Synthesis of a deuterated analogue of bacteriohopanetetrol-glucosamine, a probe of complex hopanoid biosynthesis

Article abstract of DOI:10.1039/b803365j

This study reports the straightforward preparation of a deuterated biohopanoid that will be used to probe a new biosynthetic pathway. This multistep hemisynthesis required a stereoselective glycosylation, a regioselective deuteration as well as a properly defined strategy for the final deprotections and the purification of the amphiphilic final glycoside.

Full text of DOI:10.1039/b803365j

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of a deuterated analogue of bacteriohopanetetrol–glucosamine,
a probe of complex hopanoid biosynthesis
Weidong Pan and Ste´phane P. Vincent*
Received 27th February 2008, Accepted 28th March 2008
First published as an Advance Article on the web 6th May 2008
DOI: 10.1039/b803365j
This study reports the straightforward preparation of a deuterated biohopanoid that will be used to
probe a new biosynthetic pathway. This multistep hemisynthesis required a stereoselective
glycosylation, a regioselective deuteration as well as a properly defined strategy for the final
deprotections and the purification of the amphiphilic final glycoside.
In contrast with most other common functionalities present in
biomolecules, there is no general biochemical reaction explain-
ing the formation of natural ethers. Surprisingly, some living
organisms produce polyhydroxylated ethers along with their
corresponding glycosides. This is the case for calditol, found in the
archaean Sulfolobus solfactaricus,1 and bacteriohopane derivatives
1 and 2 (Fig. 1), isolated from Zymomonas mobilis.2–4 Triter-
penoids of the hopane series are widespread amongst eubacteria.
Composite hopanoids are the major constituents of the lipids of
Z. mobilis, by far the most abundant being bacteriohopanetetrol
derivatives 1 and 2. We thus recently developed synthetic methods
to construct biohopanoids to investigate both their biosynthesis
and their biological function which are poorly understood.
A preliminary biosynthetic investigation led us to hypothesize
that a glycoside such as 2 could be the precursor of ether 1 through
a so far unknown enzymatic ring contraction (Scheme 1).5 As a
matter of fact we are also investigating the mechanism of the
mycobacterial galactofuranose–pyranose interconversion, which
is also a ring contraction.6–10 At first sight, the process depicted
in Scheme 1 appears analogous to the well-established inositol–
synthase mechanism,11 with, however, a very significant difference:
the putative mechanism implied in the biosynthesis of cyclitol
1 from glycoside 2 would involve an endo opening of the sugar
ring giving rise to an oxonium intermediate whereas the inositol
synthase pathway involves the formation of an intermediate
aldehyde leading to an intramolecular aldol process.11
University of Namur (FUNDP), Laboratoire de Chimie Bio-Organique,
rue de Bruxelles 61, 5000, Namur, Belgium. E-mail: stephane.vincent@
fundp.ac.be; Fax: +32 (0) 81 72 45 17
Fig. 1 Natural hopanoids (1–3) isolated from Z. mobilis and deuterated analog 4.
Scheme 1 Putative biosynthesis of bacteriohopane–carbapseudopentose 1.
2394 | Org. Biomol. Chem., 2008, 6, 2394–2399 This journal is The Royal Society of Chemistry 2008
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The isotopic labeling of a molecule used in feeding experiments
is exploited as a tracer of a specific biosynthetic pathway. In the
case of the pentacyclic hopane skeleton of natural molecules 1–3,
feeding experiments with 13C- and deuterium-labelled molecules
led Rohmer et al. to discover the long overlooked non-mevalonate
pathway for isoprenoid biosynthesis.4,12–15 More recently, a prelimi-
nary feeding experiment using deuterated N-acetyl-D-glucosamine
(6,6ꢀ-D2-GlcNAc) proved that glycoside 2 and ether 1 share the
same biosynthetic pathway derived from GlcNAc.5 Moreover,
the similar isotope abundance found in the glycon moieties
of hopanoids 1 and 2, after feeding experiments, suggested
a precursor to product relationship between glycoside 2 and
cyclopentitol 1.
In order to demonstrate or rule out this biosynthetic assump-
tion, we now need to show that glycoside 2 can be transformed
into ether 1 by Z. mobilis enzymatic machinery. Therefore, the
synthesis of deuterated glycoside 4 is required to unambiguously
demonstrate that 2 is the direct biosynthetic precursor of 1
(Fig. 1).
Recently, we have reported a concise strategy centered on the
copper activation of the C30 hopane moiety for the synthesis
of bacteriohopanetetrol 3 and its glycosylated derivative 2.16
Then, we developed a more general approach based on a LiDBB
lithiation of phenyl sulfide 5 (Scheme 2) for the synthesis of a wide
range of biohopanoids including bacteriohopanepentols bearing
an additional hydroxyl group at position C-31.17,18
The synthesis of deuterated analog 4 thus started from phenyl
sulfide 5 (Scheme 2), easily obtained in a few steps from the com-
mercially available Dammar resin.17,19 Diol 6 was then prepared by
the new coupling procedure we recently developed.17 The properly
protected glucosamine donor 7 was prepared by a quantitative
acetylation of the known20 primary alcohol. The acetate group was
chosen to allow its selective deprotection, after the glycosylation
step.
were in complete agreement with the assigned structure, especially
the expected b-configuration.
