Evidence That Trimethyllysine Hydroxylase Catalyzes the Formation of (2S,3S)-3-Hydroxy-Nε-trimethyllysine

Article abstract of DOI:10.1021/acs.orglett.6b03608

Trimethyllysine hydroxylase (TMLH) is an Fe(II) and 2-oxoglutarate (2OG) dependent oxygenase involved in the biomedically important carnitine biosynthesis pathway. A combination of synthetic and NMR studies provides direct evidence that human TMLH catalyzes the stereoselective conversion of (2S)-N^ε-trimethyllysine to (2S,3S)-3-hydroxy-N^ε-trimethyllysine.

Full text of DOI:10.1021/acs.orglett.6b03608

Letter
pubs.acs.org/OrgLett
Evidence That Trimethyllysine Hydroxylase Catalyzes the Formation
of (2S,3S)‑3-Hydroxy‑Nε‑trimethyllysine
Y. Vijayendar Reddy, Abbas H. K. Al Temimi, Paul B. White, and Jasmin Mecinovic*
́
Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands
S
* Supporting Information
ABSTRACT: Trimethyllysine hydroxylase (TMLH) is an Fe(II) and
2-oxoglutarate (2OG) dependent oxygenase involved in the biomedi-
cally important carnitine biosynthesis pathway. A combination of
synthetic and NMR studies provides direct evidence that human
TMLH catalyzes the stereoselective conversion of (2S)-Nε-trimethylly-
sine to (2S,3S)-3-hydroxy-Nε-trimethyllysine.
nzymes have time and again served as an immense source
TMLH ultimately confirmed that the enzymatic hydroxylation
Eof inspiration for organic chemists, most notably in the occurs at the C-3 site of (2S)-Nε-trimethyllysine substrate;
rational design of small molecular systems that catalyze
challenging chemical transformations.1 In the past decade, a
special effort has been made in developing bioinspired, selective
transformations of highly abundant and inert C−H bonds.2
Members of Fe(II) and 2-oxoglutarate-dependent oxygenases
catalyze stereoselective hydroxylations of unactivated C−H
bonds in various (bio)molecules, including proteins, DNA, and
small molecule metabolites.3 Pioneering biochemical studies on
trimethyllysine hydroxylase (TMLH), an Fe(II)/2OG oxygen-
ase involved in the first step of physiologically important
carnitine biosynthesis in humans, revealed that the enzyme
catalyzes C-3 hydroxylation of (2S)-Nε-trimethyllysine (L-
trimethyllysine, 1) to produce 3-hydroxy-Nε-L-trimethyllysine
(2), which then undergoes three additional enzymatic steps to
the final L-carnitine (Scheme 1).4 Recent advanced NMR
spectroscopic studies on recombinantly expressed human
however, such analyses did not unravel the stereochemistry of
the hydroxy group at the C-3 position of the product,
presumably due to the freely rotatable C2−C3 single bond.5
Due to the intrinsic nature of enzymes to efficiently catalyze
transformations of molecules in a highly stereoselective fashion,
it is likely that TMLH-catalyzed C-3 hydroxylation of (2S)-Nε-
trimethyllysine leads to only one product (i.e., 3R or 3S). In
this regard, it is noteworthy that the final step in carnitine
biosynthesis is catalyzed by γ-butyrobetaine hydroxylase
(BBOX), another Fe(II)/2OG oxygenase, that specifically
produces enantiopure L-carnitine (Scheme 1).6
In order to unquestionably assign the stereochemistry of the
TMLH-catalyzed formation of 3-hydroxy-Nε-L-trimethyllysine,
it is essential to carry out comparative NMR spectroscopic
studies on the enzymatically produced (2S)-3-hydroxy-Nε-
trimethyllysine and the two enantiopure synthetic standards
that possess 3R and 3S stereochemistry, respectively. Herein,
we report concise syntheses of enantiopure (2S,3R)-3-hydroxy-
Nε-trimethyllysine (2a) and (2S,3S)-3-hydroxy-Nε-trimethylly-
sine (2b) that enabled full elucidation of the stereochemistry at
the C-3 site of the TMLH-catalyzed formation of the 3-
hydroxy-Nε-trimethyllysine product (Scheme 1). Our studies
provide direct evidence that TMLH exclusively catalyzes the
formation of (2S,3S)-3-hydroxy-Nε-trimethyllysine, not forming
any (2S,3R)-3-hydroxy-Nε-trimethyllysine diastereoisomer.
We conceived that both diastereoisomers of (2S)-3-hydroxy-
Nε-trimethyllysine could be synthesized from readily available
Garner’s aldehyde 3 via a sequential set of transformations
(Scheme 2 and Supporting Information).7 The enantiopure
alkyne 4 was prepared using a literature protocol by metal-
mediated diastereoselective propargylation of Garner’s alde-
hyde followed by benzyl protection of the newly formed
alcohol in good yield.8 To incorporate the dimethyl group on
Scheme 1. Role of Trimethyllysine Hydroxylase (TMLH) in
a
the Carnitine Biosynthesis Pathway
a
Structures of two potential TMLH-catalyzed products 2a and 2b are
Received: December 2, 2016
shown on the right.
