Dichloromaleimide (diCMI): A small and fluorogenic reactive group for use in affinity labeling

Article abstract of DOI:10.1248/cpb.c16-00501

Chemical probes comprising a ligand moiety, a reactive group (e.g. epoxide, haloacetyl or photoreactive group) and a tag unit (e.g. fluorophore or radioisotope) are widely used in affinity labeling to identify the target proteins of bioactive molecules. However, design and synthesis of highly functionalized chemical probes are often time-consuming. In this paper, we propose a simple design strategy for chemical probes bearing a small 2,3-dichloromaleimide (diCMI) unit, which serves as a combined reactive group and tag unit by reacting with a nucleophilic lysine residue near the ligand-binding site of the target protein to generate the 2-amino-3-chloromaleimide fluorophore. Model ligand-protein experiments confirmed that the diCMI unit has suitable reactivity and fluorogenic capability for efficient affinity labeling.

Full text of DOI:10.1248/cpb.c16-00501

Vol. 64, No. 11
Chem. Pharm. Bull. 64, 1647–1653 (2016)
1647
Note
Dichloromaleimide (diCMI): A Small and Fluorogenic Reactive Group
for Use in Affinity Labeling
Kosuke Chiba, Yuichi Hashimoto, and Takao Yamaguchi*
Institute of Molecular and Cellular Biosciences, The University of Tokyo; 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113–0032,
Japan.
Received June 21, 2016; accepted August 17, 2016
Chemical probes comprising a ligand moiety, a reactive group (e.g. epoxide, haloacetyl or photoreactive
group) and a tag unit (e.g. fluorophore or radioisotope) are widely used in affinity labeling to identify the tar-
get proteins of bioactive molecules. However, design and synthesis of highly functionalized chemical probes
are often time-consuming. In this paper, we propose a simple design strategy for chemical probes bearing
a small 2,3-dichloromaleimide (diCMI) unit, which serves as a combined reactive group and tag unit by
reacting with a nucleophilic lysine residue near the ligand-binding site of the target protein to generate the
2
-amino-3-chloromaleimide fluorophore. Model ligand–protein experiments confirmed that the diCMI unit
has suitable reactivity and fluorogenic capability for efficient affinity labeling.
Key wordsꢀ affinityꢀlabeling;ꢀdichloromaleimide;ꢀproteinꢀmodification
Affinityꢀlabelingꢀisꢀaꢀpowerfulꢀmethodꢀtoꢀlabelꢀandꢀvisual- toꢀ exhibitꢀ strongꢀ fluorescenceꢀ atꢀ >400ꢀnmꢀ withꢀ largeꢀ Stokesꢀ
1,2)
19)
ize target proteins of bioactive molecules. This method gen- shifts (>100 nm). We thus initially expected that 2,3-dibro-
erally utilizes a chemical probe composed of a ligand moiety, momaleimideꢀ wouldꢀ beꢀ availableꢀ asꢀ aꢀ fluorogenicꢀ reactiveꢀ
a reactive group and a tag unit (Fig. 1a). The reactive group groupꢀforꢀlysineꢀresidues.ꢀHowever,ꢀtoꢀourꢀknowledge,ꢀreactionꢀ
servesꢀtoꢀformꢀaꢀcovalentꢀbondꢀwithꢀtheꢀtargetꢀprotein.ꢀForꢀef- of 2,3-dibromomaleimide and amine in aqueous media (or al-
ficientꢀ andꢀ specificꢀ targetꢀ labeling,ꢀ theꢀ reactiveꢀ groupꢀ shouldꢀ cohols) has not been reported. On the other hand, 2,3-dichlo-
2
0)
beꢀ stableꢀ inꢀ aqueousꢀ media,ꢀ haveꢀ lowꢀ reactivityꢀ towardsꢀ romaleimidesꢀ reactꢀ withꢀ amineꢀ inꢀ alcohols. ꢀ Therefore,ꢀ weꢀ
non-targetꢀ molecules,ꢀ andꢀ reactꢀ appropriatelyꢀ withꢀ theꢀ targetꢀ selectedꢀ diCMIꢀ asꢀ aꢀ candidateꢀ fluorogenicꢀ reactiveꢀ groupꢀ forꢀ
protein after ligand–target binding. The tag unit serves to vi- useꢀinꢀaffinityꢀlabeling.
sualize the target protein. Fluorophores are often used as a tag
ToꢀevaluateꢀtheꢀreactivityꢀofꢀtheꢀdiCMIꢀunit,ꢀweꢀfirstꢀexam-
unitꢀ becauseꢀ ofꢀ theꢀ easeꢀ ofꢀ detection.ꢀ However,ꢀ aꢀ majorꢀ ob- ined the reaction of N-n-butyl-2,3-dichloromaleimide (1)ꢀwithꢀ
stacleꢀtoꢀaffinityꢀlabelingꢀisꢀoftenꢀsynthesisꢀofꢀtheꢀhighlyꢀfunc- n-propylamine in H O–THF (1:2) (Table 1, entry 1). Nearly
2
tionalized chemical probe. More importantly, the ligand must 50%ꢀ ofꢀ theꢀ startingꢀ materialꢀ wasꢀ convertedꢀ toꢀ theꢀ desired
retainꢀitsꢀbindingꢀaffinityꢀafterꢀintroductionꢀofꢀtheseꢀtwoꢀfunc- product 2aꢀwithinꢀ1ꢀh,ꢀandꢀ3aꢀwasꢀobtainedꢀinꢀ17%ꢀyield.ꢀIm-
tional groups (reactive group and tag unit). For these reasons, portantly,ꢀthisꢀresultꢀrevealedꢀthatꢀtheꢀdiCMIꢀunitꢀisꢀstableꢀin
aꢀ smallꢀ alkyneꢀ tagꢀ isꢀ oftenꢀ usedꢀ inꢀ affinityꢀ labeling,ꢀ sinceꢀ aqueousꢀ mediaꢀ andꢀ exhibitsꢀ moderateꢀ reactivityꢀ withꢀ amine.
itꢀ canꢀ beꢀ subsequentlyꢀ conjugatedꢀ withꢀ aꢀ detectionꢀ unitꢀ (e.g. Itꢀ isꢀ noteworthyꢀ thatꢀ thisꢀ reactionꢀ wasꢀ carriedꢀ outꢀ atꢀ high
azide-fluorophore,ꢀ azide-biotin),ꢀ despiteꢀ theꢀ inconvenienceꢀ concentration (0.10M),ꢀ whichꢀ mimicsꢀ aꢀ reactionꢀ promotedꢀ by
3)
ofꢀ theꢀ additionalꢀ conjugationꢀ step(s). We recently reported ligand–target interaction. To our surprise, dichloromaleimide
a small alkoxy nitrobenzoxadiazole (O-NBD, 180Da) unit as 1ꢀ showedꢀ noꢀ reactivityꢀ withꢀ 1-propanethiolꢀ evenꢀ inꢀ theꢀ pres-
4)
aꢀ fluorogenicꢀ reactiveꢀ groupꢀ forꢀ affinityꢀ labeling. ꢀ Here,ꢀ weꢀ ence of excess thiol (entries 2, 3). We further tested these
showꢀthatꢀ2,3-dichloromaleimideꢀ(diCMI,ꢀ164ꢀDa)ꢀcanꢀserveꢀasꢀ reactions in phosphate buffer (pH 7.0)/THF considering the
anꢀevenꢀsmallerꢀfluorogenicꢀreactiveꢀgroupꢀ(Fig.ꢀ1b).
possible pH effect (entries 4, 5). As a result, amine adduct 2a
and thiol adduct 2bꢀwereꢀobtainedꢀinꢀ12ꢀandꢀ73%,ꢀrespective-
ly.ꢀThisꢀmeansꢀthatꢀdichloromaleimideꢀreactsꢀwithꢀbothꢀamine
Results and Discussion
Molecular Design, Model Reaction and Spectrometric and thiol under neutralized aqueous conditions.
