Facile chemoenzymatic strategies for the synthesis and utilization of S-adenosyl-L-methionine analogues

Article abstract of DOI:10.1002/anie.201308272

A chemoenzymatic platform for the synthesis of S-adenosyl-L-methionine (SAM) analogues compatible with downstream SAM-utilizing enzymes is reported. Forty-four non-native S/Se-alkylated Met analogues were synthesized and applied to probing the substrate specificity of five diverse methionine adenosyltransferases (MATs). Human MAT II was among the most permissive of the MATs analyzed and enabled the chemoenzymatic synthesis of 29 non-native SAM analogues. As a proof of concept for the feasibility of natural product alkylrandomization , a small set of differentially-alkylated indolocarbazole analogues was generated by using a coupled hMAT2-RebM system (RebM is the sugar C4′-O-methyltransferase that is involved in rebeccamycin biosynthesis). The ability to couple SAM synthesis and utilization in a single vessel circumvents issues associated with the rapid decomposition of SAM analogues and thereby opens the door for the further interrogation of a wide range of SAM utilizing enzymes. Mix and MATch: Methionine adenosyltransferase (MAT) was used to synthesize S-adenosylmethionine (SAM) analogues in a method directly compatible with downstream SAM-utilizing enzymes. As a proof of concept for the feasibility of natural product alkylrandomization by using this method, a coupled strategy in which MAT was applied in conjunction with the methyltransferase RebM was used to generate a small set of indolocarbazole analogues.

Full text of DOI:10.1002/anie.201308272

Angewandte
Chemie
DOI: 10.1002/anie.201308272
Biocatalysis
Facile Chemoenzymatic Strategies for the Synthesis and Utilization of
S-Adenosyl-l-Methionine Analogues**
Shanteri Singh,* Jianjun Zhang, Tyler D. Huber, Manjula Sunkara, Katherine Hurley,
Randal D. Goff, Guojun Wang, Wen Zhang, Chunming Liu, Jꢀrgen Rohr, Steven G. Van Lanen,
Andrew J. Morris, and Jon S. Thorson*
Abstract: A chemoenzymatic platform for the synthesis of S-
adenosyl-l-methionine (SAM) analogues compatible with
downstream SAM-utilizing enzymes is reported. Forty-four
non-native S/Se-alkylated Met analogues were synthesized and
applied to probing the substrate specificity of five diverse
methionine adenosyltransferases (MATs). Human MAT II
was among the most permissive of the MATs analyzed and
enabled the chemoenzymatic synthesis of 29 non-native SAM
analogues. As a proof of concept for the feasibility of natural
product “alkylrandomization”, a small set of differentially-
alkylated indolocarbazole analogues was generated by using
a coupled hMAT2–RebM system (RebM is the sugar C4’-O-
methyltransferase that is involved in rebeccamycin biosynthe-
sis). The ability to couple SAM synthesis and utilization in
a single vessel circumvents issues associated with the rapid
decomposition of SAM analogues and thereby opens the door
for the further interrogation of a wide range of SAM utilizing
enzymes.
natural product differential alkylation (alkylrandomization)
would thus be anticipated to dramatically expand the
potential scope of NP chemical diversity.
The typical alkyl donor for MT-catalyzed alkylation is S-
adenosyl-l-methionine (SAM) and importantly, we and
others have demonstrated that NP,[5] protein,[6] and nucleic
acid[7] MTs transfer alternative alkyl groups in the presence of
suitably modified SAM analogues. However, reminiscent of
the restriction imposed upon glycorandomization by the
availability of sugar nucleotides, access to arrays of stable
SAM analogues similarly restricts the development of NP
alkylrandomization. Current state-of-the-art for the chemical
synthesis of SAM analogues relies upon multistep syntheses
of diastereomeric mixtures of SAM analogues and requires
HPLC purification to remove the starting material, S-
adenosyl-homocysteine (SAH, a strong inhibitor of MTs).[8]
The purified SAM analogues from this process are also
markedly unstable, therby limiting their practical develop-
ment as stand-alone synthetic reagents and/or biological
probes. Within this context, a general platform to enable the
generation and direct utilization of SAM analogues, starting
from stable precursors, would be advantageous.
