OleD Loki as a Catalyst for Tertiary Amine and Hydroxamate Glycosylation

Article abstract of DOI:10.1002/cbic.201600676

We describe the ability of an engineered glycosyltransferase (OleD Loki) to catalyze the N-glycosylation of tertiary-amine-containing drugs and trichostatin hydroxamate glycosyl ester formation. As such, this study highlights the first bacterial model catalyst for tertiary-amine N-glycosylation and further expands the substrate scope and synthetic potential of engineered OleDs. In addition, this work could open the door to the discovery of similar capabilities among other permissive bacterial glycosyltransferases.

Full text of DOI:10.1002/cbic.201600676

DOI: 10.1002/cbic.201600676
Communications
OleD Loki as a Catalyst for Tertiary Amine and
Hydroxamate Glycosylation
Ryan R. Hughes,[a] Khaled A. Shaaban,[a] Jianjun Zhang,[a] Hongnan Cao,[b]
George N. Phillips, Jr.,[b] and Jon S. Thorson*[a]
We describe the ability of an engineered glycosyltransferase
(OleD Loki) to catalyze the N-glycosylation of tertiary-amine-
containing drugs and trichostatin hydroxamate glycosyl ester
formation. As such, this study highlights the first bacterial
model catalyst for tertiary-amine N-glycosylation and further
expands the substrate scope and synthetic potential of engi-
neered OleDs. In addition, this work could open the door to
the discovery of similar capabilities among other permissive
bacterial glycosyltransferases.
tial of enhanced OleDs and presents a complement to conven-
tional synthetic strategies to access quaternary amino-N-glyco-
sides.[11]
Inspired by the general reversibility of GT-catalyzed reac-
tions,[12] we recently used simple aromatic glycosides as effi-
cient donors in GT-catalyzed sugar nucleotide synthesis and
coupled transglycosylation reactions.[9a,13] Importantly, the use
of 2-chloro-4-nitrophenyl (ClNP) glycoside donors in this con-
text also offered a convenient colorimetric screen to enable
the directed evolution of enhanced GTs with broad substrate
permissivity and the identification of new GT substrates (Fig-
ure 1A).[9a] Using ClNP-b-d-Glc and the OleD variant Loki,[9a] this
colorimetric assay was applied to a panel of 28 representative
aliphatic tertiary-amine-containing drugs (Figure S1 the Sup-
porting Information). Assays [1 mm putative acceptor, 2 mm
ClNP-b-d-Glc, 0.1 mm UDP, 25 mm Tris (pH 8.0), 5 mm MgCl2,
0.25 mm OleD Loki, 20 mL total volume, 308C, 8 h] were con-
ducted in triplicate in 384-well plates, and progress was moni-
tored through the change in absorbance at 410 nm (DA410).
Each plate also contained positive (4-methylumbeliferone; 4-
MeUmb)[14] and negative (no acceptor, DMSO) comparator con-
trols. Nine putative primary hits were identified (DA410 >2 stan-
dard deviations above the negative control) in this first-phase
screen. Reaction mixtures identified as hits were subsequently
subjected to HPLC, desalted,[15] and analyzed by LC-MS to con-
firm or refute glycoside formation. This streamlined strategy re-
vealed eight tertiary amines as validated OleD substrates (Fig-
ure 1B), none of which contained prototypical GT acceptor O-,
S-, or N-nucleophiles. To elucidate the nature of the glycosides
formed, substrates 1 and 2 were selected for subsequent
scale-up and full structure elucidation based on relative turn-
over in analytical scale reactions (Figure 1C).
Glycosyltransferases (GTs), among which sugar nucleotide-de-
pendent GTs (also referred to as Leloir GTs) are the most preva-
lent, mediate the regio- and stereospecific glycoconjugation of
diverse sugars to a broad range of acceptors.[1] Although Leloir
GT-catalyzed formation of O-glycosides is the most common,
corresponding microbial GTs involved in the biosynthesis of
C-,[2] S-,[3] and N-glycosides have also been characterized. Bio-
chemically characterized representative native N-GTs include
the indole N-modifying GTs involved in the biosynthesis of in-
dolocarbazoles (AtmG, RebG, NokL, and StaG),[4] the strepto-
thricin guanidino N-modifying GT StnG[5] and bacterial protein/
ansamitocin asparagine side chain amide-modifying N-GTs
(HMWC, NGT, and Asm25).[6] The gene encoding the putative
mannopeptimycin guanidino N-modifying GT has also been
