An asymmetric synthesis of l -pyrrolysine

Article abstract of DOI:10.1021/ol300045c

An efficient asymmetric synthesis of the 22nd amino acid l-pyrrolysine has been accomplished. The key stereogenic centers were installed by an asymmetric conjugate addition reaction. A Staudinger/aza-Wittig cyclization was used to form the acid-sensitive

Full text of DOI:10.1021/ol300045c

ORGANIC
LETTERS
2012
Vol. 14, No. 6
1378–1381
An Asymmetric Synthesis of L-Pyrrolysine
Margaret L. Wong,† Ilia A. Guzei,† and Laura L. Kiessling*,†,‡
Departments of Chemistry and Biochemistry, University of Wisconsin;Madison,
Madison, Wisconsin 53706, United States
kiessling@chem.wisc.edu
Received January 10, 2012
ABSTRACT
An efficient asymmetric synthesis of the 22nd amino acid L-pyrrolysine has been accomplished. The key stereogenic centers were installed by an
asymmetric conjugate addition reaction. A Staudinger/aza-Wittig cyclization was used to form the acid-sensitive pyrroline ring. Pyrrolysine was
synthesized in 13 steps in 20% overall yield.
Pyrrolysine is the 22nd genetically encoded amino acid.1
It consists of a (4R,5R)-4-methyl-5-carboxypyrroline ring
linked to the ε-nitrogen of L-lysine.2,3 Pyrrolysine was
identified by X-ray crystallography, when it was first
observed in the structure of Methanosarcina barkeri mono-
methylamine methyltransferase.1 It has been hypothesized
that this unique amino acid plays an important role in
methane production in some archaeal species.4
Pyrrolysine is encoded by an in-frame UAG codon,
which is nonterminating, and its incorporation is mediated
by a dedicated tRNA and cognate tRNA synthetase.5 The
mechanism of pyrrolysine incorporation into proteins
offers a platform for developing new protein labeling
methods.6 Small molecules, such as fluorophores,6a biotin,5e
ubiquitin,6b and oligosaccharides,5e have been used for
labeling pyrrolysine and pyrrolysine-surrogate residues.7
Although pyrrolysine has been exploited for protein label-
ing, an understanding of its biosynthesis,5e,8 evolutionary
purpose, and distribution in the proteome is incomplete.
New synthetic methods that provide sufficient quantities of
this novel amino acid would facilitate an understanding of
pyrrolysine biochemistry.
Pyrrolysine is a synthetic target.2,9 One reported chemi-
cal synthesis2 involves the coupling of (4R,5R)-4-methyl-
pyrroline-5-carboxylic acid to lysine. While this innovative
route affords the desired product, the yield is modest (9%
overall). Moreover, we and others5a,9 have found that the
reactions were irreproducible. We therefore sought to
devise an alternative synthetic route.
† Department of Chemistry.
‡ Department of Biochemistry.
(1) (a) Hao, B.; Gong, W.; Ferguson, T. K.; James, C. M.; Krzycki,
J. A.; Chan, M. K. Science 2002, 296, 1462. (c) Srinivasan, G.; James,
C. M.; Krzycki, J. A. Science 2002, 296, 1459.
(2) Hao, B.; Zhao, G.; Kang, P. T.; Soares, J. A.; Ferguson, T. K.;
Gallucci, J.; Krzycki, J. A.; Chan, M. K. Chem. Biol. 2004, 11, 1317.
(3) Soares, J. A.; Zhang, L.; Pitsch, R. L.; Kleinholz, N. M.; Jones,
R. B.; Wolff, J. J.; Amster, J.; Green-Church, K. B.; Krzycki, J. A.
J. Biol. Chem. 2005, 280, 36962.
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Int. Ed. 2009, 48, 1633. (b) Li, X.; Fekner, T.; Ottesen, J. J.; Chan, M. K.
Angew. Chem., Int. Ed. 2009, 48, 9184. (c) Nguyen, D. P.; Lusic, H.;
Neumann, H.; Kapadnis, P. B.; Deiters, A.; Chin, J. W. J. Am. Chem.
(4) Hand, C. E.; Honek, J. F. J. Nat. Prod. 2005, 68, 293. Rother, M.;
Krzycki, J. A. Archaea 2010, 2010, 453642.
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Soc. 2009, 131, 8720. (d) Ou, W.; Uno, T.; Chiu, H.-P.; Grunewald, J.;
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Chang, E.; Zhao, G.; Kang, P. T.; Green-Church, K. B.; Chan, M. K.;
Krzycki, J. A. Nature 2004, 431, 333. (b) Polycarpo, C.; Ambrogelly, A.;
Berube, A.; Winbush, S. M.; McCloskey, J. A.; Crain, P. F.; Wood, J. L.;
Soll, D. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 12450. (c) Kobayashi,
T.; Yanagisawa, T.; Sakamoto, K.; Yokoyama, S. J. Mol. Biol. 2009,
385, 1352. (d) Namy, O.; Zhou, Y.; Gundllapalli, S.; Polycarpo, C. R.;
Denise, A.; Rousset, J. P.; Soll, D.; Ambrogelly, A. FEBS Lett. 2007,
581, 5282. (e) Longstaff, D. G.; Larue, R. C.; Faust, J. E.; Mahapatra,
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(9) Berube, A. Progress Toward the Total Syntheses of the Polycyclic
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r
10.1021/ol300045c
Published on Web 03/06/2012
2012 American Chemical Society

