Protein modification by strain-promoted alkyne-nitrone cycloaddition

Article abstract of DOI:10.1002/anie.201000408

(Figure Presented) Quicker and slicker: An efficient metalfree 1,3-dipolar cycloaddition of dibenzocyclooctynes with nitrones proceeded with rate constants of up to 39 M^-1S^-1, or up to 300 times faster than similar reactions with azi

Full text of DOI:10.1002/anie.201000408

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Chemie
DOI: 10.1002/anie.201000408
Bioorthogonal Reactions
Protein Modification by Strain-Promoted Alkyne–Nitrone
Cycloaddition**
Xinghai Ning, Rinske P. Temming, Jan Dommerholt, Jun Guo, Daniel B. Ania,
Marjoke F. Debets, Margreet A. Wolfert, Geert-Jan Boons,* and Floris L. van Delft*
The bioorthogonal chemical reporter strategy is emerging as a
versatile method for the labeling of biomolecules, such as
nucleic acids, lipids, carbohydrates, and proteins.[1] In this
approach, an abiotic chemical functionality (reporter) is
incorporated into a target biomolecule and can then react
with a complementary bioorthogonal functional group linked
to one of a diverse set of probes.
The azide functional group, which is the most commonly
employed reporter, can react in a Staudinger ligation with
modified phosphines,[2] in a copper(I)-catalyzed cycloaddition
with terminal alkynes (CuAAC),[3] or in a strain-promoted
alkyne–azide cycloaddition (SPAAC).[4] The last type of
Figure 1. Ring-strained cyclooctynes for bioorthogonal cycloaddition
reaction[5] is attractive because it does not require a cytotoxic
metal catalyst and therefore provides unique opportunities
for the labeling of cell-surface glycans[4b,6] and proteins[7] of
living cells, the decoration of polymeric nanostructures,[8] the
labeling of lipids,[9] proteomics,[10] and tissue reengineering.[11]
The first generation of cyclooctynes suffered from rela-
tively slow reaction rates; however, it has been found that the
rate of strain-promoted cycloaddition can be increased by
appending electron-withdrawing groups adjacent to the triple
bond. For example, reactions of difluorinated cyclooctynes,
such as 1 (Figure 1), with azides proceed approximately 60
times faster than the corresponding reactions of unsubstituted
derivatives.[5a] We have found that derivatives of the
4-dibenzocyclooctynol 2 react fast with azido-containing
saccharides and amino acids and can be employed for the
visualization of metabolically labeled glycans of living cells.[12]
Attractive features of dibenzocyclooctynols include easy
synthetic access, nontoxicity, and the straightforward attach-
ment of a variety of probes. Recently, we introduced the more
polar azacyclooctyne 3,[13] which exhibits a higher rate of
reactions with azides.
reaction. Despite these advances, there is an urgent need for
new and faster bioorthogonal reactions for labeling at low
concentration.[1b]
We report herein a novel bioorthogonal reaction pair
based on strain-promoted alkyne–nitrone cycloaddition
(SPANC) to give N-alkylated isoxazolines with exceptionally
fast reaction kinetics. The new methodology was used in a
Table 1: Rate constants for the cycloaddition of dibenzocyclooctynol 2
with nitrones 4a–f.[a]
4
R1
R2
R3
k[b] [mÀ1 sÀ1
]
k’
Yield [%]
[*] X. Ning, J. Guo, M. A. Wolfert, G.-J. Boons
Complex Carbohydrate Research Center, University of Georgia
315 Riverbend Road, Athens, GA 30602 (USA)
E-mail: gjboons@ccrc.uga.edu
a
b
c
d
e
f
H
H
H
Me
H
Ph
Me
Me
Ph
Me
Me
Me
1.3ꢀ10À2
3.2ꢀ10À2
1
3
95
80
89
33
92
93
CH2CH2Ph
Ph
CH2CH2CO2Et
CO2Et
>0.2[c]
>17
<0.1
330
180
<1ꢀ10À3
3.9
R. P. Temming, J. Dommerholt, D. B. Ania, M. F. Debets,
F. L. van Delft
H
C(O)NHBn
2.2
Radboud University Nijmegen
[a] The nitrone substrates (except 4d) were formed as pure Z isomers.