reported,[7] and recent studies of the macrolide-inactivating
O-GT OleD,[8] and corresponding engineered/evolved variants,
revealed OleD-catalyzed N-glucosylation of model aromatic/
benzyl primary and secondary amines and alkoxyamines.[9] Al-
though examples of tertiary-amine N-glucuronidation of drugs
have been reported as part of phase II metabolism,[10] to the
best of our knowledge, bacterial/fungal comparators are un-
precedented. Within this context, herein we describe the dis-
covery that engineered OleDs can catalyze the N-glycosylation
of a set of model tertiary-amine-containing drugs and the for-
mation of hydroxamate glycosyl esters. As such, the work put
forth further expands the substrate scope and synthetic poten-
Scaled-up reactions for the chemoenzymatic synthesis of the
1 and 2 glucosides were conducted in a total volume of 30 mL
[1 mm aglycon, 2 mm ClNP-b-d-Glc, 0.1 mm UDP, 25 mm Tris
(pH 8.0), 5 mm MgCl2, 0.25 mm OleD Loki, 308C, 24 h]. Reaction
progress was monitored in real time through DA410, and upon
completion, reactants and products were captured by XAD-16
solid-phase extraction, and the resulting glucosides were sub-
sequently purified by HPLC and size-exclusion chromatogra-
phy. The molecular formulae of the corresponding products
were established as C23H30ClN2O5S and C25H34ClN2O5 by HR-
ESIMS; these are consistent with the glucosylation of 1 and 2,
[a] R. R. Hughes, Dr. K. A. Shaaban, Dr. J. Zhang, Prof. J. S. Thorson
Center for Pharmaceutical Research and Innovation
College of Pharmacy, University of Kentucky
789 South Limestone Street, Lexington, KY 40536 (USA)
E-mail: jsthorson@uky.edu
[b] Dr. H. Cao, Prof. G. N. Phillips, Jr.
1
respectively. Interestingly, H and 13C NMR spectra of 1a and
Department of Chemistry, Rice University
P.O. Box 1892, MS 60, Houston, TX 77251 (USA)
2a also revealed signatures that are consistent with atypical
glucoside formation. Closer analysis revealed 3’-CH2, 4’-CH3,
and 5’-CH3 1H and 13C NMR chemical shifts and clear HMBC cor-
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/cbic.201600676.
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Figure 1. A) Schematic of colorimetric high-throughput screen. B) Tertiary-amine-containing pharmacophores identified as putative OleD Loki substrates:
chlorpromazine (1), clomipramine (2), cyclobenzaprine (3), triflupromazine (4), imipramine (5), amitriptyline (6), doxepin (7), and diltiazem (8). C) Reaction
progress as monitored by DA410 in the standard 50 mL assay format [see (A) and main text]. Vehicle without acceptor served as the negative control, and
4-MeUmb served as the positive control. Assays were conducted in triplicate with less than 5% error between replicates.
relations that were consistent with quaternary aminogluco-
sides 3’-N-b-d-glucosylchlorpromazine (1a) and 3’-N-b-d-gluco-
sylclomipramine (2a; Scheme 1, Tables S1 and S2). Additional
COSY and HMBC correlations (Figure S5) were also consistent
with this assignment.
The discovery that OleD Loki could catalyze tertiary-amine
N-glucosylation prompted a reassessment of additional terti-
ary-amine-bearing substrates previously identified as OleD Loki
substrates—the naturally occurring HDAC inhibitor trichosta-
tin A (9);[16] the c-Raf inhibitor ZM 336372 (10)[17] and the mac-
Scheme 1. Key HMBC and COSY correlations for compounds 1a and 2a.
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rolide antibiotic timulcosin (11; Scheme 2)[18]—through colori-
metric screening and LC-MS.[9a] For these selected targets, the
strategies employed for reaction scale-up, product isolation,
and characterization paralleled those described for 1a and 2a.
The molecular formulae of the purified glycosides were estab-
lished by HR-ESIMS to be C23H32N2O8, C29H33N3O8, and
C52H90N2O18; these are consistent with the glucosylation of 9,
10, and 11, respectively. Unlike for 1a and 2a, the 1H and
13C NMR spectra of the isolated glucosides revealed signatures
that were consistent with O-glucoside formation. Consistent
with this, 1D and 2D NMR data revealed 9a to be the unique
glucopyranosyl hydroxamate trichostatin C and notably high-
light the first one-step synthesis of this previously reported