The 6ꢀ-acetate selective deprotection was readily achieved by
carefully treating glycoside 8 with MeONa in a mixed solvent
of THF–MeOH at room temperature. Under these conditions,
the phthalimido group was not deprotected and the desired 6ꢀ-
hydroxy glycoside 9 was obtained in 94% yield. Intermediate 9
could then undergo an oxidation followed by a LiAlD4 reduction
to introduce the two deuterium atoms in the target molecule 11.
Many methods are available for the conversion of primary hydroxy
groups into carboxylic acids. However, most of them are not
appropriate for highly functionalized structures such as compound
9 bearing acid and base sensitive protective groups. Moreover, we
wanted to achieve the regioselective oxidation of the glucosamine
primary alcohol in presence of the secondary alcohol of the ribitol
side-chain. After the screening of the best oxidation reagents, we
found that the TEMPO oxidation procedure developed by Davis
and Flitsch gave, from far, the best yields.21 From the known
procedure, we only modified the esterification condition, using an
excess amount of iodomethane in presence of sodium bicarbonate
and tetrabutylammonium chloride. The desired ester 10 was thus
obtained in 69% yield for two steps (Scheme 2).
As expected, the following reduction was much more difficult
to optimize. The use of LiAlD4 was essential for the complete
bisdeuteration of ester 10, since NaBD4 is generally not efficient
for the reduction of carboxylic esters. Not surprisingly, the main
side-reaction was the removal of the phthalimido group. The
desired deuterated alcohol 11 was then isolated in a moderate
but reproducible yield.
At this stage, the bisdeuteration was easily confirmed by the
comparison of 1H, 13C NMR and mass spectra of derivative 11 with
those of the corresponding non-deuterated molecule 9. A small
multiplet was observed for the carbon C-6ꢀ of the glucosamine
subunit due to the coupling between the carbon and the two
deuterium atoms. In addition, the expected a- and b-shifts were
also observed for the C-6ꢀ (Dd = 0.4 ppm, see Experimental section)
and C-5ꢀ atoms (Dd = 0.2 ppm), respectively.
The regio- and stereoselective coupling of diol 6 and phenyl
sulfide 7 was conducted in the presence of NIS and a catalytic
amount of TfOH in CH2Cl2 at a temperature ranging from −78 ◦C
to −30 ◦C. The reaction proceeded smoothly and furnished the
desired b-D-glucosaminylhopanetetrol derivative 8 as the main
product in 84% yield (Scheme 2). The 1H and 13C NMR spectra of 8
The sequence of deprotections of 11 was similar to that we
had developed for the non-labelled one (Scheme 3).16 Thus, the
acetonide group in 11 was removed using hydrochloric acid (1% in
Scheme 2 Reagents and conditions: (a) NIS, TfOH, MS 4 A, CH2Cl2, −78 to −30 ◦C, 84%; (b) MeONa, THF–MeOH, rt, 94%; (c) TEMPO, NaOCl,
NaHCO3, KBr, Bu4NCl, CH2Cl2, 0 ◦C; then MeI, NaHCO3, Bu4NCl, rt, 69%; d) LiAlD4 THF, −40 to −20 ◦C, 36%.
˚
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Scheme 3 Reagents and conditions: (a) HCl (1%), MeOH–THF, rt, 83%; (b) H2N–NH2·H2O, EtOH–H2O, 80 ◦C, 88%; (c) Pd black, H2, THF–AcOH–H2O,
rt.
MeOH) at room temperature yielding bisdeuterated glycoside 12
in 83% yield. The hydrazine promoted phthalimido deprotection
afforded the desired amine 13. The purification of this product
proved more difficult than its tri-O-benzyl analogue16 due to
its higher polarity which complicated the required silica gel
chromatography. Given the glycolipidic nature of molecule 13,
purification using standard reversed phase preparative HPLC or
ion exchange chromatography was not appropriate. A successful
purification was finally achieved in two steps: a silica gel chro-
matography using a polar solvent system (CH2Cl2–MeOH, 20 : 1),
followed by a size-exclusion chromatography (CH2Cl2–CH3OH,
2 : 1) to afford pure 13 in 88% yield. After screening catalyst and
solvent systems, the final bisdeuterated glycoside 4 was obtained
after the hydrogenolysis of 13 in the presence of palladium black
under a hydrogen atmosphere in 93% yield.