could be installed by conjugate addition to a compound
such as 4. The Feringa group has developed efficient
catalysts for this type of transformation.11 Accordingly,
the Josiphos catalyst gave rise to the 1,4-addition of methyl
Grignard to R,β-unsaturated thioester 4 (Scheme 2). At-
tempts to functionalize the R-position with an electrophilic
azide source or by an asymmetric R-halogenation12 and
subsequent nucleophilic displacement with an azide13 did
not afford the desired products. A more significant draw-
back of this approach was our observation that the most
robust R-halogenation methods14 were incompatible with
the thioester required for the Josiphos-catalyzed reaction.
As a result, the conjugate addition and the selective
halogenation reactions demanded different carbonyl deriv-
atives (thioester versus aldehyde), thereby necessitating
multiple changes in oxidation state.
Scheme 1. Retrosynthesis of Pyrrolysine
A drawback of the reported pyrrolysine synthesis is that
it entails exposure of the sensitive carboxypyrroline ring to
strongly acidic conditions, during both imine formation
and lysine coupling. We focused on generating the pyr-
roline ring in the penultimate step of the synthesis. In this
way, we hoped to avoid reactions that could lead to
pyrroline ring degradation. Specifically, we envisioned
forming the cyclic imine from azido aldehyde 2 via a
tandem Staudinger/aza-Wittig reaction10 (Scheme 1).
Azido aldehyde 2 could be obtained from coupling of 3
with a selectively protected lysine derivative. We consid-
ered two different approaches to compound 3: an asym-
metric conjugate addition of a methylcarbanion followed
by R-substitution (“Route I”) or a Michael addition with a
glycine enolate (“Route II”).
Scheme 3. Pyrroline Precursors via Michael Addition
Scheme 2. Attempts To Synthesize Pyrrolysine Precursors via
Asymmetric Methylcarbanion 1,4-Addition and R-Substitution
The roadblocks encountered pursuing Route I
prompted us to focus on Route II. In this approach, we
plannedtoinstall the pyrrolinestereochemistry by carrying
out Michael addition of a glycine enolate in the presence of
a chiral catalyst. Calcium ion promotedconjugateaddition
of N-(tert-butylphenylmethylidene)glycine tert-butyl ester
to methyl crotonate produced 915 in 92% yield with 99%
enantiomeric excess and greater than 99:1 diastereomeric
excess (Scheme 3). This reaction was not only stereo-
selective and efficient but also robust. These favorable
ꢀ
ꢀ
ꢀ~
(11) Ruiz, B. M.; Geurts, K.; Fernandez-Ibanez, M. A.; ter Horst, B.;
Minnaard, A. J.; Feringa, B. L. Org. Lett. 2007, 9, 5123.
(12) (a) Wack, H.; Taggi, A. E.; Hafez, A. M.; Drury, W. J.; Lectka, T.
J. Am. Chem. Soc. 2001, 123, 1531. (b) Brochu, M. P.; Brown, S. P.;
MacMillan, D. W. C. J. Am. Chem. Soc. 2004, 126, 4108. (c) Halland, N.;
Braunton, A.; Bachmann, S.; Marigo, M.; Jorgensen, K. A. J. Am. Chem.
Soc. 2004, 126, 4790.
We pursued these strategies in parallel. For Route I, we
anticipated the methyl group of pyrrolysine precursor 3
(13) Papa, A. J. J. Org. Chem. 1966, 31, 1426.
(14) Brochu, M. P.; Brown, S. P.; MacMillan, D. W. C. J. Am. Chem.
Soc. 2004, 126, 4108.
(10) (a) Mulzer, J.; Meier, A.; Buschmann, J.; Luger, P. Synthesis
1996, 123. (b) Molina, P.; Vilaplana, M. J. Synthesis 1994, 1994, 1197.
(15) Kobayashi, S.; Tsubogo, T.; Saito, S.; Yamashita, Y. Org. Lett.
2008, 10, 807.
Org. Lett., Vol. 14, No. 6, 2012
1379

