γ-Butyrobetaine hydroxylase catalyses a Stevens type rearrangement

Article abstract of DOI:10.1016/j.bmcl.2012.06.024

γ-Butyrobetaine hydroxylase (BBOX) is a 2-oxoglutarate and Fe(II)-dependent oxygenase that catalyses the final step of l-carnitine biosynthesis in animals. BBOX catalyses the oxidation of 3-(2,2,2- trimethylhydrazinium)propionate (THP), a clinically used BBOX inhibitor, to form multiple products including 3-amino-4-(methyamino)butanoic acid (AMBA), which is proposed to be formed via a Stevens type rearrangement mechanism. We report the synthesis of AMBA and confirm that it is a product of the BBOX catalysed oxidation of THP. AMBA reacts with formaldehyde, which is produced enzymatically by BBOX, to give a cyclic adduct.

Full text of DOI:10.1016/j.bmcl.2012.06.024

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4975–4978
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Bioorganic & Medicinal Chemistry Letters
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c-Butyrobetaine hydroxylase catalyses a Stevens type rearrangement
⇑
⇑
Luc Henry , Ivanhoe K. H. Leung , Timothy D. W. Claridge , Christopher J. Schofield
Department of Chemistry, University of Oxford, Chemistry Research Laboratory, 12 Mansfield Road, Oxford OX1 3TA, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
c
-Butyrobetaine hydroxylase (BBOX) is a 2-oxoglutarate and Fe(II)-dependent oxygenase that catalyses
Received 4 May 2012
Revised 10 June 2012
Accepted 11 June 2012
Available online 16 June 2012
the final step of -carnitine biosynthesis in animals. BBOX catalyses the oxidation of 3-(2,2,2-trim-
L
ethylhydrazinium)propionate (THP), a clinically used BBOX inhibitor, to form multiple products including
3-amino-4-(methyamino)butanoic acid (AMBA), which is proposed to be formed via a Stevens type rear-
rangement mechanism. We report the synthesis of AMBA and confirm that it is a product of the BBOX
catalysed oxidation of THP. AMBA reacts with formaldehyde, which is produced enzymatically by BBOX,
to give a cyclic adduct.
Keywords:
BBOX
Carnitine
Ó 2012 Elsevier Ltd. All rights reserved.
THP
Stevens rearrangement
Formaldehyde
Fatty acid oxidation
2-Oxoglutarate (2OG) oxygenase
Trimethyl L-lysine
c
-Butyrobetaine hydroxylase (BBOX) is a 2-oxoglutarate (2OG)
and Fe(II)-dependent oxygenase that catalyses the final step of
-carnitine (
-Car) biosynthesis in animals (Scheme 1).1 3-(2,2,2-
being produced by BBOX-catalysed N-demethylation.3 To test the
proposed structure of 2, we synthesised both enantiomers of AMBA
1 ((S)-AMBA 1a and (R)-AMBA 1b) and studied their reaction with
formaldehyde. The results validate the proposed production of
AMBA 1 by BBOX-catalysed THP oxidation. They also reveal that
formaldehyde reacts with AMBA 1 to form the cyclic adduct 3,
which likely exists in equilibrium with the previously proposed
hemiaminal 2.3
Enantiomerically enriched (3S)-AMBA 1a and (3R)-AMBA 1b
were obtained in six steps with overall yields of ꢀ50% from com-
mercially available (3S) and (3R)-aspartic acid 4-tert-butyl esters
4a and 4b (Scheme 3). Thus, following protection of the amino
group of 4 with a N-tert-butoxycarbonyl group, the carboxylic acid
of 5 was reacted with iso-butyl chloroformate to give a mixed
anhydride, which was reduced in situ with sodium borohyride to
give the homoserine derivative 6.6 b-Aminoalcohol 6 was oxidised
to aldehyde 7, using the Dess–Martin periodinane. Attempted
reductive amination using monomethylamine and sodium triacet-
oxyborohydride did not yield detectable amounts of the desired
product 8. However, use of neat benzylmethylamine and sodium
triacetoxyborohydride gave (S)-tert-butyl 4-(benzyl(methyl)ami-
no)-3-(tert-butoxycarbonylamino)butanoate 9 in good yield. The
benzyl group of 9 was removed by hydrogenation to give 8 which
was converted to the hydrochloride salt of AMBA 1.
L
L
Trimethylhydrazinium)propionate (THP) is a BBOX inhibitor that
is used clinically for the treatment of angina and myocardial infarc-
tion.2 THP was recently demonstrated to be a substrate for recom-
binant BBOX,3 an observation that is consistent with rat tissue
studies on THP metabolism.4 Our NMR and mass spectrometric
studies identified malonic acid semialdehyde, formaldehyde, di-
methyl amine and an unanticipated compound, 3-amino-4-(meth-
ylamino)butanoic acid 1 (AMBA), as products of the oxidation of
THP as catalysed by recombinant human BBOX (Scheme 1).3 The
structures were assigned on the basis of NMR and mass spectro-
metric analyses using 13C-labelled THP.3 The proposed formation
of AMBA 1 suggests that BBOX catalyses an oxidative rearrange-
ment reaction involving N–N bond cleavage and C–C bond forma-
tion, likely via a radical mechanism in a process related to a
Stevens type rearrangement (Scheme 2).5 However, under the
incubation conditions, free AMBA 1 was not observed. Instead,
the product was provisionally assigned as an AMBA-formaldehyde
adduct 2 or derivative there of (Scheme 1), with the formaldehyde
Abbreviations: AMBA, 3-amino-4-(methylamino)butanoic acid; BBOX, c-butyr-
obetaine hydroxylase; THP, 3-(2,2,2-trimethylhydrazinium)propionate.
⇑
Corresponding authors. Tel.: +44 (0)1865 275 625; fax: +44 (0)1865 275 674.
E-mail addresses: tim.claridge@chem.ox.ac.uk (T.D.W. Claridge), christopher.
schofield@chem.ox.ac.uk (C.J. Schofield).
The synthetic AMBA enantiomers 1a and 1b were then sepa-
rately analysed by NMR in the absence and in the presence of form-
aldehyde under buffered conditions identical to those used for the
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.024