The structure of 4 was confirmed by comparing all analytical
data with its non-deuterated analogue 2,16 and also by the
comparison of the 1H-NMR of the peracetates of 4 and 2.22,23
In conclusion, we have developed a straightforward preparation
of deuterated glycoside 4 that will be used as a biosynthetic probe
for the discovery of a new enzymatic reaction. This multistep
synthesis required a stereoselective glycosylation, a regioselective
deuteration as well as a properly defined strategy for the final
deprotections and the purification of the amphiphilic glycoside 4.
Feeding experiments using deuterated molecule 4 are in progress
and the results will be reported in due course.
J1,2 = 10.6 Hz, 1-H), 4.47–4.94 (4H, m, BnCH2), 4.50 (1H, dd,
J5,6b = 2.1 Hz, Jgem = 11.9 Hz, 6-Hb), 4.46 (1H, dd, J2,3 = 10.3 Hz,
J3,4 = 8.6 Hz, 3-H), 3.44 (1H, t, J = 10.3 Hz, 2-H), 3.43 (1H,
dd, J5,6a = 5.2 Hz, Jgem = 11.9 Hz, 6-Ha), 3.81 (1H, ddd, J5,6b
=
2.1 Hz, J5,6a = 5.2 Hz, J4,5 = 9.9 Hz, 5-H), 3.71 (1H, J3,4 = 8.6 Hz,
J4,5 = 9.9 Hz, 4-H), 2.12 (3H, s, OAc). 13C NMR (101 MHz):
d = 170.5 (CH3C=O), 133.8, 133.7, 132.6, 128.7, 128.5, 128.0,
127.8, 127.4, 123.4, 123.3, 83.1 (C-1), 80.4 (C-3), 79.1 (C-4), 77.0
(C-5), 75.01 and 75.00 (BnCH2), 62.9 (C-6), 54.7 (C-2), 20.8
+
=
(CH3C O). HRMS (DCI): m/z 641.2319 [M + NH4 ], calcd for
C36H37O7N2S: 641.2321.
Acetate 8
A suspension of diol 6 (53.0 mg, 0.09 lmol), glycoside 7 (78.9 mg,
˚
126 lmol) and molecular sieves (4 A, 30.0 mg) in anhydrous
CH2Cl2 (3 mL) under an atmosphere of argon was stirred at room
temperature for 15 min, then NIS (51.0 mg, 0.225 mmol) was
added at room temperature, followed by a slow addition of TfOH
(0.40 lL, 0.0045 lmol) at −78 ◦C. The reaction temperature was
◦
allowed to warm up to −30 C gradually within ca. 3 h and the
reaction was monitored by TLC during that period, until diol
6 could not be detected any more. The reaction mixture was
quenched with Et3N at −20 ◦C before it was diluted with CH2Cl2.
Then the organic phase was washed successively with saturated
aqueous Na2S2O3, brine, and dried over MgSO4, filtered through
cotton and the filtrates were evaporated under reduced pressure.
The resulting crude product was chromatographed on silica gel
(toluene–EtOAc, 5 : 1) to yield 8 as a light yellow syrup (83.3 mg,
84%, Rf 0.47, toluene–EtOAc, 5 : 1). [a]1D9 +19 (c 0.35, CHCl3).
1H NMR (400 MHz, CDCl3): d = 6.92–7.71 (14H, m, ArH), 5.29
Experimental
Phenyl 6-O-acetyl-3,4-di-O-benzyl-1,2-dideoxy-2-phthalimido-1-
thio-b-D-glucopyranoside 7
(1H, d, J1 ,2 = 8.5 Hz, 1ꢀ-H), 4.45–4.93 (4H, m, BnCH2), 4.46 (1H,
ꢀ
ꢀ
m, 6ꢀ-Hb), 4.39 (1H, dd, J2 ,3 = 10.7 Hz, J3 ,4 = 8.0 Hz, 3ꢀ-H),
ꢀ
ꢀ
ꢀ ꢀ