Analysis The maleimide motif is a highly reactive Michael
Next,ꢀ UV-Visꢀ andꢀ fluorescenceꢀ propertiesꢀ ofꢀ 1, 2a and b
acceptorꢀandꢀhasꢀbeenꢀwidelyꢀusedꢀforꢀchemicalꢀmodificationꢀ wereꢀ evaluatedꢀ (Figs.ꢀ 2a,ꢀ b).ꢀ Asꢀ shownꢀ inꢀ Fig.ꢀ 2a,ꢀ theꢀ amineꢀ
5
–7)
of thiols of biomolecules. ꢀ Forꢀ instance,ꢀ fluorophore-conju- adduct 2aꢀ wasꢀ foundꢀ toꢀ showꢀ aꢀ maximumꢀ absorbanceꢀ atꢀ ca.
gatedꢀmaleimidesꢀhaveꢀbeenꢀemployedꢀforꢀtheꢀmodificationꢀofꢀ 375ꢀnmꢀ similarꢀ toꢀ aꢀ reportedꢀ 2-aminomaleimideꢀ fluorophore,ꢀ
8
,9)
19)
cysteine residues. Recently, 2-bromomaleimide and 2,3-di- 2-bromo-3-n-butylamino-N-methylmaleimide (4). As expect-
bromomaleimideꢀ wereꢀ developedꢀ asꢀ novelꢀ cysteine-labelingꢀ ed, 2aꢀexhibitedꢀstrongꢀfluorescenceꢀ(Fig.ꢀ2b,ꢀλ : 374nm, λ :
ex
em
reagents,ꢀwhichꢀreactꢀwithꢀthiolꢀinꢀanꢀaddition-eliminationꢀse- 482ꢀnmꢀinꢀ1,4-dioxane),ꢀwhereasꢀ1ꢀshowedꢀnoꢀsignificantꢀfluo-
10–18)
quence (nucleophilic substitution) to afford thiomaleimide.
rescenceꢀ(dataꢀnotꢀshown).ꢀMoreover,ꢀtheꢀthiolꢀadductꢀ2bꢀwasꢀ
Inꢀ anꢀ organicꢀ solventꢀ suchꢀ asꢀ tetrahydrofuranꢀ (THF),ꢀ foundꢀ toꢀ showꢀ weakꢀ fluorescenceꢀ (λ : 372nm, λ : 471nm).
ex
em
2
2
,3-dibromomaleimideꢀ alsoꢀ reactsꢀ withꢀ amine,ꢀ affordingꢀ Theseꢀfluorescenceꢀdata,ꢀasꢀwellꢀasꢀtheꢀaboveꢀreactivityꢀdata,ꢀ
-amino-3-bromomaleimideꢀ asꢀ anꢀ amine-conjugationꢀ prod- indicateꢀthatꢀtheꢀdiCMIꢀunitꢀwouldꢀbeꢀsuitableꢀforꢀfluorogenicꢀ
uct.ꢀ Interestingly,ꢀ 2-aminomaleimidesꢀ wereꢀ recentlyꢀ reported labelingꢀ ofꢀ aminesꢀ andꢀ thiols,ꢀ withꢀ muchꢀ higherꢀ fluorescenceꢀ
*
ꢀToꢀwhomꢀcorrespondenceꢀshouldꢀbeꢀaddressed.ꢀ e-mail:ꢀyamaguchi@iam.u-tokyo.ac.jp
©
2016 The Pharmaceutical Society of Japan

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Chem. Pharm. Bull.
Vol. 64, No. 11 (2016)
Fig.ꢀ 1.ꢀ SchematicꢀRepresentationꢀofꢀAffinityꢀLabeling
(a)ꢀGeneralꢀaffinityꢀlabelingꢀusingꢀaꢀreactiveꢀgroupꢀandꢀaꢀtagꢀunit.ꢀAAꢀmeansꢀanꢀaminoꢀacidꢀresidueꢀnearꢀtheꢀligand-bindingꢀsite.ꢀ(b)ꢀSimpleꢀaffinityꢀlabelingꢀmethodꢀ
usingꢀtheꢀsmallꢀdiCMIꢀunit.ꢀTheꢀdiCMIꢀunitꢀreactsꢀwithꢀaꢀnucleophilicꢀlysineꢀ(Lys)ꢀresidueꢀnearꢀtheꢀligand-bindingꢀsiteꢀtoꢀaffordꢀtheꢀ2-amino-3-chloromaleimideꢀfluoro-
phore.
Table 1. Reaction of 1ꢀwithꢀn-Propylamine/1-Propanethiol
Yield (%)
a)
Entry
Solvent
X
2
3
1
1
2
3
4
5
A
A
A
B
B
NH
S
(1eq)
(1eq)
(10eq)
(1eq)
(1eq)
47 (2a)
—
17 (3a)
—
21
98
93
81
—
S
—
—
b)
NH
S
12 (2a)
73 (2b)
Trace (3a)
—
1
a) Solvent A: H O–THF (1:2), solvent B: phosphate buffer (0.6M, pH 7.0)–THF (1:2). b)ꢀThisꢀcompoundꢀwasꢀobservedꢀinꢀtheꢀ H-NMR spectrum of crude material. Reac-
2
tion conditions: 1 (0.10M (entries 1–3), 20mM (entries 4, 5)), n-propylamine/1-propanethiol (0.10 or 1.0M (entries 1–3), 20mM (entries 4, 5)), r.t., and 1h.
Fig. 2. Spectral Analysis of 1, 2a and b
1
9)
2
-Bromo-3-n-butylamino-N-methylmaleimide (4)ꢀ wasꢀ usedꢀ asꢀ aꢀ standard. (a) UV-Vis spectra of 1, 2a, b and 4. Measurement conditions: 100µM compound in
1
−
−1
−1
−1
1
3
,4-dioxane.ꢀ Theꢀ molarꢀ extinctionꢀ coefficientsꢀ wereꢀ calculatedꢀ fromꢀ theꢀ maximumꢀ absorptionꢀ (εmax: 3400M cm at 375nm for 2a, 1501M cm at 372nm for 2b and
500 M cm at 376nm for 4). (b) Fluorescence spectra of 2a, b and 4. Measurement conditions: 100µM compound in 1,4-dioxane. The standard compound 4ꢀwasꢀexcitedꢀ
−
1
−1
1
9
)
atꢀ374ꢀnmꢀasꢀreported.ꢀTheꢀrelativeꢀfluorescenceꢀquantumꢀyieldsꢀ(ϕ ) of 2a and bꢀwereꢀfoundꢀtoꢀbeꢀ0.40ꢀ(λ : 375nm) and 0.018 (λ : 372nm), respectively.
f
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Chem. Pharm. Bull.
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Fig.ꢀ 3.ꢀ TheꢀDesignedꢀBiotin–diCMIꢀProbeꢀMoleculesꢀforꢀLabelingꢀofꢀStreptavidin
2
2)
(
a)ꢀ Structureꢀ modelꢀ ofꢀ biotin–streptavidinꢀ complexꢀ (PDBꢀ ID:ꢀ 1STP,ꢀ monomerꢀ stateꢀ isꢀ shown ).ꢀ Theꢀ biotinꢀ moleculeꢀ andꢀ lysineꢀ 119ꢀ (K119)ꢀ areꢀ shownꢀ inꢀ black.ꢀ Theꢀ
distanceꢀbetweenꢀtheꢀbiotinꢀcarbonylꢀgroupꢀandꢀstreptavidinꢀK119ꢀwasꢀestimatedꢀbyꢀPyMOLꢀsoftware.ꢀ(b)ꢀStructuresꢀofꢀtheꢀdesignedꢀbiotin–diCMIꢀprobeꢀmolecules.ꢀTheꢀ
linkerꢀlengthsꢀwereꢀcalculatedꢀusingꢀChemBioꢀ3DꢀUltraꢀ13.0ꢀsoftwareꢀwithꢀMM2ꢀenergyꢀminimization.
Reagents and conditions: (a) N-Hydroxysuccinimide,ꢀEDCI,ꢀDIPEA,ꢀN-Boc-1,3-diaminopropane or N-Boc-1,8-diamino-3,6-dioxaoctane, DMF, r.t., 46% for 6a, 36% for
6
b;ꢀ(b)ꢀTFA,ꢀDCM,ꢀ0°Cꢀtoꢀr.t.,ꢀquant.;ꢀ(c)ꢀ2,3-Dichloromaleicꢀanhydride,ꢀAcOH,ꢀreflux,ꢀ24%ꢀforꢀ5a, 57% for 5b.
