A versatile chemoenzymatic synthesis for the discovery of potent cryptophycin analogs

Article abstract of DOI:10.1021/acschembio.9b00998

The cryptophycins are a family of macrocyclic depsipeptide natural products that display exceptionally potent antiproliferative activity against drug-resistant cancers. Unique challenges facing the synthesis and derivatization of this complex group of molecules motivated us to investigate a chemoenzymatic synthesis designed to access new analogs for biological evaluation. The cryptophycin thioesterase (CrpTE) and the cryptophycin epoxidase (CrpE) are a versatile set of enzymes that catalyze macrocyclization and epoxidation of over 20 natural cryptophycin metabolites. Thus, we envisioned a drug development strategy involving their use as standalone biocatalysts for production of unnatural derivatives. Herein, we developed a scalable synthesis of 12 new unit A-B-C-D linear chain elongation intermediates containing heterocyclic aromatic groups as alternatives to the native unit A benzyl group. N-Acetyl cysteamine activated forms of each intermediate were assessed for conversion to macrocyclic products using wild type CrpTE, which demonstrated the exceptional flexibility of this enzyme. Semipreparative scale reactions were conducted for isolation and structural characterization of new cryptophycins. Each was then evaluated as a substrate for CrpE P450 and its ability to generate the epoxidized products from these substrates that possess altered electronics at the unit A styrenyl double bond position. Finally, biological evaluation of the new cryptophycins revealed a des-β-epoxy analog with low picomolar potency, previously limited to cryptophycins bearing epoxide functionality.

Full text of DOI:10.1021/acschembio.9b00998

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Article
A Versatile Chemoenzymatic Synthesis for the
Discovery of Potent Cryptophycin Analogs
Jennifer J. Schmidt, Yogan Khatri, Scott I. Brody, Catherine Zhu,
Halina Pietraszkiewicz, Frederick A. Valeriote, and David H. Sherman
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00998 • Publication Date (Web): 21 Jan 2020
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ACS Chemical Biology
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A Versatile Chemoenzymatic Synthesis for the Discovery of Potent
Cryptophycin Analogs
Jennifer J. Schmidt1,2, Yogan Khatri1, Scott I. Brody1, Catherine Zhu1,3, Halina Pietraszkiewicz6,
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Frederick A. Valeriote6, David H. Sherman1, 2, 3, 4, 5*
1Life Sciences Institute, 2Department of Medicinal Chemistry, 3Program in Chemical Biology, 4Department of Chemistry,
5Department of Microbiology & Immunology, University of Michigan, Ann Arbor, Michigan 48109, United States
6Department of Internal Medicine, Henry Ford Cancer Institute, Henry Ford Health System, Detroit, Michigan 48202, United
States
ABSTRACT: The cryptophycins are a family of macrocyclic depsipeptide natural products that display exceptionally potent anti-
proliferative activity against drug-resistant cancers. Unique challenges facing the synthesis and derivatization of this complex group
of molecules motivated us to investigate a chemoenzymatic synthesis designed to access new analogs for biological evaluation. The
cryptophycin thioesterase (CrpTE) and the cryptophycin epoxidase (CrpE) are a versatile set of enzymes that catalyze macrocycliza-
tion and epoxidation of over twenty natural cryptophycin metabolites. Thus, we envisioned a drug development strategy involving
their use as standalone biocatalysts for production of unnatural derivatives. Herein, we developed a scalable synthesis of 12 new unit
A-B-C-D linear chain elongation intermediates containing heterocyclic aromatic groups as alternatives to the native unit A benzyl
group. N-acetyl cysteamine activated forms of each intermediate were assessed for conversion to macrocyclic products using wild
type CrpTE, which demonstrated the exceptional flexibility of this enzyme. Semi-preparative scale reactions were conducted for
isolation and structural characterization of new cryptophycins. Each was then evaluated as a substrate for CrpE P450 and its ability
to generate the epoxidized products from these substrates that possess altered electronics at the unit A styrenyl double bond position.
Finally, biological evaluation of the new cryptophycins revealed a des--epoxy analog with low picomolar potency, previously lim-
ited to cryptophycins bearing epoxide functionality.
Polyketide synthases (PKSs), non-ribosomal peptide synthe-
tases (NRPSs), and their hybrids (PKS/NRPS) are modular pro-
teins that generate a vast array of complex natural products.
These proteins select a group of relatively simple chemical
building blocks (malonate, malonate derivatives, amino acids,
and/or amino acid derivatives) and construct diverse scaffolds
with a wide range of biological activities.1, 2 A large class of
these natural products, macrocycles,3 contain a constrained ring
structure which locks them into their biologically active confor-
mation and protects the constituent peptide bonds from degra-
dation.4 The biosynthesis of these important molecules is typi-
cally terminated by a thioesterase (TE) domain, an ,-hydro-
lase that utilizes a serine, histidine, aspartic acid catalytic triad
to affect regio- and stereospecific cyclization.4, 5 Finally, these
macrocyclic structures are often further tailored by cyto-
chromes P450 to produce the mature, biologically active natural
product.
(Figure 1) that contains a geminal dimethyl functionality in unit
C entered clinical trials for both the treatment of platinum re-
sistant ovarian cancer and non-small cell lung cancer.10-12 De-
spite showing significant disease stabilization and an overall
positive result, the trials were discontinued due to dose limiting
peripheral neuropathy and a lack of broad in vivo efficacy.10, 13
Although the cryptophycin-52 clinical trial was terminated, this
class of metabolites continues to represent a compelling scaf-
fold for further lead optimization as it is particularly effective
in difficult-to-treat, drug-resistant cancers.14 Recently, the cryp-
tophycins have been investigated extensively as potential
An interesting family of macrocycles, the cryptophycins are
a class of 16-membered ring depsipeptides natural products
generated by a mixed PKS/NRPS biosynthetic system.6, 7 These
molecules were first identified in Nostoc sp. ATCC 53789 as
antifungals,8 and were subsequently rediscovered in Nostoc sp.
GSV 224 as one of the most potent anti-proliferative, microtu-
bule binding agents.9 An initial medicinal chemistry screening
effort indicated that the -epoxide functionality was necessary
for maximal activity and that most modifications were detri-
mental to potency. A synthetic analog, cryptophycin-52
Figure 1. Select cryptophycin molecules.
