Multiple-turnover isotopic labeling of Fmoc- and boc-protected amino acids with oxygen isotopes

Article abstract of DOI:10.1021/ol902519g

Chemical Equation Presented An efficient method for the selective isotopic labeling of carboxylic acids is reported. By reacting an amino acid with excess carbodiimide and ¹⁸OH₂, a kinetically enhanced multiple turnover reaction provides the ¹⁸O-labeled product in high yield and excellent isotopic enrichment. This reaction is fully compatible with standard Fmoc, Boc, Trt, and OtBu protecting groups and provides a means to selectively label the α-carboxylic acids of functionalized amino acids with stable oxygen isotopes

Full text of DOI:10.1021/ol902519g

ORGANIC
LETTERS
2010
Vol. 12, No. 1
104-106
Multiple-Turnover Isotopic Labeling of
Fmoc- and Boc-Protected Amino Acids
with Oxygen Isotopes
Martin S. Seyfried,† Birgit S. Lauber,‡ and Nathan W. Luedtke*,†
Institute of Organic Chemistry, UniVersity of Zu¨rich, Winterthurerstrasse 190,
CH-8057 Zu¨rich, Switzerland, and Swiss Federal Institute of Technology (ETH)
Ho¨nggerberg, CH-8093 Zu¨rich, Switzerland
luedtke@oci.uzh.ch
Received October 31, 2009
ABSTRACT
An efficient method for the selective isotopic labeling of carboxylic acids is reported. By reacting an amino acid with excess carbodiimide and
18OH2, a kinetically enhanced multiple turnover reaction provides the 18O-labeled product in high yield and excellent isotopic enrichment. This
reaction is fully compatible with standard Fmoc, Boc, Trt, and OtBu protecting groups and provides a means to selectively label the r-carboxylic
acids of functionalized amino acids with stable oxygen isotopes.
Isotopically labeled amino acids have found wide application
in structure elucidation of peptides and proteins. In addition
to 17O NMR studies,1-5 2D-IR techniques are emerging as
important new tools for probing protein folding and ligand
binding.6-10 Due to its time resolution in the subpicosecond
range, time-resolved 2D-IR spectroscopy can report the rapid
evolution of nonequilibrium conformational states to reveal
folding pathways.7-10 These techniques rely upon site-
specific incorporation of 18O-labeled amino acids into pep-
tides and proteins to serve as spectroscopic probes.8-10 To
date, only aliphatic, aromatic, and sulfur-containing amino
acids have been utilized in such studies because currently
(7) Ihalainen, J. A.; Bredenbeck, J.; Pfister, R.; Helbing, J.; Chi, L.;
Stokkum, I. H. M.; Woolley, G. A.; Hamm, P. Proc. Natl. Acad. Sci. U.S.A.
2007, 104, 5383. Hamm, P.; Manho, L.; Hochstrasser, R. M. J. Phys. Chem.
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Moroder, L.; Wachtveitl, J.; Hamm., P. J. Phys. Chem. B 2003, 107, 8654.
Woutersen, S.; Hamm, P. J. Phys.: Condens. Matter 2002, 14, R1035.
(8) Ihalainen, J. A.; Paoli, B.; Muff, S.; Backus, E. H.; Bredenbeck, J.;
Woolley, G. A.; Caflisch, A.; Hamm, P. Proc. Natl. Acad. Sci. U.S.A. 2008,
105, 9588. Shim, S. H.; Gupta, R.; Ling, Y. L.; Strasfeld, D. B.; Raleigh,
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Mukherjee, P.; Kass, I.; Arkin, I. T.; Zanni, M. T. J. Phys. Chem. B 2006,
110, 24740.
† University of Zu¨rich.
‡ ETH Ho¨nggerberg.
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48. Goc, R.; Ponnusamy, E.; Tritt-Goc, J.; Fiat, D. Int. J. Pept. Protein Res
1988, 31, 130. Steinschneider, A.; Burgar, M. I.; Buku, A.; Fiat, D. Int. J.
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10.1021/ol902519g  2010 American Chemical Society
Published on Web 11/30/2009