Chart 1. Synthesis of the Designed Probe Molecules
intensityꢀofꢀtheꢀamineꢀadduct.ꢀTheꢀrelativeꢀfluorescenceꢀquan- tert-butoxycarbonylꢀ (Boc)ꢀ group,ꢀ andꢀ reactionꢀ withꢀ dichloro-
tum yields (ϕ ) of 2a and bꢀwereꢀfoundꢀtoꢀbeꢀ0.40ꢀandꢀ0.018,ꢀ maleic anhydride to afford the probes 5a and b (Chart 1).
f
19)
respectively,ꢀ byꢀ comparisonꢀ withꢀ 4 (ϕ =0.20). 2-Amino-
-chloromaleimideꢀfluorophoresꢀareꢀlikelyꢀtoꢀhaveꢀbetterꢀfluo- atedꢀ fluorescenceꢀ labelingꢀ ofꢀ streptavidin,ꢀ theꢀ modelꢀ targetꢀ
Evaluation of Streptavidin Labeling We next evalu-
f
3
rescence properties than 2-amino-3-bromomaleimides because protein, by using 2,3-dichloromaleimides 1, 5a and b (Fig. 4).
of the intramolecular heavy atom effect (Cl versus Br), as seen Theꢀlabelingꢀreactionꢀwasꢀperformedꢀinꢀphosphateꢀbufferꢀ(pHꢀ
21)
in 7-(diethylamino)coumarin derivatives. We also note that 7.0) at 0°C. After the reaction, sodium dodecyl sulfate (SDS)
2
2
aꢀ showsꢀ aꢀ solvatochromicꢀ characterꢀ (dataꢀ notꢀ shown),ꢀ likeꢀ sampleꢀ bufferꢀ wasꢀ addedꢀ andꢀ eachꢀ sampleꢀ wasꢀ subjectedꢀ toꢀ
-aminomaleimide.
polyacrylamideꢀ gelꢀ electrophoresisꢀ (PAGE).ꢀ Labeledꢀ strep-
Probe Design and Synthesisꢀ ꢀT oꢀconfirmꢀtheꢀusefulnessꢀofꢀ tavidinꢀ wasꢀ visualizedꢀ usingꢀ anꢀ in-gelꢀ fluorescenceꢀ imagerꢀ
19)
theꢀdiCMIꢀunitꢀforꢀaffinityꢀlabeling,ꢀweꢀselectedꢀbiotin–strep- andꢀ thenꢀ stainedꢀ withꢀ Coomassieꢀ Brilliantꢀ Blueꢀ (CBB).ꢀ Asꢀ
tavidin as a model ligand–target protein because of their ready shownꢀinꢀFig.ꢀ4a,ꢀbothꢀ5a and bꢀwereꢀfoundꢀtoꢀlabelꢀtheꢀtargetꢀ
availability.ꢀBiotin–diCMIꢀprobesꢀwereꢀdesignedꢀbasedꢀonꢀtheꢀ streptavidin (lanes 3, 4). The probe 5bꢀshowedꢀstrongerꢀfluo-
reported X-ray crystal structure of biotin–streptavidin com- rescence than 5a,ꢀ whichꢀ probablyꢀ meansꢀ thatꢀ theꢀ diCMIꢀ unitꢀ
2
2)
plexꢀ(PDBꢀID:ꢀ1STP,ꢀaꢀmonomerꢀstateꢀisꢀshown )ꢀ(Fig.ꢀ3).ꢀItꢀ of 5bꢀ hadꢀ betterꢀ accessꢀ toꢀ K119.ꢀ Inꢀ theꢀ caseꢀ ofꢀ compoundꢀ 1
isꢀwellꢀknownꢀthatꢀtheꢀcarboxylicꢀacidꢀendꢀofꢀbiotinꢀhasꢀaꢀlowꢀ (diCMIꢀwithoutꢀtheꢀligandꢀunit),ꢀalmostꢀnoꢀlabelingꢀtookꢀplaceꢀ
impactꢀonꢀtheꢀbindingꢀaffinity.ꢀTherefore,ꢀtheꢀdiCMIꢀunitꢀwasꢀ under the same conditions (lane 2). Furthermore, the labeling
attached to biotin at this position via a linker unit (Fig. 3b). of streptavidin by 5bꢀwasꢀinhibitedꢀbyꢀadditionꢀofꢀbiotinꢀasꢀaꢀ
Theꢀ distancesꢀ betweenꢀ theꢀ diCMIꢀ unitꢀ andꢀ theꢀ amidesꢀ ofꢀ 5a competitor (Fig. 4b). These results indicate that the labeling
and bꢀwereꢀcalculatedꢀtoꢀbeꢀ6.7ꢀandꢀ12.6ꢀÅ,ꢀrespectively.ꢀSinceꢀ reactionꢀwasꢀacceleratedꢀbyꢀtheꢀbiotin–streptavidinꢀinteraction.ꢀ
lysine 119 (K119), the nearest lysine residue to the ligand- WeꢀthenꢀexaminedꢀtheꢀtargetꢀselectivityꢀofꢀdiCMIꢀprobeꢀmol-
bindingꢀ site,ꢀ isꢀ locatedꢀ 12.9–17.6ꢀÅꢀ fromꢀ theꢀ carboxylicꢀ acidꢀ ecules in the presence of non-target proteins, bovine serum
end of biotin, 5bꢀ wasꢀ expectedꢀ toꢀ reactꢀ moreꢀ efficientlyꢀ thanꢀ albuminꢀ(BSA)ꢀandꢀcarbonicꢀanhydraseꢀIIꢀ(CAII).ꢀUnderꢀtheseꢀ
5
aꢀwithꢀstreptavidin.ꢀWeꢀnoteꢀthatꢀstreptavidinꢀhasꢀnoꢀcysteineꢀ conditions, both 5a and b labeled the target streptavidin (Fig.
residues;ꢀthusꢀthisꢀlabelingꢀtakesꢀplaceꢀwithoutꢀinvolvingꢀthiolꢀ 4c,ꢀ lanesꢀ 1,ꢀ 3),ꢀ butꢀ sufficientꢀ target-selectivityꢀ wasꢀ observedꢀ
species.ꢀ Importantly,ꢀ theꢀ synthesisꢀ ofꢀ theꢀ designedꢀ biotin– withꢀ theꢀ designedꢀ 5b.ꢀ Althoughꢀ non-targetꢀ BSAꢀ wasꢀ alsoꢀ
diCMIꢀ probesꢀ wasꢀ achievedꢀ inꢀ onlyꢀ threeꢀ stepsꢀ fromꢀ biotin;ꢀ labeled in some degree, competition analysis importantly re-
conjugationꢀ ofꢀ aꢀ linkerꢀ unit,ꢀ followedꢀ byꢀ deprotectionꢀ ofꢀ theꢀ vealedꢀthatꢀtheꢀtargetꢀstreptavidinꢀwasꢀ specificallyꢀlabeledꢀbyꢀ

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Fig.ꢀ 4.ꢀ In-GelꢀFluorescenceꢀAnalysisꢀofꢀStreptavidinꢀLabeledꢀwithꢀBiotin–diCMIꢀProbeꢀMoleculesꢀ(λ : 440–500nm, λem> 540 nm)
ex
(a)ꢀ Labelingꢀ ofꢀ streptavidinꢀ withꢀ 1, 5a, or b.ꢀ Labelingꢀ efficienciesꢀ wereꢀ determinedꢀ fromꢀ theꢀ fluorescenceꢀ intensities.ꢀ Labelingꢀ conditions:ꢀ 50ꢀµM streptavidin, 50µM
probe, 20mMꢀphosphateꢀbuffer,ꢀpHꢀ7.0,ꢀ0°C,ꢀandꢀ30ꢀmin.ꢀ(b)ꢀCompetitionꢀanalysisꢀwithꢀbiotin.ꢀLabelingꢀconditions:ꢀ10ꢀµM streptavidin, 10µM 5b, 20mM phosphate buffer,
pHꢀ7.0,ꢀ0°C,ꢀandꢀ30ꢀmin.ꢀStreptavidinꢀwasꢀpre-incubatedꢀwithꢀ20ꢀorꢀ100ꢀµM biotin for 30min at 0°C before addition of 5b. (c) Selective labeling of the target protein (strep-
tavidin)ꢀinꢀaꢀmixtureꢀofꢀstreptavidin,ꢀBSAꢀandꢀcarbonicꢀanhydraseꢀIIꢀ(CAII).ꢀLabelingꢀconditions:ꢀ5ꢀµM streptavidin, 1µM BSA, 1µMꢀCAII,ꢀ5ꢀµM 5a or b, 20mM phosphate
buffer,ꢀpHꢀ7.0,ꢀ0°C,ꢀandꢀ30ꢀmin.ꢀForꢀcompetitionꢀanalysisꢀ(lanesꢀ2,ꢀ4),ꢀproteinꢀmixtureꢀwasꢀpre-incubatedꢀwithꢀ50ꢀµM biotin for 30min at 0°C before an addition of 5a or b.
