00, 1-3 | 11

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Page 12 of 15

ARTICLE

hydroxyhexyl)carbamate 4 (38 mg, 0.15 mmol, 1.0 equiv.) in a

Journal Name

Cycloadduct furan-chiral maleimide (25). To _Va_iewso_Al_ru_tict_li_eo_On_nlio_nef

DOI: 10.1039/D0OB00403K

solution of THF/H₂O (1 mL, 2:1 v/v) was added a solution of 19 furan-2-ylmethanol 6 (24.5 mg, 0.25 mmol, 1.0 equiv.) in a

(30 mg, 0.15 mmol, 1.0 equiv.) in THF/H₂O (1 mL, 2:1 v/v). The solution of THF/H₂O (1 mL, 2:1, v/v) was added a solution of 19

mixture was stirred at room temperature for 3 days. After (50 mg, 0.25 mmol, 1.0 equiv.) in THF/H₂O (1 mL, 2:1, v/v). The

completion, the reaction mixture was diluted with water (10 mixture was stirred at room temperature for 48 h. After

mL) and the aqueous layer extracted with EtOAc (3 × 15 mL). completion, the reaction mixture was diluted with water (10

The organic layers were combined, dried over MgSO₄, and mL) and the aqueous layer extracted with EtOAc (2 × 15 mL).

solvents were removed under vacuum. The crude product was The organic layers were combined and dried over MgSO₄, and

purified twice by flash-column chromatography (silica gel, solvents were removed under vacuum. The crude product was

cyclohexane/EtOAc as gradient from 100:0 to 90:10, 80:20, purified by flash-column chromatography (silica gel,

70:30 and 60:40 v/v) to furnish the title compound (48 mg, 0.11 cyclohexane/EtOAc as gradient from 100:0 to 70:30 and finally

mmol, 70 % yield) as a white foam. Two stereoisomers were 50:50, v/v) to furnish the title compound (60 mg, 0.20 mmol, 80

observed by NMR in a ratio of 1:1. IR (neat): 3358, 2931, 2860, % yield) as a colorless oil. Three stereoisomers were observed

1687, 1528, 1453, 1361, 1234, 1190, 1056, 1024, 698. ¹H NMR by NMR in a ratio of 0.7:1:1. IR (neat): 3449, 2925, 2857, 1691,

(300 MHz, CDCl₃): δ 7.39 – 7.30 (m, 5H), 7.30 – 7.27 (m, 3H), 1378, 1356, 1219, 1188, 1025, 983, 969, 730, 697. ¹H NMR (300

7.25 – 7.19 (m, 2H) 6.20 – 5.88 (m, 4H), 5.30 (q, J = 7.3 Hz, 2H), MHz, CDCl₃): δ 7.41 – 7.27 (m, 13.2H), 6.60 (dd, J = 5.7, 1.5 Hz,

4.64 (bs, 2H), 3.79 (dd, J = 10.8, 5.8 Hz, 2H), 3.64 (t, J = 6.5 Hz, 0.7H), 6.55 – 6.50 (m, 0.7H), 6.28 (dd, J = 5.8, 1.7 Hz, 1H), 6.20

2H), 3.64 (t, J = 6.5 Hz, 2H), 3.59 – 3.47 (m, 3H), 3.25 – 3.03 (m, (d, J = 5.8 Hz, 1H), 6.12 (dd, J = 5.8, 1.7 Hz, 1H), 6.02 (d, J = 5.8

9H), 2.86 – 2.67 (m, 4H), 2.18 – 1.96 (m, 2H), 1.94 – 1.74 (m, 1H), Hz, 0.9H), 5.39 (q, J = 7.3 Hz, 0.9H), 5.30 – 5.15 (m, 4.8H), 4.29 –

1.68 (d, J = 7.3 Hz, 6H), 1.61 – 1.44 (m, 10H), 1.44 – 1.27 (m, 4.06 (m, 5.4H), 4.03 – 3.83 (m, 1.4H), 3.58 (dd, J = 7.8, 5.4 Hz,

10H), 1.21 (t, J = 7.0 Hz, 3H), 1.14 (d, J = 6.6 Hz, 3H) 0.98 – 0.70 1.9H), 3.32 (dd, J = 7.8, 1.7 Hz, 2H), 2.95 (dd, J = 6.6, 1.0 Hz,