Isoxazoline 5 was formed as a mixture of regio- and diastereoisomers.
See the Supporting Information for reaction conditions. [b] Method A:
The rate constant was determined by 1H NMR spectroscopy in CD3CN/
D2O (3:1); [2a]=18 mm, [4a–f]=16.4 mm. Method B: The rate con-
stant was determined[18] by UV spectroscopy in CH3CN/H2O (3:1); [2a]=
0.33 mm, [4a–f]=0.30 mm. These reactions were too fast for monitoring
by NMR spectroscopy. [c] The reaction was too fast for accurate
determination of the rate constant by NMR spectroscopy. Determination
by UV spectroscopy was not possible owing to overlapping absorptions.
Bn=benzyl.
Institute for Molecules and Materials
Heijendaalseweg 135, 6525 AJ, Nijmegen (The Netherlands)
E-mail: f.vandelft@science.ru.nl
[**] This research was supported financially (in part) by the Council for
Chemical Sciences of The Netherlands Organization for Scientific
Research (NWO-CW, F.L.v.D.) and the National Cancer Institute of
the US National Institutes of Health (Grant No. RO1 CA88986,
G.-J.B.).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201000408.
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one-pot three-step protocol for the site-specific modification
of peptides and proteins.
(1.1 equiv) to rapidly generate aldehyde 11, which was first
treated with p-methoxybenzenethiol (6.6 equiv, 30 min), and
then with N-methylhydroxylamine (2.2 equiv), p-anisidine
(5 equiv), and 2 (2.2 equiv) to give the desired isoxazoline 13
via nitrone 12 (Scheme 1). We found that treatment with
p-MeOC6H4SH was essential to avoid the conversion of
Nitrones 4a–f were readily prepared by the condensation
of appropriate aldehydes with N-methylhydroxylamine.
Cycloaddition reactions of 4a–f with cyclooctynol 2 in a
mixture of acetonitrile and water gave the corresponding
stable[14] isoxazolines, in most cases in high yield (Table 1). We
measured the rate constants of the cycloaddition
reactions by 1H NMR or UV spectroscopy at
258C and found that the substituents on the
nitrone greatly influenced the reaction kinetics.
For example, the replacement of an N-methyl
with a phenyl group (to give 4c) led to a faster
reaction,[15] whereas nitrone 4d, derived from a
ketone, exhibited reaction kinetics that were too
slow for accurate determination of the rate
constant. Exceptionally high reaction rates
were measured for the cycloaddition of 2 with
a-carboxynitrones 4e and 4 f. These reactions
proceeded 18 and 32 times as fast, respectively,
as the cycloaddition of 2 with benzyl azide
(0.12 mÀ1 sÀ1).[16] Also, we found that a high water
content increased the reaction rate constants
(e.g. 12.8 mÀ1 sÀ1 for a derivative of 2 in aceto-
N-methylhydroxylamine
into
nitrosomethane
dimer
nitrile/water (1:9); see the Supporting Informa-
tion).[17] Finally, we determined a rate constant
for the cycloaddition of azacyclooctyne 3 with
4 f. As expected,[13] a further enhancement of the
reaction rate (39 mÀ1 sÀ1) was observed when 3
was used in place of the carbon analogue 2.
Next, the challenge was to find a strategy for the
incorporation of nitrones into biomolecules. We first focused
our attention on metabolic labeling with monosaccharide
derivatives bearing a nitrone moiety.[19] Unfortunately, the
incubation of Jurkat cells in the presence of nitrones 6–9 (10,
20, 50, and 100 mm; Figure 2), followed by labeling with
dibenzocyclooctyne–biotin and staining with an avidin–fluo-
rescein isothiocyanate (FITC) conjugate, led to no detectable
fluorescence labeling of the cells.[12] Presumably, either the
biosynthetic glycosylation machinery does not accept nitrone
modifications, or nitrones undergo intracellular hydrolysis in
acidic compartments.