Phenyl 3,4-di-O-benzyl-1,2-dideoxy-2-phthalimido-1-thio-b-D-
glucopyranoside13 (1.84 g, 3.16 mmol) was acetylated (pyridine–
Ac2O, 2 : 1, v/v, 30 mL) at room temperature for 1 h. The
excess of Ac2O was decomposed by pouring the reaction mixture
into ice–water. The resulting mixture was extracted three times
with CH2Cl2. The combined extracts were washed with brine,
dried (MgSO4) and filtered through cotton. The solvents were
concentrated to dryness under reduced pressure. The crude
acetate was chromatographed on silica gel (cyclohexane–EtOAc,
3 : 1) to yield 7 as a light yellow syrup (2.04 g, quant., Rf 0.32,
4.30 (1H, dd, J5 ,6 a = 3.9 Hz, Jgem = 11.8 Hz, 6ꢀ-Ha), 4.26 (1H, m,
ꢀ
ꢀ
34-H), 4.22 (1H, dd, J1 ,2 = 8.5 Hz, J2 ,3 = 10.7 Hz, 2ꢀ-H), 3.83
(2H, m, 33-H and 35-Hb), 3.74 (2H, m, 5ꢀ-H and 4ꢀ-H), 3.63 (1H,
t, J34,35a = Jgem = 9.6 Hz, 35-Ha), 3.35 (1H, m, 32-H), 2.55 (1H,
brs, J = 3.0 Hz, 32-OH), 2.13 (3H, s, AcO), 1.28 and 1.26 (6H, 2 s,
acetal-Me), 1.00 and 0.98 (6H, 2 s, 8b- and 14a-Me), 0.89 (3H, s,
4a-Me), 0.86 (3H, s, 10b-Me), 0.84 (3H, s, 4b-Me), 0.82 (3H, d,
J22,29 = 6.4 Hz, 22-Me), 0.71 (3H, s, 18a-Me), 1.11–1.80 (29H, m,
hopane), 0.72–0.97 (3H, m, hopane); 13C NMR (101 MHz): d =
170.6 (CH3C=O), 137.5, 133.3, 128.5, 128.0, 127.8, 127.4, 123.3,
108.2 (acetal-C), 97.9 (C-1ꢀ), 80.3 (C-33), 79.1 (C-3ꢀ), 79.0 (C-4ꢀ),
ꢀ
ꢀ
ꢀ ꢀ
1
cyclohexane–EtOAc, 3 : 1). [a]2D5 +92 (c 1.00, CHCl3). H NMR
(400 MHz, CDCl3): d = 6.92–7.73 (19H, m, ArH), 5.58 (1H, d,
2396 | Org. Biomol. Chem., 2008, 6, 2394–2399
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75.02 and 74.95 (BnCH2), 74.8 (C-34), 73.1 (C-5ꢀ), 69.0 (C-32),
67.4 (C-35), 62.5 (C-6ꢀ), 56.1 (C-5), 55.3 (C-2ꢀ), 54.4 (C-17), 50.4
(C-9), 49.2 (C-13), 45.6 (C-21), 44.3 (C-18), 42.0 (C-3), 41.7 (C-14),
41.6 (C-8), 41.5 (C-19), 40.3 (C-1), 37.3 (C-10), 36.7 (C-22), 33.6
(C-15), 33.4 (C-24), 33.22 and 33.19 (C-4 and C-7), 30.3 (C-30),
30.2 (C-20), 28.0 (acetal-Me), 27.6 (C-31), 25.4 (acetal-Me), 23.9
continued overnight. The resulting dark red mixture was extracted
three times with CH2Cl2, and the combined extracts were washed
with brine, dried over MgSO4 and filtered through cotton. The
resulting filtrates were concentrated to dryness and the residue
was chromatographed on silica gel (cyclohexane–EtOAc, 4 : 1) to
afford 10 as a syrup (24.6 mg, 69%, Rf 0.49, cyclohexane–EtOAc,
1
(C-12), 22.8 (C-16), 21.6 (C-23), 20.9 (C-11), 20.8 (CH3C O), 20.1
2 : 1). [a]1D9 +40 (c 0.80, CHCl3). H NMR (400 MHz, CDCl3):
=
d = 6.89–7.71 (14H, m, ArH), 5.31 (1H, d, J1 ,2 = 8.4 Hz, 1ꢀ-H),
ꢀ
ꢀ
(C-29), 18.6 (C-2 and C-6), 16.5 and 16.4 (C-26 and C-27), 15.9
(C-25 and C-28). HRMS (FAB): m/z 1122.6663 [M + Na+], calcd
for C66H91O10NNa: 1122.6646.