(m, 4H). ¹³C NMR (75 MHz, CDCl₃): δ 178.4, 178.4, 178.21, 156.4, 0.8H), 2.92 – 2.89 (m, 0.8H), 2.76 (dt, J = 28.2, 7 Hz, 1H), 2.12 –

139.4, 133.0, 132.8, 130.1, 129.9, 128.1, 127.5, 127.4, 66.9, 2.02 (m, 2H), 1.80 (d, J = 7.4 Hz, 2H), 1.70 (d, J = 7.6 Hz, 5.7H).

62.6, 49.9, 43.8, 43.8, 43.4, 43.3, 40. 8, 35.9, 33.5, 32.5, 32.0, ¹³C NMR (75 MHz, CDCl₃): δ 175.9, 175.8, 175.7, 175.7, 175.1,

29.9, 27.8, 27.8, 26.3, 25.3, 16.4, 16.4. HRMS (ESI+) Calculated 175.0, 174.6, 170.6, 167.7, 142.4, 140.2, 139.0, 138.9, 138.9,

for C₂₆H₃₅N₂O₅[M+H]⁺: 455.2546 ; found : 455.2538. HPLC 138.8, 138.4, 138.3, 136.9, 136.8, 135.3, 135.2, 134.6, 134.5,

System A (λ = 254 nm) t_R= 4.09 min.

133.9, 132.4, 130.8, 128.4, 128.4, 128.31, 128.1, 128.1, 127.8,

127.6, 127.6, 127.5, 127.1, 127.0, 126.9, 110.3, 107.6, 92.2,

92.2, 91.5, 91.4, 81.0, 80.7, 79.6, 79.6, 68.1, 67.9, 61.3, 61.3,

60.7, 60.5, 57.3, 50.3, 50.1, 50.1, 49.6, 49.6, 47.7, 47.7, 47.5,

47.47, 45.6, 45.5, 38.6, 30.3, 29.6, 28.8, 25.5, 23.7, 22.9, 17.6,

16.4, 15.9, 15.9, 14.0, 10.9. HRMS (ESI+) Calculated for

C₁₇H₁₈NO₄[M+H]⁺: 300.1236 ; found : 300.1240. HPLC System

A (λ = 254 nm) t_R= 3.20 min, 3.25 min.

