Biosynthesis of 4-methylproline in cyanobacteria: Cloning of nosE and nosF genes and biochemical characterization of the encoded dehydrogenase and reductase activities

Article abstract of DOI:10.1021/jo026479q

The biosynthesis of the unusual amino acid 4-methylproline in the Nostoc genus of cyanobacteria was investigated on the genetic and enzymatic level. Two genes involved in the biosynthesis were cloned and the corresponding enzymes, a zinc-dependent long-chain dehydrogenase and a Δ¹-pyrroline-5-carboxylic acid (P5C) reductase homologue, were overexpressed in Escherichia coli and biochemically characterized. Putative substrates were synthesized to test enzyme substrate specificities, and deuterium labeling studies were carried out to reveal the stereospecificities of the enzymatic reactions with respect to the substrates as well as to the coenzymes.

Full text of DOI:10.1021/jo026479q

Biosyn th esis of 4-Meth ylp r olin e in Cya n oba cter ia : Clon in g of
n osE a n d n osF Gen es a n d Bioch em ica l Ch a r a cter iza tion of th e
En cod ed Deh yd r ogen a se a n d Red u cta se Activities
Hendrik Luesch, Dietmar Hoffmann, J oan M. Hevel, J ulia E. Becker,
Trimurtulu Golakoti, and Richard E. Moore*
Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii 96822
moore@gold.chem.hawaii.edu
Received September 25, 2002
The biosynthesis of the unusual amino acid 4-methylproline in the Nostoc genus of cyanobacteria
was investigated on the genetic and enzymatic level. Two genes involved in the biosynthesis were
cloned and the corresponding enzymes, a zinc-dependent long-chain dehydrogenase and a
∆1-pyrroline-5-carboxylic acid (P5C) reductase homologue, were overexpressed in Escherichia coli
and biochemically characterized. Putative substrates were synthesized to test enzyme substrate
specificities, and deuterium labeling studies were carried out to reveal the stereospecificities of
the enzymatic reactions with respect to the substrates as well as to the coenzymes.
In tr od u ction
Cyanobacteria are prolific producers of secondary
metabolites of peptidic nature that are commonly modi-
fied by the incorporation of polyketide portions and then
considered peptide-polyketide hybrids.1 Their small size,
modification, cyclic nature, N-methylation, and the pres-
ence of hydroxy acids, unusual amino acids, D-amino
acids, or unusual residues indicate that those metabolites
are presumably produced nonribosomally via large modu-
lar enzyme complexes, called nonribosomal peptide syn-
thetases (NRPSs)2 and type I polyketide synthases
(PKSs).3 For most biosynthetic gene clusters, the orga-
nization and order of the modules on the chromosome or
plasmid maps in a 1:1 manner to the sequence of the
product (collinearity rule).2,3 The clustering of biosyn-
thetic genes and the involvement of PKS (type I) and
NRPSs in the biosynthesis of cyanobacterial metabolites
has recently been demonstrated for microcystin.4
We have isolated the NRPS/PKS hybrid gene cluster
coding for the biosynthesis of nostopeptolides A5 (Gen-
Bank accession no. AF204805) in the terrestrial cyano-
bacterium Nostoc sp. GSV224.6 The biosynthetic gene
cluster appears to follow the collinearity rule as evidenced
(1) For a recent review, see: Gerwick, W. H.; Tan, L. T.; Sitachitta,
N. In The Alkaloids; Cordell, G. A., Ed.; Academic Press: San Diego,
CA, 2001; Vol. 57, pp 75-184.
(2) For reviews, see: (a) Marahiel, M. A.; Stachelhaus, T.; Mootz,
H. D. Chem. Rev. 1997, 97, 2651-2673. (b) von Do¨hren, H.; Keller,
U.; Vater, J .; Zocher, R. Chem. Rev. 1997, 97, 2675-2705.
(3) For review, see: Khosla, C. Chem. Rev. 1997, 97, 2577-2590.
(4) Tillett, D.; Dittmann, E.; Erhard, M.; von Do¨hren, H.; Bo¨rner,
T.; Neilan, B. A. Chem. Biol. 2000, 7, 753-764.
(5) Golakoti, T.; Yoshida, W. Y.; Chaganty, S.; Moore, R. E.
Tetrahedron 2000, 56, 9093-9102.
(6) Hoffmann, D.; Hevel, J . M.; Moore, R. E. Manuscript in prepara-
tion.
by either predicted (substrate-binding pocket model)7 or
experimentally determined (substrate specificity of over-
10.1021/jo026479q CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/28/2002
J . Org. Chem. 2003, 68, 83-91
83

Luesch et al.
SCHEME 1. P r esu m ed Biosyn th esis of th e
(2S,4S)-4-Meth ylp r olin e Moiety in th e
Nostop ep tolid es A
reactions. More recently we have also isolated and
sequenced the gene cluster encoding the biosynthesis of
another MePro-containing cyanobacterial metabolite,
nostocyclopeptide A12 from Nostoc sp. ATTC53789 (Gen-
Bank accession no. AY167420).13 Genes homologous to
nosE (ncpD, 97% identical and 98% positive on protein
level) and nosF (ncpE, 88% identical and 92% positive
on protein level) were identified, and the enzyme activity
of NcpD is also presented in this paper.
Resu lts a n d Discu ssion
Over exp r ession of NosE a n d NosF . Proteins en-
coded by the nosE and nosF genes were overexpressed
in E. coli as N-His6-tagged fusion proteins and purified
on Ni-NTA resin (Figure 1). NosE and NosF were easily
purified to >85% purity, were stable, and migrated as
distinct bands of ∼35 (NosE) and ∼30 kDa (NosF).
Ch a r a ct er iza t ion of NosE . Scheme 2 outlines the
synthetic strategy applied to efficiently produce the
putative natural substrate of NosE, (2S,4S)-5-hydroxy-
leucine (17) (based on the MePro stereochemistry in the
nostopeptolides), and its stereoisomers 18-20 in enan-
tiomerically pure form. The reaction sequence is derived
from a synthesis reported for 4-methylprolines.14 The
corresponding nonmethylated compounds, (2S)- and (2R)-
5-hydroxy-2-aminovaleric acid (15 and 16, Hava), were
to be evaluated as well for their potential to serve as
NosE substrates. For Hava 15 and 16, the alkylation step
was simply omitted (Scheme 2).
Compounds 17-20 were tested as NosE substrates in
activity assays that followed NADH formation. The 2S,4S
isomer 17 was the only stereoisomer accepted by NosE.