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 isomer for this late stage transformation, demonstrating the
payloads for antibody drug conjugates (as well as other direct
targeting agents including conjugation with RGD peptides15 and
folic acid16), as this could circumvent the systemic toxicity ob-
served with these compounds.17-21 Although there is still signif-
icant interest in cryptophycins as anticancer agents, challenges
in lead optimization as well as costly synthetic efforts for their
production have stymied exploration.
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need for alternative methodologies, including biocatalytic ones
that can efficiently complete this regio- and stereo-specific pro-
cess. Thus, we embarked on an effort to generate a series of
novel cryptophycin chain elongation intermediates designed to
probe substrate tolerance for both CrpTE and CrpE, and to pro-
duce high value analogs that may address some of the limita-
tions identified previously (dose limiting peripheral neuropathy
and broad in vivo efficacy).10, 13 Herein, we report the synthesis
of a series of heterocyclic cryptophycin A subunits, elaboration
of synthetic unit A-B-C-D N-acetyl cysteamine (NAc) chain
elongation intermediates, analysis of CrpTE’s ability to cata-
lyze macrolactonization, semi-preparative scale-up, and biolog-
ical evaluation of the corresponding macrocyclic products. The
cryptophycin analogs were then assessed as substrates for the
CrpE P450, giving us valuable insight into the chemical and bi-
ochemical features that likely govern its catalytic competency.
In this process, we identified a new styrenyl cryptophycin de-
rivative that possesses low picomolar activity against human
colorectal carcinoma (HCT-116), previously observed only in
-epoxy cryptophycins.
The penultimate step in cryptophycin biosynthesis is the ste-
reospecific macrocyclization of the chain elongation intermedi-
ate via the cryptophycin thioesterase (CrpTE).6 Finally, the sty-
rene moiety of the cryptophycin macrocycle is further tailored
by an epoxidizing cytochrome P450 (CrpE) to produce the main
isolate of this class cryptophycin 1 (Figure 1). In a previous
study, we demonstrated that excised CrpTE is able to catalyze
facile cyclization to varying degrees of native and modified sub-
strates to generate cryptophycin 3, 51 (Figure 1) and an unnat-
ural cryptophycin containing a terminal olefin in unit A, origi-
nally synthesized as LY404291 at Eli Lilly.22 Moreover, the
CrpTE was employed in conjunction with the late stage, P450
epoxidase (CrpE) to provide both native and non-native struc-
tures.7, 23 These initial studies demonstrated the unique, inher-
ent flexibility of both CrpTE and CrpE, and their potential as a
versatile biocatalysts for the production of novel cryptophycin
analogs of medicinal importance. Synthetically, the two most
difficult steps in the formation of complex natural products are
regio- and stereo- specific macrocyclization and epoxidation.
Despite excellent methodology for macrocyclization via cross
metathesis, biocatalytic head to tail cyclization would be an ex-
cellent addition to the synthetic toolbox as chemically, this
method is often complicated by low yields. In the cryptophy-
cins, the most difficult synthetic step is epoxidation as current
methodologies display inefficient stereoselectivity towards the
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RESULTS AND DISCUSSION
Based on the progress made from our previous work ,22, 23 we
initially sought to continue interrogating the selectivity of the
CrpTE towards unnatural substrates with the goal of expanding
to late stage oxidation via the CrpE upon successfully generated
macrolactones. Pioneering work on PKS and NRPS TEs com-
prised of both structural studies and in vitro biochemical analy-
sis have yielded important insights into the complex catalytic
mechanisms that mediate cyclization.24-31 PKS TEs have re-
cently been implicated as one of the major bottlenecks in
Scheme 1. A. Diversifiable synthesis of unit AB intermediates with altering aryl groups. B. Synthesis of units CD incor-
porating either native -alanine or the gem dimethyl analog. C. Elaboration of seco-cryptophycins from unit AB and unit
CD intermediates.
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formation of unnatural polyketide macrocycles.32 However,
With diversifiable substrates 8 in hand, coupling with 14 (a
1
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4
5
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7
8
these results contrast with earlier studies on NRPS TEs that
identified inherent substrate pre-organization as a major driving
force in cyclization of certain peptides. 28, 33, 34 Despite the iden-
tification of different catalytic mechanisms employed for cy-
clization between different TE subtypes, little is known about
the factors employed by mixed PKS/NRPS systems, underscor-
ing the importance of continued investigations into this enzyme
family. The unusual aryl starter unit of cryptophycins served as
an intriguing site of exploration for the CrpTE and CrpE en-
zymes as it is exocyclic to the macrocycle yet conjugated to the
double bond that is epoxidized by CrpE. The unit A terminus
also represents an underexplored site for cryptophycin struc-
ture-activity relationship studies (SAR). Previously, alkylated,
halogenated, and phenolic moieties of unit A have been ex-
plored with little emphasis placed on heterocyclic aryl
groups.35-38 Moreover, it represents an attractive position for use
of these as tactical bioisosteres that could increase water solu-
bility, leading to better in vivo efficacy as well as potentially
decreased toxicity.11, 13
Synthesis of Seco Cryptophycin Chain Elongation Inter-
mediates. In order to test the substrate scope of the CrpTE as
well as produce novel cryptophycin macrolactones, a scalable
synthesis of the N-Acetylcysteamine-activated (NAc) linear
chain elongation intermediate was developed that was amena-
ble to late-stage diversification of the unit A aryl group. Ana-
logs were synthesized using two key intermediates, including
unit AB (Scheme 1A, 8) and unit CD-NAc (Scheme 1B, 14a
or b). Formulation of units AB took advantage of chiral auxil-
iary chemistry as well as a Suzuki coupling strategy reported
previously.39 A final Horner Wadsworth Emmons olefination
(HWE) was employed to form the key junction between units
A and B. Units CD with the NAc recognition element was gen-
erated via peptide coupling of commercially and readily manip-
ulated amino acid derivatives.
or b) and successive Suzuki diversification were investigated.
Labile functional groups and racemization precluded diversifi-
cation prior to the final peptide coupling. Screening Suzuki con-
ditions for substrate 8, yielded a procedure that utilized
Pd2(dba)3 and K3PO4 to produce a suite of novel unit-AB cryp-
tophycin analogs 9a-j (Scheme 1A), in good to excellent yields
with no detectable racemization. Elaboration of the final seco
cryptophycin chain elongation intermediates was accomplished
via the coupling of units AB with units CD (Scheme 1C). Sa-
ponification of the methyl esters 9a-j also proved to be
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O
Ar
O
O
HN
Cl
O
O
O
OMe
N
H
Crp TE
R1 R2
16a-j
15a-j
100 mM Sodium Phosphate
Buffer, 7% DMSO
+
O
O
H
N
OH
N
H
O
Ar
R1 R2
OH
O
O
17a-j
OMe
Cl
R1 = CH3
R2 = H
a.