Cycloadduct anthracene-chiral maleimide (24). To

a

solution of anthracen-9-ylmethanol 5 (52 mg, 0.25 mmol, 1.0

equiv.) in a solution of THF/H₂O (1 mL, 2:1 v/v) was added a

solution of 19 (50 mg, 0.25 mmol, 1.0 equiv.) in THF/H₂O (1 mL,

2:1 v/v). The mixture was stirred at room temperature for 120

h. After completion, the reaction mixture was diluted with

water (10 mL) and the aqueous layer extracted with EtOAc (2 ×

15 mL). The organic layers were combined, dried over MgSO₄,

and solvents were removed under vacuum. The crude product

was purified by flash-column chromatography (silica gel,

cyclohexane/EtOAc as gradient from 100:0 to 50:50, v/v) to

provide the title compound (56 mg, 0.14 mmol, 54 % yield) as a

colorless solid. Two stereoisomers were observed by NMR in a

ratio of 0.8:1. IR (neat): 3448, 3065, 3031, 2969, 2941, 1688,

Azaphthalimide (26). To a solution of 6-(5-ethoxy-2-

methyloxazol-4-yl)hexanoic acid 8 (60 mg, 0.25 mmol, 1.0

equiv.) in a solution of THF/H₂O (1 mL, 2:1 v/v) were added a

solution of 19 (50 mg, 0.25 mmol, 1.0 equiv.) in THF/H₂O ((1 mL,

2:1 v/v) and TFA (19 µL, 0.25 mmol, 1.0 equiv.). The mixture was

stirred at room temperature for 24 h. After completion, the

reaction mixture was diluted with water (10 mL) and the

aqueous layer extracted with EtOAc (3 × 15 mL). The organic

layers were combined, dried over MgSO₄, and solvents were

removed under vacuum. The crude product was purified by RP-

HPLC (System D) and fractions were collected and lyophilized to

furnish the title compound (61 mg, 0.12 mmol, 48 % yield) as a

yellow powder. One stereoisomer was observed by NMR. IR

(neat): 3375, 2941, 2865, 1699, 1393, 1335, 1197, 1146, 1061,

759, 698. ¹H NMR (300 MHz, CD₃CN + 5% methanol-d4): δ 7.47

– 7.23 (m, 5H), 5.49 (q, J = 7.4 Hz, 1H), 2.89 (t, J = 7.6 Hz, 2H),

2.66 (s, 3H), 2.27 (t, J = 7.4 Hz, 2H), 1.85 (d, J = 7.3 Hz, 3H), 1.72

(dd, J = 15.1, 7.7 Hz, 2H), 1.66 – 1.54 (m, 2H), 1.47 – 1.34 (m,

2H). ¹³C NMR (75 MHz, CD₃CN + 5% methanol-d4): δ 176.2,

168.8, 160.4, 146.8, 146.5, 141.7, 129.6, 128.7, 128.1, 122.8,

122.1, 118.6, 50.5, 34.5, 32.7, 29.7, 28.7, 25.6, 19.5, 17.9. HRMS

(ESI-) Calculated for C₂₂H₂₃N₂O₅[M-H]^-: 395.1607 ; found :

395.1617. HPLC System A (λ = 254 nm) t_R= 4.22 min.

1

1457, 1359, 1221, 1195, 907, 728, 696. H NMR (300 MHz,

CDCl₃): δ 7.61 (dd, J = 5.2, 3.6 Hz, 1.7H), 7.40 – 7.30 (m, 2.7H),

7.29 – 7.26 (m, 1.1H), 7.26- 7.22 (m, 2.4H) 7.21 – 7.09 (m,

13.4H), 6.99 – 6.91 (m, 2H), 6.90 – 6.82 (m, 1.6H), 5.13 (dt, J =

12.8, 6.7 Hz, 1.3H), 5.07 – 4.84 (m, 4.2H), 4.76 (d, J = 3.3 Hz,

0.8H), 4.73 (d, J = 2.9 Hz, 0.9H), 3.35 – 3.20 (m, 3.6H), 2.70 (t, J

= 6.3 Hz, 1.1H), 2.67 (t, J = 6.3 Hz, 1H), 1.31 (d, J = 7.3 Hz, 2.4H),

1.24 (d, J = 6.2 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃): δ 176.7, 176.6,

176.5, 176.4, 142.2, 142.2, 142.2, 139.3, 139.3, 139.0, 138.5,

138.5, 128.2, 127.3, 127.2, 126.9, 126.8, 126.8, 126.7, 126.5,

126.4, 125.2, 123.9, 123.9, 123.2, 123.2, 122.3, 60.2, 60.1, 49.6,

49.5, 49.3, 49.3, 47.4, 47.4, 45.6, 45.5, 45.5, 15.84, 15.7. HRMS

(ESI+) Calculated for C₂₇H₂₄NO₄[M+H]⁺: 410.1756 ; found :

410.1757. HPLC System A (λ = 254 nm) t_R= 4.67 min.

12 | J. Name., 2012, 00, 1-3

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Organic & Biomolecular Chemistry

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ARTICLE

Azaphtalimide model (AM).To a solution of 4-benzyl-5- stabilization by an internal or external source of protons to

View Article Online

DOI: 10.1039/D0OB00403K

ethoxy-2-methyloxazole 9 (54 mg, 0.25 mmol, 1.0 equiv.) in a protect them from long-term air N-oxidation. From this

solution of THF/H₂O (1 mL, 2:1 v/v) were successively added a observation, it resulted the preparation of a novel and storable

solution of 19 (50 mg, 0.25 mmol, 1.0 equiv) in THF/H₂O (1 mL, conjugable tyrosine-based oxazole, obtained within three steps