Due to the anticipated instability of the product (3S,5S)-
3-methyl-P5C (MeP5C), the assay product was not iso-
lated but was directly converted to (2S,4S)-MePro by
addition of NosF (coupled assay). The product of the
coupled enzyme assay was isolated and identified as
(2S,4S)-MePro by NMR and chiral HPLC analysis. The
2S isomer of Hava (15) was accepted as a substrate,
demonstrating a similar kcat, but a Km,app that was about
5 times greater than that for the methylated natural
substrate 17. The enzymatic oxidation was again proven
by a coupled assay as above and subsequent isolation of
L-proline. The 2R isomer of Hava (16) was not a sub-
strate. Michaelis-Menten parameters are provided in
Table 1. NosE activity was dependent on Zn2+ as a
cofactor, which is consistent with the characteristic small
subunit size and the homology of NosE to other zinc-
dependent (group I, long-chain) dehydrogenases.15 NosE
activity employed NAD+ and not NADP+ as a coenzyme
and exhibited the highest activity at pH 10 and 42 °C.
In summary, the dehydrogenase activity of NosE was
confirmed. The (2S,4S) stereochemistry of the substrate
is an absolute requirement for NosE while the lack of
the methyl group at C-4 is tolerated. The same conclusion
was drawn from data obtained for the NosE homologue,
expressed NRPSs measured by an ATP-[32P]PPi exchange
enzyme assay)6 data. The unusual amino acid (2S,4S)-
4-methylproline (MePro) is incorporated into the chemical
structures of the nostopeptolides. Some of the genes
responsible for the biosynthesis of this unusual amino
acid appeared to be embedded in the 3′ end of the
nostopeptolide gene cluster. The nosE gene showed strong
homology to zinc-dependent long-chain alcohol dehydro-
genases and the nosF gene to ∆1-pyrroline-5-carboxylic
acid (P5C) reductases.6 This suggested that MePro bio-
synthesis proceeded via a P5C homologue (Scheme 1) in
analogous fashion to 3-methylproline biosynthesis. Feed-
ing experiments have demonstrated that 3-methylproline
originates from isoleucine in the cyanobacterial metabo-
lite scytonemin A8,9 and in fungal metabolites of the
paraherquamide family.10 This suggested that MePro
probably arises ultimately from leucine (Scheme 1). A
feeding experiment with GSV224 using [1-13C]L-leucine
supported this hypothesis since label was incorporated
into C-1 of the MePro unit in nostopeptolide A1. Fur-
thermore, the enrichment was comparable with incorpo-
ration into C-1 of the leucine unit and C-3 of the modified
leucine unit (1%). However, the only uncharacterized
open-reading frame in the nostopeptolides A gene cluster,
ORF1, showed no homology to any type of oxidizing
enzyme. Enzymes for the biosynthesis of the presumed
NosE substrate, (2S,4S)-5-hydroxyleucine, could also be
encoded by (a) gene(s) outside the cluster, and ORF1
might have a different, e.g., regulatory function. The
primary NosE reaction product would presumably cyclize
spontaneously as described for glutamic acid γ-semial-
dehyde in proline biosynthesis.11 Finally, MePro synthe-
sis would be completed by the NosF-catalyzed reduction
of the pyrroline ring.
In this paper we report the overexpression and kinetic
characterization of NosE and NosF, two proteins involved
in the biosynthesis of MePro. The focus is on the
determination of the substrate specificity of these en-
zymes and of the stereospecificity of the enzymatic
(7) (a) Stachelhaus, T.; Mootz, H. D.; Marahiel, M. A. Chem. Biol.
1999, 6, 493-505. (b) Challis, G. L.; Ravel, J .; Townsend, C. A. Chem.
Biol. 2000, 7, 211-224.
(8) Helms, G. L.; Moore, R. E.; Niemczura, W. P.; Patterson, G. M.
L. J . Org. Chem. 1988, 53, 1298-1307.
(12) Golakoti, T.; Yoshida, W. Y.; Chaganty, S.; Moore, R. E. J . Nat.
Prod. 2001, 64, 54-59.
(13) Becker, J . E.; Moore, R. E. Manuscript in preparation.
(14) Koskinen, A. M. P.; Rapoport, H. J . Org. Chem. 1989, 54, 1859-
1866.
(15) For review, see: Reid, M. F.; Fewson, C. A. Crit. Rev. Microbiol.
1994, 20, 13-56.
(9) Hemscheidt, T. K. (University of Hawaii at Manoa), personal
communication.
(10) Stocking, E. M.; Martinez, R. A.; Silks, L. A.; Sanz-Cervera, J .
F.; Williams, R. M. J . Am. Chem. Soc. 2001, 123, 3391-3392.
(11) Kramer, J . J .; Henslee, J . G.; Wakabayashi, Y.; J ones, M. E.
Methods Enzymol. 1985, 113, 113-120.
84 J . Org. Chem., Vol. 68, No. 1, 2003

Biosynthesis of 4-Methylproline in Cyanobacteria
F IGURE 1. Heterologous expression and purification of NosE and NosF: (a) N-His6-tagged NosE and (b) N-His6-tagged NosF
overproduced in E. coli BL21(DE3)pLysS. Lane 1, MW standard; lane 2, soluble cell extract without IPTG induction; lane 3,
soluble proteins after IPTG induction; lane 4, Ni-NTA column flow-through; lane 5, first wash; lane 6, second wash; lanes 7 and
8, purified proteins (eluates 1 and 2).
SCHEME 2. Syn th esis of P u ta tive NosE Su bstr a tesa
a
Conditions: (a) AcOCl, MeOH; (b) pyridine; (c) TMSCl; (d) Et3N, Pb(NO3)2, PhFlBr; (e) O-tert-butyl N,N′-diisopropylisourea; (f) KHMDS,
-78 °C; (g) MeI; (h) LiAlH4, -78 °C; (i) TFA; (j) Dowex-50W (H+), 4 N NH4OH.
TABLE 1. Kin etic Da ta for th e En zym a tic Oxid a tion of NosE a n d Ncp D Su bstr a tesa
NosE
NcpD
substrate
K
m,app (M)
kcat/Km,app (M-1 s-1
)
Km,app (M)
kcat/Km,app (M-1 s-1
)
(2S,4S)-5-OH-Leu (17)
(S)-Hava (15)
(2.4 ( 0.16) × 10-4
(2.5 ( 0.45) × 103
(6.6 ( 1.1) × 10-4
(1.3 ( 0.42) × 10-3
(7.6 ( 1.3) × 102
(1.3 ( 0.40) × 102
(1.2 ( 0.078) × 10-3
(5.0 ( 0.90) × 102
a
At 2 mM NAD+, 42 °C, 1 mM ZnSO4, in 100 mM glycine buffer (pH 10).