used methods for preparing 18O-labeled amino acids result
in uniform 18O-labeling of side chain and R-carboxylic
acids.11-13
enrichments of approximately 92-95% for both oxygen
atoms of the carboxylic acid (Table 1).
18O-labeling is normally conducted by heating the car-
boxylic acid with 18OH2 in the presence of a strong acid to
catalyze exchange (Scheme 1). By using an excess of 18O
Table 1. 18O-Enrichment and Isolated Yieldsa
Scheme 1.
Typical Equilibrium-Based 18O Labeling Reaction
water, the reported yields for isotopic enrichments are
typically 75-85%.6,11-13 A labeling procedure for Fmoc-
protected amino acids with multiple rounds of acid-catalyzed
18OH2 equilibration recently reported 18O enrichments of
90-96% for Phe, Ala, Gly, and Phe.6 This method can be
very efficient with respect to 18OH2 consumption, but the
protecting groups typically used for functionalized side chains
(Boc, Trt, tBu, etc.) are not stable under the strongly acidic
conditions used to catalyze exchange. These conditions can
therefore cause uniform labeling of Asp, Glu, Asn, and Gln
residues.13
Single-turnover 17OH2 saponification reactions of pen-
tafluorophenyl esters have recently been reported.4 These
reactions tolerate acid labile (tBu) protecting groups and can
selectively label R-carboxylic acids with isotopic enrichments
ranging from 20% to 85% for products containing a single
17O atom.4
We are interested in developing mild and selective
reactions for the isotopic labeling of amino acids, peptides,
and proteins to provide highly enriched materials for NMR,
IR, and MS studies. Here we report a nonequilibrium
multiple-turnover reaction where a carboxylic acid is reacted
with a carbodiimide to form an O-acylisourea that is
hydrolyzed by 18O water (abstract). After one round of
activation and hydrolysis, the mixed 16O/18O carboxylic acid
will be reactivated and hydrolyzed again and again (Scheme
2). Since carbodiimides react more rapidly with carboxylates
than with water,14 one-pot reactions containing an excess of
coupling reagent and 18OH2 result in multiple rounds of
O-acylisourea formation and hydrolysis to provide isotopic
a Total enrichment for both 18O oxygen atoms is indicated.
While heavy atom isotope effects are typically considered
negligible for preparative-scale reactions, a modest kinetic
bias should favor formation of the double-labeled 18O
product. The 16O atom in the mixed carboxylic acid (Scheme
2) should react faster than 18O to form the corresponding
16O-acylisourea.15 The resulting effect on efficiency depends
on the kinetic isotope effect (KIE) to the exponential power
of reaction turnovers (n), i.e., (KIE)n. In theory, a very small
KIA of 1.010 would result in cumulative KIA’s of about
11% and 270% after 10 and 100 turnovers, respectively. This
should serve to decrease 18OH2 consumption, especially for
large-scale reactions. In the current set of examples only six
turnovers/equivalents of 18OH2 (if 100% enriched) are
theoretically sufficient for making amino acids with 98%
isotopic enrichment, even without taking kinetic isotope
effects into account.
Scheme 2.
Nonequilibrium, Multiple-Turnover 18O Labeling Reaction
Org. Lett., Vol. 12, No. 1, 2010
105