2:1 v/v) and TFA (20 µL, 0.25 mmol, 1.0 equiv.). The mixture was and a good overall yield. In addition, while cyclopentadiene

stirred at room temperature for 24 h. After completion, the proved particularly unstable, a recent study reported in situ

reaction mixture was diluted with water (10 mL) and the deprotection of cyclopentadienes to overcome their important

aqueous layer extracted with EtOAc (2 × 15 mL). The organic lack of stability.⁵²

layers were combined, dried over MgSO₄, and solvents were With regard to the kinetic study, it was established that

removed under vacuum. The crude product was purified by cyclopentadiene conjugate reacts with maleimides between

flash-column chromatography (silica gel, cyclohexane/EtOAc as one and three orders of magnitude faster than the other diene

gradient from 100:0 to 95:5) to yield the title compound (70 mg, families. The second order kinetic rate constants were found to

0.19 mmol, 75 % yield) as a yellow oil. One stereoisomer was be between 10^-4and 10 M^-1.s^-1, comparatively, the Staudinger

observed by NMR. IR (neat): 3430, 3088, 3064, 3029, 2979, ligation has a rate constant of 10^-3M^-1. s^-1.⁵³Dienes may be

2933, 1764, 1695, 1602, 1395, 1362, 1328, 1146, 953, 906, 726, ranked in descending order in terms of their reactivity as

695. ¹H NMR (300 MHz, CDCl₃): δ 7.50 – 7.41 (m, 3H), 7.38 – 7.14 follows: cyclopentadiene, fulvene, 5-ethoxyoxazoles family and

(m, 7H), 5.48 (q, J = 7.3 Hz, 1H), 4.24 (s, 2H), 2.74 (s, 3H), 1.89 anthracene, cyclic and linear dienes and finally furan based

(d, J = 7.3 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃): δ 169.0, 167.6, derivatives. Such difference was leveraged in a proof-of-

156.9, 147.5, 145.3, 139.6, 137.6, 129.1, 128.5, 128.4, 127.9, concept for sequential labeling strategy. The chemical

127.3, 126.5, 120.4, 119.7, 49.7, 38.8, 20.1, 17.3. HRMS (ESI+) compatibility observed between the cyclopentadiene and 5-

Calculated for C₂₀H₂₁N₂O₃[M+H]⁺: 373.1552 ; found : 373.1550. ethoxyoxazole could further be exploited in the design of novel

HPLC System A (λ = 254 nm) t_R= 5.09 min.

heterobifunctional cross-linking reagents with useful

applications in bioconjugation and materials science.

Preparation of Michael adducts

The formation of multiple isomeric products was observed with

all dienes, except for oxazole whose aromatization step leads to

the formation of a single product, with unique fluorescent

properties.

In addition, stability experiments revealed that only furan

cycloadduct underwent cleavage through a rD-A process

leading to the dissociation of the reaction partners under

physiological conditions, whereas unbridged fused systems

were mainly subject to monohydrolysis in HBP.

To conclude, since none of the dienes combines all the

appropriate aforementioned properties (i.e. starting material

commercially available and indefinitely stable at room

temperature, ligation rate faster than 1 M^-1.s^-1, single isomeric

product, etc…), we hope this article will be a useful guide for the

reader in choosing the most convenient diene according his

targeted application and the (bio)molecular system which will

be involved, in particular with regard to the

accessibility/availability of dienes, reaction rates, products

isolation and stability or fluorescence properties.

Thio Michael adduct (27). To a solution of 19 (25 mg, 0.12

mmol, 1.0 equiv) in a solution of MeCN/PBS pH 7.4 (1 mL, 1:1,

v/v) was added N-acetylcysteine (23 mg, 0.14 mmol, 1.2 equiv).

The mixture was stirred at room temperature for 2 h. After

completion, the reaction mixture was diluted with water (10

mL) and the aqueous layer extracted with EtOAc (2 × 15 mL).