NcpD, catalyzing the oxidation step in the biosynthesis
of MePro in nostocyclopeptide A (see Table 1 for kinetic
data).
behavior (Km,app ) 150 µM, kcat/Km,app ) 3.5 × 104 M-1
s-1, pH 8). At this point it was still unclear if both
enantiomers of P5C (21 and 22) were substrates of NosF.
Consequently, the proline generated in enzymatic reac-
Ch a r a cter iza tion of NosF . Due to the high homology
of NosF to P5C reductases and the ease of generating
P5C (NaIO4 oxidation of δ-hydroxylysine),16 P5C was
evaluated as a potential substrate for NosF. Addition of
DL-P5C (21/22) to enzyme reactions resulted in the
oxidation of NADH or NADPH in a concentration-
dependent fashion, although NADH was the preferred
nicotinamide. NosF activity displayed Michaelis-Menten
tions was isolated and shown to be exclusively of
L
stereochemistry by chiral HPLC analysis. Therefore it
was concluded that L-P5C (21) is a substrate for NosF,
while D-P5C (22) is not. Since the ratio of 21 and 22 was
1:1 and assuming the D-isomer is not an inhibitor, then
one can estimate the Km,app of the pure L-P5C (21) at
roughly 75 µM. The deduced specificity of NosF was
confirmed by employing the enantiomerically pure com-
pounds in NosF assays (data not shown). Herefore, L-P5C
(21) and D-P5C (22) were synthesized by chromic acid
(16) (a) Williams, I.; Frank, L. Anal. Biochem. 1975, 64, 85-97. (b)
Wu, G. Y.; Seifter, S. Anal. Biochem. 1975, 67, 413-421.
J . Org. Chem, Vol. 68, No. 1, 2003 85

Luesch et al.
SCHEME 3. Deter m in a tion of th e Ster eosp ecificity of th e NADH Red u ction by NosF Usin g
Ster eosp ecifica lly Deu ter a ted NADH
oxidation of Hava 15 and 16, respectively, similarly as
described for R-aminoadipic-δ-semialdehyde.17
assays; however, no catalysis was observed. This also
indicated that there is no significant equilibrium between
both tautomers under assay conditions.
In summary, the predicted reductase activity of NosF
was demonstrated. The L-configuration (5S) of the sub-
strate and the S stereochemistry at C-3 of MeP5C are
essential for activity. On the other hand, the absence of
the C-3 methyl group is tolerated by NosF.
Ster eosp ecificity of NADH Red u ction by NosF .
For reasons that become obvious below, the stereospeci-
ficity of NosF was investigated prior to the stereospeci-
ficity of NosE. To determine whether NosF was A-side
or B-side specific, (4R-2H)- and (4S-2H)-NADH (A-side
and B-side NADD) were synthesized. The stereospeci-
ficities of horse liver dehydrogenase and glucose dehy-
drogenase were exploited to generate A-side and B-side
NADD, respectively, using established procedures.19
Since the stereospecificity of an enzyme is independent
of its substrate,20 stereospecificity studies were carried
out with the nonmethylated substrate, L-P5C (21), em-
ployed as a racemic mixture with 22. When NosF was
incubated with DL-P5C (21/22) and A-side NADD, the
label was retained in the oxidized coenzyme (see Scheme
3). Inspection of the 1H NMR spectrum of the crude
product led to this conclusion. The aromatic region lacked
the signal for H-4 of the pyridine moiety of NAD+; thus
NAD+ contained deuterium at this position.21 This result
was confirmed after isolation of L-proline, which was
deuterium-free (see Scheme 3 and Figure 2a), indicating
that a hydride was transferred during the reaction.
Chromic acid oxidation of 5-hydroxyleucines (17-20)
provided the corresponding methylated compounds (23-
26, MeP5C). Although preparations of both the natural
substrate (23) and the diastereomer 24 supported NosF
activity, the activity observed with the natural substrate
was approximately three times faster at any given
concentration of substrate (data not shown). Since (2S,4S)-
MePro was the only product formed in both NosF
reactions, i.e., starting from 23 and 24 (determined by
NMR and chiral HPLC of the isolated assay product), it
appears that the apparent activity of (3R,5S)-MeP5C (24)
is due to partial epimerization at C-3 (∼30%) under the
strong acidic conditions of the substrate synthesis (4 N
HCl, 80 °C, 1 h). Preliminary data suggest that compound
24 may be functioning as an inhibitor of NosF activity,
such that more studies will be required to obtain kinetic
parameters.
PDC oxidation of compound 11 or of the corresponding
selectively N-deprotected compound led to formation of
the enamine tautomers of P5C derivatives, 27 and 28,
respectively.18 Following TFA-mediated deprotection, the
resulting enamine 29 was tested as a substrate in NosF
(19) Viola, R. E.; Cook, P. F.; Cleland, W. W. Anal. Biochem. 1979,
96, 334-340.
(20) You, K.; Arnold, L. J ., J r.; Allison, W. S.; Kaplan, N. O. Trends
Biochem. Sci. 1978, 3, 265-268.
(17) J ones, E. E.; Broquist, H. P. J . Biol. Chem. 1965, 240, 2531-
2536.
(18) Luesch, H. Ph.D. Thesis, University of Hawaii, 2002.
(21) For a similar NMR analysis and spectra, see: Ottolina, G.; Riva,
S.; Carrea, G.; Danieli, B.; Buckmann, A. F. Biochim. Biophys. Acta
1989, 998, 173-178.
86 J . Org. Chem., Vol. 68, No. 1, 2003

Biosynthesis of 4-Methylproline in Cyanobacteria
Since NosF is known to be B-side specific, L-proline would
contain two deuterium atoms at its δ carbon if NosE were
B-side specific as well. Only one deuterium atom would
be found if NosE had the opposite stereospecificity, i.e.,
were A-side specific (Scheme 4). Due to the instability of
NADH/NADD but ease of proline isolation, the latter
alternative was selected. The coupled assay was run in
NosE buffer at pH 10; under these conditions NosF was
active for only a few minutes. Because the L-proline
subsequently isolated contained only one deuterium
atom, NosE had to be A-side specific (Figure 3a). The
deuterium label was cis to the carboxyl group since the
deuterium must have been present in the intermediate
(Scheme 4) and NosF transferred a hydride trans to the
carboxyl group (compare Figure 3, parts a and b). The
potential risk of scrambling if two NAD(H)-dependent
enzymes of opposite stereospecificity are coupled was not
observed, as NosF showed activity only for a short time
period as mentioned. There was no need to remove or
denature NosE prior to NosF addition.