N
R1 = CH3
R2 = H
b.
c.
N
N
R1 = CH3
R2 = H
R1 = CH3
R2 = CH3
*
c.
R1 = CH3
R2 = H
d.
d.
N
R1 = CH3
R2 = CH3
As a starting point, unit A was synthesized (Scheme 1A) us-
ing an Evans asymmetric aldol with N-crotonyl oxazolidinone
1 and aldehyde 2, which proceeded in excellent yields with a
high dr (>20:1) to afford the desired (2R, 3S) adduct. Subse-
quent silation with TBS trifluoromethanesulfonate produced 3,
which was reductively cleaved form the chiral auxiliary furnish-
ing 4. The primary alcohol was then tosylated and a consecutive
reductive deoxygenation with lithium triethyl borohydride fur-
nished the desired intermediate 5.40 At this stage, a vinyl pinacol
boronic ester was introduced via Hoveyda-Grubbs cross me-
tathesis to give the desired Suzuki handle for future diversifica-
tion.39 Removal of the p-methoxy benzyl (PMB) protecting
group with DDQ and subsequent Des Martin Periodinane
(DMP) oxidation to 6 furnished the unit A aldehyde fragment
necessary for the HWE olefination.41 Utilizing phosphonate 7,
HWE olefination conditions were explored, yielding optimal
conversion with sodium hydride in THF, which furnished the
diversifiable unit AB fragment 8 in a 57% yield of the correct
isomer (70% overall yield, with ~ 5:1 E:Z product ratio).
*
N
N
R1 = CH3
R2 = H
e.
f.
N
N
R1 = CH3
R2 = H
N
N
R1 = CH3
R2 = H
N
N
g.
h.
R1 = CH3
R2 = H
N
R1 = CH3
R2 = CH3
N
N
*
h.
i.
R1 = CH3
R2 = H
N
N
R1 = CH3
R2 = H
O
N
j.
Unit CD-NAc was readily synthesized beginning from com-
mercially available leucic acid 10 (Scheme 1B). Initial benzyl
protection of the acid functionality produced 11, which was
subsequently coupled with -amino acids 12 (a or b) to produce
the desired esters. These were readily deprotected via H2/Pd hy-
drogenolysis to furnish acids 13 (a or b).42 Finally, these mole-
cules were coupled with N-acetyl cysteamine, to yield the de-
sired unit CD fragments 14 (a or b).
Figure 2. Analysis of analytical scale CrpTE cyclization reactions
for analogs 14a-m with starting material peaks in black, product
peaks in blue, and hydrolytic byproducts in green as assessed by
TOF-MS. Minor peaks in traces a, c, c*, and i represent likely di-
astereomers but were not characterized. * Indicate gem dimethyl
analogs.
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Page 4 of 10
susceptible to racemization and a screen of different hydrolysis compared to the native benzyl substrate for the four alternate
1
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8
procedures yielded trimethyltin hydroxide, which provided the
corresponding acids in good yield with no detectable racemiza-
tion.43 Simultaneous Boc deprotection of 14 (a or b) with 4M
HCl/Dioxane and subsequent peptide coupling at 0 C with
HATU produced the TBS protected intermediates in good
yields with minimal racemization (7:1 to 12:1 dr). Diastere-
omers were resolved using reverse phase HPLC prior to re-
moval of the TBS group. Finally, deprotection of the silyl group
using aqueous HF in acetontrile furnished the desired seco-
cryptophycin NAc analogs (Scheme 1C, 15a-j).
six membered aromatic rings tested. The native benzyl substrate
(Table 1, 16a) shows an overall conversion of 73% and TTN of
110. Unreacted starting material as well as the competing hy-
drolysis reaction observed in a 9:1 ratio in comparison to cy-
clization account for all peaks observed. In contrast, the 2-, 3-,
4-pyridyl and pyrazine substrates showed nearly complete sub-
strate consumption as well as nearly undetectable levels of hy-
drolytic byproducts (Figure 2, 15b-e). This is reflected in
higher percent conversion, TTNs, and cyclization:hydrolysis
(Table 1, 16b-e), all of which are significantly greater (91 -96%
conversion, >10:1 cyclization:hydrolysis ratio) when compared
to the native substrate 16a.
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Analytical Substrate Conversion Assay of CrpTE with
Unit A Heterocycles. With a diversifiable synthesis in hand we
began to explore the flexibility of CrpTE against the newly gen-
erated unit A chain elongation intermediates (Figure 1 15a-j).
The initial set of CrpTE analogs contained six membered ring
heterocycles in place of the native benzene ring (Figure 2, 15b-
e). Analytical scale reactions revealed an increase in conversion
to product as well as higher total turnover numbers (TTN)
The set of unnatural unit A analog was expanded to include
five membered ring aromatic heterocycles with varying alkyl
chains. The 2-, 3-, and 4-methyl pyrazole derivatives (15f-j)
were synthesized and tested in the same analytical assay, utiliz-
ing the benzyl substrate 15a as a control. The 2-methyl pyrazole
(16f) and 3-methyl pyrazole (16g) groups showed slightly lower
Table 1. Percent conversion, isolation yield, total turnover numbers (TTN)a, and cyclization to hydrolysis analysis of CrpTE
reactions.
