Asymmetric synthesis and translational competence of L-α-(1- Cyclobutenyl)glycine

Article abstract of DOI:10.1021/ol049026b

(Chemical Equation Presented) L-α-(1-Cyclobutenyl)glycine (1-Cbg) was targeted as a potentially translatable analogue of isoleucine and valine and as a useful building block for peptides. An enantioselective synthesis was executed in which the key step was diastereoselective addition of 1-cyclobutenylmagnesium bromide to the sulfinimine 2b derived from (S)-t-butanesulfinimide and tert-butyl glyoxylate. 1-Cbg was found to substitute efficiently for isoleucine and valine, but not leucine, in the translation of green fluorescent protein in vitro.

Full text of DOI:10.1021/ol049026b

ORGANIC
LETTERS
2004
Vol. 6, No. 21
3659-3662
Asymmetric Synthesis and Translational
Competence of
L
-r-(1-Cyclobutenyl)glycine
Lasanthi P. Jayathilaka, Mahua Deb, and Robert F. Standaert*
Department of Chemistry (M/C 111), UniVersity of Illinois at Chicago,
845 West Taylor Street, Room 4500 Chicago, Illinois 60607
rfs@uic.edu
Received May 26, 2004
ABSTRACT
L
-r-(1-Cyclobutenyl)glycine (1-Cbg) was targeted as a potentially translatable analogue of isoleucine and valine and as a useful building block
for peptides. An enantioselective synthesis was executed in which the key step was diastereoselective addition of 1-cyclobutenylmagnesium
bromide to the sulfinimine 2b derived from (S)-t-butanesulfinimide and tert-butyl glyoxylate. 1-Cbg was found to substitute efficiently for
isoleucine and valine, but not leucine, in the translation of green fluorescent protein in vitro.
Unnatural amino acids are important tools for the preparation
of novel peptides, proteins, and derived materials such as
bioconjugates and polymers. Among the amino acids, an
interesting subset comprises translatable ones, that is,
analogues of the standard 20 amino acids that are accepted
by the protein biosynthetic machinery.1 Such analogues can
allow for the production of large quantities of analogue-
labeled polypeptides by microbial expression, and the
introduction of unique functional groups can allow for
selective posttranslational chemistry. For the full potential
of this approach to be realized, it will be necessary to expand
the pool of functionalized amino acids that are translationally
competent, an effort that entails both modification of the
biosynthetic machinery2 and synthesis of novel amino acids.
Cyclobutenes have recently attracted heightened interest
because of their high reactivity in olefin metathesis, making
them valuable as substrates for ring-opening metathesis
polymerization3 and as synthetic intermediates.4 However,
little effort has been directed toward their applications in
peptide and protein chemistry. The first cyclobutene-contain-
ing amino acid, (()-â-(1-cyclobutenyl)alanine, was prepared
in 19615 but has not appeared subsequently in the literature.
In addition, there is one citation to the isomeric â-(3-
cyclobutenyl)alanine, which was found to be a translationally
competent analogue of leucine (Leu).6
(1) Reviews: (a) Link, A. J.; Mock, M. L.; Tirrell, D. A. Curr. Opin.
Biotechnol. 2003, 14, 603-609. (b) Wang, L.; Schultz, P. G. Chem.
Commun. (Cambridge) 2002, 1-11. (c) van Hest, J. C. M.; Tirrell, D. A.
Chem. Commun. (Cambridge) 2001, 1897-1904. (d) Budisa, N.; Minks,
C.; Alefelder, S.; Wenger, W.; Dong, F.; Moroder, L.; Huber, R. FASEB J.
1999, 13, 41-51. (e) Kiick, K. L.; Tirrell, D. A. Mater. Sci. Technol. 1999,
20, 571-594. (f) Kothakota, S.; Dougherty, M. J.; Fournier, M. J.; Mason,
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This laboratory maintains an interest in translatable
analogues of isoleucine (Ile) such as the natural product
furanomycin.7,8 Recognizing the chemical value of cyclo-
butenes and that the cyclobutenyl group is a reasonable
isostere for the sec-butyl side chain of Ile or the isopropyl
side chain of Val, we targeted R-(1-cyclobutenyl)glycine (1-
Cbg, 1) as an analogue of potentially high value. Especially
in light of the translational competence of (3-cyclobutenyl)-
alanine as a substitute for Leu, it seemed plausible that 1-Cbg
10.1021/ol049026b CCC: $27.50
© 2004 American Chemical Society
Published on Web 09/11/2004