F IGURE 2. 1H NMR spectra (500 MHz) of L-proline disclosing
the stereospecificity of the NosF-mediated reduction with
respect to coenzyme and substrate. (a) Nondeuterated L-proline
from reaction of L-P5C (21) with NosF and A-side NADD, and
(b) (2S,5R)-5-2H-proline () trans) from reaction with NosF and
B-side NADD.
To determine whether the 5pro-R or the 5pro-S hy-
drogen is abstracted in the NosE-catalyzed oxidation
step, a stereoselective synthesis of either C-5 monodeu-
terated (2S)-Hava or (2S,4S)-5-hydroxyleucine was re-
quired. Again, this study was undertaken employing the
nonmethylated substrate. Stereospecifically deuterated
substrates as outlined in Scheme 5 were synthesized
starting from N-benzyloxycarbonyl-L-glutamic acid (31),
which was reacted with paraformaldehyde to give oxazo-
lidinone acid 32.24 Action of oxalyl chloride on compound
32 yielded the acyl chloride 33, which was converted to
the deuterated aldehyde 34 via a modified Rosenmund
reduction.25 The two protecting groups on the amino
functionality prevented an intramolecular cyclization.
The stable aldehyde 34 was then subjected to Midland
reduction,26 i.e., reduction with (R)- or (S)-Alpine borane,
to yield the monodeuterated compounds 35 and 36,
respectively. It has been established that (R)-Alpine
borane gives the (S)-alcohol while (S)-Alpine borane gives
the (R)-alcohol, and no exceptions are reported.27 Acid
hydrolysis28 liberated the stereospecifically labeled target
compounds 37 and 38 which were subsequently used in
coupled assays with NosE and NosF. The 1H NMR
spectra of the isolated prolines (Figure 4) indicated not
only the stereospecificity of NosE with respect to the
substrate, but also that the Midland reduction proceeded
in a highly stereoselective fashion. (S)-Alcohol 37 was
converted to regular (nondeuterated) L-proline (Figure
4a), while (R)-alcohol 38 reacted to monodeuterated
L-proline with the deuterium cis to the carboxyl group
(Figure 4b), as when dideuterated Hava 30 was used
(Figure 3a). The fact that deuterium was retained during
the reaction sequence clearly indicated that NosE is pro-S
specific with respect to the substrate.
When NosF was incubated with DL-P5C (21/22) and
B-side NADD, the aromatic region of the 1H NMR
spectrum of the crude product mixture exhibited the
complete set of signals for the pyridine ring of NAD+,
suggesting that a deuteride had been transferred (Scheme
3). Indeed the isolated L-proline displayed a 1H NMR
spectrum that lacked the signal for one δ-proton (see
Figure 2b). Comparison with L-proline from reaction with
A-side NADD at the same pH (∼5) demonstrated that
the upfield signal of the δ-protons was missing (Figure
2), which is known to arise from the proton trans to the
carboxyl group in proline.22 The presence of a deuteron
2
at this position was confirmed by H NMR spectroscopy
and the signal assignment in the 1H NMR spectrum was
confirmed by 1D NOE experiments.23
In summary, NosF was shown to be B-side specific, i.e.,
to transfer the (4pro-S)-hydrogen of NADH. Furthermore,
it was demonstrated that NosF mediates the hydride
transfer to the face of the pyrroline ring that is opposite
to the carboxyl group in the substrate.
Ster eosp ecificity of NAD+ Oxid a tion by NosE. As
with NosF, the stereospecificity of the nicotinamide
oxidation by NosE was ascertained based on reactions
with the nonmethylated substrate. Our strategy required
the dideuterated (S)-Hava 30 that is easily accessible via
the route described in Scheme 2, but by using LiAlD4 in
the reduction of methyl ester 3. Incubation with NosE
and NAD+ under established conditions would result in
generation of either A-side or B-side NADD, which are
diastereomers and thus distinguishable by 1H NMR
spectroscopy.21 Alternatively, the NosE assay could be
coupled with an NosF assay and the final product
1
(24) (a) Baldwin, J . E.; Lee, E. Tetrahedon 1986, 42, 6551-6554.
(b) Lee, B. H.; Miller, M. J . Tetrahedron Lett. 1984, 25, 927-930.
(25) Burgstahler, A. W.; Weigel, L. O.; Shaefer, C. G. Synthesis 1976,
767-768.
L-proline isolated and its H NMR spectrum inspected.
(22) (a) Pogliani, L.; Ellenberger, M.; Valat, J . Org. Magn. Reson.
1975, 7, 61-71. (b) Altman, L. J .; Silberman, N. Anal. Biochem. 1977,
79, 302-309. (c) Anteunis, M. J . O.; Borremans, F. A. M.; Becu, C.;
Sleeckx, J . Int. J . Peptide Protein Res. 1979, 14, 445-450.
(23) For standard proline at identical pH, irradiation of the R-proton
shows an NOE to the upfield δ-proton, indicating that the latter proton
is located trans to the carboxyl group.
(26) Midland, M. M.; Greer, S.; Tramontano, A.; Zderic, S. A. J . Am.
Chem. Soc. 1979, 101, 2352-2355.
(27) Prabhakaran, P. C.; Gould, S. J .; Orr, G. R.; Coward, J . K. J .
Am. Chem. Soc. 1988, 110, 5779-5784.
(28) Bold, G.; Steiner, H.; Moesch, L.; Walliser, B. Helv. Chim. Acta
1990, 73, 405-410.
J . Org. Chem, Vol. 68, No. 1, 2003 87

Luesch et al.
SCHEME 4. Cou p led NosE/NosF Assa y To Deter m in e th e Ster eosp ecificity of th e NAD+ Oxid a tion by NosE
P r ior to NosF Ad d ition
3-methylproline in the paraherquamide family of fungal
metabolites has been investigated and shown to proceed
via a similar intermediate.10 The second to last enzymatic
steps in MePro and Pro biosynthesis are quite different.
While glutamic acid γ-semialdehyde is commonly gener-
ated by two-electron reduction of the ω-carboxyl func-
tionality of glutamic acid in Pro biosynthesis,11 the
corresponding C-4 methylated γ-semialdehyde (Scheme
1) originates from an oxidation step.
The relaxed substrate specificity of NosE and NosF
may suggest that the specific incorporation of MePro into
nostopeptolides A resides in the specificity of the NRPS
module. Alternatively, specific incorporation of MePro
may be governed by the reaction preceding NosE oxida-
tion. Further studies are required to investigate the
earlier step(s) in MePro biosynthesis.
