16-Aza-ent-beyerane and 16-Aza-ent-trachylobane
A R T I C L E S
structures of 12 and 13, it seems more likely that the reduced
affinity of 13 relative to 12 (in either the absence or presence
of PPi) is a function of the lower basicity of the aziridine
carbocation intermediates close to the negative charge, particu-
larly in the absence of other stabilizing influences in the active
11
site. Finally, the fact that ent-beyeran-16-yl PP does not appear
to be a free intermediate further emphasizes the tight control
KS must exert over the ionized carbocation intermediates and
PP‚Mg anion within its active site to prevent recombination of
the closely associated ion pair.
44
nitrogen (parent aziridinium ion pKa ∼ 8.0) relative to that
for the bridged pyrrolidine in 12 (pyrrolidinium ion pKa )
1
1.3).44 Thus azabeyerane would exist exclusively in the
ammonium form in the incubation medium at pH 7.4, and it is
likely bound to the enzyme in the protonated state, as seems to
be the case for azabornane in the active site of bornyl PP
synthase.17 By contrast, the proportion of the bridged aziridine
in 13 existing in the binding-competent, protonated state would
Experimental Section (see Supporting Information for
procedures and data for other compounds)
Methyl 16-ent-Azabeyerane-16-carboxylate (23). A literature
25a
procedure was followed with modification. A solution of com-
mercially available mercuric trifluoroacetate2 (65 mg, 0.15 mmol) and
carbamate 20a (44 mg, 0.132 mmol) in 3 mL of THF was purged with
nitrogen, covered with aluminum foil, and stirred at room temperature
be much lower. Since aziridinium ions are known to act as
5b
electrophiles in solution,3
7,38,45
there is a distinct possibility that
the protonated form of 13 reacts slowly with an active site
nucleophile, thus inactivating the enzyme and explaining the
increased inhibition observed in extended incubations mentioned
above.
for 16 h. The reaction was judged to be complete by TLC analysis (R
.22, 1:4 EtOAc:petroleum ether). A solution of NaBH (6 mg, 0.14
mmol) in 0.1 mL of 2.5 M NaOH was added dropwise. After another
h, aqueous Na CO (1 mL) was added, and stirring was continued
f
0
4
The relatively weak affinity observed for ent-beyeranyl PP
8
2
3
(
11) implies that the beyeranyl+ carbocation and the PP‚Mg
for an additional 4 h. The suspension was concentrated to remove THF,
anion leaving group are further separated in the KS active site
and the residue was extracted with ether (2 × 20 mL). The combined
4
6
than a typical covalent C-O bond length (1.42 Å). This
disparity presumably arises from separation of the PP‚Mg anion
and the hydrocarbon moieties of 5 following ionization by
changes in their relative positions and/or orientations. From
previous structural studies it seems most likely that absence of
covalent bond formation reflects repositioning of the hydrocar-
bon moiety, since the bound PP‚Mg counterions occupy a
relatively constant position in various terpene synthase-
substrate analogue cocrystals.19
ethereal extracts were washed with brine (2 × 10 mL), dried (MgSO
4
),
and concentrated. The resulting solid material (42 mg, 96%) showed
the following properties for a 1.7:1 mixture of N-CO Me rotamers:
TLC R 0.23 (1:9 EtOAc:hexane); FTIR (CHCl ) νmax 3017, 2950, 1682,
2
f
3
1
0
1
1
449, 1387 cm- ; H NMR (400 MHz, CDCl
.85 (s, 3H, CH ), 0.93 (s, ca. 1.9 H, CH ), 0.94 (s, ca. 1.1H CH
.34 (s, ca. 1.1H, CH ), 1.20-1.72 (m, 12.5 H), 1.44 (s, 1.9H, CH
1 1
3
) δ 0.80 (s, 3H, CH
3
),
3
),
3
),
3
3
3
.98 (br d, ∼0.4 H, J = 8 Hz, H12R), 2.16 (br d, ∼0.6 H, J = 8 Hz,
H12R), 2.90 (app d, ∼0.6 H, J =10 Hz, H15 endo), 2.97 (app d, ∼0.4
H, J = 11 Hz, H15 endo), 3.64 (s, ∼1.9 H, CO CH ), 3.66 (s, ∼1.1 H,
CO CH
2
3
2
3
), 3.89 (app dd, ∼0.6 H, J = 11, 2 Hz, H15 exo), 3.98 (app
Conclusions
dd, ∼0.4H, J = 11, 2 Hz, H15 exo). The crude product was hydrolyzed
to azabeyerane (12) without further purification.
The lack of any detectable conversion of ent-beyeran-16-yl
PP (11) to ent-kaurene in incubations with ent-kaurene synthase
appears to exclude this secondary PP as a free intermediate in
the enzyme-catalyzed bicyclization of ent-copalyl PP (5). The
enhanced affinities of 16-aza-ent-beyerane and 16-aza-ent-
trachylobane for KS in the presence of inorganic PP, in
comparison to the binding of the bicyclic substrate, indicate that
the heterocycles behave as transition state inhibitors and are
good candidates for active site probes for this key enzyme
associated with gibberellin phytohormone biosynthesis in plants.
