Cyclization of synthetic seco-proansamitocins to ansamitocin macrolactams by actinosynnema pretiosum as biocatalyst

Article abstract of DOI:10.1002/cbic.201000422

Ring closure is possible with seco-proansamitocin and two activated SNAC esters, which can be processed to ansamitocin P3 and 19-deschloro-20-demethoxy AP-3, respectively, by an AHBA-blocked mutant of Actinosynnema pretiosum. This work sheds light on the

Full text of DOI:10.1002/cbic.201000422

DOI: 10.1002/cbic.201000422
Cyclization of Synthetic seco-Proansamitocins to Ansamitocin Macrolactams
by Actinosynnema pretiosum as Biocatalyst
Kirsten Harmrolfs,[a] Marco Brꢀnjes,[a] Gerald Drꢁger,[a] Heinz G. Floss,[b] Florenz Sasse,[c] Florian Taft,[a] and
Andreas Kirschning*[a]
The plant-derived maytansinoids 4[1] and their microbial coun-
terparts, the ansamitocins P-2 to P-4 5–7 (Scheme 1)[2] exert
strong in vitro and in vivo antitumor activity by blocking the
assembly of tubulin into functional microtubules.[3] Ansamito-
cin biosynthesis involves construction of the carbon framework
on a type I modular polyketide synthase (PKS)[4] from 3-amino-
5-hydroxybenzoic acid 1 (AHBA)[5] via chain extension by one
“glycolate”, three propionate, and three acetate units. The last
PKS module holds the seco-proansamitocin 2a, which is re-
leased and cyclized, presumably by an ansamycin amide syn-
thase (gene asm9),[6] to yield the 19-membered macrocyclic
lactam, proansamitocin 3 (Scheme 1).[7] The latter enzyme is
particularly interesting because the corresponding chemical
macrolactamizations are known to be challenging, due, in part,
to the lack of nucleophilicity of the aniline moiety.[7–9] The
amide synthases are not part of the PKS,[10] but are separate
enzymes with homology to arylamine acyl transferases.[4,11,12]
We have studied the substrate specificity of the ansamitocin
biosynthetic machinery[13,14] using a mutant strain (HGF073) of
Actinosynnema pretiosum blocked in the biosynthesis of the
Scheme 1. Biosynthesis of ansamitocins 5–7 via seco-proansamitocin 2a and
proansamitocin 3, and comparison with maytansine 4.
PKS starter unit AHBA 1.[7] Through mutasynthetic approaches,
we could show that the PKS, the amide synthase, and the tai-
loring enzymes accept several substrates that are modified in
the aromatic moiety of the biosynthetically advancing ansami-
tocins. As the amide synthase is thought to use PKS-bound
seco-proansamitocin 2a as substrate, it is unclear whether
simple thioester analogues or free carboxylic acids can also act
as substrates in mutabiosyntheses. Acceptance of very ad-
vanced biosynthetic intermediates and analogues would be
highly desirable because it would allow one to bypass the
“conservative” PKS with modified substrates that the PKS ma-
chinery has difficulty processing. In addition, this approach
would also shed light on mechanistic aspects of the amide
synthase and on its substrate specificity. So far, the difficulty in
accessing potential substrates of the amide synthase has been
a major hurdle in this research. Although it is a time-consum-
ing task, total synthesis is a reliable strategy including access
to analogues.
We previously reported the total synthesis of N-acetylcystea-
mine thioester (SNAC ester) 2b.[13] Herein we describe the syn-
thesis of seco-acid 2c and of the SNAC ester of the new seco-
proansamitocin derivative 16, as well as the use of all three ad-
vanced substrates in conversion experiments with A. pretiosum
mutant HGF073.
SNAC ester 2b (2.3 mg, 4.1 mmol) was added to a culture
(50 mL) of A. pretiosum strain HGF073 (Scheme 2).[15] The cul-
ture was harvested after seven days, and extracts were ana-
lyzed by UPLC–MS (ultra-high-performance LC-coupled ESI-
MS). The spectra of the biosynthetic samples were compared
with those of authentic AP-3 (6) and found to be identical,
with a parent ion at m/z 657 [M+Na]+ and collision-induced
fragmentation giving daughter ion spectra with a base peak at
m/z 569 (from m/z 657) due to loss of the ester function at C3.