rare natural product.[19] In a similar fashion, the 1D and 2D
NMR data revealed 10a to be 4-O-b-d-glucosyl-ZM336372 and
11 a to be 2’-O-b-d-glucosyltilmicosin, the latter is consistent
with the native activity of wtOleD as a macrolide-inactivating
glucosyltransferase.[8b]
To the best of our knowledge, this study highlights the first
reported example of a microbial GT that is capable of catalyz-
ing tertiary-amine N-glycosylation. A comparison of the kinetic
parameters reveals that the catalytic competencies of tertiary
amine 1 (kcat/Km =2.2ꢁ10À4 mmÀ1 sÀ1) and hydroxamate 9 (kcat/
Km =1.4ꢁ10À4 mmÀ1 sÀ1) rival that of the parental OleD Loki
acceptor 4-MeUmb (kcat/Km =2.2ꢁ10À4 mmÀ1 À1). Although ter-
s
tiary-amine N-glucuronidation has been reported in the con-
text of phase II metabolism,[10] fundamental biochemical study
of the corresponding glucuronsyltransferases has been ham-
pered by limited access to suitable in vitro models. Likewise,
although sugar conjugation is known to influence small-mole-
cule mechanisms, potency, and ADMET,[1a,e,20] a lack of practical
synthetic or chemoenzymatic access has limited studies to
probe the fundamental properties of quaternary N-glyco-
sides.[10,11] Our discovery offers a convenient new model for GT-
catalyzed tertiary-amine N-glycosylation and a potential com-
plementary synthetic platform for efficient one-step synthesis
of quaternary N-glycosides. In a similar fashion, the discovery
that OleD Loki is an efficient catalyst for hydroxamate glycosyl
ester synthesis further extends the demonstrated synthetic util-
ity of this permissive catalyst. Analysis of the established sub-
strates 1–8 highlights a common alkyl N,N-dimethyl acceptor
nucleophile that extends from a hydrophobic aromatic core
reminiscent of the longer unsaturated spacer separating the 9
pharmacophore and warhead or the previously reported ability
of OleD ASP to catalyze asymmetric 4’-O-glycosylation of one
“arm” of mitoxantrone.[21] The lack of detectable tertiary-amine
N-glycosylation with substrates 9–11 may be attributed to sub-
strate orientation in the enzyme-bound complex (as suggested
by the determined OleD:macrolide complex, Figure 2) and/or
poor nucleophilicity (as anticipated in the context of the N,N-
dimethyl aniline moiety of 9 and 10).[22] Although these exam-
ples suggest that certain substrate specificity features might
infringe on OleD Loki-catalyzed tertiary-amine reactant scope,
the underlying discovery also importantly implicates N,N-dime-
thylamino- and/or hydroxamate-functionalized small molecules
as a potential acceptors to evaluate in the context of other
permissive GTs.[23]
Acknowledgements
This work was supported, in part, by National Institute of Health
grants R37 AI52218 (J.S.T.) and U01 GM098248 (G.N.P.), the Na-
Scheme 2. Additional tertiary-amine-containing OleD substrates trichostatin
A (9), ZM 336372 (10), and tilmicosin (11) and their products. Tertiary amines
within 9–11 are shaded yellow, and key HMBC and COSY correlations are
highlighted.
tional
Center
for
Advancing
Translational
Sciences
(UL1TR001998), the University of Kentucky College of Pharmacy
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Keywords: antidepressants
·
drug
metabolism
·
glucuronidation · glycosyltransferases · trichostatin
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Manuscript received: December 16, 2016
Final Article published: && &&, 0000
ChemBioChem 2017, 18, 1 – 6
www.chembiochem.org
5
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

COMMUNICATIONS
R. R. Hughes, K. A. Shaaban, J. Zhang,
H. Cao, G. N. Phillips, Jr., J. S. Thorson*
Bacterial model catalyst: The use of
a high-throughput colorimetric screen
to identify new substrates revealed the
engineered macrolide glycosyltransfer-
ase OleD Loki as the first reported bac-
terial glycosyltransferase capable of ter-
tiary-amine N-glycosylation. This study
also revealed the same enzyme to cata-
lyze trichostatin hydroxamate glycosyl
ester formation.
&& – &&
OleD Loki as a Catalyst for Tertiary
Amine and Hydroxamate
Glycosylation
&
ChemBioChem 2017, 18, 1 – 6
www.chembiochem.org
6
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!