ꢀ
ꢀ
ꢀ ꢀ
4.44–4.85 (4H, m, BnCH2), 4.37 (1H, dd, J2 ,3 = 10.6 Hz, J3 ,4
=
8.7 Hz, 3ꢀ-H), 4.26 (1H, dd, J1 ,2 = 8.4 Hz, J2 ,3 = 10.6 Hz, 2ꢀ-H),
ꢀ
ꢀ
ꢀ ꢀ
4.25 (1H, m, 34-H), 4.15 (1H, d, J4 ,5 = 9.7 Hz, 5ꢀ-H), 3.99 (1H,
ꢀ
ꢀ
dd, J3 ,4 = 8.7 Hz, J4 ,5 = 9.7 Hz, 4ꢀ-H), 3.87 (1H, dd, J34,35b
=
ꢀ
ꢀ
ꢀ ꢀ
Alcohol 9
3.6 Hz, Jgem = 9.6 Hz, 35-Hb), 3.81 (1H, m, 33-H), 3.80 (3H, s,
OMe), 3.62 (1H, dd, J34,35a = Jgem = 9.6 Hz, 35-Ha), 3.29 (1H, m,
32-H), 2.39 (1H, brs, 32-OH), 1.16 and 1.13 (6H, 2 s, acetal-Me),
0.87 (6H, 2 s, 8b- and 14a-Me), 0.78 (3H, s, 4a-Me), 0.75 (3H, s,
10b-Me), 0.72 (3H, s, 4b-Me), 0.68 (3H, d, J22,29 = 6.4 Hz, 22-Me),
0.59 (3H, s, 18a-Me), 1.02–1.75 (29H, m, hopane), 0.50–0.85 (3H,
m, hopane); 13C NMR (101 MHz): d = 168.4 (ester-C=O), 137.4,
133.6, 131.6, 128.4, 128.0, 127.9, 127.4, 123.3, 108.2 (acetal-C),
98.1 (C-1ꢀ), 81.0 (C-4ꢀ), 80.3 (C-33), 78.1 (C-3ꢀ), 75.0, 74.8, 74.7
(C-34), 74.3 (C-5ꢀ), 69.1 (C-32), 67.4 (C-35), 56.1 (C-5), 55.0 (C-2ꢀ),
54.4 (C-17), 52.6 (OMe), 50.4 (C-9), 49.2 (C-13), 45.5 (C-21), 44.3
(C-18), 42.1 (C-3), 41.7 (C-14), 41.6 (C-8), 41.5 (C-19), 40.2 (C-1),
37.3 (C-10), 36.6 (C-22), 33.6 (C-15), 33.3 (C-24), 33.20 and 33.17
(C-4 and C-7), 30.2 (C-30), 30.1 (C-20), 28.0 (acetal-Me), 27.5 (C-
31), 25.3 (acetal-Me), 23.9 (C-12), 22.8 (C-16), 21.5 (C-23), 20.9
(C-11), 20.0 (C-29), 18.6 (C-2 and C-6), 16.5 and 16.4 (C-26 and
C-27), 15.8 (C-25 and C-28). HRMS (FAB): m/z 1108.6479 [M +
Na+], calcd for C67H91O11NNa: 1108.6490.
To a solution of compound 8 (79.0 mg, 72 lmol) in anhydrous
THF (2 mL) under an atmosphere of argon was added a freshly
prepared solution of MeONa in MeOH (0.03 M, 4 mL) at room
temperature. The resulting mixture was then stirred for 70 min. The
reaction mixture was neutralized (pH = 7) by adding Amberlyst
A-120 resin (H+ form) before it was filtered through cotton. The
filtrates were evaporated under reduced pressure and the residue
was chromatographed on silica gel (cyclohexane–acetone, 7 :
1) affording the desired product 9 as a white amorphous solid
(71.0 mg, 94%, Rf 0.28, cyclohexane–EtOAc, 2 : 1). [a]1D8 +52 (c
1
1.0, CHCl3). H NMR (400 MHz, CDCl3): d = 6.91–7.70 (14H,
m, ArH), 5.32 (1H, d, J1 ,2 = 8.6 Hz, 1ꢀ-H), 4.45–4.92 (4H, m,
ꢀ
ꢀ
BnCH2), 4.38 (1H, dd, J2 ,3 = 10.7 Hz, J3 ,4 = 8.7 Hz, 3ꢀ-H), 4.27
ꢀ
ꢀ
ꢀ ꢀ
(1H, m, 34-H), 4.18 (1H, dd, J1 ,2 = 8.6 Hz, J2 ,3 = 10.7 Hz, 2ꢀ-H),
ꢀ
ꢀ
ꢀ ꢀ
3.97 (1H, dd, J5 ,6 b = 2.1 Hz, Jgem = 12.1 Hz, 6ꢀ-Hb), 3.76–3.85 (4H,
m, 33-H, 6ꢀ-Ha, 35-Hb, 4ꢀ-H), 3.67 (1H, t, J34,35a = Jgem = 9.6 Hz, 35-
Ha), 3.61 (1H, m, 5ꢀ-H), 3.36 (1H, m, 32-H), 2.58 (1H, brs, 32-OH),
2.19 (1H, brs, 6ꢀ-OH), 1.29 and 1.26 (6H, 2 s, acetal-Me), 0.99 and
0.98 (6H, 2 s, 8b- and 14a-Me), 0.89 (3H, s, 4a-Me), 0.86 (3H, s,
10b-Me), 0.84 (3H, s, 4b-Me), 0.82 (3H, d, J22,29 = 6.4 Hz, 22-Me),
0.71 (3H, s, 18a-Me), 1.10–1.79 (29H, m, hopane), 0.72–0.97 (3H,
m, hopane); 13C NMR (101 MHz): d = 137.7, 133.7, 128.5, 128.0,
127.8, 127.4, 123.3, 108.4 (acetal-C), 98.1 (C-1ꢀ), 80.3 (C-33), 79.0
(C-4ꢀ), 78.8 (C-3ꢀ), 75.5 (C-5ꢀ), 75.02 (BnCH2), 74.96 (C-34), 74.8
(BnCH2), 69.1 (C-32), 67.7 (C-35), 61.5 (C-6ꢀ), 56.1 (C-5), 55.5
(C-2ꢀ), 54.4 (C-17), 50.4 (C-9), 49.2 (C-13), 45.7 (C-21), 44.4 (C-
18), 42.1 (C-3), 41.7 (C-14), 41.61 (C-8), 41.56 (C-19), 40.3 (C-1),
37.3 (C-10), 36.7 (C-22), 33.6 (C-15), 33.4 (C-24), 33.23 and 33.21
(C-4 and C-7), 30.4 (C-30), 30.2 (C-20), 28.0 (acetal-Me), 27.6 (C-
31), 25.4 (acetal-Me), 23.9 (C-12), 22.8 (C-16), 21.6 (C-23), 20.9
(C-11), 20.1 (C-29), 18.7 (C-2 and C-6), 16.5 and 16.4 (C-26 and
C-27), 15.9 (C-25 and C-28). HRMS (FAB): m/z 1080.6553 [M +
Na+], calcd for C66H91O10NNa: 1080.6541.