O
O
O
H
Ar
N
SNAc
O
O
HN
Cl
N
H
O
Crp TE
O
Ar
R1 R2
OMe
OH
O
O
O
O
OMe
N
H
Na2PO4, 7% DMSO
R1 R2
16a-m
15a-m
N
Cl
Unit C monomethyl analogues:
O
O
O
O
N
O
O
HN
Cl
O
O
HN
Cl
O
O
HN
Cl
N
O
O
HN
Cl
O
O
O
O
O
O
OMe
O
O
OMe
O
O
OMe
N
H
N
H
N
H
O
O
OMe
N
H
73% conversion
49% isolated yield
TTN: 110
9:1 Cyc:Hydrob
16a
91% conversion
66% isolated yield
TTN: 137
12:1 Cyc:Hydro
16b
96% conversion
63% isolated yield
TTN: 145
96% conversion
66% isolated yield
TTN: 144
27:1 Cyc:Hydro
16d
33:1 Cyc:Hydro
16c
N
N
O
O
O
O
N
N
N
N
N
O
O
HN
Cl
O
O
HN
Cl
O
O
HN
Cl
O
O
HN
Cl
N
O
O
O
O
O
O
OMe
N
H
O
O
OMe
O
O
OMe
O
O
OMe
N
H
N
H
N
H
95% conversion
62% isolated yield
85% conversion
58% isolated yield
84% conversion
61% isolated yield
TTN: 126
10:1 Cyc:Hydrob
16g
97% conversion
69% isolated yield
TTN: 145
39:1 Cyc:Hydro
16h
TTN: 143
TTN: 127
23:1 Cyc:Hydro
16e
17:1 Cyc:Hydrob
16f
O
O
N
N
N
O
O
O
HN
Cl
O
O
HN
Cl
O
O
O
O
OMe
O
O
OMe
N
H
N
H
83% conversion
59% isolated yield
TTN: 124
69% conversion
55% isolated yield
TTN: 104
40:1 Cyc:Hydro
16i
15:1 Cyc:hydro
16j
Unit C Gem Dimethyl Analogues:
O
O
O
O
N
N
O
O
HN
Cl
O
O
HN
Cl
N
O
O
HN
Cl
O
O
HN
Cl
N
O
O
O
O
O
O
OMe
O
O
OMe
N
H
O
O
OMe
N
H
O
O
OMe
N
H
N
H
67% conversion
71% conversion
54% isolated yield
TTN: 106
12:1 Cyc:Hydro
16d*
69% conversion
49% isolated yield
TTN: 103
8:1 Cyc:hydro
16c*
68% conversion
Not Isolated
7:1 Cyc:Hydro
Cryptophycin 51
49% isolated yield
TTN: 100
11:1 Cyc:Hydrob
16h*
Reaction Conditions: 75 uM substrate, 0.5 uM Crp TE, 7% DMSO, 100 mM NaPO4 Buffer, 150 mM NaCl, pH 7.2, 30 ºC, 10 h
aTTN = moles of product / moles of TE, bnumbers estimated from extrapolated peaks due to co-elution of starting material and hydrolysis
peaks
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percent conversion than the previous six membered ring heter- Evaluation of CrpE with Newly Generated, Unit A Heter-
1
2
3
4
5
6
7
8
ocycles at 85% and 84%, with unreacted starting material ac-
counting for the lower conversion (Table 1, 16f and g). Inter-
estingly, incorporation of a 4-methyl pyrazole ring (15h),
showed nearly complete conversion to product with no measur-
able starting substrate or hydrolytic byproducts.
ocyclic Analogs. Following the generation of nine new crypto-
phycin unit A analogs 16b-j with the CrpTE, we next sought to
investigate their binding and conversion to -epoxy derivatives
using the wild type CrpE P450. Initial characterization of this
enzyme was performed using a CrpE-MBP fusion protein23
however, for this study, an optimized, histidine tagged form of
CrpE was generated (Supplemental Figure X). This new variant
was recharacterized utilizing the native substrate cryptophycin
3 (16a) which demonstrated a Soret band maximum at 390–394
nm resulting in a high‐spin (HS) state (the so‐called type I
shift).44 A titration of this substrate gave a Kd value of 0.53 ±
0.15 M, lower than that of the MBP-fusion previously charac-
terized (Figure 3A). Next, Kd values were calculated for ana-
logs 16b-j to determine each compounds viability as substrates.
All but one analog, 16d, displayed characteristic type I binding,
with varied Kd values. The binding of the 4-pyridyl containing
cryptophycin 16d displayed the opposite spectrum with a max-
imum at 430 nm and a negative minimum at 415 nm (the so‐
called type II shift) suggesting the formation of a six‐coordi-
nated complex with the ligand, generally considered as a P450
inhibitor (Figure 3B).45
Incorporating a larger alkyl chain, the 4-isopropyl pyrazole
group (Figure 2, 16i) provided important insight into potential
size restrictions of the CrpTE binding pocket. Although hydro-
lytic products were not observed, incomplete consumption of
substrate after conclusion of the reaction produced a lower per-
cent conversion (83%) compared to its methyl counterpart,
which showed almost quantitative conversion to product. Alt-
hough the enzyme showed a slight preference for the smaller
alkyl substituent overall conversion observed with this analog
rivals the native indicating the preference is not substantial. Fi-
nally, a dimethyl isoxazole substrate 15j was investigated. This
compound showed similar percent conversion and TTN com-
pared with native substrate (Table 1, 16j) with a significant
amount of starting substrate remaining.
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Isolation and Characterization of Unit A Cryptophycin
Analogs. All reactions were conducted on semi-preparative
scale (using the same conditions as the analytical reactions) of
10 mg in order to obtain isolated yields, full structural charac-
terization, and for subsequent CrpE analysis as well as biologi-
cal evaluation. These results corresponded closely with percent
conversions observed in the analytical reactions. The six mem-
bered heterocycles 16b-e were isolated in good yields from 62
– 66% (Table 1). The five membered rings 16f-j were also con-
ducted on 10 mg scale and isolated in yields varying from 55%
- 69% (Table 1). All novel cryptophycin analogs generated
from these chemoenzymatic reactions were confirmed by
HRMS, 1H NMR, and 13C NMR.
With the knowledge that eight of the new analogs bind as po-
tential substrates, we sought to conduct an in vitro conversion
assay. In the absence of autologous redox partners of CrpE from
Nostoc. sp. ATCC 53789, we investigated two separate surro-
gate systems: the RhFRED system previously described46 as
well as spinach ferredoxin and ferredoxin reductase for in vitro
reconstitution. These initial tests demonstrated no conversion
with the RhFRED system however, significant conversion
(38%) was observed with the spinach redox system on crypto-
phycin 3 (Figure 3A, 16a).
Analysis of CrpE P450 with the heterocyclic cryptophycin
analogs resulted in distinct epoxidized products with varying
A. CrpE P450 Reaction
B. CrpE Spectral Shift
O
O
O
Fdr
Fdx
Ar
Ar
CrpE
O
O
O
HN
Cl
O
O
O
HN
Cl
O
O
O
OMe
O
OMe
N
H
N
H
18a-j
16a-j
%
%
Kd [µM]
Kd [µM]
Entry
Entry
Conversion
Conversion
N
a.
f.
N
39%
18%
1.30 ± 0.10
0.53 ± 0.15
N
N
b.
c.
d.
g.