To address this need, we herein describe the broad
substrate specificity assessment of a representative set of
five distinct methionine adenosyltransferases (MATs) from
various sources. MAT (EC 2.5.1.6; also sometimes referred to
as AdoMet synthetase or SAM synthetase) is the primary
catalyst for the biosynthesis of SAM from adenine triphos-
phate (ATP) and l-methionine (Met; Scheme 1a) and there is
preliminary evidence to suggest that certain MATs are
capable of utilizing Met analogues.[6e,9] Consistent with this
notion, the studies highlighted herein reveal that human
MAT II (hMAT2) enables the cumulative synthesis of a broad
E
nzyme-catalyzed late-stage group-transfer-based tailoring
reactions contribute to the structural and functional diversity
of many complex natural products (NPs).[1] Representative
examples include amination, acylation, alkylation, glycosyla-
tion, halogenation, phosphorylation, and sulfation. In some
cases, the enzymes responsible for such transformations
display notable flexibility with respect to their substrate
scope.[2] Such promiscuity is an enabling feature of chemo-
enzymatic NP diversification platforms as exemplified by NP
glycorandomization (a platform for the differential glycosyl-
ation of natural products/drugs).[3] Enzyme-catalyzed alkyl-
ation is a highly prevalent modification that leads to the N, O,
S, and/or C alkylation of NPs (Figure 1).[4] A platform to co-
opt natural product methyltransferases (MTs) for broad
[*] Dr. S. Singh, Dr. J. Zhang, T. D. Huber, Prof. J. S. Thorson
Center for Pharmaceutical Research and Innovation
College of Pharmacy, University of Kentucky
Lexington, KY 40536 (USA)
M. Sunkara, Prof. A. J. Morris
Division of Cardiovascular Medicine, Gill Heart Institute
University of Kentucky, Lexington, KY 40536 (USA)
K. Hurley
E-mail: ssingh555@uky.edu
Department of Biochemistry, University of Wisconsin-Madison
Madison, WI 53705 (USA)
jsthorson@uky.edu
Dr. S. Singh, Dr. J. Zhang, T. D. Huber, Dr. G. Wang, Prof. J. Rohr,
Prof. S. G. Van Lanen, Prof. J. S. Thorson
Department of Pharmaceutical Sciences, College of Pharmacy,
University of Kentucky
Dr. R. D. Goff
Western Wyoming Community College
2500 College Dr., Rock Springs, WY 82902-0428 (USA)
[**] This work was supported by NIH RO1 CA84374 (JST), U01
GM098248and the National Center for Advancing Translational
Sciences (UL1TR000117).
789 South Limestone Street, Lexington, KY 40536 (USA)
W. Zhang, Prof. C. Liu
Molecular and Cellular Biochemistry, University of Kentucky
College of Medicine, University of Kentucky
Lexington, KY 40536 (USA)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201308272.
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strate the feasibility of NP alkylrandomiza-
tion, this study also highlights the subse-
quent generation of a small set of indolo-
carbazole analogues by using a coupled
hMAT2–RebM system, where RebM is the
sugar C4’-O-MT that is involved in rebec-
camycin biosynthesis.[5a,10] This study both
offers a strategic advance in terms of NP
diversification and reveals that hMAT2
displays surprising substrate permissivity.
Given the conflicting reports regarding
the relative stability of S- versus Se-
SAM[6c,d,11] and the fact that Se-SAM is
considered a better alkyl donor than S-
SAM in MT-catalyzed reactions owing to
À
the inherently longer and weaker Se C
bond,[11b] both S- and Se-Met analogues
were synthesized for this study. The general
strategy for the synthesis of the S- and Se-
Met analogue panel employed direct or
reductive alkylation of l-homocystine, the
oxidized dimer of l-homocysteine (l-Hcy)
[1 or 1-Se, Scheme 1b, (i) and (iii), respec-
tively], l-Hcy [Scheme 1b, (ii)], or 2-Se
[Scheme 1b, (iv)] following simple modifi-
cations of previously reported strategies.[12]
The desired S- and Se-Met analogues were
purified from the crude reaction mixtures
by reverse-phase high-pressure liquid chro-
matography (RP-HPLC), C18 RP flash
Figure 1. Representative methylated natural products in which methyl groups deriving from
SAM through MT-catalyzed methylation are highlighted as spheres. AT3=AT2433; CLC=col-
chicine; ESP=esperamicin; EVM=evernimicin; GLM=geldanamycin; MGM=megalomi-
cin; NGM=nogalamycin.