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L. Henry et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4975–4978
7
BBOX
Fe (II)
N+
N+
O
HN
N
6
2
OH
3
HO
4
O-
O-
3
2OG
O2
Succinate
CO2
- H2O
O
OH
O
O
OH
L-Car
NH2
γ-BB
O
HN
H
N
N
HO
HO
2
+ HCHO
O
BBOX
Fe (II)
N+
HN
HO
OH
O
NH2
H
+
+
+
NH3
+
HCHO
HN
N
O-
HO
O-
2OG
O2
Succinate
CO2
AMBA 1
O
THP
Scheme 1. BBOX catalyses the hydroxylation of
c
-butyrobetaine (c-BB) in the final step of L-carnitine (L-Car) biosynthesis in animals (shaded box). The clinically used
compound 3-(2,2,2-trimethylhydrazinium)propionate (THP) is a BBOX substrate. The unexpected product of BBOX catalysis, AMBA 1, reacts with formaldehyde to give
heterocycle 3, likely via the branched hemiaminal 2.
BBOX assay (Fig. 1 and Supplementary Figs. S1–S4). The NMR anal-
yses validate the proposal that AMBA 1 is a product of the oxida-
tion of THP by BBOX and forms a cyclic adduct 3 by reaction
with formaldehyde (Supplementary Figs. S1 and S2). When
comparing free AMBA 1 and the AMBA-formaldehyde adduct 3,
striking variations are observed in the tertiary C-3 chemical shifts
13
13
(d
C
1 = 46.1 ppm; d
C
2 = 55.1 ppm) and N-
AMBA
AMBA-formaldehyde
13
13
methyl C-6 (d
C
= 33.3 ppm; d
C
= 39.4 ppm),
AMBA 1
AMBA-formaldehyde 2
Hi s
His
His
Asp
OH
O
His
O
His
Asp
O
O
Asp
OH
O
His
O
Fe III
H
FeIII
FeIV
H
O-
O-
O-
O
H
H
NH
N+
Succinate
NH
N+
Succinate
Succinate
NH
H
N+
THP
1,2-H shift
O
- BBOX-FeII
- Succinate
His
His
His
O
Asp
Asp
His
O-
A
O
HO
FeIII
O
+ HCHO
7
+ HN
FeII
O-
H
OH
O
+ 2 H2O
- NH3
OH
O-
O
H
NH
Succinate
Succinate
H
N+
B
NH
H
N+
A
O
H
HN
N
4
6
2
3
HO
B
3
- H2O
O
- BBOX-FeII
- Succinate
His
His
OH
O
Asp
His
His
Asp
OH
H
NH2
1,5-H shift
O
FeIII
FeIII
H
N
O-
NH2
H
HO
O
OH
O
O-
NH
2
Succinate
Succinate
N+
H
- HCHO
O
H
N+
H
H
NH2
H
H
H
N
HO
AMBA 1
Scheme 2. Proposed mechanism for the BBOX catalysed oxidative rearrangement of THP. The proposed Fe(IV)-oxo intermediate (shaded box) is formed by oxidative
decarboxylation of 2OG likely via the consensus mechanism for 2OG and Fe(II)-dependent oxygenases.11 The operation of two divergent reaction pathways A and B give rise
to all the observed THP derived products. In pathway A, the methylene radical rebounds to the iron-hydroxyl intermediate to give the hydroxylation product. In pathway B,
the methylene radical adds into the imine to generate the rearranged C–C framework for AMBA. Although not observed, NH3 is expected to be formed in path A, according to
the proposed mechanism.