ꢀ
ꢀ
Bisdeuterated alcohol 11
To a solution of 10 (95.0 mg, 87 lmol) in freshly distilled THF
(8 mL) under an atmosphere of dry argon was added LiAlD4
(5.8 mg, 138 lmol) portionwise in four amounts at a temperature
varying from −40 ◦C to −20 ◦C until the starting material could
not be detected any more by TLC. Excess LiAlD4 was reacted
with EtOAc (2 mL) at −78 ◦C before the reaction mixture was
concentrated to dryness in vacuo, the residue was purified by
column chromatography (cyclohexane–EtOAc, 4 : 1) yielding 11
as a white amorphous solid (33.4 mg, 36%, Rf 0.28, cyclohexane–
1
EtOAc, 2 : 1). [a]1D8 +59 (c 1.25, CHCl3). H NMR (400 MHz,
ꢀ
ꢀ
CDCl3): d = 6.91–7.70 (14H, m, ArH), 5.32 (1H, d, J1 ,2 = 8.5 Hz,
1ꢀ-H), 4.45–4.92 (4H, m, BnCH2), 4.38 (1H, dd, J2 ,3 = 10.7 Hz,
ꢀ
ꢀ
J3 ,4 = 8.7 Hz, 3ꢀ-H), 4.27 (1H, m, 34-H), 4.18 (1H, dd, J1 ,2
=
ꢀ
ꢀ
ꢀ ꢀ
8.5 Hz, J2 ,3 = 10.7 Hz, 2ꢀ-H), 3.81 (3H, m, 33-H, 35-Hb, 4ꢀ-H),
ꢀ
ꢀ
ꢀ
ꢀ
3.67 (1H, t, J34,35a = Jgem = 9.6 Hz, 35-Ha), 3.60 (1H, d, J4 ,5
=
Ester 10
9.8 Hz, 5ꢀ-H), 3.35 (1H, m, 32-H), 2.55 (1H, brd, 32-OH), 2.08
(1H, s, 6ꢀ-OH), 1.29 and 1.26 (6H, 2 s, acetal-Me), 0.99 and 0.98
(6H, 2 s, 8b- and 14a-Me), 0.89 (3H, s, 4a-Me), 0.86 (3H, s, 10b-
Me), 0.84 (3H, s, 4b-Me), 0.82 (3H, d, J22,29 = 6.4 Hz, 22-Me),
0.71 (3H, s, 18a-Me), 1.10–1.79 (29H, m, hopane), 0.72–0.97 (3H,
m, hopane); 13C NMR (101 MHz): d = 137.7, 133.7, 128.5, 128.0,
127.8, 127.4, 123.3, 108.4 (acetal-C), 98.1 (C-1ꢀ), 80.4 (C-33), 79.0
(C-4ꢀ), 78.8 (C-3ꢀ), 75.3 (C-5ꢀ, b-shift), 75.02 (BnCH2), 74.97 (C-
34), 74.85 (BnCH2), 69.1 (C-32), 67.7 (C-35), 60.9 (m, C-6ꢀD2,
a-shift), 56.1 (C-5), 55.5 (C-2ꢀ), 54.4 (C-17), 50.4 (C-9), 49.3 (C-
13), 45.7 (C-21), 44.4 (C-18), 42.1 (C-3), 41.8 (C-14), 41.63 (C-8),
To a solution of 9 (35.0 mg, 33 lmol) in CH2Cl2 (5 mL) was
◦
added TEMPO (1.0 mg, 7 lmol) at 0 C, followed by a mixture
of saturated aqueous NaHCO3 (1 mL), KBr (5.0 mg) and Bu4NCl
(6.0 mg). The resulting mixture was stirred vigorously for 20 min
at 0 ◦C before a mixture of saturated aq. NaCl, saturated aq.