The organic layers were combined, dried over MgSO₄, and

solvents were removed under vacuum. The crude product was

purified twice by RP-HPLC (System D) and fractions were

collected and lyophilized to furnish the title compound (20 mg,

0.055 mmol, 46 % yield) as a lyophilized white powder. Two

stereoisomers were observed by NMR in a ratio of 1:1. IR (neat)

3342, 3276, 2985, 2941, 2106, 1695, 1538, 1362, 1219, 1183,

1

765, 698. H NMR (300 MHz, methanol-d4): δ 7.42 – 7.20 (m,

10H), 5.37 (q, J = 7.3 Hz, 2H), 4.71 (dd, J = 9.1, 4.4 Hz, 1H), 4.64

(t, J = 6.3 Hz, 1H), 3.94 (t, J = 3.6 Hz, 1H), 3.91 (t, J = 3.6 Hz, 1H),

3.51 (dd, J = 13.9, 4.4 Hz, 1H), 3.24 – 3.17 (m, 2H), 3.17 – 3.10

(m, 2H), 2.91 (dd, J = 13.9, 9.2 Hz, 1H), 2.50 (dd, J = 18.6, 3.9 Hz,

1H), 2.38 (dd, J = 18.6, 3.6 Hz, 1H), 2.00 (s, 3H), 1.99 (s, 3H), 1.79

(d, J = 0.8 Hz, 3H), 1.77 (d, J = 0.8 Hz, 3H). ¹³C NMR (75 MHz,

methanol-d4): δ 178.5, 178.3, 176.6, 176.5, 173.4, 173.4, 173.3,

141.0, 141.0, 129.4, 129.4, 128.6, 128.2, 128.1, 53.7, 52.9, 51.6,

51.5, 41.3, 39.9, 37.1, 36.6, 34.3, 34.1, 22.4, 16.9, 16.8 . HRMS

(ESI+) Calculated for C₁₇H₂₁N₂O₅S [M+H]⁺: 365.1171 ; found :

365.1165. HPLC System A (λ = 254 nm) t_R= 3.23 min.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

Conclusions

Herein is reported a comparative study of a collection of

(hetero)diene systems involved in maleimide-based

cycloaddition reactions that had proved useful applications for

chemoselective ligation. It was observed that non-commercially

available dienes were stable for several months at -25 °C or days

at room temperature, and that 5-alkoxyoxazoles required

The authors thank Agence National pour la Recherche (ANR) for

Ph.D fellowship to A.L. and the Region Haute-Normandie

(FEDER CHIMBIO HN 0001401) for financial support to K.R. This

work was also supported by the Centre National de la

Recherche Scientifique (CNRS), Rouen University and INSA de

Rouen. The authors also acknowledge Albert Marcual (CNRS)

J. Name., 2013, 00, 1-3 | 13

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30 L.-A. Jouanno, V. Tognetti, L. Joubert, C. Sabot, and P.-Y.

and Dr Corinne Bourhis-Loutelier (Rouen University) for HMRS

analyses and Dr Pierre Bohn (Centre Henry Bequerel / CHU de

Rouen) and Dr Morgane Detraz (Rouen University) for providing

HBP. Dr Vincent Aucagne (Centre de Biophysique Moléculaire

d’Orléans) is warmly acknowledged for helpful discussions.

View Article Online

Renard. Org. Lett., 2013, 15, 2530.

DOI: 10.1039/D0OB00403K

31 L.-A. Jouanno, V. Di Mascio, V. Tognetti, L. Joubert, C. Sabot,

and P.-Y. Renard. J. Org. Chem., 2014, 79, 1303.

32 A. Lizzul-Jurse, L. Bailly, M. Hubert-Roux, C. Afonso, P.-Y.

Renard, and C. Sabot. Org. Biomol. Chem., 2016, 14, 7777.

33 K. Renault, L.-A. Jouanno, A. Lizzul-Jurse, P.-Y. Renard, and C.

Sabot, C. Chem. Eur. J., 2016, 22, 18522.

34 J. E. T. Corrie. J. Chem. Soc., Perkin Trans. 1, 1994, 2975.

35 D. Kand, A. M. Kalle, and P. Talukdar. Org. Biomol. Chem.,

2013, 11, 1691.

36 G. Viault, S. Dautrey, N. Maindron, J. Hardouin, P.-Y. Renard,

and A. Romieu. Org. Biomol. Chem., 2013, 11, 2693.

37 L. Cheng, X. Kang, D. Wang, Y. Gao, L. Yi, and Z. Xi. Org. Biomol.

Chem., 2019, 17, 5675.

38 T. Yokoi, T. Ueda, H. Tanimoto, T. Morimoto, and K. Kakiuchi.

Chem. Commun., 2019, 55, 1891.