panel of unnatural SAM analogues (29 analogues detected)
starting from synthetic S/Se-alkylated Met analogues (44
analogues) or commercial sources (3 analogues). To demon-
chromatography, or Dowex 50W8X-200 resin to afford
yields of isolated product ranging from 20–90% (Table S1 in
the Supporting Information).
A representative set of MATs from various sources,
including bacterial, archaeal, and mammalian orthologues,
were selected for the initial assessment. The exploratory set
included the human MAT II catalytic alpha and regulatory
beta subunits (hMAT2),[13] the human MAT II catalytic alpha
subunit alone (hMAT2A),[13] the human MAT I catalytic
alpha subunit (hMAT1A),[13] Escherichia coli MAT
(eMAT),[14] and the thermophilic Methanocaldococcus janna-
schii MAT (mMAT).[15] The basis for the inclusion of the
human homologues was an interest in interrogating the
substrate specificities of disease-relevant hMATs,[16] while the
additional bacterial and archaeal homologues were selected
to compare and contrast the current study with prior work
relating to their substrate specificities.[9a,17]
Standard uniform assay conditions [Met analogue (2 mm),
ATP (1 mm), MAT (5 mm), tris(hydroxymethyl)aminometh-
ane (Tris; 25 mm), MgCl2 (5 mm), KCl (50 mm), pH 8, 4 h]
were adopted to facilitate this broad assessment. For the
thermophilic counterpart (mMAT), the reactions were con-
ducted at 658C, while all other reactions were incubated at
378C. The production of the SAM analogues was determined
by a simple RP-HPLC end-point assay to afford complete
analysis of the desired SAM analogue and/or any major
corresponding SAM analogue degradation products. The
formation of the putative SAM analogues and SAM-derived
degradation products was subsequently confirmed by HRMS
Scheme 1. a) The methionine adenosyltransferase (MAT)-catalyzed
reaction, in which l-Met (R=CH3, X=S) is the native substrate and
the native product is S-adenosylmethionine (R=CH3, X=S). The
variability of both R and X are assessed within the current study.
b) General methodology for the synthesis of S- and Se-l-Met analogues
(X=S and Se, respectively): i) Na, NH3, À788C with 1 or 1-Se;
ii) K2CO3, acetone with l-Hcy; iii) NaBH4/NaOH, H2O, THF, 5 h with 1-
Se; iv) HCl, Reflux with 2-Se (see Table S1 in the Supporting Informa-
tion).
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analysis for all of the positive reactions (Table S2 in the
Supporting Information).
the reaction forward through degradation of the pyrophos-
phate coproduct did not appreciably increase turnover.
Based on these results, the corresponding MATs can be
generally classified as having broad (hMAT2/hMAT2A),
medium (mMAT), or stringent (eMAT and hMAT1A) sub-
strate specificities. Superimposition of the available X-ray
structures (hMAT1A, hMAT2A, and eMAT) reveals a root
mean square deviation (rmsd) of 0.268 ꢀ between hMAT2A
and hMAT1A (sequence identity ꢀ 85%) and approximately
0.56 ꢀ between eMAT and hMAT1A or hMAT2A (sequence
identity ꢀ 58%; Figure S3 in the Supporting Information).