NaHCO3 and 10% aq. NaOCl (2 : 1 : 2, v/v/v, 1.5 mL) was
added dropwise within 1 h at 0 ◦C. Then, to this yellowish mixture
was added successively NaHCO3 (20.0 mg), Bu4NCl (20.0 mg)
and MeI (1 mL) at 0 ◦C. The reaction mixture was allowed to
warm up gradiently to room temperature and the stirring was
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41.57 (C-19), 40.3 (C-1), 37.4 (C-10), 36.7 (C-22), 33.6 (C-15), 33.4
(C-24), 33.24 and 33.21 (C-4 and C-7), 30.4 (C-30), 30.2 (C-20),
28.1 (acetal-Me), 27.6 (C-31), 25.4 (acetal-Me), 23.9 (C-12), 22.8
(C-16), 21.6 (C-23), 20.9 (C-11), 20.1 (C-29), 18.7 (C-2 and C-6),
16.5 and 16.4 (C-26 and C-27), 15.9 (C-25 and C-28). HRMS
(FAB): m/z 1082.6656 [M + Na+], calcd for C66H89O10D2NNa:
1082.6666.
103.3 (C-1ꢀ), 84.2 (C-3ꢀ), 78.2 (C-4ꢀ), 75.7 (C-5ꢀ), 75.3 (BnCH2),
75.0 (BnCH2), 74.0 (C-32), 73.5 (C-33), 71.8 (C-34), 71.6 (C-35),
60.6 (m, C-6ꢀ), 56.4 (C-2ꢀ), 56.1 (C-5), 54.5 (C-17), 50.5 (C-9), 49.2
(C-13), 46.2 (C-21), 44.4 (C-18), 42.1 (C-3), 41.8 (C-14), 41.7 (C-
8), 41.6 (C-19), 40.3 (C-1), 37.4 (C-10), 36.9 (C-22), 33.7 (C-15),
33.4 (C-24), 33.3 and 33.2 (C-4 and C-7), 31.7 (C-30), 29.0 (C-20),
27.8 (C-31), 24.0 (C-12), 22.9 (C-16), 21.6 (C-23), 20.9 (C-11), 20.1
(C-29), 18.7 (C-2 and C-6), 16.6 and 16.5 (C-26 and C-27), 15.9
(C-25 and C-28). HRMS (FAB): m/z 912.6284 [M + Na+], calcd
for C55H83O8D2NNa: 912.6298.
Bisdeuterated tetrol 12
The diol 11 (6.5 mg, 6 lmol) was dissolved in THF–MeOH
(1 : 1, v/v, 2 mL) containing aqueous HCl (37%, 12 lL) at
Bisdeuterated 35-O-b-D-glucosaminylbacteriohopanetetrol 4
0
◦C. The resulting mixture was stirred overnight vigorously at
room temperature. Then, it was neutralized with solid NaHCO3,
filtered through cotton and concentrated to dryness. The residue
was chromatographed on silica gel (CH2Cl2–MeOH, 30 : 1)
providing 12 as a white solid (5.2 mg, 83%, Rf 0.18, CH2Cl2–
A solution of 13 (14.8 mg, 17 lmol) in THF–AcOH–H2O (4 mL,
16 : 8 : 3, v/v/v) was vigorously stirred overnight under an
atmosphere of hydrogen in presence of Pd black (20 mg) at
room temperature. The catalyst was filtered off by passing the
1
MeOH, 20 : 1). [a]1D8 +29 (c 0.61, CHCl3). H NMR (400 MHz,
R
reaction mixture through a short plug of Celiteꢀ, and the solvents
ꢀ
ꢀ
CDCl3): d = 6.91–7.72 (14H, m, ArH), 5.25 (1H, d, J1 ,2 = 8.5 Hz,
were evaporated under reduced pressure yielding the deprotected
1ꢀ-H), 4.47–4.93 (4H, m, ArCH2), 4.43 (1H, dd, J2 ,3 = 10.7 Hz,
ꢀ
ꢀ
bisdeuterated analogue 4 as a pale solid (11.0 mg, 93%, Rf 0.53,
J3 ,4 = 8.5 Hz, 3ꢀ-H), 4.18 (1H, dd, J1 ,2 = 8.5 Hz, J2 ,3 = 10.7 Hz,
2ꢀ-H), 3.88 (2H, m, 35-Ha and 35-Hb), 3.74 (2H, m, 34-H and 4ꢀ-
H), 3.63 (2H, m, 32-H and 5ꢀ-H), 3.52 (1H, m, 33-H), 3.22 (1H,
brd, 34-OH), 2.97 (1H, brd, J = 4.8, 33-OH), 2.72 (1H, s, 6ꢀ-OH),
2.47 (1H, brd, 32-OH), 0.98 (6H, 2 s, 8b- and 14a-Me), 0.92 (3H,
d, J22,29 = 6.3 Hz, 22-Me), 0.89 (3H, s, 4a-Me), 0.85 (3H, s, 10b-
Me), 0.83 (3H, s, 4b-Me), 0.72 (3H, s, 18a-Me), 1.10–1.85 (29H, m,
hopane), 0.72–0.95 (3H, m, hopane); 13C NMR (101 MHz): d =
137.8, 137.7, 133.8, 131.5, 128.6, 128.1, 128.0, 127.8, 127.4, 123.4,
98.8 (C-1ꢀ), 79.3 (C-4ꢀ), 79.0 (C-3ꢀ), 75.5 (C-5ꢀ), 75.1 (BnCH2),
74.9 (BnCH2), 73.9 (C-33), 73.3 (C-32), 71.7 (C-35), 71.6 (C-34),
56.1 (C-5), 55.8 (C-2ꢀ), 54.5 (C-17), 50.4 (C-9), 49.3 (C-13), 46.1
(C-21), 44.3 (C-18), 42.1 (C-3), 41.8 (C-14), 41.7 (C-8), 41.6 (C-
19), 40.3 (C-1), 37.4 (C-10), 36.9 (C-22), 33.7 (C-15), 33.4 (C-24),
33.3 and 33.2 (C-4 and C-7), 31.4 (C-30), 28.9 (C-20), 27.7 (C-
31), 23.9 (C-12), 22.8 (C-16), 21.6 (C-23), 20.9 (C-11), 20.0 (C-29),
18.7 (C-2 and C-6), 16.6 and 16.5 (C-26 and C-27), 15.9 (C-25
and C-28). HRMS (FAB): m/z 1042.6343 [M + Na+], calcd for
C63H85O10D2NNa: 1042.6353.