1
13
5
a or b through ligand–target interactions (lanes 2, 4). This
H-NMR and at 125MHz for C-NMR. High-resolution mass
resultꢀdemonstratedꢀthatꢀtheꢀdiCMIꢀunitꢀhasꢀsuitableꢀreactivityꢀ spectraꢀ(HR-MS)ꢀwereꢀobtainedꢀusingꢀaꢀBRUKERꢀmicrOTOFꢀ
forꢀtarget-specificꢀlabeling.
IIꢀmassꢀspectrometer.
N-n-Butyl-2,3-dichloromaleimide (1)
n-Butylamine (148µL,ꢀ 1.50ꢀmmol)ꢀ wasꢀ addedꢀ toꢀ aꢀ solutionꢀ
Conclusion
Weꢀ haveꢀ examinedꢀ theꢀ utilityꢀ ofꢀ theꢀ diCMIꢀ unitꢀ asꢀ aꢀ fluo- of dichloromaleic anhydride (250mg, 1.50mmol) in acetic acid
rogenicꢀreactiveꢀgroupꢀforꢀaffinityꢀlabeling.ꢀdiCMIꢀwasꢀstableꢀ (1.00ꢀmL)ꢀ andꢀ theꢀ mixtureꢀ wasꢀ stirredꢀ atꢀ roomꢀ temperatureꢀ
inꢀaqueousꢀmediaꢀandꢀshowedꢀsufficientꢀreactivityꢀwithꢀamine.ꢀ forꢀ 30ꢀmin,ꢀ andꢀ thenꢀ refluxedꢀ forꢀ 4ꢀh.ꢀ Afterꢀ coolingꢀ toꢀ roomꢀ
Spectrometric analysis revealed that 2-amino-3-chloromale- temperature,ꢀ theꢀ mixtureꢀ wasꢀ addedꢀ saturatedꢀ aq.ꢀ NaHCO3.
imideꢀ hasꢀ strongꢀ fluorescence.ꢀ Modelꢀ experimentsꢀ withꢀ theꢀ TheꢀwholeꢀwasꢀextractedꢀwithꢀAcOEt.ꢀTheꢀcombinedꢀorganicꢀ
biotin-streptavidinꢀ systemꢀ confirmedꢀ thatꢀ theꢀ diCMIꢀ unitꢀ hasꢀ layerꢀ wasꢀ driedꢀ overꢀ Na SO and concentrated. The residue
2
4
excellentꢀ characteristicsꢀ forꢀ target-specificꢀ labeling.ꢀ Notably,ꢀ wasꢀ purifiedꢀ byꢀ flashꢀ silica-gelꢀ chromatographyꢀ (AcOEt–n-
diCMI-basedꢀ chemicalꢀ probesꢀ areꢀ simpleꢀ toꢀ designꢀ andꢀ syn- hexane=1:20) to afford 1 (313mg, 1.41mmol, 94%) as a col-
1
thesize.ꢀSinceꢀtheꢀdiCMIꢀunitꢀisꢀsmall,ꢀtheꢀbiologicalꢀactivityꢀ orless oil. 1: H-NMR (CDCl ) δ: 0.86 (3H, t, J=7.4Hz), 1.25
3
ofꢀ theꢀ ligandꢀ isꢀ likelyꢀ toꢀ beꢀ wellꢀ retained.ꢀ Thus,ꢀ theꢀ diCMIꢀ (2H, tq, J=7.4, 7.4Hz), 1.53 (2H, tt, J=7.5, 7.5Hz), 3.53 (2H,
1
3
unitꢀisꢀexpectedꢀtoꢀbeꢀusefulꢀforꢀaffinityꢀlabeling.
t, J=7.2Hz). C-NMR (CDCl ) δ: 13.6, 20.0, 30.6, 39.3, 133.3,
3
1
63.2.ꢀ HR-MSꢀ (electrosprayꢀ ionizationꢀ (ESI))ꢀ m/z: 276.0170
+
Experimental
Chemistry
Synthetic Procedures
(Calcd for C H Cl NO (M+MeOH+Na ): 276.0170).
8
9
2
2
N-n-Butyl-2-chloro-3-(n-propylamino)maleimide (2a)
N -n-Butyl-2,3-dichloro-N -propylmaleamide (3a)
1
4
Chemicalsꢀ wereꢀ purchasedꢀ fromꢀ Sigma-Aldrichꢀ Co.ꢀ LLCꢀ
n-Propylamine (8.20µL,ꢀ 0.100ꢀmmol)ꢀ wasꢀ addedꢀ toꢀ aꢀ solu-
(
U.S.A.),ꢀ Kantoꢀ Chemicalꢀ Co.,ꢀ Inc.ꢀ (Japan),ꢀ Tokyoꢀ Chemicalꢀ tion of 1 (22.2mg, 0.100mmol) in H O–THFꢀ(0.900ꢀmL,ꢀ1ꢀ:ꢀ2)ꢀ
2
Industryꢀ Co.,ꢀ Ltd.ꢀ (Japan),ꢀ Acrosꢀ Organicsꢀ (U.S.A.)ꢀ orꢀ Wakoꢀ atꢀroomꢀtemperature,ꢀandꢀtheꢀmixtureꢀwasꢀstirredꢀforꢀ1ꢀh.ꢀAfterꢀ
PureꢀChemicalꢀIndustries,ꢀLtd.ꢀ(Japan),ꢀandꢀusedꢀwithoutꢀfur- theꢀreaction,ꢀtheꢀmixtureꢀwasꢀimmediatelyꢀconcentratedꢀunderꢀ
therꢀ purification.ꢀ Reactionsꢀ wereꢀ monitoredꢀ byꢀ TLCꢀ (Merckꢀ reducedꢀ pressure.ꢀ Theꢀ residueꢀ wasꢀ purifiedꢀ byꢀ flashꢀ silca-gelꢀ
silica gel 60F254)ꢀ plate.ꢀ Bandsꢀ wereꢀ visualizedꢀ usingꢀ UVꢀ chromatography (AcOEt–n-hexane=1:15 to 1:2) to afford 2a
lightꢀorꢀappropriateꢀreagentsꢀfollowedꢀbyꢀheating.ꢀFlashꢀchro- (11.5mg, 47.0µmol,ꢀ 47%)ꢀ asꢀ aꢀ yellowꢀ solidꢀ andꢀ 3a (4.80mg,
1
matographyꢀ wasꢀ carriedꢀ outꢀ withꢀ silicaꢀ gelꢀ (Silicaꢀ gelꢀ 60N,ꢀ 17.1 µmol,ꢀ 17%)ꢀ asꢀ aꢀ whiteꢀ solid.ꢀ 2a: H-NMR (CDCl ) δ:
3
4
0–50µm particle size) purchased from Kanto Chemical Co., 0.82 (3H, t, J=7.2Hz), 0.92 (3H, t, J=7.4Hz), 1.23 (2H, tq,
Inc.ꢀ NMRꢀ spectraꢀ wereꢀ recordedꢀ onꢀ aꢀ JEOLꢀ JNM-GX500ꢀ J=7.5, 7.5Hz), 1.48 (2H, tt, J=7.5, 7.5Hz), 1.61 (2H, tq, J=7.2,
or JNMECA-500 spectrometer, operating at 500MHz for 7.2Hz), 3.41 (2H, t, J=7.2Hz), 3.49 (2H, dt, J=7.2, 7.2Hz),

Vol. 64, No. 11 (2016)
Chem. Pharm. Bull.