Structural comparison of the permissive (hMAT2A) and
stringent (hMAT1A and eMAT) MATs highlights key
distinctions at certain residues (Figure 3). Interestingly,
while hMAT2A active-site mutagenesis has led to a slight
improvement in turnover with an unnatural analogue,[6e] the
current structural comparison suggests the broad substrate
specificity of hMAT2A to be predominately mediated by
A cumulative comparison of the substrate specificities of
all five MAT model systems based on RP-HPLC is illustrated
in Figure 2, in which 5’-methyl-thio(seleno)-5’-deoxyadeno-
sine (MTA) production (as measured by RP-HPLC) was
interpreted as product production based on the well-estab-
Figure 2. a) Turnover of S/Se-Met analogues to the corresponding
S(Se)AM analogues catalyzed by selected MATs (as measured by RP-
HPLC, average percent error ꢁ5%; see the Supporting Information).
No product formation was observed in the absence of MAT, S/Se-Met
analogues or ATP. b) Structures of the S/Se-Met analogues listed in
Figure 2a (X=S, Se).
lished SAM decay pathways that indicate that MTA is directly
derived from SAM (and not ATP).[6d,11a,18] This cumulative
analysis revealed hMAT2 to be the most permissive of the
MATs tested at 378C, while the thermophilic mMAT also
displayed notable permissivity at 658C. Of the 47 putative
substrates tested with hMAT2, 10 led to appreciable (> 50%)
production of the corresponding SAM analogue, an addi-
tional 8 led to moderate (25–50%) conversion, while 11
offered detectable product (< 25%) under the conditions
described. In general, smaller alkyl substitutions were better
tolerated, thus suggesting that steric infringement perhaps
prohibits larger substitutions. Interestingly, in the case in
which direct comparisons could be made, the degree of
unsaturation correlated with a reduction in turnover (e.g.,
propyl > allyl > propargyl). Also, in most cases, Se analogues
were preferred over their S counterparts, most notably for
hMAT2. Consistent with previous studies,[19] the regulatory
beta subunit of MAT2A improved the overall proficiency of
hMAT2 and, in some cases, this increase in proficiency
translated to a slight increase in permissivity. Finally, the
addition of inorganic pyrophosphatase in an attempt to drive
Figure 3. Mapping of the residue positions that differ between eMAT
(PDB ID:1RG9), hMAT2A (PDB ID:2PO2) and hMAT1A (PDB
ID:2OBV) on the hMAT2A homodimeric structure. Residues conserved
across all three MATs are indicated by grey ribbons; residues that
differ between the three MATs are highlighted as blue ball and stick
models; and residues that are conserved in the “stringent” MATs
(hMAT1A and eMAT) but differ in the more permissive hMAT2A are
highlighted as yellow ball and stick models. The purple and green
spheres within the active site represent K+ and Mg+2, respectively.
residues within the secondary shell and/or on the solvent-
exposed surface distal to active-site loops that likely contrib-
ute to interdomain movement/dynamics. Whether or not
these residues contribute to the hMAT2 alpha/beta interface
is currently unknown.
To assess the feasibility of “alkylrandomization” through
a single-vessel coupled MAT/MT reaction, the rebeccamycin
MT RebM was selected. RebM is the sugar C4’-O-MT that is
involved in rebeccamycin biosynthesis and has previously
been demonstrated to tolerate both alternative acceptors and
alternative alkyl donors.[5a,10] The coupled system also
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employed standard assay conditions [Met analogue (2 mm),
ATP (1 mm), rebeccamycin congener 31 (50 mm), hMAT2
(5 mm), RebM (10 mm), SAH-hydrolase (5 mm), Tris (25 mm),
MgCl2 (5 mm), KCl (50 mm), pH 8, 24 h at 378C] and included
only those S/Se-Met analogues that afforded ꢂ 20% turnover
with hMAT2 under standard conditions (Figure 2). From this
pilot study, 8 out of 18 of the selected S/Se-Met series led to
the production of the corresponding differentially alkylated
indolocarbazoles in appreciable yields (ꢂ 40%, Scheme 2 and
such, this platform circumvents a major liability of prior SAM
analogue/MT strategies, namely the decomposition of the
SAM analogues, and thereby opens the door to further
interrogation of a host of MTs that operate on macro-
molecular (protein/nucleic acid) and small-molecule (natural
products) substrates. Finally, the strategy presented unveils
a single vessel proof of concept for natural product “alkyl-
randomization”, which, while currently somewhat limited by
enzyme specificity, is expected to be further advanced
through MT/MAT directed evolution and/or structure-based
engineering in a manner reminiscent to that used for
advancing glycorandomization.[3,23]
Received: September 20, 2013
Revised: December 16, 2013
Published online: March 11, 2014
Keywords: alkylrandomization · biocatalysis · enzymes ·
.
methionine adenosyltransferase · S-adenosylmethionine
[1] a) S. S. Lamb, G. D. Wright, Proc. Natl. Acad. Sci. USA 2005,
102, 519 – 520; b) C. Olano, C. Mendez, J. A. Salas, Nat. Prod.