N
1.10 ± 0.11
Trace
18%☨
Trace☨
Trace
NC
28%
7%
2.20 ± 0.70
1.40 ± 0.22
N
N
N
h.
i.
9.27 ± 1.50
N
N
N
3.48 ± 0.70
1.48 ± 0.21
3.59 ± 0.55
1.27 ± 0.26
N
N
O
e.
j.
29%
N
Reaction Conditions: 50 uM substrate, 0.5 uM Crp E, spinach Fdx (10 µM), FdR (1.5 µM) glucose-6-phosphate (5 mM)
and glucose-6-phosphate dehydrogenase (1 U) in 20 mM potassium phosphate buffer, pH 7.4
☨
Figure 3. A. CrpE epoxidation reaction with percent conversion and Kd values for CrpE epoxidation depicted, indicates [Mepoxida-
tion+H2O] B. Spectral shifts induced by binding of A) Crp3 and B) 4-pyridyl Crp to the P450 CrpE. CrpE displays a type I shift with a
peak maximum at 395 nm and a minimum at 425 nm (A), whereas for 4-pyridyl Crp the peak maximum and minimum were 430 and 405,
respectively, representing the type II shift.
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percent conversions. Substantial turnover to peaks correspond- engineering efforts could afford epoxidized products in high
1
2
3
4
5
6
7
8
ing to the epoxidized product were observed with the 3-pyridyl,
pyrazine, and 2-methyl pyrazole cryptophycins with varying
percent conversions (Figure 3A, 16c, e, and f) and trace con-
version seen with the 3-pyridyl and isoxazole containing sub-
strates (Figure 3C, 16g and 16j). Interestingly, despite the 4-
pyridyl analog (16d) displaying a type II shift (Kd = 3.50 µM),
a small amount of epoxidized product was observed indicating
that some of the type II ligand can also act as a substrate.47 The
4-methyl pyrazole substrate (h) also displayed significant turn-
over, although to the presumed diol product (epoxidized mass
plus water) that appears to be produced upon the mechanical
stress of the HPLC. In order to confirm this result chemical
epoxidation was attempted using mCPBA. The crude reaction
product appeared to contain a mixture of the desired  and 
epoxide product that upon HPLC purification decomposed to
the diol products. This same decomposition product is observed
in trace quantities with the isopropyl-pyrazole (16i) substrate as
well.
yield for a variety of substrates that alter the double bond elec-
tronics.
Biological Evaluation of Novel Styrene Cryptophycin An-
alogs. Each of the cryptophycin analogs were initially assessed
using an in vitro disk diffusion assay which examines the dif-
ferential cell killing as zones over a variety of both murine and
human solid tumor cell lines and leukemia.48 This data indicates
these are all very potent in both solid tumors and leukemia cell
lines, with the isoxazole containing heterocycle (16j) being the
clear outlier in terms of potency over all cell lines tested (Table
S1). Next, each analog was assessed for half minimal inhibitory
concentration (IC50) values in HCT-116 human colorectal can-
cer cell line. The potency of the initial monomethyl unit C ana-
logs displayed significant variability in activities depending on
the heterocyclic ring present (Table 2). For the cryptophycin
analog bearing six membered ring heterocycles, the IC50 values
vary dramatically with the seemingly small change of nitrogen
migration around the ring. Within the pyridyl set of analogs, the
2-pyridyl is the least potent with an IC50 of 102 nM, which is
over two orders of magnitude higher than the 3-pyridyl (16c)
and 4-pyridyl (16d) containing analog (0.860 nM and 0.51 nM,
Table 2). Cryptophycin analog bearing five membered rings
show even larger differences in IC50 values spanning over 5 or-
ders of magnitude. The inclusion of an isoxazole ring (16j)
greatly diminished activity with an IC50 value of 1.4 µM. Most
notably, the introduction of a 4-methyl pyrazole ring (16h) pro-
vided an analog with single digit picomolar activity, making it
one of the most potent cryptophycins known to date. In a direct
comparison, our des-epoxy analog displays lower IC50 values
than that of the clinically evaluated cryptophycin 52 (as well as
the glycinate pro drug ester cryptophycin 55, Table 2) in this
cell line. Interestingly, the previous SAR of these compounds
demonstrated the importance of the -epoxide moiety to
achieve low picomolar activity, however incorporation of a 4-
methyl pyrazole ring maintains this activity in the absence of an
epoxide. All substrates tested against CrpE P450 (Figure 3)
were conducted on analytical scale and biological evaluation of
products was not conducted.
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Closer analysis of the correlation between binding affinity
and percent conversion give insight into the biochemical basis
for the differences in reactivities displayed. Predominantly,
higher binding affinity appears to correlate with higher percent
conversion, however this appears to break down in some in-
stances. The 3-methyl pyrazole analog 16g displays a similar Kd
to that of the 2-methyl pyrazole, pyrazine, and 3-pyridyl ana-
logs. These three compounds showed the highest turnover how-
ever, only trace product could be detected for the 3-methyl py-
razole despite a similar Kd value. The opposite is observed with
the 4-methyl pyridyl analog, in which its binding affinity is the
lowest of the set however its percent conversion is similar to
those with the lower binding affinity. Substrate binding alone
does not appear to confer activity indicating either electronics
or double bond geometry plays a crucial role. Despite the over-
all low turnover displayed with this enzyme, product peaks are
observed for the majority of the analogs indicating a more ap-
propriate redox partner in conjunction with possible
Table 2. IC50 valuesa for cryptophycin analogs in HCT-116
human colorectal carcinoma
Chemoenzymatic Synthesis and Biological Evaluation of
Gem-dimethyl Analogs. Utilizing the above data to guide our
design of analogs to test with the CrpTE biocatalyst, the gem-
dimethyl unit C analogs were synthesized utilizing the chemis-
try described above for our top three analogs: unit A bearing a
terminal 3-pyridyl, 4-pyridyl or 4-methyl pyrazole. The ester
linkage between units C and D is known to be metabolically
unstable and addition of a second methyl group adjacent to this
labile position was utilized in the case of the clinically utilized
cryptophycin 52 to improve drug half-life.49, 50 In our previous
work, we showed there was an increase in hydrolytic byprod-
ucts (6:1 cyclization to hydrolysis versus 10:1) with the gem-
dimethyl (unit C) present, and the native benzyl containing unit
A.22 Our new analogs (15c*, d*, and h*) were first tested on an
analytical scale for direct comparison of hydrolysis:cyclization
ratios as well as percent conversion to the monomethyl counter-
parts. All three showed an increase in hydrolytic activity when
incubated with CrpTE (Figure 2 and Table 1) as well as a
higher percentage of unreacted substrate, consistent with our
previous findings. Despite the lower overall conversion to mac-
rocycle, the corresponding chain elongation intermediates
showed similar TTNs to that of the native substrate, further
demonstrating the remarkable flexibility of CrpTE against
IC50 [nM]
HCT - 116
IC50 [nM]
HCT - 116
Unit C
Unit A
Unit C
Unit A
N
R1 = CH3
R2 = H
e.
f.