1651
13
5
.32 (1H, brs). C-NMR (CDCl ) δ: 11.0, 13.7, 20.0, 24.2, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiiimide (205mg,
3
3
0.6,ꢀ 30.8,ꢀ 38.2,ꢀ 44.9,ꢀ 88.7,ꢀ 140.4,ꢀ 165.9,ꢀ 168.1.ꢀ HR-MSꢀ (ESI)ꢀ 1.07ꢀmmol)ꢀ wereꢀ addedꢀ toꢀ aꢀ solutionꢀ ofꢀ (+)-biotin (173mg,
+
m/z: 267.0871 (Calcd for C H ClN O (M+Na ): 267.0871). 0.710ꢀmmol)ꢀ inꢀ anhydrousꢀ DMFꢀ (3.00ꢀmL),ꢀ andꢀ theꢀ mix-
11
17
2
2
1
3
a: H-NMR (CDCl ) δ: 0.86–0.88 (6H, m), 1.30 (tq, J=6.0, tureꢀ wasꢀ stirredꢀ forꢀ 2ꢀhꢀ atꢀ roomꢀ temperatureꢀ underꢀ anꢀ
3
6.0Hz), 1.44–1.54 (4H, m), 3.20–3.27 (4H, m), 6.442 (2H, brs). Ar atmosphere. N-Boc-1,8-diamino-3,6-dioxaoctane (166mg,
13
C-NMR (CDCl ) δ: 11.4, 13.8, 20.1, 22.5, 31.2, 40.2, 42.2, 0.710ꢀmmol)ꢀinꢀDMFꢀ(2.00ꢀmL)ꢀandꢀN,N-diisopropylethylamine
3
1
30.8,ꢀ 130.9,ꢀ 161.6,ꢀ 161.7.ꢀ HR-MSꢀ (ESI)ꢀ m/z: 303.0651 (Calcd (367 µL,ꢀ 2.13ꢀmmol)ꢀ wereꢀ thenꢀ added,ꢀ andꢀ theꢀ resultingꢀ mix-
+
for C H Cl N O (M+ Na ): 303.0638).
tureꢀ wasꢀ stirredꢀ forꢀ 24ꢀhꢀ atꢀ roomꢀ temperature.ꢀ Afterꢀ removalꢀ
ofꢀ theꢀ solvent,ꢀ theꢀ residueꢀ wasꢀ purifiedꢀ byꢀ flashꢀ silica-gelꢀ
1
1
18
2
2
2
N-n-Butyl-2-chloro-3-(n-propylthio)maleimide (2b)
Propanethiol (9.10µL,ꢀ 0.100ꢀmmol)ꢀ wasꢀ addedꢀ toꢀ aꢀ solutionꢀ chromatography (CHCl –MeOH=10:1) to afford 6b (121mg,
3
1
of 1 (22.2mg, 0.100mmol) in phosphate buffer (0.6M, pH 7.0)– 0.256ꢀmmol,ꢀ 36%)ꢀ asꢀ aꢀ whiteꢀ solid.ꢀ 6b: H-NMR (CDCl ) δ:
3
THFꢀ (5.00ꢀmL,ꢀ 1ꢀ:ꢀ2)ꢀ atꢀ roomꢀ temperature,ꢀ andꢀ theꢀ mixtureꢀ 1.41–1.43 (11H, m), 1.63–1.76 (4H, m), 2.23 (2H, t, J=7.4Hz),
wasꢀstirredꢀforꢀ1ꢀh.ꢀThenꢀtheꢀmixtureꢀwasꢀaddedꢀH O and the 2.74 (1H, d, J=12.6Hz), 2.90 (1H, dd, J=4.9, 12.9Hz),
2
wholeꢀwasꢀextractedꢀwithꢀAcOEt.ꢀTheꢀcombinedꢀorganicꢀlayerꢀ 3.13–3.16 (2H, m), 3.30–3.32 (1H, m), 3.42 (2H, dt, J=5.0,
wasꢀ driedꢀ overꢀ Na SO ꢀ andꢀ concentrated.ꢀ Theꢀ residueꢀ wasꢀ 5.0Hz), 3.55 (2H, t, J=5.0Hz), 3.57 (2H, t, J=5.0Hz), 3.62
2
4
purifiedꢀ byꢀ flashꢀ silica-gelꢀ chromatographyꢀ (AcOEt–n-hex- (4H, brs), 4.30 (1H, dd, J=4.5, 7.5Hz), 4.50 (1H, dd, J=5.0,
ane=1:15) to afford 2b (19.2mg, 73.3µmol,ꢀ73%)ꢀasꢀaꢀyellowꢀ 7.5Hz), 5.37 (1H, brs), 6.19 (1H, brs), 6.79 (1H, brs), 7.06
1
13
oil. 2b: H-NMR (CDCl ) δ: 0.92 (3H, t, J=7.5Hz), 1.04 (3H, (1H, brs). C-NMR (CDCl ) δ: 25.6, 28.1, 28.4, 35.9, 39.1,
3
3
t, J=7.3Hz), 1.30 (2H, tq, J=7.5, 7.5Hz), 1.56 (2H, tt, J=7.4, 40.3, 40.5, 55.8, 60.2, 60.3, 61.8, 61.9, 69.9, 70.0, 70.1, 79.3,
7
.5Hz), 1.72 (2H, tq, J=7.3, 7.3Hz), 3.33 (2H, t, J=7.5Hz), 156.2,ꢀ 173.0,ꢀ 173.8.ꢀ HR-MSꢀ (ESI)ꢀ m/z: 497.2401 Calcd for
13
+
3
2
.53 (2H, t, J=7.3Hz). C-NMR (CDCl ) δ: 13.1, 13.6, 20.0, C H N O S (M+Na ): 497.2404).
3
21 38
4
6
4.1,ꢀ30.6,ꢀ32.5,ꢀ38.8,ꢀ126.7,ꢀ139.0,ꢀ164.5,ꢀ166.0.ꢀHR-MSꢀ(ESI)ꢀ
3-(Biotinylamino)propylamine (7a)
+
m/z: 284.0480 (Calcd for C H ClNO S (M+Na ): 284.0482).