In earlier studies, we could unequivocally exclude background
synthesis of AP-3 with blocked mutant HGF073.[16] The yield of
AP-3 formed from SNAC ester 2b was 0.2% based on quantita-
tion by UV absorption at l=248 nm after preparative HPLC
purification.[17] That equals 2% of the incorporation observed
with AHBA supplementation (~80 mgLꢀ1).
[a] Dr. K. Harmrolfs, Dr. M. Brꢀnjes, Dr. G. Drꢁger, Dr. F. Taft,
Prof. Dr. A. Kirschning
Institut fꢀr Organische Chemie und Biomolekulares
Wirkstoffzentrum (BMWZ), Leibniz Universitꢁt Hannover
Schneiderberg 1B, 30167 Hannover (Germany)
Fax: (+49)511-762-3011
E-mail: andreas.kirschning@oci.uni-hannover.de
[b] Prof. Dr. H. G. Floss
Department of Chemistry, University of Washington
Seattle, WA 98195-1700 (USA)
[c] Dr. F. Sasse
Abteilung Chemische Biologie
Helmholtz Zentrum fꢀr Infektionsforschung (HZI)
Inhoffenstrasse 7, 38124 Braunschweig (Germany)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201000422: Synthesis and spectra of new
compounds, protocols of feeding experiments and their analysis.
ChemBioChem 2010, 11, 2517 – 2520
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2517

from benzyl alcohol 10, intermediate propargyl aniline 12 was
prepared via the N-Boc-protected benzyl bromide, which was
subjected to a palladium-catalyzed cross-coupling with alkynyl-
indium derivative 11 (Scheme 4).[20] After desilylation, carboalu-
mination,[21] during which the Boc group was also removed,
followed by Teoc protection yielded vinyl iodide 13 in good
yield and with good stereocontrol.
In the following, the Heck reaction allowed the merging of
vinyl iodide 13 and complex alkene 14[13] under Jeffery condi-
tions[22] to furnish the coupling product 15 in 68% yield. Simul-
taneous oxidation of both hydroxy groups and further oxida-
tion of the aldehyde moiety to the carboxylic acid was fol-
lowed by SNAC ester formation. Finally, the silyl ethers were
removed by treatment with the HF·pyr complex, and deprotec-
tion of the Teoc group was achieved with ZnCl2 under ultra-
sound conditions to yield the desired SNAC ester 16. Standard
functional group manipulations led to seco-acid derivative 17
using alcohol 15 as branching point. Interestingly, we were
unable to chemically cyclize seco-proansamitocin 17 under var-
ious conditions, including those of the Mukaiyama (N-methyl-
2-chloropyridinium iodide, Et3N)[23] and Kita (ethoxyacetylene,
cat. [RuCl2(p-cymene)]2)[24] protocols. The difficulty of smoothly
achieving macrolactamization in ansamycin synthesis has been
encountered before, for which the decreased nucleophilicity of
the arylamino group is likely responsible.[8]
Scheme 2. Feeding experiments with seco-proansamitocin 2c and SNAC
ester 2b to yield ansamitocin P3 6 and proposed activated intermediates of
seco-acids 8a and 8b.
The SNAC ester 2b may have been accepted as substrate by
the amide synthase directly; alternatively, 2b could have been
initially loaded onto the last PKS module by transesterification
before macrolactamization occurs. Surprisingly, seco-proansa-
mitocin 2c (0.4 mg, 0.9 mmol), prepared from a published
advanced synthetic precursor 9[13] via an established sequence
(Scheme 3),[18] also gave AP-3 (6) in 1.5% yield relative to
AHBA supplementation (0.13% absolute yield), when incubat-
ed with A. pretiosum strain HGF073 (50 mL). Macrolactamiza-
tion of 2c can only occur after activation of the carboxylate to
the CoA ester 8a, for example, or the adenylate 8b. The amide
synthase then promotes macrolactamization either directly or
following transfer onto the final PKS module.