Exp er im en ta l Section
F IGURE 3. 1H NMR spectra (500 MHz) of L-proline disclosing
Gen er a l. E. coli BL21(DE3)pLysS (Stratagene, La J olla,
CA) was used as an expression host and pRSETB (Invitrogen,
Carlsbad, CA) as an expression vector. H NMR spectra were
the stereospecificity of the NosE-mediated oxidation with
respect to the coenzyme: (a) (2S,5S)-5-2H-proline () cis), the
reaction product of the coupled assay starting from dideuter-
ated (S)-Hava 30, and (b) (2S,5R)-5-2H-proline () trans), the
reaction product from reaction with NosF and B-side NADD
(see Figure 2b) for comparison.
1
recorded at 300 or 500 MHz, and 13C NMR spectra at 125 MHz.
In cor p or a tion of [1-13C]L-leu cin e in to n ostop ep tolid e
A1. The labeled amino acid (120 mg) was fed to a 20-L culture
in eight equal doses starting from day 12 after inoculation.
The culture was harvested on day 30 and the cyanobacterium
lyophilized to give 10 g of freeze-dried material from which 1
mg of labeled nostopeptolide A1 was isolated. The 13C NMR
spectrum showed equally enhanced singlet signals (1%) at
175.3 (C-1 of Leu unit), 206.0 (C-3 of LeuAc unit), and 174.7
ppm (C-1 of MePro unit).
In summary, NosE is A-side specific with respect to
the coenzyme, i.e., it mediates the hydride transfer from
the substrate to the re-face of the pyridine moiety of
NAD+. This finding is consistent with the prediction that
zinc-dependent dehydrogenases29 or metal-dependent
dehydrogenases in general30 are pro-R specific. Further-
more, NosE catalyzes specifically the abstraction of the
5pro-S hydrogen of the substrate.
DNA Isola tion , Ma n ip u la tion s, a n d Sequ en cin g. In
vitro DNA manipulations, cloning, and transformation of
competent E. coli BL21(DE3)pLysS cells were performed
according to Sambrook et al.31 DNA sequencing was performed
using the ABI PRISM BigDye Terminator Cycle Sequencing
Ready Reaction Kit with AmpliTaqDNA Polymerase, FS (PE
Applied Biosystems, Foster City, CA) according to the manu-
facturer’s protocol and analyzed on a DNA sequencer. DNA
sequences were aligned with the LASERGENE program
Seqman (DNASTAR, London, United Kingdom).
P CR Am p lifica tion a n d Clon in g of n osE a n d n osF for
Over exp r ession . The coding sequences for NosE and NosF6
were amplified by PCR using Pfu polymerase (Stratagene),
primers with 5′ tails encoding restriction sites, and in-frame
stop codons (Operon, Alameda, CA) and cosmid pREMc4 as
DNA template.6 The oligonucleotide sequences were as follows
(5′ tails are underlined and restriction sites are in boldface):
nosEFor[BamHI, NdeI], 5′-GGGATCCACAT ATG CCC TTA
GCA GCT GTG ATG AC-3′; nosERev[PstI], 5′-GCTGCAG TCA
AGA TAG ATT AGG TTG AAT CAC AG-3′; nosFFor[BamHI,
Con clu sion
The characterization of two enzymes involved in the
biosynthesis of 4-methylproline (MePro) in cyanobacteria
has been described in this paper. The biosynthetic
pathway leading to MePro has parallels in proline (Pro)
biosynthesis, yet also displays some substantial differ-
ences. The intermediates in both pathways are structur-
ally related: MeP5C for MePro and P5C for Pro. The high
homology of NosF to other P5C reductases and the
relaxed substrate specificity of NosF, also accepting
L-P5C (21), might suggest that the nosF gene originated
by duplication of the gene encoding the last step of the
primary Pro anabolism. Recently, the biosynthesis of
(29) Schneider-Bernlo¨hr, H.; Adolph, H.-W.; Zeppezauer, M. J . Am.
Chem. Soc. 1986, 108, 5573-5576.
(30) Glasfeld, A.; Benner, S. A. Eur. J . Biochem. 1989, 180, 373-
375.
(31) Sambrook, J .; Fritsch, E. F.; Maniatis, T. Molecular Cloning, a
Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory Press:
Cold Spring Harbor, NY, 1989.
88 J . Org. Chem., Vol. 68, No. 1, 2003

Biosynthesis of 4-Methylproline in Cyanobacteria
SCHEME 5. Ster eoselective Syn th esis of C-5 Mon od eu ter a ted (S)-Ha va a
a
Conditions: (a) paraformaldehyde, p-TsOH, toluene, reflux, 100 min, Dean-Stark apparatus; (b) (COCl)2, cat. DMF, CH2Cl2, rt, 30
min; (c) D2, 5% Pd/BaSO4, THF, lutidine, rt, 2 h; (d) (R)-Alpine borane, THF, rt, 10 h; (e) (S)-Alpine borane, THF, rt, 10 h; (f) 5 N HCl,
100 °C, 2 h.
tent cells of E. coli BL21(DE3)pLysS. Transformed E. coli
BL21(DE3)pLysS were grown in LB medium at 37 °C in the
presence of 50 µg/mL of carbenicillin, 34 µg/mL of chloram-
phenicol, and 2.5 mM betaine. Cultures were induced with
0.4-0.8 mM IPTG at an A600 of 0.5-0.7 and allowed to grow
for an additional 2-4 h at 30 °C. Cells were harvested at 8000
× g (10 min) and resuspended in wash buffer (50 mM NaH2-
PO4, pH 8.0, 300 mM NaCl, 20 mM imidazole) containing 0.1
mM PMSF and Protease Inhibitor Cocktail for the purification
of poly(Histidine)-tagged proteins according to the manufac-
turers protocol (Sigma, St. Louis, MO), sonicated on ice (4
times for 15 s each), and then centrifuged for 20 min at 12,000
× g and 4 °C. Crude lysates were applied to Ni-NTA columns
(Qiagen, Valencia, CA), the columns were washed twice with
wash buffer, and the His6-tagged proteins were eluted with
elution buffer (50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 250
mM imidazole). The eluates were applied onto an equilibrated
Econo-Pac 10DG desalting column (Bio-Rad), and the fusion
proteins were eluted with buffer A (50 mM Tris, 1 mM EDTA,
5% glycerol, pH 7.3, filtered; 1 mM DTT added before use).
Expression level and protein purity were analyzed by SDS-
polyacrylamide electrophoresis using Brilliant Blue G-Colloidal
F IGURE 4. 1H NMR spectra (500 MHz) of L-proline disclosing
the stereospecificity of the NosE-mediated oxidation with
respect to the substrate: (a) nondeuterated L-proline, the
stain (Sigma). Protein concentration was determined by the
Bradford Protein assay32 or Micro Bradford Protein assay (Bio-
reaction product of the coupled assay starting from (2S,5S)-
Rad, Hercules, CA) using bovine serum albumin as a standard.