would likewise substitute for Ile and perhaps Val. Even if
this proved not to be the case, we expected that 1-Cbg would
be of high value in chemically synthesized peptides.
An asymmetric synthesis of 1-Cbg was designed around
a stereoselective addition of the cyclobutenyl component to
a chiral sulfinimine.9 Davis10 has demonstrated high dia-
stereoselectivity in the addition of Grignard reagents to
sulfinimine 2a derived from condensation of t-butane-
sulfinamide11 with ethyl glyoxylate. In accord with his
findings, we found that 1-cyclobutenylmagnesium bromide
(prepared by lithiation of 1-bromocyclobutene12 and trans-
metalation with MgBr2) likewise added smoothly to provide
a 9:1 mixture of diastereomers. Unfortunately, attempted
saponification of the ethyl ester led to extensive epimerization
and decomposition.
To circumvent the need for strong base in deprotection,
we sought an acid-labile ester and accordingly prepared the
corresponding sulfinimine 2b from tert-butyl glyoxylate,
easily obtained through ozonolysis of di-tert-butyl fumarate.13
As with the ethyl glyoxylate-derived sulfinimine 2a, addition
to 2b proceeded smoothly to afford a 9:1 mixture of
diastereomers, from which the major isomer 3b could be
crystallized in 40-50% yield and >97% de from hexane.
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D. S.; Wang, L.; Schultz, P. G. Proc. Natl. Acad. Sci. U.S.A. 2002, 99,
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I.; Kirshenbaum, K.; Tirrell, D. A. J. Am. Chem. Soc. 2003, 125, 7512-
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A. B.; King, D. S.; Horn, D. M.; Schultz, P. G. J. Am. Chem. Soc. 2003,
125, 935-939. (s) Wang, L.; Zhang, Z.; Brock, A.; Schultz, P. G. Proc.
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Scheme 1. Synthesis of l-(1-Cyclobutenyl)glycine
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To obtain the parent amino amino acid for use in ribosomal
protein translation (and potentially other applications), both
N- and C-protecting groups had to be removed. The simplest
path appeared to be simultaneous deprotection of both the
tert-butylsulfinamide and the tert-butyl ester. In practice, the
tert-butyl ester proved to be much more resistant to acid,
and best results were obtained via two-stage deprotection;
thus, treatment of 3b with a 1:1 mixture of methanol and 4
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V. Angew. Chem., Int. Ed. 2000, 39, 910-912.
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acids, see: (a) Ellman, J. A. Pure Appl. Chem. 2003, 75, 39-46. (b) Ellman,
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Org. Lett., Vol. 6, No. 21, 2004