L. Henry et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4975–4978
4977
Ot-Bu
Ot-Bu
Ot -Bu
Ot-Bu
O
NH2
O
vi
O
ii
O
iii
iv
O
H
N
N
O
O
OH
HO
HN
Boc
R = Bn 9
R
HN
HN
Boc
HN
Boc
R
OH
R = H
R = Boc 5
4
6
7
AMBA 1
v
i
R = H
8
Scheme 3. Synthesis of 3-amino-4-(methyamino)butanoic acid 1 (AMBA). Reagents: (i) (Boc)2O, Na2CO3; (ii) i-BuOCOCl, NaBH4; (iii) Dess–Martin periodinane; (iv) BnMeNH,
NaBH(OAc)3; (v) H2 (1 bar), Pd/C; (vi) 2 M HCl in Et2O. Compounds 1a, 4a–9a correspond to the (3S)-series of compounds; 1b, 4b–9a correspond to the (3R)-series of
compounds.
A
B
2/2'
7/7'
N
O
NH2
3
O
HN
3
H
N
2/2'
6
6
6
HO
HO
4/4'
4/4'
2/2'
2/2'
6
4/4'
3
3
4
4'
Tris-D11
Tris-D11
7
7'
Figure 1. Reaction of 3-amino-4-(methyamino)butanoic acid 1 (AMBA) with formaldehyde. Addition of [12C]-formaldehyde to AMBA induces substantial chemical shift
differences, indicating substitution on both AMBA nitrogen atoms. (A) 2D 1H–13C HSQC NMR for AMBA in the absence of formaldehyde; (B) 2D 1H–13C HSQC NMR for AMBA in
the presence of stoichiometric [12C]-formaldehyde. Except for C-2, all aliphatic carbons show a large positive
Dd. The same results were obtained for both (S)- and (R)-AMBA.
An impurity from the formaldehyde solution is indicated by an asterisk (Ã).
which differ by ꢀ9 ppm and ꢀ6 ppm, respectively (Fig. 1). These
observations suggest that both nitrogen atoms of the AMBA-form-
aldehyde adduct 3 are substituted. However, when [12C]-formalde-
hyde is added to AMBA 1, no HMBC correlations were observed
from H-3 or H4/4’ to the aminal/hemiaminal C-7 (Supplementary
Fig. S3), likely due in part to the fast transverse relaxation of the
broad resonances (see below). In an attempt to observe the HMBC
correlations predicted for a cyclic structure, AMBA 1 was titrated
with a stoichiometric amount of [13C]-labelled formaldehyde. Sub-
sequently 1H–13C HSQC analysis revealed that the only [13C]-en-
riched position in the resultant adduct is the aminal/hemiaminal
C-7 carbon (Supplementary Fig. S4). Notably, HMBC correlations
are observed between H-3 and H4/4’ and the aminal/hemiaminal
13C-7 of the formaldehyde adduct 3, implying ring formation (Sup-
plementary Fig. S4). It has previously been observed that the reac-
tions between formaldehyde and amines are highly pH
dependent,7,8 and the broad 1H resonances of the aminal/hemiami-
nal CH2 suggest that this is a dynamic system, which may reflect
the pKa of a protonated amino group of AMBA (pKa ꢀ7.2; Supple-
mentary Fig. S5). Indeed, the 1H resonances of the N–CH2–N group
in 3 sharpen significantly when treated with deuterated hydro-
chloric acid (Supplementary Fig. S6). Thus, at pH 7.5 the AMBA-
formaldehyde adduct probably exists as an equilibrium between
the favoured cyclic aminal 3 and acyclic hemiaminal forms such
as 2 (Scheme 2).
enantiomers of aspartic acid 4-tert-butyl ester 4. By reacting both
enantiomers of the synthetic AMBA 1 with stoichiometric amounts
of [12C]- or [13C]-formaldehyde and using NMR for analyses, our re-