In contrast, when SNAC ester 16 (1.2 mg, 2.2 mmol) was incu-
bated with HGF073, the new deschloro-desmethoxy-AP-3 18
(14 mg, 1.1%) was formed (Scheme 5). Again, UPLC–MS served
to identify the metabolite, revealing the parent ion at m/z 571
[M+H]+ and collision-induced fragmentation that gave daugh-
ter ion spectra with a base peak at m/z 483 (from m/z 571). To
confirm the structure of 18, we conducted another mutational
biosynthesis with A. pretiosum HGF073 using bromoaminoben-
zoic acid 20 as mutasynthon, which yielded 19-bromo-20-de-
methoxy-AP-3 21[15] in good yield (up to 15 mgLꢀ1). Palladium-
catalyzed debromination finalized the alternative synthesis of
19-deschloro-20-demethoxy-AP-3 18. The product was identi-
cal in all respects to the fermentation product collected from
feeding SNAC ester 16. The semisynthetic material also served
to quantify the feeding experiment with SNAC ester 16. This
was achieved with an authentic standard using diagnostic UV
absorption at l=248 nm.[17] The degree of incorporation for all
three seco-acid derivatives is rather low, and is similar in mag-
nitude to incorporation yields for tri- or tetraketide PKS inter-
mediates.[16] This does not necessarily mean that their further
processing is inefficient. In fact, we encountered
As a second complex substrate, we prepared the SNAC ester
of 20-deoxy-seco-proansamitocin 16, which was chosen be-
cause we had observed that supplementing a culture of
HGF073 with aminobenzoic acid 19 (260 mg, 1.5 mmol) unex-
pectedly gave the corresponding ansamitocin derivative 18 in
very low yield, as determined by UPLC–MS (see Scheme 5
below).[19] The total synthesis approach relates to the success-
ful preparation of SNAC ester 2b (Scheme 4).[13] Thus, starting
severe stability problems with all three open-chain
substrates 2b, 2c, and 16 during the final steps of
total synthesis. These included dehydration (at C2–
C3), retro-aldol reaction (at C3) and double bond iso-
merization (C10 to C14), just to name those we could
clearly identify. Clearly, only when cyclization has oc-
curred, are undesired degradations suppressed.[25]
Therefore, it cannot be excluded that substantial
Scheme 3. Synthesis of seco-proansamitocin 2c: a) 9,[13] DMSO, (COCl)2, ꢀ608C, 1 h then
Et3N, ꢀ408C (75%); b) NaClO2, NaH2PO4, H2O, tBuOH, 2-methyl-2-butene, 08C!RT,
30 min (86%); c) HF·pyr, THF, RT, 6 h, (48%); d) ZnCl2, MeNO2, ultrasound, RT, 1 h, (47%).
TBS=tert-butyldimethylsilyl, Teoc=trimethylsilylethoxycarbonyl.
amounts of the free seco-acid derivatives decom-
posed during the seven days in the fermentation
broth.
2518
www.chembiochem.org
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2010, 11, 2517 – 2520

Scheme 5. Preparation of deschloro-desmethoxy-AP-3 18:
a) Bu3SnH, Pd[PtBu3]2, toluene, 608C (80%).
These results demonstrate for the first time that
open-chain seco-acid precursors can, in principle, be
cyclized by A. pretiosum when a mutant blocked in
AHBA biosynthesis is used. We also showed that not
only activated thioesters are cyclized but also the
free carboxylic acid can be accepted and is trans-
formed into the corresponding macrolactam. Overex-
pression and isolation of the amide synthase should
pave the way to study its mechanism in detail and
will allow synthetic chemists to explore the use of
the enzyme for preparative macrolactamizations
using other long-chain amino acids as substrates.
Such investigations are currently pursued in our labo-
ratories.
Scheme 4. Total synthesis of SNAC ester 16 and seco-acid 17, and feeding experiments
with 16 toward deschloro-desmethoxy-AP-3 18: a) Boc2O, Et3N, dioxane/H2O (2.5:1), RT,
12 h, (82%); b) PPh3, CBr4, CH2Cl2, RT, 1 h (83%); c) Pd(dppf)Cl2, THF, 658C, 4 h (98%);
d) TBAF·3H2O, THF, ꢀ208C, 1 h (88%); e) 1. AlMe3, Cp2ZrCl2, (CH2Cl)2, RT, 1 h, then addi-
tion of 11[13] at 08C, RT, 72 h, 2. I2, THF, ꢀ308C!08C, 1.5 h (53%); f) TeocCl, NaHCO3,
CH2Cl2, RT, 10 min (91%); g) 14, Pd(OAc)2, Cs2CO3, Bu4NBr, NEt3, DMF, RT, 2 h (68%);
h) DMSO, (COCl)2, ꢀ608C, 1 h then Et3N, ꢀ408C (90%); i) NaClO2, NaH2PO4, H2O, tBuOH,
2-methyl-2-butene, 08C!RT, 30 min (85%); j) AcNH(CH2)2SH, DIC, DMAP, CH2Cl2, RT, 2 h
(56%); k) HF·pyr (70% HF)/THF, RT, 24 h (62%); l) ZnCl2, MeNO2, ultrasound, RT, 80 min
(63%; toward 17: 11%). TMS=trimethylsilyl, Boc=tert-butyloxycarbonyl; dppf=bis(di-
phenylphosphino)ferrocene, TBAF= tetra-n-butylammonium fluoride; DIC=diisopropyl-
carbodiimide, DMAP=4-dimethylamino pyridine.