5-2H-Hava (37), and (b) (2S,5S)-5-2H-proline () cis), the
NosE Assa ys. Typical reaction mixtures contained 100 mM
glycine (pH 10), 2 mM â-NAD, substrate, 1 mM ZnSO4, and
approximately 3 µg of NosE (N-His6-tagged). Typical reaction
volumes were 500 µL. NADH formation was monitored spec-
trophotometrically (A340 increase) at 42 °C. Initial rates were
dependent upon protein concentration and were fitted to the
Michaelis-Menten equation using nonlinear regression.
NosF Assa ys. Typical reaction mixtures contained 200 mM
Tris (pH 8), substrate, 0.2 mM â-NADH (â-NADPH), and
approximately 5 µg of NosF (N-His6-tagged). Reaction volumes
were usually 1 mL. NAD(P)H consumption was monitored
spectrophotometrically (A340 decrease) at 42 °C. Initial rates
were dependent upon protein concentration and were fitted
to the Michaelis-Menten equation using nonlinear regression.
reaction product of the coupled assay starting from (2S,5R)-
5-2H-Hava (38).
NdeI], 5′-GGGATCCACAT ATG CTC GAA GAT TTA CAA
ATT GC-3′; nosFRev[PstI], 5′-GCTGCAG TTA ACT GAT ATT
ACC TAA TTG TTG AG-3′.
The obtained PCR products were cloned using the PCR-
Script Amp Cloning Kit (Stratagene), sequenced, and sub-
cloned into the predigested (BamHI/PstI) expression vector
pRSETB (Invitrogen). To verify the correct insert orientation
(the pPCR-Script AMP SK(+) multiple cloning site contains
additional BamHI and PstI sites), the recombinant expression
plasmids were sequenced with the following primer: RSET,
5′-CTC ACT ATA GGG AGA CCA CAA C-3′.
Over exp r ession a n d P u r ifica tion of N-His6-Ta gged
NosE a n d NosF . The recombinant pRSET derivatives des-
ignated pNosEx and pNosFx were used to transform compe-
(32) Bradford, M. M. Anal. Biochem. 1976, 72, 248-254.
J . Org. Chem, Vol. 68, No. 1, 2003 89

Luesch et al.
For determination of NosF stereospecificity, several reac-
tions using 0.64 mM DL-P5C (21/22) were conducted simulta-
neously at 42 °C. After the absorption at 340 nm had decreased
and reached a plateau, the reaction mixtures were combined,
treated with CHCl3, and centrifuged. The aqueous solution was
concentrated and typically applied to a Dowex-50W column
(H+ form, 1.4 × 25 cm). Elution was done with 1 N HCl.
Proline-containing fractions were identified by 1H NMR,
concentrated to dryness, reconstituted in H2O, and subjected
to chiral HPLC [Chirex phase 3126 (D)-Penicillamine (4.6 ×
250 mm), Phenomenex; 2 mM CuSO4-MeCN (95:5); detection
at 254 nm]. L-Proline eluted at tR 11.5 min. No D-proline was
detected (tR of standard, 22.2 min). The proline-containing
fraction was concentrated and the residue redissolved in 1 N
HCl. To remove copper ions from the solutions, hydrogen
sulfide gas was bubbled through the solution for approximately
1 min. After centrifugation, the supernatant was concentrated
to dryness and taken up in the NMR solvent to determine the
deuterium content and position.
washed with H2O. Elution with 4 N NH4OH and evaporation
of the eluant gave 17 (6.5 mg, 84%): 1H NMR (300 MHz, D2O)
δ 0.99 (d, J ) 6.2 Hz, 3H), 1.74-1.88 (m, 3H), 3.47-3.53 (m,
2H), 3.77 (dd, J ∼ 10, 4 Hz, 1H).
(2S,4R)-5-Hyd r oxyleu cin e (18). Analogous treatment of
12.0 mg (27.1 µmol) of compound 12 yielded 18 (3.5 mg, 88%):
1H NMR (300 MHz, D2O) δ 1.00 (d, J ) 6.7 Hz, 3H), 1.71 (m,
1H), 1.84 (m, 1H), 1.95 (m, 1H), 3.49 (dd, J ) -11.1, 6.3 Hz,
1H), 3.53 (dd, J ) -11.1, 5.7 Hz, 1H), 3.80 (t, J ) 6.6 Hz, 1H).
(2R,4R)-5-Hyd r oxyleu cin e (19). Analogous treatment of
compound 13 (15.0 mg, 33.8 µmol) yielded 19 (4.0 mg, 80%).
For NMR data, see compound 17.
(2R,4S)-5-Hyd r oxyleu cin e (20). Analogous treatment of
compound 14 (12.0 mg, 27.1 µmol) yielded 20 (3.2 mg, 80%).
For NMR data, see compound 18.
(S)-5-Hydr oxy-2-am in ovaler ic Acid (15). Analogous treat-
ment of 30.0 mg (69.8 µmol) of compound 9 yielded 15 (8.5
mg, 91%): 1H NMR (300 MHz, D2O) δ 1.61 (m, 2H), 1.88 (m,
2H), 3.62 (t, J ) 6 Hz, 1H), 3.74 (t, J ) 6 Hz, 1H).
For the determination of the specificity of NosF with respect
to the stereochemistry at C-3 of L-MeP5C, assays using
(3S,5S)-MeP5C (23) and (3R,5S)-MeP5C (24) were carried out
(both slightly epimerized at C-3). MePro was isolated and
stereochemically characterized similarly as described above
by chromatography on Dowex-50W and chiral HPLC. (2S,4S)-
MePro eluted at tR 27.0 min. No (2S,4R)-MePro was found after
any assay (tR of standard, 25.0 min).
Cou p led Assa ys. Coupled assays, performed at 42 °C, were
carried out to determine the stereospecificity of NosE. The
reaction mixture contained 100 mM glycine (pH 10), 3 mM
deuterated Hava, 2 mM â-NAD, 1 mM ZnSO4, and ap-
proximately 10 µg of NosE. After 5 h of incubation at 42 °C,
approximately 10 µg of NosF was added, resulting in a
decrease in absorption at 340 nm for the following 15 min.
Proline was then isolated as described above (see NosF assay)
and deuterium content and position determined by NMR.