Compound 7aꢀ wasꢀ preparedꢀ differentlyꢀ fromꢀ theꢀ reportedꢀ
11
16
2
2
4)
2
-Bromo-3-n-butylamino-N-methylmaleimide (4)
procedure. ꢀ Trifluoroaceticꢀ acidꢀ (TFA)ꢀ (739ꢀµL,ꢀ 9.60ꢀmmol)ꢀ
Compound 4ꢀ wasꢀ preparedꢀ differentlyꢀ fromꢀ theꢀ reportedꢀ wasꢀaddedꢀdropwiseꢀtoꢀaꢀsolutionꢀofꢀ6a (384mg, 0.960mmol)
19)
procedure. n-Butylamine (18.4µL,ꢀ 0.190ꢀmmol)ꢀ wasꢀ addedꢀ in CH Cl ꢀ (20.0ꢀmL)ꢀ atꢀ 0°C,ꢀ andꢀ theꢀ mixtureꢀ wasꢀ stirredꢀ atꢀ
2
2
to a solution of 2,3-dibromo-N-methylmaleimide (50.0mg, roomꢀ temperatureꢀ forꢀ 3ꢀh.ꢀ Theꢀ solventꢀ wasꢀ thenꢀ removedꢀ
.190ꢀmmol)ꢀ inꢀ methanolꢀ (2.00ꢀmL)ꢀ atꢀ roomꢀ temperature,ꢀ under reduced pressure to afford 7a (463mg, TFA adduct,
0
andꢀ theꢀ mixtureꢀ wasꢀ stirredꢀ forꢀ 5ꢀh.ꢀ Theꢀ mixtureꢀ wasꢀ thenꢀ quant.)ꢀasꢀanꢀorangeꢀoil.ꢀThisꢀcompoundꢀwasꢀusedꢀforꢀtheꢀnextꢀ
1
concentratedꢀ andꢀ theꢀ residueꢀ wasꢀ purifiedꢀ byꢀ flashꢀ silica-gelꢀ reactionꢀ withoutꢀ furtherꢀ purification.ꢀ 7a: H-NMR (CD OD)
3
chromatography (AcOEt–n-hexane=1:15 to 1:10) to afford δ: 1.40 (2H, tt, J=7.4, 7.4Hz), 1.52–1.71 (4H, m), 1.80 (2H, tt,
1
4
(
ꢀ(23.5ꢀmg,ꢀ0.0900ꢀmmol,ꢀ47%)ꢀasꢀaꢀyellowꢀsolid.ꢀ4: H-NMR J=6.9, 6.9Hz), 2.19 (2H, t, J=7.4Hz), 2.66 (1H, d, J=12.6Hz),
CDCl ) δ: 0.97 (3H, t, J=7.4Hz), 1.42 (2H, tq, J=7.5, 7.5Hz), 2.87–2.90 (3H, m), 3.16 (1H, dt, J=4.9, 9.3Hz), 3.23 (2H,
3
1
.64 (2H, tt, J=7.5, 7.5Hz), 3.02 (3H, s), 3.64 (2H, dt, J=7.0, dt, J=2.0, 6.8Hz), 4.26 (1H, dd, J=4.5, 7.5Hz), 4.46 (1H,
7
.0ꢀHz).ꢀ HR-MSꢀ (ESI)ꢀ m/z: 283.0050 (Calcd for C H BrN O
dd, J=5.0,ꢀ 7.5ꢀHz).ꢀ HR-MSꢀ (ESI)ꢀ m/z: 323.1512 Calcd for
9
13
2
2
+
+
(
M+ Na ): 283.0053).
-Biotinylamino-N-(tert-butoxycarbonyl)propylamine (6a)
N-Hydroxysuccinimide (380mg, 3.30mmol) and 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide (863mg, 4.50mmol) procedure. TFA (200µL,ꢀ 2.60ꢀmmol)ꢀ wasꢀ addedꢀ dropwiseꢀ
wereꢀaddedꢀtoꢀaꢀsolutionꢀofꢀ(+)-biotin (733mg, 3.00mmol) in to a solution of the compound 6b (121mg, 0.260mmol) in
anhydrous N,N-dimethylformamideꢀ(DMF)ꢀ(13.0ꢀmL),ꢀandꢀtheꢀ CH Cl ꢀ (2.00ꢀmL)ꢀ atꢀ 0°C,ꢀ andꢀ theꢀ mixtureꢀ wasꢀ stirredꢀ atꢀ
C H N O S (M+Na ): 323.1512).
13
24
4
2
3
2-(2-(2-(Biotinylamino)ethoxy)ethoxy)ethylamine (7b)
Compound 7bꢀ wasꢀ preparedꢀ differentlyꢀ fromꢀ theꢀ reportedꢀ
2
3)
2
2
mixtureꢀwasꢀstirredꢀforꢀ6ꢀhꢀatꢀroomꢀtemperatureꢀunderꢀanꢀArꢀ roomꢀ temperatureꢀ forꢀ 6ꢀh.ꢀ Theꢀ solventꢀ wasꢀ thenꢀ removedꢀ
atmosphere. N-Boc-1,3-diaminopropane (575mg, 3.30mmol) under reduced pressure to afford 7b (129mg, TFA adduct,
1
inꢀ DMFꢀ (2.00ꢀmL)ꢀ andꢀ N,N-diisopropylethylamineꢀ (1.55ꢀmL,ꢀ quant.)ꢀ asꢀ aꢀ yellowꢀ oil.ꢀ 7b: H-NMR (CD OD) δ: 1.35–1.37
3
9
.00ꢀmmol)ꢀ wereꢀ thenꢀ added,ꢀ andꢀ theꢀ resultingꢀ mixtureꢀ wasꢀ (4H, m), 1.47–1.67 (4H, m), 2.14 (2H, t, J=7.2Hz), 2.63
stirred for 21h at room temperature. After removal of the (1H, d, J=12.6Hz), 2.84 (1H, dd, J=4.6, 12.6Hz), 3.04 (2H,
solvent,ꢀtheꢀresidueꢀwasꢀpurifiedꢀbyꢀflashꢀsilica-gelꢀchromatog- brs), 3.12–3.15 (1H, m), 3.29 (2H, t, J=4.5Hz), 3.47 (2H, t,
raphy (CHCl –MeOH=20:1) to afford 6a (550mg, 1.37mmol, J=5.0Hz), 3.57 (4H, brs), 3.62 (2H, t, J=4.5Hz), 4.24 (1H,
3
1
4
6%)ꢀ asꢀ aꢀ whiteꢀ solid.ꢀ 6a: H-NMR (CDCl ) δ: 1.44–1.46 brꢀs),ꢀ 4.43ꢀ (1H,ꢀ brꢀs).ꢀ HR-MSꢀ (ESI)ꢀ m/z: 397.1872 Calcd for
3
+
(11H, m), 1.66–1.72 (6H, m), 2.22 (2H, t, J=7.7Hz), 2.73 (1H, C H N O S (M+Na ): 397.1880).
1
6
30
4
4
d, J=13.2Hz), 2.91 (1H, dd, J=5.2, 12.6Hz), 3.13–3.17 (3H,
m), 3.28 (2H, tt, J=6.6, 6.6Hz), 4.32 (1H, dd, J=4.8, 7.8Hz),
N-(3-(Biotinylamino)propyl)-2,3-dichloromaleimide (5a)
solution of 2,3-dichloromaleic anhydride (22.0mg,
A
4
6
2
.52 (1H, dd, J=4.8, 7.8Hz), 5.08 (1H, brs), 5.83 (1H, brs), 0.130mmol) and 7a (39.5mg, 0.130mmol) in acetic acid
13
.65 (1H, brs), 6.77 (1H, brs). C-NMR (CDCl ) δ: 25.9, 28.1, (1.00ꢀmL)ꢀ wasꢀ stirredꢀ atꢀ roomꢀ temperatureꢀ forꢀ 15ꢀminꢀ underꢀ
3
8.3, 28.5, 30.1, 36.0, 36.2, 37.4, 40.6, 55.8, 60.3, 61.8, 79.4, anꢀ Arꢀ atmosphere.ꢀ Theꢀ mixtureꢀ wasꢀ thenꢀ stirredꢀ atꢀ 120°Cꢀ
1
56.7,ꢀ 164.3,ꢀ 173.8.ꢀ HR-MSꢀ (ESI)ꢀ m/z: 423.2051 (Calcd for forꢀ 3ꢀhꢀ andꢀ theꢀ solventꢀ wasꢀ removedꢀ underꢀ reducedꢀ pressure.ꢀ
+
C H N O S (M+Na ): 423.2036).
Theꢀ residueꢀ wasꢀ purifiedꢀ byꢀ flashꢀ silica-gelꢀ chromatogra-
18
32
4
4
N-tert-Butoxycarbonyl-2-(2-(2-(biotinylamino)ethoxy)- phy (CHCl –MeOH=20:1 to 15:1) to afford 5a (14.3mg,
3
1
ethoxy)ethylamine (6b)
0.0318ꢀmmol,ꢀ 24%)ꢀ asꢀ aꢀ whiteꢀ solid.ꢀ 5a: H-NMR (CD OD)
3
Compound 6bꢀ wasꢀ preparedꢀ differentlyꢀ fromꢀ theꢀ reportedꢀ δ:ꢀ1.35ꢀ(2Η,ꢀtt,ꢀJ=7.7, 7.7Hz), 1.49–1.65 (4H, m), 1.72 (2H, tt,
2
3)
procedure.
N-Hydroxysuccinimide (90.0mg, 0.780mmol) J=7.0, 7.0Hz), 2.10 (2H, J=7.0Hz), 2.60 (1H, d, J=12.6Hz),

1
652
Chem. Pharm. Bull.