Acknowledgements
We thank the US National Institutes of Health (grant
CA 76461), the Deutsche Forschungsgemeinschaft
(grant Ki 397/13-1), and the Fonds der Chemischen In-
dustrie for financial support. We are grateful to Dr. T.
Knobloch for helpful discussions. We thank M. Quit-
schalle and Dr. T. Frenzel for expert synthetic support.
Keywords: amide synthase
·
ansamitocin
·
mutabiosynthesis · SNAC esters · total synthesis
The new AP-3 derivative 18[26] was tested for its inhibitory
effects toward the proliferation of various cancer cell lines rela-
tive to AP-3 6 (Table 1). It showed very strong activity in the
pgmLꢀ1 range against all cancer cell lines tested. In the case of
cervix and prostate carcinoma, 18 had an even higher activity
than AP-3 6.
[1] a) S. M. Kupchan, Y. Komoda, W. A. Court, G. J. Thomas, R. M. Smith, A.
Karim, C. J. Gilmore, R. C. Haltiwanger, R. F. Bryan, J. Am. Chem. Soc.
1972, 94, 1354–1356; b) S. M. Kupchan, Y. Komoda, A. R. Branfman, A. T.
Sneden, W. A. Court, G. J. Thomas, H. P. J. Hintz, R. M. Smith, A. Karim,
G. A. Howie, A. K. Verma, Y. Nagao, R. G. Dailey, Jr., V. A. Zimmerly, W. C.
Sumner, Jr., J. Org. Chem. 1977, 42, 2349–2357; c) R. F. Bryan, C. J. Gil-
more, R. C. Haltiwanger, J. Chem. Soc. Perkin Trans. 2 1973, 897–901.
[2] E. Higashide, M. Asai, K. Ootsu, S. Tanida, Y. Kozai, T. Hasegawa, T. Kishi,
Y. Sugino, M. Yoneda, Nature 1977, 270, 721–722.
[3] Reviews: a) A. Kirschning, K. Harmrolfs, T. Knobloch, C. R. Chimie 2008,
1523–1543; b) J. M. Cassady, K. K. Chan, H. G. Floss, E. Leistner, Chem.
Pharm. Bull. 2004, 52, 1–26.
Table 1. Antiproliferative activity of 18 compared with that of 6 toward
various cancer cell lines.[a]
[4] T.-W. Yu, L. Bai, D. Clade, D. Hoffmann, S. Toelzer, K. Q. Trinh, J. Xu, S. J.
Moss, E. Leistner, H. G. Floss, Proc. Natl. Acad. Sci. USA 2002, 99, 7968–
7973.
[5] K. Hatano, S.-L. Akiyama, M. Asai, R. W. Rickards, J. Antibiot. 1982, 35,
1415–1417.
[6] P. Spiteller, L. Bai, G. Shang, B. J. Carroll, T.-W. Yu, H. G. Floss, J. Am.
Chem. Soc. 2003, 125, 14236–14237.
[7] A. Meyer, M. Brꢀnjes, F. Taft, T. Frenzel, F. Sasse, A. Kirschning, Org. Lett.
2007, 9, 1489–1492.
IC50 [ngmLꢀ1
]
KB-3-1
cervical
U-937
lymphoma
PC-3
SK-OV-3 A-431
A-549
lung
prostate
ovarian
epidermal
18
6
0.022
0.11
0.015
0.0035
0.020
0.035
0.040
0.030
0.080
0.050
0.095
0.095
[a] The IC50 values toward primary HUVEC (from LONZA) were 0.021 and
0.031 ngmLꢀ1 for 18 and 6, respectively.
ChemBioChem 2010, 11, 2517 – 2520
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
2519

[8] D. Kashin, A. Meyer, R. Wittenberg, K.-U. Schçning, S. Kamlage, A.
Kirschning, Synthesis 2007, 304–319.