P r ep a r a tion of A-Sid e a n d B-Sid e NADD. A-side and
B-side NADD were synthesized and purified similarly as
described by Viola et al.19
(R)-5-Hydr oxy-2-am in ovaler ic Acid (16). Analogous treat-
ment of 30.0 mg (69.8 µmol) of compound 10 yielded 16 (7.5
mg, 81%). For NMR data, see compound 15.
ter t-Bu tyl (S)-5-Hyd r oxy-2-(N-(9-(9-p h en ylflu or en yl))-
a m in o)-5,5-d id eu ter iop en ta n oa te. Compound 3 (56.4 mg,
0.12 mmol) was reduced as described for the synthesis of 9,
except that lithium aluminum deuteride was used as the
reducing agent, to afford the target compound (35.6 mg,
1
67%): Rf 0.12 (20% EtOAc in hexanes); H NMR (300 MHz,
CDCl3) δ 1.19 (s, 9H), 1.47 (m, 1H), 1.53 (m, 2H), 1.55 (m, 1H),
2.52 (dd, J ) 6.6, 4.4 Hz, 1H), 7.18-7.70 (m, 13H).
(S)-5-Hyd r oxy-2-a m in o-5,5-d id eu ter iova ler ic Acid (30).
As described for the synthesis of 15 from 9, tert-butyl (S)-5-
hydroxy-2-(N-(9-(9-phenylfluorenyl))amino)-5,5-dideuteriopen-
tanoate (35.5 mg, 8.2 µmol), generated in the previous step,
was converted to 30 (10.9 mg, 98%). 1H NMR (300 MHz, D2O)
δ 1.61 (m, 2H), 1.88 (m, 2H), 3.73 (t, J ) 6 Hz, 1H).
The stereospecifically labeled NosE substrates were syn-
thesized in the following manner:
(4S)-3-(Ben zyloxyca r b on yl)-4-(ca r b oxyet h yl)-1,3-ox-
a zolid in -5-on e (32). The reaction was carried out analogously
as described for the aspartic acid derivative.24a To a suspension
of N-Cbz-L-glutamic acid (31) (2.16 g, 7.7 mmol) in 150 mL of
toluene were added paraformaldehyde (1.46 g) and p-TsOH
(154 mg). The mixture was heated under reflux for 100 min
using a Dean-Stark apparatus for azeotropic removal of
water. The product mixture was filtered through silica gel and
the product eluted with Et2O (400 mL). The solvent was
evaporated to yield 32 as a colorless oil (2.25 g, 100%). 1H NMR
(300 MHz, CDCl3) δ 2.10-2.60 (m, 4H), 4.38 (t, J ) 6 Hz, 1H),
5.10-5.30 (m, 3H), 5.45 (br, 1H), 7.30-7.40 (m, 5H).
(4S)-3-(Ben zyloxyca r bon yl)-4-(3-ch lor o-3-oxop r op yl)-
1,3-oxa zolid in -5-on e (33). A solution of oxazolidinone acid
32 (2.26 g, 7.7 mmol) in CH2Cl2 (7.7 mL) was treated with
redistilled oxalyl chloride (1.04 mL, 11.7 mmol) at room
temperature. One drop of anhydrous DMF was added as
catalyst. After 30 min the solvent and the excess reagent were
removed under water aspirator vacuum and DMF under high
vacuum to give 33 as an off-white solid residue (2.40 g, 100%).
1H NMR (300 MHz, CDCl3) δ 2.10-2.40 (m, 4H), 3.07 (m, 2H),
4.34 (t, J ) 6 Hz, 1H), 5.10-5.30 (m, 3H), 5.45 (br, 1H), 7.30-
7.40 (m, 5H).
(4S)-3-(Ben zyloxyca r bon yl)-4-(3-d eu ter iofor m yleth yl)-
1,3-oxa zolid in -5-on e (34). Acyl chloride 33 (1.58 g, 5.0 mmol)
was dissolved in 4 mL of THF and added over 3 min to the
deuterium-equilibrated catalyst (5% Pd/BaSO4, 0.30 g) in 20
mL of THF containing 1 equiv of redistilled lutidine (0.58 mL,
5.0 mmol). After being stirred for 2 h at room temperature,
the mixture was filtered. The filtrate was diluted with Et2O
(60 mL) and washed with dilute HCl (pH 3, 20 mL), H2O (20
mL), saturated NaHCO3 (2 × 20 mL), H2O (20 mL), and
saturated NaCl (20 mL). The organic phase was dried (MgSO4),
and the solvent evaporated to yield aldehyde 34 (0.68 g, 48%).
Syn th esis of NosE Su bstr a tes. Intermediates 3-8 and
11-14 were synthesized as described starting from L- or
D-glutamic acid (1 or 2).5,14 Those compounds were converted
into putative NosE substrates as described below:
ter t-Bu tyl (S)-5-Hyd r oxy-2-(N-(9-(9-p h en ylflu or en yl))-
a m in o)p en ta n oa te (9). The synthesis was carried out simi-
larly as described by Koskinen and Rapoport.14 Compound 3
(56.4 mg, 0.12 mmol) was dissolved in 3 mL of THF and cooled
to -78 °C. Lithium aluminum hydride (11.4 mg, 0.30 mmol)
was added. After 4 h of stirring at this temperature, 100 µL
of saturated aqueous Na2SO4 was added to quench the reaction
mixture. The mixture was allowed to warm to room temper-
ature, at which an additional 5 mL of saturated aqueous Na2-
SO4 was added. The product was extracted into EtOAc (4 ×
10 mL), and the combined organic layers were dried over
MgSO4, filtered, and evaporated. The crude product mixture
was subjected to flash silica gel chromatography. Elution was
initiated with 10% EtOAc in hexanes, and the product 9 eluted
with 20% EtOAc in hexanes (40.5 mg, 76%): Rf 0.12 (20%
EtOAc in hexanes); 1H NMR (300 MHz, CDCl3) δ 1.19 (s, 9H),
1.47 (m, 1H), 1.53 (m, 2H), 1.55 (m, 1H), 2.52 (dd, J ) 6.5, 4.5
Hz, 1H), 3.55 (br t, J ∼ 6.5 Hz, 2H), 7.18-7.70 (m, 13H).
ter t-Bu tyl (2R)-5-Hyd r oxy-2-(N-(9-(9-p h en ylflu or en yl))-
a m in o)p en ta n oa te (10). Compound 4 was converted analo-
gously to 10 (35.8 mg, 68%). For NMR data, see compound 9.
