The synthesis of the vitamers of biotin

Article abstract of DOI:10.1006/bioo.1998.1094

An efficient synthetic pathway for the preparation of the vitamers of biotin which provides easy access to optically pure KAPA as well as diastereomeric mixtures of DAPA, DTB, and analogs thereof has been developed.

Full text of DOI:10.1006/bioo.1998.1094

BIOORGANIC CHEMISTRY 26, 157–168 (1998)
ARTICLE NO. BH981094
The Synthesis of the Vitamers of Biotin
Abraham Nudelman,* Ayelet Nudelman,* Dana Marcovici-Mizrahi,†
and Dennis Flint‡
*Department of Chemistry, Bar Ilan University, Ramat Gan 52900, Israel; †Department of Organic
Chemistry, Israel Institute for Biological Research, P.O. Box 19, Ness Ziona 74100, Israel; and
‡Central Research and Development, Experimental Station, E. I. Du Pont de Nemours and Company,
Wilmington, Delaware 19880-0328
Received April 7, 1998
An efficient synthetic pathway for the preparation of the vitamers of biotin which provides
easy access to optically pure KAPA as well as diastereomeric mixtures of DAPA, DTB, and
analogs thereof has been developed.
1998 Academic Press
INTRODUCTION
Biotin is a water-soluble vitamin that functions as a coenzyme in certain carboxyla-
tion and transcarboxylation reactions (1). Perhaps the most important of these, the
carboxylation of acetyl-CoA to form malonyl-CoA, is catalyzed by acetyl-CoA
carboxylase and is an initial step in fatty acid and polyketide biosynthesis. Since
the proper function of at least one of the reactions in which biotin participates is
a prerequisite for the growth and development of almost all organisms, biotin is
an essential nutrient. Many organisms, including the majority of plants and microbes,
synthesize their own biotin; others, including animals and certain microbes such as
yeast and Lactobacillus, must obtain the vitamin from external sources. The later
intermediates in the biosynthetic path, the so-called vitamers 7-keto-8-amino pelar-
gonic acid (KAPA), 7,8-diamino pelargonic acid (DAPA), and desthiobiotin (DTB)
(Scheme 1), have been identified.
Earlier reports (2) described the synthesis of racemic KAPA and DAPA without
reporting any spectral data. Moreover, while a very large number of publications
(3) have described the partial or total synthesis of biotin, none of these synthetic
routes have been based on the natural biosynthetic path.
The final stage in the biosynthesis, at which DTB is converted into biotin, involves
insertion of a S atom between two nonactivated positions (methyl and methylene
groups) to form the tetrahydrothiophene ring and is of particular interest. This
reaction is exceedingly difficult from a chemical standpoint. The mechanism of the
enzymatic reaction is unknown. Although the enzymatic reaction has been observed
in vitro (4), the sulfur donor has not been established (5). There is evidence that
the reaction catalyzed by biotin synthase can be the rate-limiting step in the biotin
biosynthetic pathway.
We were interested in developing convenient syntheses of the natural vitamers
157
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NUDELMAN ET AL.
SCHEME 1. Biosynthetic pathway of biotin.
and in extending the methodology developed, for the synthesis of analogs that may
be used to study the mechanism of this last biosynthetic step. We describe herein
a new total synthesis of the three vitamers.
RESULTS AND DISCUSSION
Synthesis of KAPA. Our initial approach involved acylation of the anion of
2-nitroethane with a suitable derivative of pimelic acid. However, hydrolytic workup
of the reaction gave only the monoester of pimelic acid (Scheme 2).
This problem was overcome by initial conversion of the acyl chloride 3 to the
corresponding acyl imidazole 4 that led to the desired 7-keto-8-nitro-pelargonate
5. Reduction of the latter with Raney Ni gave the KAPA ethyl ester 6 in poor yield
(Scheme 3).
This approach suffered from several deficiencies: (a) The products obtained were
racemic, (b) the yield in the reduction step was very low, and (c) pimelic acid, as