1
EtOAc–i-PrOH–H2O, 4.5 : 3 : 2). [a]2D2 +5 (c 0.52, pyridine). H
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ ꢀ
ꢀ
ꢀ
NMR (400 MHz, pyridine-D5): d = 4.92 (1H, d, J1 ,2 = 8.0 Hz,
1ꢀ-H), 4.68 (1H, m, 34-H), 4.67 (1H, m, 35-Hb), 4.58 (1H, m, 35-
ꢀ
ꢀ
Ha), 4.42 (1H, m, 32-H), 4.30 (1H, m, 33-H), 4.21 (1H, dd, J3 ,4
=
9.1 Hz, J4 ,5 = 9.6 Hz, 4ꢀ-H), 4.03 (1H, dd, J2 ,3 = 9.4 Hz, J3 ,4
=
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ ꢀ
ꢀ
9.1 Hz, 3ꢀ-H), 3.88 (1H, d, J4 ,5 = 9.6 Hz, 5 -H), 3.32 (1H, m, 2 -H),
1.06 (3H, d, J22,29 = 5.7 Hz, 22-Me), 0.95 and 0.94 (6H, 2 s, 8b- and
14a-Me), 0.86 (3H, s, 4a-Me), 0.80 (6H, 2 s, 10b-Me and 4b-Me),
0.69 (3H, s, 18a-Me), 1.08–2.20 (29H, m, hopane), 0.70–0.91 (3H,
m, hopane). 13C NMR (101 MHz): d = 105.2 (C-1ꢀ), 78.3 (C-5ꢀ,
b-shift), 77.7 (C-3ꢀ), 75.4 (C-33), 73.5 (C-32), 73.3 (C-34), 73.1 (C-
35), 71.4 (C-4ꢀ, c-shift), 58.4 (C-2ꢀ), 56.1 (C-5), 54.4 (C-17), 50.3
(C-9), 49.3 (C-13), 46.5 (C-21), 44.2 (C-18), 41.9 (C-3), 41.6 (C-
14), 41.5 (C-8 and C-19), 40.1 (C-1), 37.2 (C-10), 37.0 (C-22), 33.6
(C-15), 33.2 (C-24), 33.0 (C-4 and C-7), 32.1 (C-30), 29.8 (C-20),
27.7 (C-31), 23.9 (C-12), 22.6 (C-16), 21.5 (C-23), 21.1 (C-11), 20.2
(C-29), 18.7 (C-2 and C-6), 16.54 and 16.45 (C-26 and C-27), 15.8
(C-25 and C-28). HRMS (FAB): m/z 732.5379 [M + Na+], calcd
for C41H71D2O8NNa: 732.5359.
ꢀ
ꢀ
ꢀ
Bisdeuterated amine 13
Acknowledgements
To a suspension of 12 (5.2 mg, 5 lmol) in EtOH (2 mL) and H2O
(3 drops) under an atmosphere of argon was added hydrazine
monohydrate (100%, 120 lL), the reaction mixture was refluxed
overnight at 80 ◦C. The resulting mixture was concentrated to
dryness and the crude product was chromatographed on silica gel
(CH2Cl2–MeOH–Et3N, 20 : 1 : 0.02) followed by a gel filtration
on a size-exclusion Sephadex LH-20 chromatography (CH2Cl2–
CH3OH, 2 : 1) to afford 13 as a colorless solid (4.0 mg, 88%,
This work was supported by the University of Namur (Post-doc
grant to W.P.).
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