In addition, the strong synergy exhibited by these compounds
with inorganic PP suggests substantial stabilization of the
1
6-Aza-ent-beyerane (12). The carbamate hydrolysis conditions
47
were modeled after a literature procedure. A mixture of 23 (44 mg,
.13 mmol), 1,2-propylene glycol (1 mL), water (0.1 mL), and KOH
336 mg, 6 mmol) was heated at reflux for 18 h, cooled to room
0
(
temperature, diluted with water (5 mL), and extracted with ether (3 ×
1
1
5 mL). The combined organic extracts were washed with water (1 ×
0 mL) and dried (MgSO ). Evaporation of the solvent afforded
0.22 (10:89:1
EtOAc:hexane:triethylamine); [R]25 -10.6 (c ) 2.5, CHCl ); FTIR
4
azabeyerane (12, 33 mg, 91%) as a clear oil: TLC R
f
D
3
-
1 1
(neat) νmax 3312, 2922, 1699, 1454 cm ; H NMR (400 MHz, CDCl
δ 0.79 (s, 3H, CH ), 0.78-0.88 (m, 2H), 0.85 (s, 3H, CH ), 0.93 (s,
H, CH ), 1.05-1.26 (m, 4H), 1.16 (s, 3H, CH ), 1.33-1.71(m, 12H),
2.56 (d, 1H, J ) 10.8 Hz, H15endo), 3.40 (d, 1H, J ) 10.8 Hz, H15exo);
3
)
3
3
3
3
3
+
corresponding intermediates, in particular beyeran-16-yl , by
13
C NMR (125.64 MHz, CDCl ) δ 15.1, 18.6, 20.3, 20.4, 20.6, 22.0,
3
ion pairing with the PP‚Mg released in the initial ionization of
the ent-copalyl PP substrate. Similar findings have been reported
with other terpene synthases,1 suggesting that the use of the
charged PP‚Mg complex as a counterion to stabilize proximal
secondary carbocation intermediates may be a relatively com-
mon enzymatic mechanism utilized by these enzymes. However,
such counterion stabilization seems to be limited to carbocations
located relatively close to the original PP position, as indicated
by the weaker synergy exhibited by distal aza analogues and
2
5
2
2.1, 26.9, 33.3, 33.9, 37.9, 39.1, 39.8, 39.9, 42.1, 45.5, 55.3, 56.1,
+
6.5; HRMS (ESI) m/z calcd for C19H34N (M + H) 276.2691, found
7,18
76.2687.
48
ent-8â-Aminomethyl-13-methyl-12-podocarpene (21). The hy-
47
drolysis was modeled after a literature procedure. A solution of 20a
(150 mg, 0.45 mmol) and KOH (1 g, 17.85 mmol) in 1.8 mL of
methanol and water (0.2 mL) was heated at reflux for 8 h, cooled to
room temperature, diluted with water (8 mL), and extracted with ether
(3 × 20 mL). The combined organic extracts were washed with water
4
1
7,19
(2 × 10 mL) and dried (MgSO ). Evaporation of the solvent afforded
structural studies.
Indeed, it has been suggested that the
2
5
primary amine 21 (117 mg, 95%) as a clear oil: [R]
D
-14 (c ) 2.3,
PP‚Mg anion may drive reactions toward localization of the
1
3 3
CHCl ); H NMR (400 MHz, CDCl ) δ 0.75-0.98 (m, 2H), 0.81 (s,
(
44) Dean, J. A. Lange’s Handbook of Chemistry, 15th ed.; McGraw-Hill: New
York, 1999; p 8.30.
(47) Angle, S. R.; Arnaiz, D. O. Tetrahedron Lett. 1989, 30, 515-518.
(48) The IUPAC nomenclature was followed according to which the R or â
configuration of substituents in ent-diterpenes is that designated in the
systematic name for the “normal” antipode. See Rigaudy, J.; Klesney, S.
P. Nomenclature of Organic Chemistry, Sections A, B, C, D, E, F, and H;
Pergamon Press: Oxford, 1979; p 511.
(45) (a) Chuang, T.-H.; Sharpless, K. B. Org. Lett. 2000, 2, 3555-3557. (b)
Crist, D. R.; Leonard, N. J. Angew. Chem. Int. Ed. Engl. 1969, 8, 962-
9
74.
(
46) Carey, F. A.; Sundberg, R. J. AdVanced Organic Chemistry Part A, 4th
ed; Kluwer Academic: New York, 2000; p 13.
J. AM. CHEM. SOC.
9
VOL. 129, NO. 41, 2007 12459
Roy, Arnab