Vol. 64, No. 11 (2016)
2
.83 (1H, dd, J=5.2, 12.6Hz), 3.08 (2H, t, J=6.9Hz), 3.12 (1H, 30min at 0°C before addition of test compound, and the ex-
®
dt, J=5.8, 8.5Hz), 3.51 (2H, t, J=7.2Hz), 4.22 (1H, dd, J=4.3, cessꢀ probesꢀ wereꢀ removedꢀ byꢀ Amicon ꢀ Ultra-0.5ꢀmLꢀ (Merckꢀ
13
7
.8Hz), 4,39 (1H, dd, J=4.8, 7.8Hz). C-NMR (CD OD) δ: MilliporeꢀLtd.,ꢀGermany),ꢀafterꢀtheꢀlabelingꢀreaction.ꢀTheꢀre-
3
26.8, 29.1, 29.5, 29.7, 36.8, 37.7, 37.9, 41.1, 57.0, 61.6, 63.3, sultingꢀsampleꢀwasꢀmixedꢀwithꢀ5xꢀSDS-PAGEꢀloadingꢀbuffer,ꢀ
1
34.2,ꢀ 164.5,ꢀ 166.1,ꢀ 176.2.ꢀ HR-MSꢀ (ESI)ꢀ m/z: 471.0630 Calcd heatedꢀforꢀ20ꢀminꢀatꢀ120°C,ꢀandꢀsubjectedꢀtoꢀSDS-PAGE.ꢀTheꢀ
+
for C H Cl N O S (M+Na ): 471.0631).
gelꢀwasꢀvisualizedꢀwithꢀanꢀin-gelꢀfluorescenceꢀimagerꢀ(ATTOꢀ
17
22
2
4
4
N-(2-(2-(2-(Biotinylamino)ethoxy)ethoxy)ethyl)-2,3- Ez-Captureꢀ II)ꢀ (diaphragm:ꢀ 3.0,ꢀ exposureꢀ time:ꢀ 5ꢀs)ꢀ equippedꢀ
dichloromaleimide (5b) withꢀ WSE-5500ꢀ VariRaysꢀ (λ : 440–500nm, peak: 470nm)
solution of 2,3-dichloromaleic anhydride (8.90mg, andꢀ stainedꢀ withꢀ Commassieꢀ Brilliantꢀ Blueꢀ (seeꢀ Pico™ CBB
.0534mmol) and 7b (20.0mg, 0.0534mmol) in acetic acid Stain Kit).
0.200ꢀmL)ꢀ wasꢀ stirredꢀ atꢀ roomꢀ temperatureꢀ forꢀ 15ꢀminꢀ underꢀ
anꢀ Arꢀ atmosphere.ꢀ Theꢀ mixtureꢀ wasꢀ thenꢀ stirredꢀ atꢀ 120°Cꢀ
ex
A
0
(
Acknowledgments Theꢀworkꢀdescribedꢀinꢀthisꢀpaperꢀwasꢀ
forꢀ 4ꢀhꢀ andꢀ theꢀ solventꢀ wasꢀ removedꢀ byꢀ air-flow.ꢀ Theꢀ residueꢀ partiallyꢀ supportedꢀ byꢀ Grants-in-Aidꢀ forꢀ Scientificꢀ Researchꢀ
wasꢀ purifiedꢀ byꢀ flashꢀ silica-gelꢀ chromatographyꢀ (CHCl3– (KAKENHI,ꢀ Grant-in-Aidꢀ forꢀ Youngꢀ Scientistsꢀ (B),ꢀ No.ꢀ
MeOH=20:1 to 15:1) to afford 5b (15.8mg, 0.0302mmol, 26810091 and No. 16K17930 to T.Y.) from The Ministry of
1
5
7%)ꢀ asꢀ aꢀ whiteꢀ solid.ꢀ 5b: H-NMR (CDCl ) δ:ꢀ 1.39ꢀ (2Η,ꢀ tt,ꢀ Education, Culture, Sports, Science and Technology (MEXT)
3
J=7.6, 7.6Hz), 1.58–1.71 (4H, m), 2.22 (2H, t, J=7.4Hz), 2.70 of Japan, and the Japan Society for the Promotion of Science
(
1H, d, J=13.2Hz), 2.85 (1H, dd, J=4.9, 12.9Hz), 3.10 (1H, dt, (JSPS),ꢀ andꢀ Platformꢀ forꢀ Drugꢀ Discovery,ꢀ Informatics,ꢀ andꢀ
J=4.5, 7.3Hz), 3.36 (2H, t, J=4.6Hz), 3.46 (2H, t, J=5.2Hz), StructuralꢀLifeꢀScience.
.49–3.50 (2H, m), 3.53–3.55 (2H, m) 3.61 (2H, t, J=5.4Hz),
.75 (2H, t, J=5.4Hz), 4.28 (1H, dd, J=4.6, 8.0Hz), 4.47 (1H,
3
3
Conflict of Interest Theꢀ authorsꢀ declareꢀ noꢀ conflictꢀ ofꢀ
1
3
dd, J=4.9, 7.7Hz), 6.68 (2H, brs). C-NMR (CDCl ) δ: 25.7, interest.
2
3
8.1, 28.3, 35.8, 38.7, 39.3, 40.5, 55.7, 60.7, 62.2, 67.6, 69.9,
7
0.1,ꢀ 133.5,ꢀ 163.2,ꢀ 164.2,ꢀ 173.9.ꢀ HR-MSꢀ (ESI)ꢀ m/z: 545.1005 References
+
Calcd for C H Cl N O S (M+Na ): 545.0999).
ꢀ 1)ꢀ Takaokaꢀ Y.,ꢀ Ojidaꢀ A.,ꢀ Hamachiꢀ I.,ꢀ Angew. Chem. Int. Ed., 52,
088–4106 (2013).
Park J., Koh M., Park S. B., Mol. Biosyst., 9, 544–550 (2013).
3)ꢀ ParkꢀJ.,ꢀKohꢀM.,ꢀKooꢀJ.ꢀY.,ꢀLeeꢀS.,ꢀParkꢀS.ꢀB.,ꢀACS Chem. Biol., 11,
4–52 (2016).
2
0
28
2
4
6
4
Spectrometric Analysis Methanol for spectrochemical
analysisꢀwasꢀpurchasedꢀfromꢀWakoꢀPureꢀChemicalꢀIndustries,ꢀ
Ltd.ꢀ andꢀ 1,4-dioxaneꢀ forꢀ spectrochemicalꢀ analysisꢀ wasꢀ pur-
chasedꢀ fromꢀ Kantoꢀ Chemicalꢀ Co.,ꢀ Inc.ꢀ Theꢀ absorptionꢀ spec-
traꢀ wereꢀ recordedꢀ onꢀ aꢀ Shimadzuꢀ UV-2400PCꢀ spectrometerꢀ
equippedꢀ withꢀ aꢀ 50ꢀWꢀ halogenꢀ lamp,ꢀ andꢀ aꢀ deuteriumꢀ lampꢀ
2)
ꢀ
ꢀ
4
4)
Yamaguchi T., Asanuma M., Nakanishi S., Saito Y., Okazaki M.,
Dodo K., Sodeoka M., Chem. Sci., 5, 1021–1029 (2014).