Our protocol for the selective labeling of R-carboxyl
groups of amino acids utilizes commercially available,
protected amino acids and coupling reagents.16 To activate
the carbodiimide and suppress racemization of the O-
acylisourea intermediate, an excess of 3,5-dimethylpyri-
dinium bromide (pKa ≈ 5) is included as a dry proton
source.16 We conducted isotope labeling experiments with
three different Fmoc-protected amino acids containing a
variety of acid-labile protecting groups (Table 1). In a typical
reaction, 50 equiv of 18OH2 (95% 18O), 30 equiv of 1-ethyl-
3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC),
and 20 equiv of 3,5-dimethylpyridinium bromide in dry DMF
were reacted overnight at room temperature.17 While other
carbodiimides (DCC, DIC, etc.) worked well for this reaction,
the water solubility of EDC greatly simplified purification
as the resulting 16O- and 18O-containing ureas were washed
away with water. Isolated yields for 1 and 3 were nearly
quantitative (94-95%), while the yield for Fmoc-Trp(Boc)-
18OH was slightly lower (88%) due to the extreme acid
sensitivity of the Boc-protected indole (Table 1). Each
product 1-3 was characterized by MS, IR, NMR, and
polarimetry.18 Selective 18O labeling of the R-carboxylic
acids was observed by 13C NMR by using mixtures of
starting materials and isolated products. Consistent with
previous reports of other carboxylic acids,11,19 the 18O-labeled
R-carbon of products 1-3 were shifted upfield by ∼0.05
ppm as compared to 16O-containing starting materials (see
Figure S1 of the Supporting Information). Selective labeling
was also indicated by mass spectrometry as exactly two 18
O
atoms were incorporated into each product. Quantitative ESI
mass spectrometry of 1-3 revealed total isotopic enrichments
of 92-95% (Figure S2, Supporting Information). This
enrichment is very similar to the purity of the 18OH2 (95%
18O) added to each reaction. On the basis of these results,
we estimate that five or more cycles of activation and
hydrolysis occurred during each reaction. Similar results were
also obtained for relatively simple amino acids like Fmoc-
Gly-18OH. To the best of our knowledge, these are the first
reported examples of kinetically enhanced multiple turnover
reactions used for preparative isotopic labeling.
The CdO stretching frequency in IR spectroscopy is
strongly influenced by the relative mass of each atom.20
A
shift of approximately -20 cm-1 is observed for 18OdC
stretching frequencies as compared to 16OdC. It has been
shown that 2D-IR experiments conducted in peptides and
proteins are best resolved when using 13C/18O doubled labeled
amino acids.6,8-10 Given the vast selection of commercially
available 13C Fmoc-protected amino acids, our mild labeling
strategy can now provide a wide variety of selectively labeled
18O/17O amino acids in a single step.
(11) Ponnusamy, E.; Jones, C. C.; Fiat, D. J. Labelled Compd.
Radiopharm. 1987, 24, 773.
(12) Hollenstein, H.; Scha¨r, R. W.; Schwizgebel, N.; Grassi, G.;
Gu¨nthard, H. H. Spectrochim. Acta, Part A 1983, 39, 193.
(13) Murphy, R. C.; Clay, K. L. Biomed. Mass Spectrom. 1979, 6, 309.
Gervais, C.; Dupree, R.; Pike, K. J.; Bonhomme, C.; Profeta, M.; Pickard,
C. J.; Mauri, F. J. Phys. Chem. A 2005, 109, 6960. Wu, G.; Dong, S. J. Am.
Chem. Soc. 2001, 123, 9119.
Acknowledgment. This work was made possible by
generous support from the Swiss National Science Founda-
tion (grant no. 116868) and the University of Zu¨rich.
(14) Wrobel, N.; Schinkinger, M.; Mirsky, V. M. Anal. Biochem. 2002,
305, 135.
Supporting Information Available: Detailed synthetic
procedures and compound characterization. This material is
available free of charge via the Internet at http://pubs.acs.org.
(15) Cassano, A. G.; Anderson, V. E.; Harris, M. E. Biochemistry 2004,
43, 10547.
(16) Starting materials were extensively dried and mixed in DMF under
N2. EDC was added in three equal portions over 40 h. See the Supporting
Information for detailed procedures.
OL902519G
(17) Reactions containing 18 equiv of 18OH2 and 10 equiv of carbodi-
imide furnished products with only 55% enrichment.
(18) No changes in optical rotation were observed upon labeling,
indicating efficient suppression of racemization.
(19) Risley, J. M.; Van Etten, R. L. J. Am. Chem. Soc. 1980, 102, 4609
(20) Laulicht, I.; Pinchas, S.; Samuel, D.; Wasserman, I. J. Phys. Chem.
1966, 70, 2719.
.
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Org. Lett., Vol. 12, No. 1, 2010