© XXXX American Chemical Society
A
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Scheme 2. Synthesis of (2S,3R)-3-Hydroxy-Nε-
trimethyllysine (2a)
Scheme 3. Synthesis of (2S,3S)-3-Hydroxy-Nε-
trimethyllysine (2b)
the side chain, the TBS protecting group on the primary
alcohol was cleaved by TBAF in THF. The resulting alcohol 5
was then treated with MsCl and Et3N in CH2Cl2 to deliver the
sulfonate, thus setting the stage for incorporation of
dimethylamino functionality. The SN2 attack on the mesylate
by (Me)2NH afforded compound 6 in very good yield. In order
to generate the required carboxylic acid functionality at the C-1
position of the final (2S,3R)-3-hydroxy-Nε-trimethyllysine, the
1,2-acetonide protecting group was cleaved by pTSA in CH2Cl2
to yield the enantio- and diastereopure alcohol 7 (>96%, based
on the 19F NMR analysis of the corresponding Mosher’s ester,
Figure S1). Alcohol 7 was transformed to a carboxylic acid in
the presence of BIAB and TEMPO in a mixture of CH3CN and
H2O. Subsequently, the formed crude acid was converted to
tert-butyl ester 9 (no racemization at the Cα was observed) in
the presence of 2-tert-butyl-1,3-diisopropylisourea 8 in CH2Cl2
at 45 °C for 5 h. Finally, a simultaneous one-step reduction of
the alkyne and deprotection of the benzyl group of the alcohol
was achieved by hydrogenation with Pd−C in MeOH for 24 h.
The treatment of the resulting Nα-Boc protected (2S,3R)-3-
hydroxy-Nε-dimethyllysine with MeI under basic conditions
followed by acid-promoted hydrolysis afforded the targeted
diastereopure product (2S,3R)-3-hydroxy-Nε-trimethyllysine
(2a).
After successfully accomplishing the stereoselective synthesis
of (2S,3R)-3-hydroxy-Nε-trimethyllysine (2a), we envisaged the
synthesis of (2S,3S)-3-hydroxy-Nε-trimethyllysine (2b) by the
same protocol. Although we were able to proceed with the
preparation of the 2S,3S diastereoisomer of 9 starting from
Garner’s aldehyde 3, the last step in the synthesis of 2b failed,
presumably due to decomposition of the final product 2b under
highly acidic conditions. Thus, we established an alternative
approach for the preparation of the (2S,3S)-isomer that relies
on the Sharpless asymmetric dihydroxylation methodology
(Scheme 3).
Aldehyde 10 was prepared from ethyl bromobutyrate using an
established protocol; treatment of ethyl bromobutyrate by
DIBAL-H at −78 °C led to the formation of 10,9 which was
subjected to Horner−Emmons olefination with tert-butyl
diethylphosphonoacetate to give the requisite olefin 11 in
72% yield. The asymmetric dihydroxylation of 11 proceeded as
expected, affording the syn-dihydroxy ester 12 with an excellent
level of enantioselectivity.10
In order to incorporate the amine functionality at the C-2
position, the diol was treated with SOCl2 and Et3N to afford the
cyclic sulfite, which was, upon treatment with NaIO4 and
catalytic amount of RuCl3, further oxidized to sulfate 13. In
accordance with Corey’s method,10b the regiospecific (Cα)
nucleophilic opening of the electrophilic cyclic sulfate 13 with
sodium azide in acetone afforded the azidosulfate, which
underwent further acidic hydrolysis and Pd−C mediated
hydrogenation to afford an exclusive formation of the
aminoalcohol 14 that possesses the desired 2S,3S stereo-
chemistry. 19F NMR analyses of the Mosher’s amide of 14 and
the Mosher’s ester of the preceding azidoalcohol confirmed
high degrees (>98.5%) of enantiopurity and diastereopurity of
both compounds (Figure S2). Finally, treatment of 14 with
trimethylamine and KHCO3 in EtOH, followed by saponifica-
tion with LiOH in THF/H2O, afforded the final diastereopure
product (2S,3S)-3-hydroxy-Nε-trimethyllysine (2b).
Having synthesized enantiopure (2S,3R)-3-hydroxy-Nε-tri-
methyllysine (2a) and (2S,3S)-3-hydroxy-Nε-trimethyllysine
(2b), we aimed to provide a conclusive answer about the
stereochemistry at the C-3 position of the product of TMLH-
catalyzed C-3 hydroxylation of (2S)-Nε-trimethyllysine. An
incubation of (2S)-Nε-trimethyllysine, FeSO4, 2-oxoglutarate,
ascorbate, and TMLH in buffered solution of TRIS-d11 (20
mM, pD = 7.5) for 1 h at 37 °C resulted in almost quantitative
formation (>95%) of (2S)-3-hydroxy-Nε-trimethyllysine prod-
The preparation of tert-butyl-protected α,β-unsaturated ester
11 and its subsequent multistep conversion to the (2S,3S)-3-
hydroxy-Nε-trimethyllysine (2b) are outlined in Scheme 3.