Rep. 2010, 27, 571 – 616.
[2] a) J. J. Banik, J. W. Craig, P. Y. Calle, S. F. Brady, J. Am. Chem.
Soc. 2010, 132, 15661 – 15670; b) S. J. Kwon, M. Mora-Pale, M. Y.
Lee, J. S. Dordick, Curr. Opin. Chem. Biol. 2012, 16, 186 – 195;
c) T. L. Li, Y. C. Liu, S. Y. Lyu, Curr. Opin. Chem. Biol. 2012, 16,
170 – 178.
[3] a) R. W. Gantt, P. Peltier-Pain, J. S. Thorson, Nat. Prod. Rep.
2011, 28, 1811 – 1853; b) R. W. Gantt, P. Peltier-Pain, W. J.
Cournoyer, J. S. Thorson, Nat. Chem. Biol. 2011, 7, 685 – 691;
c) R. W. Gantt, P. Peltier-Pain, S. Singh, M. Zhou, J. S. Thorson,
Proc. Natl. Acad. Sci. USA 2013, 110, 7648 – 7653; d) M. Zhou,
A. Hamza, C.-G. Zhan, J. S. Thorson, J. Nat. Prod. 2013, 76, 279 –
286; e) P. Peltier-Pain, K. Marchillo, M. Zhou, D. R. Andes, J. S.
Thorson, Org. Lett. 2012, 14, 5086 – 5089.
Scheme 2. Products of the hMAT2A–RebM coupled reaction in the
presence of 3/3-Se, 7/7-Se, 4/4-Se, and 6/6-Se (see Figure 2). The
percentage product formation for each S/Se-Met analogue utilized is
noted in parentheses (average percent error ꢁ5%, see the Supporting
Information).
[4] D. K. Liscombe, G. V. Louie, J. P. Noel, Nat. Prod. Rep. 2012, 29,
1238 – 1250.
Figure S4b). Importantly, inclusion of SAH-hydrolase
(SAHH) in the coupled reaction to prevent the product
inhibition associated with most if not all MTs[20] improved the
overall product yields by 15–40%. Notably, this is the first
report of MT-catalyzed acetonitrilylation (35; derived from l-
Met analogue 6 or 6-Se).
[5] a) C. Zhang, R. L. Weller, J. S. Thorson, S. R. Rajski, J. Am.
Chem. Soc. 2006, 128, 2760 – 2761; b) H. Stecher, M. Tengg, B. J.
Ueberbacher, P. Remler, H. Schwab, H. Griengl, M. Gruber-
Khadjawi, Angew. Chem. 2009, 121, 9710 – 9712; Angew. Chem.
Int. Ed. 2009, 48, 9546 – 9548; c) J. M. Winter, G. Chiou, I. R.
Bothwell, W. Xu, N. K. Garg, M. Luo, Y. Tang, Org. Lett. 2013,
15, 3774 – 3777.
In conclusion, the broad substrate specificity analysis
presented reveals that hMAT2/2A and mMAT display
notably broad substrate tolerance. Of these, hMAT2A is
overexpressed in a number of tumor types,[21] in which
inhibition of hMAT2A by small-molecule inhibitors or
siRNA affords dramatic tumor reduction.[22] The discovered
substrate malleability of hMAT2A, in conjunction with the
known aberrant cancer-specific hMAT2A overexpression,
may offer the potential to employ suitable S/Se-Met ana-
logues as cell-based metabolic probes for investigating the
role of methylation in cancer. In addition, the work high-
lighted herein enables one of the first facile syntheses of SAM
analogues directly compatible with downstream SAM-utiliz-
ing enzymes, including MTs, in a method with significant
advantages over other recently reported chemoenzymatic
strategies that depend on synthetic non-native alkylated
amino acid donors and non-native nucleoside acceptors.[6f] As
[6] a) K. Islam, W. Zheng, H. Yu, H. Deng, M. Luo, ACS Chem.