M HCl in dioxane14 removed the tert-butylsulfinyl group
exclusively, providing tert-butyl ester 4b in 99% yield.
The enantiomeric purity and absolute configuration of the
amino ester were confirmed by converting it to both (R)-
and (S)-MTPA (Mosher) amides.15 The diastereomeric
amides had well-resolved resonances for the vinylic hydrogen
To test the translational competence of 1-Cbg, we em-
ployed a novel approach in which the expression of green
fluorescent protein (GFP) in vitro was monitored. In vitro
translation has been used previously to assess the translational
competence of amino acid analogues and was found to
provide a useful indicator of the analogues’ abilities to
substitute in vivo.19 The in vitro approach offers several
advantages over in vivo expression. One is that it allows for
determination of an analogue’s ability to substitute for any
amino acid of interest, as the amino acids are added at known
concentrations, and it is a simple matter to substitute the
analogue for one of the standard amino acids or to perform
direct competition experiments. In addition, the method is
operationally simple, obviates the need for expensive micro-
chemical analysis of expressed proteins, and avoids a variety
of complications associated with living cells (discussed
further below).
One drawback to the in vitro approach as originally
implemented was the need for electrophoretic separation,
blotting, and radioisotopic detection of the expressed protein.
These steps were circumvented in the present work by using
GFP as the reporter for translation. GFP is a small (27 kDa)
protein in which a fluorescent chromophore forms spontane-
ously and without assistance by enzymes or other catalysts
(Scheme 2).20,21 The natural fluorescence of the protein
1
and methoxy protons in the H NMR spectrum and for the
trifluoromethyl group in the 19F NMR. In both derivatives,
the diastereomeric excess was judged to be >95% from the
19F NMR, indicating an ee of >95% in the parent amino
ester. Further, the chemical shifts were in excellent accord
with the findings of Kusumi et al. for L-amino acid methyl
esters, specifically the strong upfield shift of the OCH3
protons and slight upfield shift of HR in the (R)-MTPA-L-
16
Val-OMe diastereomer.
Subsequent removal of the tert-butyl ester was effected
with 50% TFA in dichloromethane to afford 1-Cbg (1) as
its trifluoroacetic acid salt in 72% yield. The stereochemical
integrity of the amino acid was confirmed by converting the
amino acid into both diastereomeric Mosher amides; NMR
confirmed no loss of stereochemical purity in the amino acid.
Use of the tert-butyl glyoxylate-derived sulfinimine 2b
should therefore also prove to be useful in the synthesis of
other highly racemization-prone amino acids.
Having the free amino acid in hand, we performed
preliminary tests of 1-Cbg’s usefulness in its intended
applications. First among these was its ability to support
ribosomal protein translation. As mentioned previously,
1-Cbg is a reasonable isostere for either of the â-branched
amino acids Val and Ile, but not the γ-branched Leu (isobutyl
side chain). Thus, we tested for the ability of 1-Cbg to
substitute for these amino acids in protein translation, with
the expectation that substutition might be observed for Val
or Ile but not Leu.
Scheme 2. Spontaneous Formation of the GFP Chromophore
Translatability demands two properties of the amino acid.
First, it must be a substrate for an aminoacyl tRNA synthetase
(AARS), the enzyme that couples an amino acid with its
corresponding tRNA. Second, the aminoacylated tRNA must
be accepted by the ribosome. Of these two, the former is
more important, as AARSs are highly selective among the
standard amino acids,17 whereas the ability of the ribosome
to accept a vast array of unnatural amino acids has been
amply demonstrated.18
allows its relative concentration to be monitored quantita-
tively over time; in addition, it has been demonstrated that
chromophore formation occurs cotranslationally, so that the
rate of development of fluorescence is a useful proxy for
the rate of protein synthesis and folding.22
Using in vitro-translated GFP as the reporter, 1-Cbg was
tested as a substitute for Ile, Val, and Leu. To our gratifica-
tion, the rate and maximal accumulation of fluorescence was
essentially the same using either 1-Cbg or Ile in the
translation mixture; when 1-Cbg was substituted for Val,
approximately half as much expression was obtained, and
when it was substituted for Leu, no expression above
background was obtained.23
(14) Cogan, D. A.; Ellman, J. A. J. Am. Chem. Soc. 1999, 121, 268-
269.
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2001, 48, 323-335. (b) Carter, C. W., Jr. Annu. ReV. Biochem. 1993, 62,
715-748. (c) Soll, D. Experientia 1990, 46, 1089-1096. (d) Schimmel, P.
R.; So¨ll, D. Annu. ReV. Biochem. 1979, 48, 601-648.
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L. E.; Chamberlin, A. R. Methods Mol. Biol. (Clifton, NJ) 1998, 77, 325-
354. (c) Hohsaka, T.; Sisido, M. Curr. Opin. Chem. Biol. 2002, 6, 809-
815. (d) Gilmore, M. A.; Steward, L. E.; Chamberlin, A. R. Top. Curr.
Chem. 1999, 202, 77-99. (e) Sisido, M.; Hohsaka, T. Bull. Chem. Soc.
Jpn. 1999, 72, 1409-1425. (f) Thorson, J. S.; Cornish, V. W.; Barrett, J.
E.; Cload, S. T.; Yano, T.; Schultz, P. G. Methods Mol. Biol. (Clifton, NJ)
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The origin of the reduced fluorescence observed with Cbg
substituted for Val vs Ile is uncertain. One possibility we
(19) Kothakota, S.; Yoshikawa, E.; Murphy, O. J.; Mason, T. L.; Tirrell,
D. A.; Fournier, M. J. J. Polym. Sci., Part A: Polym. Chem. 1995, 33,
1267-1274.
(20) Cody, C. W.; Prasher, D. C.; Westler, W. M.; Prendergast, F. G.;
Ward, W. W. Biochemistry 1993, 32, 1212-1218.
(21) Chalfie, M.; Tu, Y.; Euskirchen, G.; Ward, W. W.; Prasher, D. C.
Science (Washington, DC) 1994, 263, 802-805.
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Nat. Biotechnol. 1999, 17, 691-695.
Org. Lett., Vol. 6, No. 21, 2004
3661