Crp 52
10.2
6.3
0.034
N
N
R1 = CH3
R2 = H
Crp 55
0.067
102
N
N
N
R1 = CH3
R2 = H
R1 = CH3
R2 = H
b.
c.
84
f.
N
N
N
R1 = CH3
R2 = H
R1 = CH3
R2 = H
N
0.008
h.
h.*
i.
0.86
2.06
0.50
N
R1 = CH3
R2 = CH3
R1 = CH3
R2 = CH3
N
c.*
d.
0.94
6.3
N
R1 = CH3
R2 = H
R1 = CH3
R2 = H
N
N
N
N
R1 = CH3
R2 = CH3
R1 = CH3
R2 = H
j.
1400
d.*
1.16
O
N
a IC50 values represented as an average of two replicates
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ACS Chemical Biology
1.
Weissman, K. J., Genetic engineering of modular PKSs:
substrates containing non-native functional groups in both the
1
2
3
4
5
6
7
8
from combinatorial biosynthesis to synthetic biology. Nat Prod
Rep 2016, 33, 203-230.
PKS and NRPS derived portions of the molecule.
These dimethyl cryptophycin analogs were evaluated for bi-
ological activity using both the in vitro disk diffusion assay de-
scribed above and IC50 determination in the HCT-116 cell line.
Incorporation of the gem-dimethyl moiety resulted in attenu-
ated potency for all three analogs in this cell line, indicating al-
ternative methods for blocking metabolism at this site is not uni-
versally tolerated and alternative methods could be explored.
2.
Walsh, C. T., Insights into the chemical logic and
enzymatic machinery of NRPS assembly lines. Nat Prod Rep
2016, 33, 127-135.
3.
Driggers, E. M.; Hale, S. P.; Lee, J.; Terrett, N. K.,
The exploration of macrocycles for drug discovery - an
underexploited structural class. Nat. Rev. Drug Discov. 2008, 7,
608-624.
4.
Horsman, M. E.; Hari, T. P.; Boddy, C. N., Polyketide
Conclusion. In this work we have described the synthesis,
biocatalytic cyclization and epoxidation of a library of crypto-
phycin analogs. This work has demonstrated the utility of these
enzymes as standalone biocatalysts for production of a new set
of unit A variants of these potent tubulin-binding compounds.
Furthermore, we have demonstrated the viability of this biocat-
alytic strategy as a useful addition to the medicinal chemistry
toolbox. Biological evaluation of the newly generated analogs
has enabled identification of one of the most potent cryptophy-
cin analogs to date, which contains a styrene functionality and
obviates the need for the -epoxide group to achieve low
picomolar potency. The valuable insights regarding selectivity
and specificity of CrpTE and CrpE P450 toward unnatural sub-
strates will facilitate future efforts to engineer more robust and
broader scope biocatalysts. These enzymes are especially at-
tractive for mediating late-stage assembly of complex chain
elongation intermediates derived through synthetic chemistry,
or as a part of larger, enzymatic cascade for the production of
complex bioactive molecules.
9
synthase and non-ribosomal peptide synthetase thioesterase
selectivity: logic gate or a victim of fate? Nat Prod Rep 2016, 33,
183-202.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5.
Kohli, R. M.; Walsh, C. T., Enzymology of acyl chain
macrocyclization in natural product biosynthesis. ChemComm
2003, 7, 297-307.
6.
Magarvey, N. A.; Beck, Z. Q.; Golakoti, T.; Ding, Y.;
Huber, U.; Hemscheidt, T. K.; Abelson, D.; Moore, R. E.;
Sherman, D. H., Biosynthetic characterization and
chemoenzymatic assembly of the cryptophycins. Potent anticancer
agents from cyanobionts. ACS Chem Biol 2006, 1, 766-779.
7.
Ding, Y.; Rath, C. M.; Bolduc, K. L.; Hakansson, K.;
Sherman, D. H., Chemoenzymatic synthesis of cryptophycin
anticancer agents by an ester bond-forming non-ribosomal peptide
synthetase module. J Am Chem Soc 2011, 133, 14492-14495.
8.
Schwartz, R. E.; Hirsch, C. F.; Sesin, D. F.; Flor, J. E.;
Chartrain, M.; Fromtling, R. E.; Harris, G. H.; Salvatore, M. J.;
Liesch, J. M.; Yudin, K., Pharmaceuticals from Cultured Algae. J
Ind Microbiol 1990, 5, 113-123.
9.
Smith, C. D.; Zhang, X. Q.; Mooberry, S. L.;
Patterson, G. M. L.; Moore, R. E., Cryptophycin - a New
Antimicrotubule Agent Active against Drug-Resistant Cells.
Cancer Res 1994, 54, 3779-3784.
ASSOCIATED CONTENT
Supporting Information
10.
D'Agostino, G.; del Campo, J.; Mellado, B.;
Izquierdo, M. A.; Minarik, T.; Cirri, L.; Marini, L.; Perez-
Gracia, J. L.; Scambia, G., A multicenter phase II study of the
cryptophycin analog LY355703 in patients with platinum-
resistant ovarian cancer. Int J Gynecol Cancer 2006, 16, 71-76.
The Supporting Information is available free of charge on the ACS
Publications website at DOI:
Full synthetic and experimental details, NMR spectra, tables, and
figures.
11.
Stevenson, J. P.; Sun, W.; Gallagher, M.; Johnson, R.;
Vaughn, D.; Schuchter, L.; Algazy, K.; Hahn, S.; Enas, N.;
Ellis, D.; Thornton, D.; O'Dwyer, P. J., Phase I trial of the
cryptophycin analogue LY355703 administered as an intravenous
infusion on a day 1 and 8 schedule every 21 days. Clin Cancer
Res 2002, 8, 2524-2529.
AUTHOR INFORMATION
Corresponding Author
* davidhs@umich.edu
12.