Biol. 2011, 6, 679 – 684; b) R. Wang, W. Zheng, H. Yu, H. Deng,
M. Luo, J. Am. Chem. Soc. 2011, 133, 7648 – 7651; c) I. R.
Bothwell, K. Islam, Y. Chen, W. Zheng, G. Blum, H. Deng, M.
Luo, J. Am. Chem. Soc. 2012, 134, 14905 – 14912; d) S. Willnow,
M. Martin, B. Luscher, E. Weinhold, ChemBioChem 2012, 13,
1167 – 1173; e) R. Wang, K. Islam, Y. Liu, W. Zheng, H. Tang, N.
Lailler, G. Blum, H. Deng, M. Luo, J. Am. Chem. Soc. 2013, 135,
1048 – 1056; f) M. Thomsen, S. B. Vogensen, J. Buchardt, M. D.
Burkart, R. P. Clausen, Org. Biomol. Chem. 2013, 11, 7606 –
7610.
ˇ
ˇ
[7] a) C. Dalhoff, G. Lukinavicius, S. Klimasauskas, E. Weinhold,
ˇ
Nat. Protoc. 2006, 1, 1879 – 1886; b) C. Dalhoff, G. Lukinavicius,
ˇ
S. Klimasauskas, E. Weinhold, Nat. Chem. Biol. 2006, 2, 31 – 32;
ˇ
c) S. Klimasauskas, E. Weinhold, Trends Biotechnol. 2007, 25,
ˇ
99 – 104; d) G. Lukinavicius, V. Lapiene, Z. Stasevskij, C.
ˇ
Dalhoff, E. Weinhold, S. Klimasauskas, J. Am. Chem. Soc.
2007, 129, 2758 – 2759; e) C. Dalhoff, M. Huben, T. Lenz, P. Poot,
E. Nordhoff, H. Koster, E. Weinhold, ChemBioChem 2010, 11,
3968 www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 3965 –3969

Angewandte
Chemie
256 – 265; f) Y. Motorin, J. Burhenne, R. Teimer, K. Koynov, S.
Willnow, E. Weinhold, M. Helm, Nucleic Acids Res. 2011, 39,
1943 – 1952.
[18] J. L. Hoffman, Biochemistry 1986, 25, 4444 – 4449.
[19] H. L. LeGros, Jr., A. B. Halim, A. M. Geller, M. Kotb, J. Biol.
Chem. 2000, 275, 2359 – 2366.
[8] P. M. Ueland, Pharmacol. Rev. 1982, 34, 223 – 285.
[9] a) Z. J. Lu, G. D. Markham, J. Biol. Chem. 2002, 277, 16624 –
16631; b) O. M. Ottink, F. H. Nelissen, Y. Derks, S. S. Wijmenga,
H. A. Heus, Anal. Biochem. 2010, 396, 280 – 283.
[20] J. K. Coward, E. P. Slisz, J. Med. Chem. 1973, 16, 460 – 463.
[21] a) H. Yang, Z. Z. Huang, J. Wang, S. C. Lu, FASEB J. 2001, 15,
1507 – 1516; b) H. Chen, M. Xia, M. Lin, H. P. Yang, J.
Kuhlenkamp, T. Li, N. M. Sodir, Y. H. Chen, H. Josef-Lenz,
P. W. Laird, S. Clarke, J. M. Mato, S. C. Lu, Gastroenterology
2007, 133, 207 – 218; c) T. Zhang, Z. C. Zheng, Y. Q. Liu, J. J.
Zhang, Y. Zhao, Y. Liu, H. T. Zhu, G. H. Zhao, J. W. Liang, Q.
Li, H. M. Xu, Acta Histochem. 2013, 115, 48 – 55.
[22] a) Q. Liu, K. Wu, Y. Zhu, Y. He, J. Wu, Z. Liu, Hepatology Res.