5)ꢀ Shenꢀ B.ꢀ Q.,ꢀ Xuꢀ K.,ꢀ Liuꢀ L.,ꢀ Raabꢀ H.,ꢀ Bhaktaꢀ S.,ꢀ Kenrickꢀ M.,ꢀ
Parsons-ReponteꢀK.ꢀL.,ꢀTienꢀJ.,ꢀYuꢀS.ꢀF.,ꢀMaiꢀE.,ꢀLiꢀD.,ꢀTibbittsꢀJ.,ꢀ
Baudys J., Saad O. M., Scales S. J., McDonald P. J., Hass P. E., Ei-
genbrotꢀC.,ꢀNguyenꢀT.,ꢀSolisꢀW.ꢀA.,ꢀFujiꢀR.ꢀN.,ꢀFlagellaꢀK.ꢀM.,ꢀPatelꢀ
D.,ꢀ Spencerꢀ S.ꢀ D.,ꢀ Khawliꢀ L.ꢀ A.,ꢀ Ebensꢀ A.,ꢀ Wongꢀ W.ꢀ L.,ꢀ Vandlenꢀ
R.,ꢀKaurꢀS.,ꢀSliwkowskiꢀM.ꢀX.,ꢀSchellerꢀR.ꢀH.,ꢀPolakisꢀP.,ꢀJunutulaꢀ
J. R., Nat. Biotechnol., 30, 184–189 (2012).
(slitꢀwidth:ꢀ0.2ꢀnm).ꢀTheꢀfluorescenceꢀmeasurementsꢀwereꢀper-
formedꢀ onꢀ aꢀ JASCOꢀ FP-6500ꢀ equippedꢀ withꢀ aꢀ 150ꢀWꢀ xenonꢀ
lampꢀ(slitꢀwidth:ꢀ3ꢀnm).ꢀAꢀ1.00ꢀcmꢀquartzꢀcellꢀwasꢀusedꢀforꢀtheꢀ
measurements.
Biological Assay
Material Preparation
6
7
)
)
Gunnoo S. B., Madder A., ChemBioChem, 17, 529–533 (2016).
Baslé E., Joubert N., Pucheault M., Chem. Biol., 17, 213–227 (2010).
Streptavidinꢀ wasꢀ purchasedꢀ fromꢀ Wakoꢀ Pureꢀ Chemicalꢀ In-
dustries,ꢀLtd.ꢀ(streptavidinꢀfromꢀStreptomyces avidinii, >90%,
ꢀ
ꢀ
8)ꢀ HolmesꢀK.ꢀL.,ꢀLantzꢀL.ꢀM.,ꢀMethods Cell Biol., 63, 185–204 (2001).
9)ꢀ KimꢀY.,ꢀHoꢀS.ꢀO.,ꢀGassmanꢀN.ꢀR.,ꢀKorlannꢀY.,ꢀLandorfꢀE.ꢀV.,ꢀCol-
lart F. R., Weiss S., Bioconjug. Chem., 19, 786–791 (2008).
10–20ꢀunits/mg).ꢀBSAꢀwasꢀpurchasedꢀfromꢀSigma-AldrichꢀCo.,ꢀ
LLCꢀ (Bovineꢀ Serumꢀ Albumin,ꢀ 96–99%),ꢀ CAIIꢀ wasꢀ alsoꢀ pur-
chasedꢀ fromꢀ Sigma-Aldrichꢀ Co.,ꢀ LLCꢀ (Carbonicꢀ Anhydraseꢀ
Isozymeꢀ IIꢀ fromꢀ bovineꢀ erythrocytes,ꢀ ≥3000W-A units/mg
protein).ꢀ Theꢀ proteinsꢀ wereꢀ usedꢀ withoutꢀ furtherꢀ purification.ꢀ
10)ꢀ Smithꢀ M.ꢀ E.ꢀ B.,ꢀ Schumacherꢀ F.ꢀ F.,ꢀ Ryanꢀ C.ꢀ P.,ꢀ Tedaldiꢀ L.ꢀ M.,ꢀ Pa-
paioannou D., Waksman G., Caddick S., Baker J. R., J. Am. Chem.
Soc., 132, 1960–1965 (2010).
Streptavidin,ꢀ BSAꢀ andꢀ CAIIꢀ wereꢀ dissolvedꢀ inꢀ MilliQꢀ water,ꢀ 11) Schumacher F. F., Nobles M., Ryan C. P., Smith M. E. B., Tinker
andꢀtheꢀconcentrationsꢀwereꢀdeterminedꢀbyꢀmeasuringꢀtheꢀab-
sorbanceꢀatꢀ280ꢀnm.ꢀTheꢀHEK-293ꢀcellꢀlysateꢀwasꢀpreparedꢀasꢀ
follows.ꢀHEK-293ꢀcellsꢀwereꢀculturedꢀinꢀDulbecco’sꢀmodifiedꢀ
Eagle’sꢀmediumꢀ(DMEM)ꢀcontainingꢀ10%ꢀfetalꢀbovineꢀserumꢀ
A., Caddick S., Baker J. R., Bioconjug. Chem., 22, 132–136 (2011).
2) Moody P., Smith M. E. B., Ryan C. P., Chudasama V., Baker J. R.,
Molloy J., Caddick S., ChemBioChem, 13, 39–41 (2012).
1
1
3) Robin M. P., Wilson P., Mabire A. B., Kiviaho J. K., Raymond
J.ꢀ E.,ꢀ Haddletonꢀ D.ꢀ M.,ꢀ O’Reillyꢀ R.ꢀ K.,ꢀ J. Am. Chem. Soc., 135,
875–2878 (2013).
4)ꢀ NathaniꢀR.ꢀI.,ꢀChudasamaꢀV.,ꢀRyanꢀC.ꢀP.,ꢀMoodyꢀP.ꢀR.,ꢀMorganꢀR.ꢀ
(
FBS)ꢀ andꢀ penicillin/streptomycinꢀ atꢀ 37°Cꢀ inꢀ aꢀ humidifiedꢀ
2
atmosphere of 5% CO .ꢀ Theꢀ cellꢀ pelletꢀ wasꢀ lysedꢀ withꢀ solu-
2
1
bilization buffer (20mM N-(2-hydroxyethyl)piperazine-N′-(2-
ethanesulfonic acid) sodium salt (Hepes-Na) (pH 7.0), 1.0mM
E., Fitzmaurice R. J., Smith M. E. B., Baker J. R., Caddick S., Org.
Biomol. Chem., 11, 2408–2411 (2013).
ethylenediaminetetraacetic acid (EDTA), 0.5% Triton X-100, 15)ꢀ YouzielꢀJ.,ꢀAkhbarꢀA.ꢀR.,ꢀAzizꢀQ.,ꢀSmithꢀM.ꢀE.ꢀB.,ꢀCaddickꢀS.,ꢀTin-
andꢀ completeꢀ miniꢀ (Roche,ꢀ Switzerland))ꢀ forꢀ 15ꢀminꢀ onꢀ iceꢀ
ker A., Baker J. R., Org. Biomol. Chem., 12, 557–560 (2014).
and centrifuged at 15000×g for 10min at 4°C. The resulting 16) Marculescu C., Kossen H., Morgan R. E., Mayer P., Fletcher S. A.,
TolnerꢀB.,ꢀChesterꢀK.ꢀA.,ꢀJonesꢀL.ꢀH.,ꢀBakerꢀJ.ꢀR.,ꢀChem. Commun.,
supernatantꢀwasꢀcollectedꢀandꢀusedꢀasꢀHEK-293ꢀcellꢀlysate.
GeneralꢀLabelingꢀProcedure
Streptavidinꢀ wasꢀ incubatedꢀ withꢀ testꢀ compoundꢀ inꢀ phos-
phate buffer (100µL,ꢀpHꢀ7.0)ꢀforꢀ30ꢀminꢀatꢀ0°C.ꢀInꢀtheꢀcompe-
titionꢀ analysis,ꢀ streptavidinꢀ wasꢀ pre-incubatedꢀ withꢀ biotinꢀ forꢀ
5
0, 7139–7142 (2014).
1
7) Smith M. E. B., Caspersen M. B., Robinson E., Morais M., Maruani
A., Nunes J. P. M., Nicholls K., Saxton M. J., Caddick S., Baker J.
R., Chudasama V., Org. Biomol. Chem., 13, 7946–7949 (2015).

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O’ReillyꢀR.ꢀK.,ꢀChem. Commun., 51, 9733–9736 (2015).
23)ꢀ GnaccariniꢀC.,ꢀBen-TaharꢀW.,ꢀLubellꢀW.ꢀD.,ꢀPelletierꢀJ.ꢀN.,ꢀKeillorꢀJ.ꢀ
W., Bioorg. Med. Chem., 17, 6354–6359 (2009).
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0) Mahle F., da Rosa Guimarães T., Vergilina Meira A., Corrêa R.,
Bella Cruz R. C., Bella Cruz A., Nunes R. J., Cechinel-Filho V., de
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