[16] F. Taft, M. Brꢀnjes, T. Knobloch, H. G. Floss, A. Kirschning, J. Am. Chem.
Soc. 2009, 131, 3812–3813.
[9] A. Meyer, A. Kirschning, Synlett 2007, 1264–1268.
[10] F. Pompeo, A. Mushtaq, E. Sim, Protein Expression Purif. 2002, 24, 138–
151.
[11] a) P. R. August, L. Tang, Y. J. Yoon, S. Ning, R. Mꢀller, T. W. Yu, M. Taylor,
D. Hoffmann, C. G. Kim, X. Zhang, C. R. Hutchinson, H. G. Floss, Chem.
Biol. 1998, 5, 69–79; b) A. Rascher, Z. Hu, N. Viswanathan, A. Schirmer,
R. Reid, W. C. Nierman, M. Lewis, C. R. Hutchinson, FEMS Microbiol. Lett.
2003, 218, 223–230.
[17] Details on quantifying the amount of fermentation products produced
are given in the Supporting Information.
[18] Details on the synthesis of 2c from 9 are provided in the Supporting
Information.
[19] Formation of 18 was determined to be 0.5% relative to supplementa-
tion with AHBA (see also ref. [17]).
[20] I. Pꢃrez, J. Pꢃrez Sestelo, L. A. Sarandeses, J. Am. Chem. Soc. 2001, 123,
4155–4160.
[12] Amide synthases perform analogous transformations to the thioesteras-
es responsible for macrolactonization and macrolactamization of poly-
ketides and nonribosomal peptides (NRP): a) F. Kopp, M. A. Marahiel,
Nat. Prod. Rep. 2007, 24, 735–749; b) J. W. Trauger, R. M. Kohli, H. D.
Mootz, M. A. Marahiel, C. T. Walsh, Nature 2000, 407, 215–218; c) J. W.
Trauger, R. M. Kohli, C. T. Walsh, Biochemistry 2001, 40, 7092–7098;
d) R. M. Kohli, J. W. Trauger, D. Schwarzer, M. A. Marahiel, C. T. Walsh,
Biochemistry 2000, 39, 7099–7108; e) R. M. Kohli, J. Takagi, C. T. Walsh,
Proc. Natl. Acad. Sci. USA 2002, 99, 1247–1252; f) R. Kohli, M. D. Burke,
J. Tao, C. T. Walsh, J. Am. Chem. Soc. 2003, 125, 7160–7161; g) R. M.
Kohli, C. T. Walsh, Chem. Commun. 2003, 297–307.
[21] a) E.-i. Negishi, D. E. Van Horn, T. Yoshida, J. Am. Chem. Soc. 1985, 107,
6639–6647; b) E.-i. Negishi, S. Y. Kondakov, D. Choueiry, K. Kasai, T. Taka-
hashi, J. Am. Chem. Soc. 1996, 118, 9577–9588.
[22] T. Jeffery, Tetrahedron 1996, 52, 10113–10130.
[23] a) K. Saigo, M. Usui, K. Kikuchi, E. Shimada, T. Mukaiyama, Bull. Chem.
Soc. Jpn. 1977, 50, 1863–1866; b) W. R. Roush, D. S. Coffey, D. J. Madar,
J. Am. Chem. Soc. 1997, 119, 11331–11332.
[24] Y. Kita, H. Maeda, K. Omori, T. Okuno, Y. Tamura, Synlett 1993, 273–274.
[25] Indeed, one may argue that macrocyclization is not only Nature’s way
of creating macrocycles that are able to adopt various defined confor-
mations to suit a given receptor’s requirements, but also allows the
chemical stabilization of open-chain polyketide metabolites.
[26] Antiproliferative activities of 21 are described in ref. [14].
[13] T. Frenzel, M. Brꢀnjes, M. Quitschalle, A. Kirschning, Org. Lett. 2006, 8,
135–138.
[14] F. Taft, M. Brꢀnjes, H. G. Floss, N. Czempinski, S. Grond, F. Sasse, A.
Kirschning, ChemBioChem 2008, 9, 1057–1060.
Received: July 23, 2010
[15] Parallel fermentations were carried out with the wild-type strain and
with mutant HGF073 supplemented with AHBA and without supple-
mentation.
Revised: October 8, 2010
Published online on November 12, 2010
2520
www.chembiochem.org
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2010, 11, 2517 – 2520