1
uct, as demonstrated by H NMR analysis (Figure 1). In
agreement with our recent study, two indicative signals at 4.07
B
DOI: 10.1021/acs.orglett.6b03608
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Letter
respectively. A minor increase in the C3-H peak intensity (at
4.07 ppm) was also observed; however, this increase derives
from a small impurity in the synthetic compound 2a (Figure
S3). Additionally, it appears that NMe3+ increases ∼2-fold upon
addition of 2a, but this is rationalized by the effect of coadding
two peaks with similar chemical shifts. The spectral
+
deconvolution of the methyl region revealed that NMe3
intensity basically remains unchanged (see Table S1 and
1
Figures S4−S6 in the Supporting Information). H−1H COSY
analysis, moreover, confirmed the appearance of new indicative
peaks upon the addition of the authentic sample 2a to the
enzymatic mixture (Figure S7). Collectively, these results
clearly indicate that (2S,3R)-3-hydroxy-Nε-trimethyllysine (2a)
is not produced in the TMLH-catalyzed reaction, suggesting
that it is very likely that the (2S,3S)-3-hydroxy-Nε-trimethylly-
sine (2b) isomer is formed in the enzymatic reaction.
Indisputable proof about the correct assignment of the
stereochemistry at the C-3 site of the enzymatic reaction was
provided by the enhancement experiment in the presence of
synthetic (2S,3S)-3-hydroxy-Nε-trimethyllysine (2b). Notably,
addition of (2S,3S)-3-hydroxy-Nε-trimethyllysine (2b) to the
enzymatic mixture resulted in no new observed peaks in the 1H
NMR spectrum (small peaks in the region of 3.95−4.05 ppm
derive from little impurities in the authentic sample 2b).
Instead, a ∼2-fold increase in peak intensity relative to the
ascorbate present was observed for C2-H, C3-H, C6-H, and
+
NMe3 (Figure 1B and Table S1). In all cases, the integrals
were normalized to ascorbate, and similar results were also
obtained when the well-resolved 2OG peaks were used instead
as a reference (Table S1). Importantly, the comparative
analyses of the multiplicity-edited 1H−13C HSQC spectra
revealed that the TMLH-catalyzed 3-hydroxy-Nε-L-trimethylly-
sine product5 and the synthetic (2S,3S)-3-hydroxy-Nε-trime-
thyllysine (2b) possess exactly the same values of 13C NMR
chemical shifts for the Cα (at 59.3 ppm) and the Cβ (at 68.8
ppm) (2a exhibits distinguishable resonances at 59.0 ppm for
Cα and at 69.4 ppm for Cβ). Taken together, these observations
clearly indicate that 3-hydroxy-Nε-trimethyllysine produced by
TMLH is not consistent with 2a but rather 2b, the (2S,3S)
diastereoisomer.
In conclusion, based on newly developed stereoselective
syntheses of (2S,3R)-3-hydroxy-Nε-trimethyllysine (2a) and
(2S,3S)-3-hydroxy-Nε-trimethyllysine (2b) and NMR doping
experiments, we have provided conclusive evidence that the 3-
hydroxy-Nε-trimethyllysine product of the human TMLH-
catalyzed reaction possesses the 2S,3S stereochemistry. It is
envisioned that this study will not only provide a valuable
insight into the rational design of small molecule inhibitors of
biomedically important carnitine biosynthesis pathway but will
also importantly contribute to a flourishing field of bioinspired
chemistry.
Figure 1. NMR spectroscopic analyses of TMLH-catalyzed hydrox-
ylation of (2S)-3-hydroxy-Nε-trimethyllysine. (A) Addition of the 2a
isomer to the enzymatic mixture. (B) Addition of the 2b isomer to the
enzymatic mixture. (Black spectrum = enzymatic reaction, red
spectrum = added authentic sample of 2a/2b to the enzymatic
reaction).
and 3.78 ppm were assigned as C3-H and C2-H, respectively.5
We then carried out H NMR-based doping experiments. To
ASSOCIATED CONTENT
* Supporting Information
■
1
S
two separate samples of the enzymatic mixture that contained
the TMLH-catalyzed (2S)-3-hydroxy-Nε-trimethyllysine prod-
uct we added an authentic sample of synthetic enantiopure (2S,
3R)-3-hydroxy-Nε-trimethyllysine (2a) and (2S,3S)-3-hydroxy-
Nε-trimethyllysine (2b), respectively.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.6b03608.
Synthetic procedures and NMR analyses (PDF)
Addition of synthetic 2a resulted in the observation of new
peaks in the enzymatic mixture spectrum (Figure 1A). The
AUTHOR INFORMATION
Corresponding Author
■
most prevalent new peaks appeared at 4.00, 3.31, and 3.06 ppm,
+
which were identified as C3,2a-H, C6,2a-H, and NMe3 2a
,
*E-mail: j.mecinovic@science.ru.nl.
C
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ORCID
Letter
Jasmin Mecinovic: 0000-0002-5559-3822
́
Notes
The authors declare no competing financial interest.
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DOI: 10.1021/acs.orglett.6b03608
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