Scheme 1. One-pot N-terminal conjugation of a hexapeptide by SPANC: a) 1. NaIO4,
NH4OAc buffer, pH 6.8, room temperature, 1 h; 2. p-MeOC6H4SH, room temperature,
1 h; then p-MeOC6H4NH2, MeHNOH·HCl, room temperature, 20 min; b) 2, room
temperature, 1 h.
À
((MeNO)2) by oxidation with iodate (IO3 ) formed in the
previous step.[21] Furthermore, the rate of nitrone formation
was greatly enhanced by the addition of p-anisidine, probably
by a similar mechanism to that described for the formation of
oximes from aldehydes and hydroxylamines.[22]
To examine whether the one-pot three-step protocol was
suitable for protein modification, we selected the chemokine
interleukin-8 (IL-8),[23] as this prototypical protein has an
N-terminal serine residue and a relatively low molecular
weight (72 amino acids, MW= 8382 Da), which facilitates
direct analysis of chemical modification by mass spectrome-
try. Current labeling methods of IL-8, for example, for the
installment of a radiolabel for scintigraphic imaging of
infections,[24] are based on random reactions of side-chain
lysine amino groups with no control over the number of
reactions that take place or the sites of reaction.
Fortunately, SPANC could be employed for efficient
peptide and protein modification by implementing a one-pot
three-step procedure. Thus, the N-terminal serine residue of
model peptide 10 was oxidized[20] with sodium periodate
Thus, IL-8 in NH4OAc buffer (2 mm, pH 6.9) was
subjected to oxidation with NaIO4 (1.1 equiv, 1 h), followed
by treatment with p-MeOC6H4SH (6.6 equiv, 2 h), then
N-methylhydroxylamine
(10 equiv)
and
p-anisidine
(10 equiv), and finally cyclooctynol 2 (25 equiv, 21 mm).
After 24 h, mass spectrometric analysis showed the presence
of a single protein with a mass corresponding to the isoxazo-
line conjugate 15 (MW= 8599 Da; Scheme 2). The one-pot
three-step SPANC protocol was also successfully employed to
PEGylate[25,26] IL-8 by using the PEG2000-modified dibenzo-
cyclooctyne 16 (PEG = poly(ethylene glycol)). Quantitative
formation of PEG-modified IL-8 17 was observed by HPLC
analysis (Figure 3).
Figure 2. Nitrone derivatives of d-mannosamine (compounds 6 and
7), sialic acid (compound 8), and d-galactose (compound 9) for
metabolic cell-surface labeling.
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[1] a) J. A. Prescher, C. R. Bertozzi, Nat. Chem.
Biol. 2005, 1, 13 – 21; b) E. M. Sletten, C. R.
Bertozzi, Angew. Chem. 2009, 121, 7108 –
7133; Angew. Chem. Int. Ed. 2009, 48,
6974 – 6998.
[2] E. Saxon, C. R. Bertozzi, Science 2000, 287,
2007 – 2010.
[3] a) C. W. Tornøe, C. Christensen, M. Meldal,
J. Org. Chem. 2002, 41, 2596 – 2599; b) V. V.
Rostovtsev, L. G. Green, V. V. Fokin, K. B.
Sharpless, Angew. Chem. 2002, 114, 2708 –
2711; Angew. Chem. Int. Ed. 2002, 41,
2596 – 2599.
[4] a) A. T. Blomquist, L. H. Liu, J. Am. Chem.
Soc. 1953, 75, 2153 – 2154; b) N. J. Agard,
J. A. Prescher, C. R. Bertozzi, J. Am. Chem.
Soc. 2004, 126, 15046 – 15047.
[5] a) J. M. Baskin, J. A. Prescher, S. T. Laughlin,
N. J. Agard, P. V. Chang, I. A. Miller, A. Lo,
J. A. Codelli, C. R. Bertozzi, Proc. Natl.
Acad. Sci. USA 2007, 104, 16793 – 16797;
b) J. F. Lutz, Angew. Chem. 2008, 120, 2212 –
2214; Angew. Chem. Int. Ed. 2008, 47, 2182 –
2184; c) C. R. Becer, R. Hoogenboom, U. S.