39 D. M. Beal, and L. H. Jones. Angew. Chem. Int. Ed., 2012, 51,

6320.

40 D. M. Patterson, and J. A. Prescher. Curr. Opin. Chem. Biol.,

2015, 28, 141.

41 C. Simon, C. Lion, C. Spriet, F. Baldacci-Cresp, S. Hawkins, and

C. Biot, C. Angew. Chem. Int. Ed., 2018, 57, 16665.

42 J. E. T. Corrie, V. R. N. Munasinghe, and W. Rettig. J.

Heterocycl. Chem., 2000, 37, 1447.

43 K. Hoogewijs, D. Buyst, J. M. Winne, J. C. Martins, and A.

Madder, A. Chem. Commun., 2013, 49, 2927.

44 J. E. Montgomery, J. A. Donnelly, S. W. Fanning, T. E. Speltz, X.

Shangguan, J. S. Coukos, G. L. Greene, and R. E. Moellering. J.

Am. Chem. Soc., 2019, 141, 16374.

45 S. D. Fontaine, R. Reid, L. Robinson, G. W. Ashley, and D. V.

Santi. Bioconjugate Chem., 2015, 26, 145.

46 D. Kalia, S. P. Pawar, and J. S. Thopate. Angew. Chem., Int. Ed.,

2017, 56, 1885.

47 L. N. Tumey, M. Charati, T. He, E. Sousa, D. Ma, X. Han, T. Clark,

J. Casavant, F. Loganzo, F. Barletta, J. Lucas, and E. I. Graziani.

Bioconjugate Chem., 2014, 25, 1871.

48 R. P. Lyon, J. R. SetterT. D. Bovee, S. O. Doronina, J. H. Hunter,

M. E. Anderson, C. L. Balasubramanian, S. M. Duniho, C. I.

Leiske, F. Li, and P. D. Senter. Nat. Biotechnol., 2014, 32, 1059.

49 K. Renault, P.-Y. Renard, and C. Sabot. Eur. J. Org. Chem.,

2018, 46, 6494.

50 J. Carneiro de Oliveira, M.-P. Laborie, and V. Roucoules.

Molecules 2020, 25, 243.

51 E. Gil de Montes, A. Istrate, C. D. Navo, E. Jiménez-Moreno, E.

A. Hoyt, F. Corzana, I. Robina, G. Jiménez-Osés, A. J. Moreno-

Vargas, and G. J. L. Bernardes. Angew. Chem., Int. Ed., 2020,

https://doi.org/10.1002/ange.201914529.

52 A. H. St. Amant, E. H. Discekici, S. J. Bailey, M. S. Zayas, J.-A.

Song, S. L. Shankel, S. N. Nguyen, M. W. Bates, A. Anastasaki,

C. J. Hawker et al. J. Am. Chem. Soc. 2019, 141, 34, 13619.

53 K. Lang, and J. W. Chin. ACS Chem. Biol., 2014, 9, 16.

54 S. N. Lam, P. Acharya, R. Wyatt, P.D. Kwong, and C. A. Bewley.

Bioorg. Med. Chem., 2008, 16, 10113.

Notes and references

1

2

L. S.Witus, and M. B. Francis. Acc. Chem. Res. 2011, 44, 774.

P. Akkapeddi, S. A. Azizi, A. M. Freedy, P. Cal, P. M. P. Gois, and

G. J. L. Bernardes. Chem. Sci., 2016, 7, 2954.

3

4

5

6

7

8

G. T. Hermanson. Bioconjugate Technique, 3rd ed. Elsevier,

Waltham, MA (2013).

K. Renault, J. W. Fredy, P.-Y. Renard, and C. Sabot.

Bioconjugate Chem., 2018, 29, 2497.

N. Joubert, C. Denevault-Sabourin, F. Bryden, and M. C. Viaud-

Massuard. Eur. J. Med. Chem., 2017, 142, 393.

V. Pozsgay, N. E. Vieira, and A. A. Yergey. Org. Lett., 2002, 4,

3191.

A. D. de Araujo, J. M. Palomo, J. Cramer, O. Seitz, K.

Alexandrov, and H. Waldmann. Chem. Eur. J., 2006, 12, 6095.