Sessa, C.; Weigang-Kohler, K.; Pagani, O.; Greim,
G.; Mora, O.; De Pas, T.; Burgess, M.; Weimer, I.; Johnson, R.,
Phase I and pharmacological studies of the cryptophycin analogue
LY355703 administered on a single intermittent or weekly
schedule. Eur J Cancer 2002, 38, 2388-2396.
Present Addresses
Funding Sources
The authors thank the National Institutes of Health (R35
GM118101) and the Hans W. Vahlteich Professorship for finan-
cial support.
13.
Edelman, M. J.; Gandara, D. R.; Hausner, P.; Israel,
V.; Thornton, D.; DeSanto, J.; Doyle, L. A., Phase 2 study of
cryptophycin 52 (LY355703) in patients previously treated with
platinum based chemotherapy for advanced non-small cell lung
cancer. Lung Cancer 2003, 39, 197-199.
Notes
14.
synthesis and anticancer activity. Med Res Rev 2002, 22, 85-101.
15. Nahrwold, M.; Weiss, C.; Bogner, T.; Mertink, F.;
Eggen, M.; Georg, G. I., The cryptophycins: their
The authors declare no competing financial interest.
ACKNOWLEDGMENT
Conradi, J.; Sammet, B.; Palmisano, R.; Royo Gracia, S.;
Preusse, T.; Sewald, N., Conjugates of modified cryptophycins
and RGD-peptides enter target cells by endocytosis. J. Med.
Chem. 2013, 56, 1853-1864.
The authors would like to thank K. Bolduc and A. N. Lowell for
helpful discussions and technical assistance with synthetic proce-
dures. We would also like to thank W. Feng for maintaining and
technical assistance with LCMS and NMR experiments.
16.
Leamon, C. P.; Wang, Y.; Vlahov, I. R.; You, F.;
Kleindl, P. J.; Santhapuram, H. K. R. Conjugates containing
hydrophilic spacer linkers. WO2009002993A1, 2008/12/31/,
2008.
REFERENCES
ACS Paragon Plus Environment

ACS Chemical Biology
Page 8 of 10
17.
Su, D.; Kozak, K. R.; Sadowsky, J.; Yu, S. F.;
Active Site Mutation in the Pikromycin Thioesterase Generates a
More Effective Macrocyclization Catalyst. J Am Chem Soc 2017,
139, 13456-13465.
1
2
3
4
5
6
Fourie-O'Donohue, A.; Nelson, C.; Vandlen, R.; Ohri, R.; Liu,
L. N.; Ng, C.; He, J. T.; Davis, H.; Lau, J.; Del Rosario, G.;
Cosino, E.; dela Cruz-Chuh, J.; Ma, Y.; Zhang, D. L.; Darwish,
M.; Cai, W. W.; Chen, C. J.; Zhou, H. X.; Lu, J. W.; Liu, Y.
C.; Kaur, S.; Xu, K. Y.; Pillow, T. H., Modulating Antibody-
Drug Conjugate Payload Metabolism by Conjugation Site and
Linker Modification. Bioconjug Chem 2018, 29, 1155-1167.
33.
Kohli, R. M.; Trauger, J. W.; Schwarzer, D.;
Marahiel, M. A.; Walsh, C. T., Generality of peptide cyclization
catalyzed by isolated thioesterase domains of nonribosomal
peptide synthetases. Biochemistry 2001, 40, 7099-7108.
34.
Trauger, J. W.; Kohli, R. M.; Walsh, C. T., Cyclization
7
8
9
18.
Verma, V. A.; Pillow, T. H.; DePalatis, L.; Li, G.;
of backbone-substituted peptides catalyzed by the thioesterase
domain from the tyrocidine nonribosomal peptide synthetase.
Biochemistry 2001, 40, 7092-7098.
Phillips, G. L.; Polson, A. G.; Raab, H. E.; Spencer, S.; Zheng,
B., The cryptophycins as potent payloads for antibody drug
conjugates. Bioorg. Med. Chem. Lett. 2015, 25, 864-868.
35.
Eissler, S.; Bogner, T.; Nahrwold, M.; Sewald, N.,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
19.
Bigot, A.; Bouchard, H.; Brun, M.-P.; Clerc, F.;
Efficient synthesis of cryptophycin-52 and novel para-
alkoxymethyl unit A analogues. Chemistry 2009, 15, 11273-87.
Zhang, J. Novel Cryptophycin Compounds and Conjugates, Their
Preparation and Their Therapeutic Use. WO2017076998 (A1),
2017/05/11/, 2017.
36.
Al-Awar, R. S.; Corbett, T. H.; Ray, J. E.; Polin, L.;
Kennedy, J. H.; Wagner, M. M.; Williams, D. C., Biological
20.
Steinkuhler, M. C.; Gallinari, M. P.; Osswald, B.;
evaluation of cryptophycin 52 fragment A analogues: effect of the
multidrug resistance ATP binding cassette transporters on
antitumor activity. Molecular cancer therapeutics 2004, 3, 1061-
7.
Sewald, N.; Ritzefeld, M.; Frese, M. Cryptophycin-Based
Antibody-Drug Conjugates with Novel Self-Immolative Linkers.
WO2016146638 (A1), 2016/09/22/, 2016.
21.
Bouchard, H.; Brun, M.-P.; Commercon, A.; Zhang, J.
37.
Al-Awar, R. S.; Ray, J. E.; Schultz, R. M.; Andis, S.
Novel Conjugates, Preparation Thereof, and Therapeutic Use
Thereof. WO2011001052 (A1), 2011/01/06/, 2011.
22.
G. I.; Sherman, D. H., Chemoenzymatic synthesis of
cryptophycin/arenastatin natural products. Biochemistry 2005, 44,
13457-13466.
L.; Kennedy, J. H.; Moore, R. E.; Liang, J.; Golakoti, T.;
Subbaraju, G. V.; Corbett, T. H., A convergent approach to
cryptophycin 52 analogues: synthesis and biological evaluation of
a novel series of fragment a epoxides and chlorohydrins. J Med
Chem 2003, 46, 2985-3007.
Beck, Z. Q.; Aldrich, C. C.; Magarvey, N. A.; Georg,
38.
deMuys, J. M.; Rej, R.; Nguyen, D.; Go, B.; Fortin,
23.
Ding, Y.; Seufert, W. H.; Beck, Z. Q.; Sherman, D. H.,
S.; Lavallee, J. F., Synthesis and in vitro cytotoxicity of
cryptophycins and related analogs. Bioorg. Med. Chem. Lett.