sults demonstrate that AMBA 1 is indeed a rearrangement product
of BBOX catalysed THP-oxidation, and that the observed adduct is
predominately present in its five-membered cyclic aminal form 3
at pH 7.5. Formaldehyde is proposed to be formed via N-methyl
hydroxylation followed by fragmentation (Scheme 2, paths A and
B). It is likely that the AMBA-formaldehyde adduct 3 is present in
equilibrium between the branched hemiaminal(s) and the cyclic
aminal forms. The observation of compound 3 is notable given
the concentrations of formaldehyde are relatively low; only small
amounts of the hemiaminal adduct formed by reaction of formal-
dehyde with tris(hydroxymethyl)aminomethane (Tris buffer),
which is present in large excess as a buffer, were observed. Form-
aldehyde is also produced by other 2OG and Fe(II)-dependent oxy-
genases, that is, histone lysine demethylases and DNA
demethylases.9 Demethylation of histone lysines is catalysed by
the ubiquitous JmjC subfamily. Given that histone proteins contain
a high content of nucleophilic/basic residues including lysines, the
results presented herein raise the possibility of the formation of
formaldehyde-mediated cross-linking of histone lysine (and other)
side-chains. Heterocycles have also been shown to be formed by
reacting formaldehyde with the tripeptide reducing agent glutathi-
one.8 Although the precise mechanisms of formaldehyde toxicity
remain unclear, it is proposed to involve the formation of DNA ad-
ducts, possibly by cross-linking.10 Thus, together with other work,
In conclusion, enantiomerically enriched (S)-AMBA 1a and (R)-
AMBA 1b were synthesised from commercially available

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L. Henry et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4975–4978
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N–CH2–N (or X–CH2–X) linkages.
Acknowledgments
3. Leung, I. K. H.; Krojer, T. J.; Kochan, G. T.; Henry, L.; von Delft, F.; Claridge, T. D.
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We thank Dr. Grazyna T. Kochan and Professor Udo Oppermann
for recombinant human BBOX and Dr. Filippo Sladojevich for chiral
HPLC analyses. We thank the Berrow Foundation, the Biotechnol-
ogy and Biological Sciences Research Council (BBSRC) and the Engi-
neering and Physical Sciences Research Council (EPSRC) for
funding.
Supplementary data
8. Hopkinson, R. J.; Barlow, P. S.; Schofield, C. J.; Claridge, T. D. W. Org. Biomol.
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ChemBioChem 2010, 11, 506; Hamm, S.; Just, G.; Lacoste, N.; Moitessier, N.; Szyf,
M.; Mamer, O. Bioorg. Med. Chem. Lett. 2008, 18, 1046.
Supplementary data (figures, experimental details of synthesis
and NMR experiments.) associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
06.024.
10. Conaway, C. C.; Whysner, J.; Verna, L. K.; Williams, G. M. Pharmacol. Ther. 1996,
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