(2S,4S)-5-Hyd r oxyleu cin e (17). Compound 11 (22.5 mg,
52.4 µmol) was dissolved in 2.5 mL of a mixture of CH2Cl2-
TFA (4:1) and stirred at room temperature for 16 h. The
mixture was dried under N2 and the residue partitioned
between isooctane (5 mL) and H2O (5 mL). The isooctane layer
was back-extracted with 2 mL of H2O, and the combined
aqueous phases were evaporated. The residue was applied to
a Dowex-50W cation-exchange resin (H+ form, 1.2 × 5 cm) and
90 J . Org. Chem., Vol. 68, No. 1, 2003

Biosynthesis of 4-Methylproline in Cyanobacteria
1H NMR (300 MHz, CDCl3) δ 2.1-2.7 (m, 2H), 4.37 (t, J ) 6
DL-P 5C (21/22). Racemic P5C was generated and purified
by cation-exchange chromatography similarly as described.16
The P5C concentration was determined by reaction with
o-aminobenzaldehyde (o-AB) and spectrophotometrical mea-
2
Hz), 5.05-5.25 (m, 3H), 5.50 (br, 1H), 7.30-7.43 (m, 5H); H
NMR (77 MHz, CHCl3) δ 9.70.
(4S)-3-(Ben zyloxycar bon yl)-4-[(3S)-3-h ydr oxy-3-deu ter -
iop r op yl]-1,3-oxa zolid in -5-on e (35). Aldehyde 34 (0.322 g,
1.16 mmol) was dissolved in 3 mL of THF and added via
syringe to a 0.5 M solution of (R)-Alpine borane in THF (2.78
mL, 1.38 mmol) under N2 atmosphere and while stirring. The
mixture was stirred overnight, and then THF was removed
by water aspirator vacuum at 35 °C bath temperature. The
residue was redissolved in Et2O (20 mL) and cooled in an ice
bath. Ethanolamine (84.2 mg, 1.38 mmol) was added and
stirring continued for 20 min on ice. The precipitate was
removed after centrifugation, and the supernatant was con-
centrated. The crude product was subjected to flash silica gel
chromatography (EtOAc-hexanes 1:1) to give the (S)-alcohol
35 as a colorless oil (59.7 mg, 18%). Rf 0.25 (EtOAc-hexanes
1:1); 1H NMR (500 MHz, CDCl3) δ 1.40-2.30 (m, 4H), 3.60
(m, 1H), 4.36 (m, 1H), 5.10-5.25 (m, 3H), 5.50 (br, 1 H), 7.30-
surement of the absorption of the adduct at 444 nm (ꢀ444
)
2940 M-1 cm-1).16a Common isolated yields of DL-P5C (21/22)
were 70-80% (∼2.5 mM solutions).
L-P 5C (21). Compound 21 was synthesized similarly as
described for R-aminoadipic-δ-semialdehyde.17 (S)-Hava (15)
(4 mg) was incubated with CrO3 (3 mg) and 4 N HCl (0.5 mL)
at 80 °C for 1 h. The reaction mixture was carefully neutralized
with NaOH solutions, and Cr(OH)3 was precipitated by
centrifugation. The supernatant was acidified (1 N HCl) to pH
<2 and stored at 4 °C. Purification could be achieved by cation-
exchange chromatography (Dowex-50W, H+ form), but the
crude product was also usable in subsequent NosF assays.
L-P5C concentration was determined as above. When the crude
product was used, after mixing with Tris buffer (pH 8), the
additional Cr(OH)3 that had formed had to be removed by
centrifugation prior to NADH and enzyme addition.
2
7.40 (m, 5H); H NMR (77 MHz, CHCl3) δ 3.60.
(4S)-3-(Ben zyloxycar bon yl)-4-[(3R)-3-h ydr oxy-3-deu ter -
iop r op yl]-1,3-oxa zolid in -5-on e (36). Compound 36 was
synthesized from 34 in the same manner as described for 35,
except that (S)-Alpine borane was used. (R)-Alcohol 36 was
obtained as a colorless oil (62.4 mg; 19%). Rf 0.25 (EtOAc-
hexanes 1:1). The NMR spectra of 35 and 36 were indistin-
guishable.
D-P 5C (22). Compound 22 was generated in the same
manner as L-P5C (21) but using D-Hava (16).
(3S,5S)-3-Meth yl-P 5C (23). The synthesis was carried out
as described above for L-P5C (21), starting from (2S,4S)-5-
hydroxyleucine (17). However, some epimerization at C-3 will
have occurred (see text). The MeP5C concentration was
estimated by color reaction with o-AB, assuming that ꢀ444 of
the adduct corresponded closely to the adduct of P5C with
o-AB. UV spectra were found to exhibit the same maximum
at ∼444 nm.
(2S,5S)-5-Hyd r oxy-5-d eu ter io-2-a m in ova ler ic a cid (37).
Compound 35 (30 mg, 0.11 mmol) was incubated with 5 N HCl
(1.5 mL) at 100 °C for 2 h. The reaction mixture was extracted
with Et2O (2 × 1.0 mL). The combined organic phases were
back-extracted with H2O (1.0 mL) and the combined aqueous
phases concentrated to dryness. The crude product was redis-
solved in a minimal amount of H2O and applied to a Dowex-
50W (H+ form) column. After washing with H2O the product
was eluted with 4 N NH4OH (11.2 mg, 78%). 1H NMR (500
MHz, D2O) δ 1.61 (m, 2H), 1.90 (m, 2H), 3.62 (t, J ) 6.1 Hz,
1H), 3.74 (t, J ) 6.0 Hz, 1H); 2H NMR (77 MHz, H2O) δ 3.62.
(2S,5R)-5-Hyd r oxy-5-d eu ter o-2-a m in ova ler ic a cid (38).
Compound 38 was generated from 36 as described for 37. (R)-
Alcohol 38 was obtained as a colorless, amorphous solid (13.1
(3R,5S)-3-Meth yl-P 5C (24). Analogous oxidation of (2S,4R)-
5-hydroxyleucine (18) yielded compound 24 and generated
minor amounts of its C-3 epimer (see text). The concentration
of MeP5C was estimated as described above.
The synthesis of enamine 29 is detailed elsewhere.18
Ack n ow led gm en t. Funding was provided by R01
grant CA12623 from the National Cancer Institute.
Thanks to J ohn Raycraft and Cheryl Phillipson for
support in protein overexpressions and running enzyme
assays, to Wesley Y. Yoshida for the acquisition of many
NMR spectra, and to Dr. Sreedhara Chaganty for
technical assistance in isolation and synthesis.
1
mg; 91%). H NMR (500 MHz, D2O) δ 1.64 (m, 2H), 1.93 (m,
2H), 3.65 (t, J ) 6.1 Hz), 3.79 (t, J ) 6.0 Hz); 2H NMR (77
MHz, H2O) δ 3.64.
Syn th esis of NosF Su bstr a tes. Syntheses under aqueous
conditions yielded the following imines while oxidation in
organic solvents (attempted in the case of the methylated
substrates) resulted in enamine formation:
J O026479Q
J . Org. Chem, Vol. 68, No. 1, 2003 91