Scheme 2. One-pot N-terminal functionalization of IL-8 by SPANC: a) 1. NaIO4, NH4OAc
buffer, pH 6.9, room temperature, 1 h; 2. p-MeOC6H4SH, room temperature, 2 h;
b) p-MeOC6H4NH2, MeNHOH·HCl, room temperature, 20 min; c) cyclooctynol 2 or PEG–
cyclooctyne 16, room temperature, 20 h.
Schubert, Angew. Chem. 2009, 121, 4998 – 5006; Angew. Chem.
Int. Ed. 2009, 48, 4900 – 4908; d) J. Kalia, T. R. Raines, Curr. Org.
Chem. 2010, 14, 138 – 147.
[6] N. J. Agard, J. M. Baskin, J. A. Prescher, A. Lo, C. R. Bertozzi,
ACS Chem. Biol. 2006, 1, 644 – 648.
[7] a) A. J. Link, M. K. S. Vink, N. J. Agard, J. A. Prescher, C. R.
Bertozzi, D. A. Tirrell, Proc. Natl. Acad. Sci. USA 2006, 103,
10180 – 10185; b) I. C. Tanrikulu, E. Schmitt, Y. Mechulam,
W. A. Goddard III, D. A. Tirrell, Proc. Natl. Acad. Sci. USA
2009, 106, 15285 – 15290.
Figure 3. HPLC traces of IL-8 (14) and crude PEGylated IL-8, 17.
[8] a) E. Lallana, E. Fernandez-Megia, R. Riguera, J. Am. Chem.
Soc. 2009, 131, 5748 – 5750; b) P. Kele, G. Mezꢀ, F. Achats, O. S.
Wolfbeis, Angew. Chem. 2009, 121, 350 – 353; Angew. Chem. Int.
Ed. 2009, 48, 344 – 347.
[9] a) M. Fernꢁndez-Suꢁrez, H. Baruah, L. Martꢂnez-Hernꢁndez,
K. T. Xie, J. M. Baskin, C. R. Bertozzi, A. Y. Ting, Nat.
Biotechnol. 2007, 25, 1483 – 1487; b) A. B. Neef, C. Schultz,
Angew. Chem. 2009, 121, 1526 – 1529; Angew. Chem. Int. Ed.
2009, 48, 1498 – 1500.
We have shown that 1,3-dipolar cycloadditions of cyclo-
octynes with nitrones that contain ester or amide a substitu-
ents exhibit much faster kinetics than similar reactions with
azides.[27] The new methodology was successfully employed
for the site-specific modification of a peptide and a protein by
implementing a one-pot three-step protocol. Besides serine or
threonine oxidation,
a variety of methods have been
described for the installment of carbonyl groups in proteins,[28]
and it is to be expected that SPANC is compatible with these
approaches. Furthermore, metal-free click reactions have
found entry into materials science.[11] SPANC will provide an
additional tool for the preparation of increasingly complex
materials by simple and flexible chemical manipulations.
Finally, we anticipate that SPANC will offer an attractive
alternative to the well-established oxime ligation[29] because
the synthesis of nitrones is simple,[19] the isoxazoline products
are stable,[14] and the combination of a functionalized nitrone
(R3 is a functional group, Table 1) with a cyclooctyne
conjugate (such as 16) will make it possible to introduce
two different functionalities in a single process.
[10] M. A. Nessen, G. Kramer, J. W. Back, J. M. Baskin, L. E. J.
Smeenk, L. J. de Koning, J. H. van Maarseveen, L. de Jong, C. R.
Bertozzi, H. Hiemstra, C. G. de Koster, J. Proteome Res. 2009, 8,
3702 – 3711.
[11] J. T. Wilson, V. R. Krishnamurthy, W. Cui, Z. Qu, E. L. Chaikof,
J. Am. Chem. Soc. 2009, 131, 18228 – 18229.
[12] X. Ning, J. Guo, M. A. Wolfert, G.-J. Boons, Angew. Chem. 2008,
120, 2285 – 2287; Angew. Chem. Int. Ed. 2008, 47, 2253 – 2255.
[13] M. F. Debets, S. S. van Berkel, S. Schoffelen, F. P. J. T. Rutjes,
J. C. M. van Hest, F. L. van Delft, Chem. Commun. 2010, 46, 97 –
99.