2007, 37, 376 – 388; b) W. Zhang, V. Sviripa, X. Chen, J. Shi, T.
Yu, A. Hamza, N. D. Ward, L. M. Kril, C. W. Vander Kooi, C. G.
Zhan, B. M. Evers, D. S. Watt, C. Liu, ACS Chem. Biol. 2013, 8,
796 – 803.
[23] a) W. A. Barton, J. Lesniak, J. B. Biggins, P. D. Jeffrey, J. Jiang,
K. R. Rajashankar, J. S. Thorson, D. B. Nikolov, Nat. Struct. Biol.
2001, 8, 545 – 551; b) W. A. Barton, J. B. Biggins, J. Jiang, J. S.
Thorson, D. B. Nikolov, Proc. Natl. Acad. Sci. USA 2002, 99,
13397 – 13402; c) D. Hoffmeister, J. Yang, L. Liu, J. S. Thorson,
Proc. Natl. Acad. Sci. USA 2003, 100, 13184 – 13189; d) J. Yang,
L. Liu, J. S. Thorson, ChemBioChem 2004, 5, 992 – 996; e) J.
Yang, X. Fu, J. Liao, L. Liu, J. S. Thorson, Chem. Biol. 2005, 12,
657 – 664; f) R. Moretti, J. S. Thorson, J. Biol. Chem. 2007, 282,
16942 – 16947; g) G. J. Williams, C. Zhang, J. S. Thorson, Nat.
Chem. Biol. 2007, 3, 657 – 662; h) R. Moretti, A. Chang, P.
Peltier-Pain, C. A. Bingman, G. N. Phillips, Jr., J. S. Thorson, J.
Biol. Chem. 2011, 286, 13235 – 13243.
[10] a) C. Sꢁnchez, I. A. Butovich, A. F. Brana, J. Rohr, C. Mendez,
J. A. Salas, Chem. Biol. 2002, 9, 519 – 553; b) C. G. Hyun, T.
Bililign, J. C. Liao, J. S. Thorson, ChemBioChem 2003, 4, 114 –
117; c) C. Zhang, C. Albermann, X. Fu, N. R. Peters, J. D.
Chisholm, G. Zhang, E. J. Gilbert, P. G. Wang, D. L. Van V-
ranken, J. S. Thorson, ChemBioChem 2006, 7, 795 – 804; d) S.
Singh, J. G. McCoy, C. Zhang, C. A. Bingman, G. N. Phillips, Jr.,
J. S. Thorson, J. Biol. Chem. 2008, 283, 22628 – 22636.
[11] a) D. F. Iwig, S. J. Booker, Biochemistry 2004, 43, 13496 – 13509;
b) D. F. Iwig, A. T. Grippe, T. A. McIntyre, S. J. Booker,
Biochemistry 2004, 43, 13510 – 13524.
[12] a) J. Jiracek, M. Collinsova, I. Rosenberg, M. Budesinsky, E.
Protivinska, H. Netusilova, T. A. Garrow, J. Med. Chem. 2006,
49, 3982 – 3989; b) Y. Yi, S. X. Fa, W. Cao, L. W. Zeng, M. X.
Wang, H. P. Xu, X. Zhang, Chem. Commun. 2012, 48, 7495 –
7497.
[13] R. L. Strausberg, et al., Proc. Natl. Acad. Sci. USA 2002, 99,
16899 – 16903.
[14] G. D. Markham, J. Deparasis, J. Gatmaitan, J. Biol. Chem. 1984,
259, 4505 – 4507.
[15] C. J. Bult, et al., Science 1996, 273, 1058 – 1073.
[16] S. C. Lu, J. M. Mato, Physiol. Rev. 2012, 92, 1515 – 1542.
[17] N. Yarlett, J. Garofalo, B. Goldberg, M. A. Ciminelli, V.
Ruggiero, J. R. Sufrin, C. J. Bacchi, Biochim. Biophys. Acta
Mol. Basis Dis. 1993, 1181, 68 – 76.
Angew. Chem. Int. Ed. 2014, 53, 3965 –3969
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 3969