1996, 6, 1111-1116.
Analysis of the cryptophycin P450 epoxidase reveals substrate
tolerance and cooperativity. J Am Chem Soc 2008, 130, 5492-
5498.
39.
Bolduc, K. L.; Larsen, S. D.; Sherman, D. H., Efficient,
24.
Bruner, S. D.; Weber, T.; Kohli, R. M.; Schwarzer,
divergent synthesis of cryptophycin unit A analogues.
ChemComm 2012, 48, 6414-6416.
D.; Marahiel, M. A.; Walsh, C. T.; Stubbs, M. T., Structural
basis for the cyclization of the lipopeptide antibiotic surfactin by
the thioesterase domain SrfTE. Structure 2002, 10, 301-310.
40.
Krishnamurthy, S., Rapid Reduction of Alkyl Tosylates
with Lithium Triethylborohydride - Convenient and
Advantageous Procedure for Deoxygenation of Simple and
Hindered Alcohols - Comparison of Various Hydride Reagents. J
Organomet Chem 1978, 156, 171-181.
25.
Samel, S. A.; Wagner, B.; Marahiel, M. A.; Essen, L.
O., The thioesterase domain of the fengycin biosynthesis cluster:
A structural base for the macrocyclization of a non-ribosomal
lipopeptide. J. Mol. Biol. 2006, 359, 876-889.
41.
Ghosh, A. K.; Swanson, L., Enantioselective synthesis
26.
Kopp, F.; Grunewald, J.; Mahlert, C.; Marahiel, M. A.,
of (+)-cryptophycin 52 (LY355703), a potent antimitotic
antitumor agent. J Org Chem 2003, 68, 9823-9826.
Chemoenzymatic design of acidic lipopeptide hybrids: New
insights into the structure-activity relationship of daptomycin and
A54145. Biochemistry 2006, 45, 10474-10481.
42.
Mast, C. A.; Eissler, S.; Stoncius, A.; Stammler, H.
G.; Neumann, B.; Sewald, N., Efficient and versatile
stereoselective synthesis of cryptophycins. Chemistry 2005, 11,
4667-4677.
27.
Yeh, E.; Lin, H. N.; Clugston, S. L.; Kohli, R. M.;
Walsh, C. T., Enhanced macrocyclizing activity of the
thioesterase from tyrocidine synthetase in presence of nonionic
detergent. Chem Biol 2004, 11, 1573-1582.
43.
Nicolaou, K. C.; Estrada, A. A.; Zak, M.; Lee, S. H.;
Safina, B. S., A mild and selective method for the hydrolysis of
esters with trimethyltin hydroxide. Angew Chem Int Edit 2005,
44, 1378-1382.
28.
Trauger, J. W.; Kohli, R. M.; Mootz, H. D.; Marahiel,
M. A.; Walsh, C. T., Peptide cyclization catalysed by the
thioesterase domain of tyrocidine synthetase. Nature 2000, 407,
215-218.
44.
Denisov, I. G.; Makris, T. M.; Sligar, S. G.;
Schlichting, I., Structure and chemistry of cytochrome P450.
Chem Rev 2005, 105, 2253-2277.
29.
Pinto, A.; Wang, M.; Horsman, M.; Boddy, C. N., 6-
Deoxyerythronolide B Synthase Thioesterase-Catalyzed
Macrocyclization Is Highly Stereoselective. Org Lett 2012, 14,
2278-2281.
45.
Gigon, P. L.; Gram, T. E.; Gillette, J. R., Studies on the
rate of reduction of hepatic microsomal cytochrome P-450 by
reduced nicotinamide adenine dinucleotide phosphate: effect of
drug substrates. Mol Pharmacol 1969, 5, 109-122.
30.
Tsai, S. C.; Miercke, L. J. W.; Krucinski, J.; Gokhale,
R.; Chen, J. C. H.; Foster, P. G.; Cane, D. E.; Khosla, C.;
Stroud, R. M., Crystal structure of the macrocycle-forming
thioesterase domain of the erythromycin polyketide synthase:
Versatility from a unique substrate channel. Proc Natl Acad Sci
USA 2001, 98, 14808-14813.
46.
Li, S. Y.; Podust, L. M.; Sherman, D. H., Engineering
and analysis of a self-sufficient biosynthetic cytochrome P450
PikC fused to the RhFRED reductase domain. J Am Chem Soc
2007, 129, 12940-12941.
47.
Pearson, J. T.; Hill, J. J.; Swank, J.; Isoherranen, N.;
31.
Hansen, D. A.; Koch, A. A.; Sherman, D. H.,
Kunze, K. L.; Atkins, W. M., Surface plasmon resonance analysis
of antifungal azoles binding to CYP3A4 with kinetic resolution of
multiple binding orientations. Biochemistry 2006, 45, 6341-6353.
Identification of a Thioesterase Bottleneck in the Pikromycin
Pathway through Full-Module Processing of Unnatural
Pentaketides. J Am Chem Soc 2017, 139, 13450-13455.
48.
Valeriote, F. A.; Tenney, K.; Media, J.;
32.
Koch, A. A.; Hansen, D. A.; Shende, V. V.; Furan, L.
Pietraszkiewicz, H.; Edelstein, M.; Johnson, T. A.; Amagata, T.;
Crews, P., Discovery and development of anticancer agents from
R.; Houk, K. N.; Jimenez-Oses, G.; Sherman, D. H., A Single
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Page 9 of 10
ACS Chemical Biology
marine sponges: perspectives based on a chemistry-experimental
therapeutics collaborative program. J Exp Ther Oncol 2012, 10,
50.
Golakoti, T.; Ogino, J.; Heltzel, C. E.; LeHusebo, T.;
1
2
3
4
5
6
Jensen, C. M.; Larsen, L. K.; Patterson, G. M. L.; Moore, R. E.;
Mooberry, S. L.; Corbett, T. H.; Valeriote, F. A., Structure
determination, conformational analysis, chemical stability studies,
and antitumor evaluation of the cryptophycins. Isolation of 18
new analogs from Nostoc sp strain GSV 224. J Am Chem Soc
1996, 117, 12030-12049.
119-134.
49.
Wagner, M. M.; Paul, D. C.; Shih, C.; Jordan, M. A.;
Wilson, L.; Williams, D. C., In vitro pharmacology of
cryptophycin 52 (LY355703) in human tumor cell lines. Cancer
Chemother Pharmacol 1999, 43, 115-125.
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