[14] For example, isoxazoline 5 f was recovered unchanged after 24 h
at 608C in CHCl3 or after being stirred at pH 3 or pH 9 in a THF/
water mixture.
[15] a) R. Huisgen, H. Seidl, I. Bruning, Chem. Ber. 1969, 102, 1102;
b) W. R. Dolbier, Jr., G. E. Wicks, C. R. Burkholder, J. Org.
Chem. 1987, 52, 2196 – 2201.
Received: January 22, 2010
Published online: March 23, 2010
[16] We accurately redetermined a rate constant of 0.12mÀ1 sÀ1 for
the reaction of 2 with benzyl azide.
[17] a) M. R. Gholami, A. H. Yangjeh, J. Chem. Res. Synop. 1999, 3,
226 – 227; b) R. N. Butler, A. G. Coyne, W. J. Cunningham, L. A.
Burke, J. Chem. Soc. Perkin Trans. 2 2002, 1807 – 1815.
Keywords: cycloaddition · cycloalkynes · kinetics · nitrones ·
protein modifications
.
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[18] A. A. Poloukhtine, N. E. Mbua, M. A. Wolfert, G. J. Boons, V. V.
[26] A. Dondoni, A. Massi, P. Nanni, A. Roda, Chem. Eur. J. 2009, 15,
11444 – 11449.
[27] During the preparation of this manuscript, the cycloaddition of
nitrones with nonfunctionalized dibenzocyclooctyne was
reported: C. S. McKay, J. Moran, J. P. Pezacki, Chem.
Commun. 2010, 46, 931 – 933.
Popik, J. Am. Chem. Soc. 2009, 131, 15769 – 15776.
[19] P. Merino in Science of Synthesis, Vol. 27 (Ed.: A. Padwa),
Thieme, Stuttgart, 2004, pp. 511 – 580.
[20] K. F. Geoghegan, J. G. Stroh, Bioconjugate Chem. 1992, 3, 138 –
146.
[28] a) J. M. Gilmore, R. A. Scheck, A. P. Esser-Kahn, N. S. Joshi,
M. B. Francis, Angew. Chem. 2006, 118, 5433 – 5437; Angew.
Chem. Int. Ed. 2006, 45, 5307 – 5311; b) I. S. Carrico, B. L.
Carlson, C. R. Bertozzi, Nat. Chem. Biol. 2007, 3, 321 – 322; c) Y.
Zeng, T. N. C. Ramya, A. Dirksen, P. E. Dawson, J. C. Paulson,
Nat. Methods 2009, 6, 207 – 209; d) K. Ebisu, H. Tateno, H.
Kuroiwa, K. Kawakami, M. Ikeuchi, J. Hirabayashi, M. Sisido,
M. Taki, ChemBioChem 2009, 10, 2460 – 2464.
[21] T. Emery, J. B. Neilands, J. Am. Chem. Soc. 1960, 82, 4903 – 4904.
[22] A. Dirksen, T. M. Hackeng, P. E. Dawson, Angew. Chem. 2006,
118, 7743 – 7746; Angew. Chem. Int. Ed. 2006, 45, 7581 – 7584.
[23] M. Baggiolini, I. Clark-Lewis, FEBS Lett. 1991, 307, 97 – 101.
[24] C. J. van der Laken, O. C. Boerman, W. J. G. Oyen, M. T. P.
van de Ven, J. W. M. van der Meer, F. H. M. Corstens, J. Nucl.
Med. 2000, 41, 463 – 469.
[25] a) I. S. Carrico, Chem. Soc. Rev. 2008, 37, 1423 – 1431; b) C. P. R.
Hackenberger, D. Schwarzer, Angew. Chem. 2008, 120, 10182 –
10228; Angew. Chem. Int. Ed. 2008, 47, 10030 – 10074.
[29] a) P. E. Dawson, S. B. H. Kent, Annu. Rev. Biochem. 2000, 69,
923 – 960; b) J. A. Borgia, G. B. Fields, Trends Biotechnol. 2002,
18, 243 – 251.
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