attributes were preserved even when the reactions were
conducted on a medium scale (50ꢀ100 mmol).
in a single step. The azide group was installed using a Zn2þ
-
mediated diazotransfer from triflyl azide,17 which afforded
coupling partner 13 in 75% yield over two steps.18 The
precursor to the pyrroline ring, R-azido carboxylic acid 13,
was appended to the epsilon nitrogen of N-trifluoroaceta-
midyl lysine methyl ester.2 The silyl protecting group was
cleaved, and the resulting primary alcohol was converted
to an aldehyde by the Swern oxidation.19 This sequence
provided compound 14 as the substrate for the Staudinger/
aza-Wittig reaction.
While the Schiff base was necessary for the high stereo-
selectivity attained,16 the imine was prone to hydrolysis
upon exposure to acid or base. Accordingly, the tert-butyl-
phenyl imine 9 was hydrolyzed, and the unmasked amine
was immediately exposed to di-tert-butyl dicarbonate. We
anticipated the tert-butyl carbamate could be removed
later concomitantly with tert-butyl ester hydrolysis. The
methyl ester was then saponified to afford 10. The three-
step interconversion of protecting groups was executed in
an overall yield of 86%. The series of transformations
proceeded with no loss of stereochemical integrity, as
revealed by X-ray crystallography (Figure 1). The struc-
ture of acid 10 confirmed it had the expected absolute
stereochemistry.
Scheme 4. Generating the Staudinger/aza-Wittig Substrate
At this point in the synthesis, we recognized that differ-
entiation of the two carboxy termini would be crucial for
accessing lysine-coupled azido aldehyde 2 (Scheme 1). To
this end, the carboxylic acid was transformed to a mixed
anhydride, which was reducedwithsodium borohydride to
affordhydroxy tert-butyrate 11 in 73% yield. The resulting
primary hydroxyl group was converted to a silyl ether to
mitigate side reactions with a free hydroxyl group. The silyl
ether 12 was generated under mildly basic conditions as
these avoided lactone formation. To minimize the forma-
tion of a disiloxane byproduct that coeluted with the product
during chromatography, the reaction was quenched under
nonaqueous conditions. Using these precautions, compound
12 could be readily obtained.
Pyrroline 15 was generated using an efficient sequence
that minimized purification steps (Scheme 5). The reduc-
tion of the azide moiety was effected using polystyrene-
supported triphenylphosphine. The putative iminophos-
phorane underwent cyclization to afford protected pyr-
rolysine derivative 15. Solution-phase triarylphosphine
reagents have been used previously to execute tandem
Staudinger reduction and intramolecular aza-Wittig
reactions.20 The popularity of this strategy is gaining,
presumably because of the efficiency and simplicity it
affords for generating even highly sensitive imines such
as 15. The use of an immobilized phosphine to effect the
transformation offers an additional advantage. It circum-
vents the need for chromatography, which can be a major
benefit in preparing sensitive imines. To generate the final
pyrrolysine product, we removed the protecting groups at