folding, or chromophore formation. Moreover, the three
proteins (wild-type, Ilef1-Cbg, and Valf1-Cbg) have
identical fluorescence emission and excitation spectra, with
an excitation maximum at 470 nm and an emission maximum
at 504 nm (not shown). Our current hypothesis is that the
substitution of 1-Cbg for Val leads to a lower quantum yield
for fluorescence. That 1-Cbg was able to subsitute in vitro
for Ile and Val shows that it is a substrate for the
corresponding Ile and Val aminoacyl tRNA synthetases and
that the aminoacylated tRNAs translate efficiently. However,
for the potential of 1-Cbg in protein engineering to be
realized, it must also substitute in vivo, which imposes
several additional requirements. First, it must enter cells;
second, it must have reasonable metabolic stability; third, it
must compete with a background of endogeneous amino acid
(even in auxotrophic strains); finally, it must not be toxic.
By using in vitro translation, the intrinsic translational
competence of the amino acid can be assessed without
interference.
Figure 1. In vitro translation of GFP. Translations were performed
using E. coli T7 S30 Extract System (Promega), a plasmid encoding
an enhanced-fluorescence mutant of GFP (pQBI-T7-GFP, QBio-
gene), and amino acids (aa) at 0.1 mM concentration. Reactions
were performed at 37 °C according to Promega’s protocol and, with
exceptions as noted, included 1 µg of plasmid and all standard aa.
Fluorescence measurements were made with an excitation filter
selecting for 485 ( 10 nm and an emission filter selecting for 528
( 10 nm. Data shown are averages of triplicate runs. Black lines:
×, -DNA/-aa; open circles, -aa; filled circles, no deletions. Red
lines: +,-Ile/+1-Cbg; no symbol, -Ile. Blue lines: +,-Val/+1-
Cbg; no symbol, -Val. Purple lines: +,-Leu/+1-Cbg; no symbol,
-Leu.
In summary, we have completed an efficient, enantio-
selective synthesis of the title compound using the tert-
butylsulfinamide chiral auxiliary and have shown that it
substitutes effectively for Val and Ile in the translation of
GFP in vitro. Efforts to introduce 1-Cbg into microbially
expressed proteins are currently underway in this laboratory,
as are efforts to explore its reactivity in synthetic peptides.
Acknowledgment. We are grateful to Dr. Jing Feng and
Prof. Gu¨nther Szeimies for their experimental procedure for
the preparation of 1-bromocyclobutene. We thank the
University of Illinois at Chicago and the National Institutes
of Health (GM 57543) for generous support of this work.
considered was that expression of the protein or formation
of the chromophore was slower for the Val-labeled protein;
however, the rate of fluorescence increase is essentially
identical in both cases. The overall reaction progress displays
pseudo-first-order kinetics, and the rate constants (k ( sk)
calculated for the 10-120 min window are 0.029 ( 0.001
m-1 for the Ile substitution and 0.031 ( 0.001 for the Val
substitution at 37 °C.24 Therefore, the reduced fluorescence
is not the result of a difference in the rate of biosynthesis,
Supporting Information Available: Experimental pro-
cedures for the synthesis of 1-Cbg (1); 1H NMR spectra for
1-Cbg (1) and intermediates 2b, 3b, and 4b; 1H and 19F NMR
spectra for the (R)- and (S)-MTPA derivatives of 1 and 4b;
and experimental procedures for in vitro transcription-
translation. This material is available free of charge via the
Internet at http://pubs.acs.org.
OL049026B
(23) Commercial S30 extracts contain varying amounts of residual amino
acids that lead to background expression even in the absence of added amino
acids. The experiments reported herein were performed with a batch of
S30 extract from Promega (Madison, WI) selected for low background
expression. Some batches were found to be unsuitable.
(24) Rate constants were calculated with the KORE program: Swain,
C. G.; Swain, M. S.; Berg, L. F. J. Chem. Inf. Comput. Sci. 1980, 20, 47-
51.
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