L.-A. Jouanno, A. Chevalier, N. Sekkat, N. Perzo, H. Castel, A.

Romieu, N. Lange, C. Sabot, and P.-Y. Renard. J. Org. Chem.,

2014, 79, 10353.

9

A. H. St Amant, D. Lemen, S. Florinas, S. Mao, C. Fazenbaker,

H. Zhong, H. Wu, C. Gao, R. J. Christie, and J. Read de Alaniz.

Bioconjugate Chem., 2018, 29, 2406.

10 A. H. St Amant, F. Huang, J. Lin, D. Lemen, C. Chakiath, S. Mao,

C. Fazenbaker, H. Zhong, J. Harper, W. Xu, et al. Bioconjugate

Chem., 2019, 30, 2340-2348.

11 B. Seelig, and A. Jäschke. Tetrahedron Lett. 1997, 38, 7729.

12 K. W. Hill, J. Taunton-Rigby, J. D. Carter, E. Kropp, K. Vagle, W.

Pieken, D. P. McGee, G. M. Husar, M. Leuck, D. J. Anziano, and

D. P. Sebesta. J. Org. Chem., 2001, 66, 5352.

13 V. Marchan, S. Ortega, D. Pulido, E. Pedroso, and A. Grandas.

Nucleic Acids Res., 2006, 34, e24.

14 V. Steven, and D. Graham. Org. Biomol. Chem. 2008, 6, 3781.

15 X. L. Sun, C. L. Stabler, C. S. Cazalis, and E. L. Chaikof.

Bioconjugate Chem., 2006, 17, 52.

16 A. Petrelli, E. Samain, S. Pradeau, S. Halila, and S. Fort.

ChemBioChem 2017, 18, 206.

17 A. H. St Amant, F. Huang, J. Lin, K. Rickert, V. Oganesyan, D.

Lemen, S. Mao, J. Harper, M. Marelli, H. Wu, et al. Angew.

Chem., Int. Ed., 2019, 58, 8489.

18 L. I. Willems, M. Verdoes, B. I. Florea, G. van der Marel, and H.

S. Overkleeft. ChemBioChem 2010, 11, 1769.

19 S. Y. Tang, J. Shi, and Q. X. Guo. Org. Biomol. Chem., 2012, 10,

2673.

20 J. S. Fell, B. N. Martin, and K. N. Houk. J. Org. Chem., 2017, 82,

1912.

21 S. Yu, H. M. de Bruijn, D. Svatunek, T. A. Hamlin, and F. M.

Bickelhaupt. ChemistryOpen 2018, 7, 995.

22 V. Borsenberger, and S. Howorka. Nucleic Acids Res., 2009, 37,

1477.

23 A. H. El-Sagheer, V. V. Cheong, and T. Brown. Org. Biomol.

Chem., 2011, 9, 232.

55 C. E. Puerto Galvis, and V. V. Kouznetsov. Org. Biomol. Chem.,

2013, 11, 407.

24 X. L. Sun, L. Yang, and E. L. Chaikof. Tetrahedron Lett., 2008,

49, 2510.

25 N. Coskun, I. Erden. Tetrahedron 2011, 67, 8607.

26 Y. Liu, R. K. Goswami, C. Liu, and S. C. Sinha. Mol. Pharm.,

2015, 12, 2544.

27 V. A. Mironov, E. V. Sobolev, and A. N. Elizarova. Tetrahedron

1963, 19, 1939.

28 C. Sabot, E. Oueis, X. Brune, and P.-Y. Renard. Chem.

Commun., 2012, 48, 768.

29 L.-A. Jouanno, C. Sabot, and P.-Y. Renard. J. Org. Chem., 2012,

77, 8549.

14 | J. Name., 2012, 00, 1-3

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DOI: 10.1039/D0OB00403K

Table of contents

Maleimide-based Diels-Alder strategies for bioconjugation are compared in terms of dienes

accessibility and stability, reactions rates, as well as products isolation and stability.

Article Doi

Maleimide-based metal-free ligation with dienes: A comparative study

DOI: 10.1039/d0ob00403k

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Authors:

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