the N- and C-termini2 of pyrroline 15 to afford the lithium
salt of L-pyrrolysine 16 in 20% overall yield.
The sequence described is high yielding and stereoselec-
tive. It affords L-pyrrolysine in 13 steps and 20% overall
yield. Through our examination of multiple strategies for
Figure 1. The stereochemistry generated in the production of
imine 9 was preserved through multiple transformations
(Scheme 3) as revealed by X-ray structure crystallographic
analysis of acid 10.
(17) (a) Alper, P. B.; Hung, S.-C.; Wong, C.-H. Tetrahedron Lett.
1996, 37, 6029. (b) Nyffeler, P. T.; Liang, C.-H.; Koeller, K. M.; Wong,
C.-H. J. Am. Chem. Soc. 2002, 124, 10773.
(18) As observed previously in literature in ref 17b, the metal-
mediated diazotransfer reaction is prone to irreproducibility in yields.
(19) (a) Omura, K.; Swern, D. Tetrahedron 1978, 34, 1651. (b)
Mancuso, A. J.; Huang, S.-L.; Swern, D. J. Org. Chem. 1978, 43, 2480.
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2, 3777. (b) Grieder, A.; Thomas, A. W. Synthesis 2003, 2003, 1707. (c)
Each functional group in ester 12 (Scheme 4) is poised
for manipulation to access compound 14, the precursor for
the tandem sequence. As anticipated, the tert-butyl ester
and carbamate protecting groups could both be removed
€
Gil, C.; Brase, S. Chem.;Eur. J. 2005, 11, 2680. (d) Baxendale, I. R.;
Deeley, J.; Griffiths-Jones, C. M.; Ley, S. V.; Saaby, S.; Tranmer, G. K.
Chem. Commun. 2006, 2566. (e) Mahdavi, H.; Amani, J. Tetrahedron
Lett. 2009, 50, 5923. (f) Smith, C. J.; Smith, C. D.; Nikbin, N.; Ley, S. V.;
Baxendale, I. R. Org. Biomol. Chem. 2011, 9, 1927.
(16) Tsubogo, T.; Saito, S.; Seki, K.; Yamashita, Y.; Kobayashi, S.
J. Am. Chem. Soc. 2008, 130, 13321.
1380
Org. Lett., Vol. 14, No. 6, 2012

effect the Staudinger/aza-Wittig cyclization. We anticipate
that this mild transformation can be used to generate
pyrrolysine analogs. Our route will also be valuable for
synthesizing isotopically labeled pyrrolysine to explore the
prevalence of this intriguing amino acid in microorganisms
and to carry out proteomic analyses.
Scheme 5. Tandem Staudinger/aza-Wittig and Protecting
Group Removal
Acknowledgment. We acknowledge Dr. Jiyoung Chang
of the Kiessling group for helpful discussions and for her
preliminary synthetic studies. This research was supported
by NIGMS (R01 GM055984). The NMR facilities at UW-
Madison are funded by the NSF (CHE-9208463) and NIH
(RR08389-01).
Supporting Information Available. Experimental pro-
cedures and full characterization of compounds 9ꢀ15 and
associated intermediate compounds and X-ray crystal-
lographic data (CIF). This material is available free of
charge via the Internet at http://pubs.acs.org.
the installation of the two stereogenic centers of pyr-
rolysine, we identified the Ca2þ-mediated conjugate addi-
tion reaction as an efficient process for generating pyrro-
line precursors. Another feature of our route is the gentle
conditions offered by a polymer-supported phosphine to
The authors declare no competing financial interest.
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