SYNTHESIS OF VITAMERS OF BIOTIN
159
SCHEME 2
well as other 7-carbon chain unit derivatives, is expensive. To overcome these
problems a new approach was taken whereby the N-containing moiety was derived
from
L-alanine. This approach provided the desired chirality and enabled condensa-
tion with readily available derivatives having shorter carbon chains. The most
suitable
L-alanine derivative in this approach was Mansour’s ͱ-keto ester 7 (6)
SCHEME 3

160
NUDELMAN ET AL.
SCHEME 4
protected by an N-Boc group (Scheme 4). Our earlier experiments, when using the
corresponding N-phthalimido derivatives, led to various rearrangement products
(7). Alkylation of 7 with ethyl 5-iodovalerate 8 in the presence of 4 equivalents of
K2CO3 in acetone (conditions found to be optimal, other bases and solvents resulting
in much lower yields) gave 9. Complete deprotection by acid hydrolysis gave opti-
cally pure KAPA, found to be identical to the natural vitamer (Scheme 4).
Synthesis of DAPA. The reductive amination of KAPA to DAPA in the biosyn-
thetic pathway involves PLP and SAM (Scheme 1). Our initial approaches were to
carry out this reaction by nonenzymatic methods taking into consideration the
stereospecificity of the reductive step. Ketones have been converted to chiral amines
by reduction of their corresponding chiral imines, such as those obtained by conden-
sation with optically pure Ͱ-methylbenzyl amine, where the imine formation takes
place under basic conditions (8). In our case, since KAPA is an Ͱ-amino ketone
unstable in basic media, an initial N-protection step was required. N-protection
reactions are commonly conducted in basic media; however, in this case the protec-
tive step required nonbasic conditions in order to prevent the formation of Ͱ-amino
ketone intermolecular condensation products. Such an N-protection was carried
out by N-formylation in the presence of formic acid/acetic anhydride (9) (Scheme
5). Unfortunately the subsequent ketimine formation under a variety of condi-
tions failed.
The next approach involved reduction of the readily formed oxime derivative of
KAPA. Oximation of KAPA or its methyl ester 10 gave a mixture of syn- and anti-
oximes (12 or 13, respectively) consisting mainly of the syn isomers. This suggested
that the chiral center at the C-8 position induced stereoselectivity and it was thus
hoped that a similar induction would be obtained in the course of the following
reduction step. However, upon reduction an approximately equimolar mixture of
diastereomers was obtained. The loss of stereospecific induction may be attributed
to initial reduction of the oxime to an intermediate imine, combined with a weaken-
ing of the H bond between the imine and the neighboring ammonium ion under

SYNTHESIS OF VITAMERS OF BIOTIN
161
SCHEME 5
the acidic conditions of the reaction. Furthermore, under the methanolic–acidic
conditions of the reaction, the corresponding DAPA methyl ester 16 was isolated
(Scheme 6).
An alternative reductive amination was carried out in the presence of NaBH3CN
SCHEME 6

162
NUDELMAN ET AL.
SCHEME 7
that gave 16 also as an approximately equimolar mixture of diastereomers (Scheme
7). This approach required double protection, both of the acid as its Me ester 10,
conveniently carried out in the presence of anhydrous HCl/MeOH (10) and of the
N as the N-Boc, 14. The latter was conducted by sonication under mild basic
conditions in order to avoid standard harsh basic media that would result in decom-
position and/or dimerization of the free Ͱ-amino ketone.
Synthesis of DTB. The formation of the imidazolidone ring of DTB was carried
out by reaction of DAPA with either triphosgene or carbonyl diimidazole (CDI).
In the former case the reaction proceeded in aqueous media in the presence of
base using the DAPA-free carboxylic acid or its ester 16. In the course of the
workup, the ester readily hydrolyzed. Alternatively the reaction was performed in
organic media from 16 and either triphosgene or CDI, from which the yields ob-
tained using triphosgene were quantitative (Scheme 8).
Summary. An efficient synthetic pathway for the synthesis of the vitamers of
biotin, which provides easy access to optically pure KAPA as well as diastereomeric
mixtures of DAPA, DTB, and analogs thereof, has been developed. As indicated,
the reduction of the oximes 12 and 13 did not proceed stereospecifically. No further
attempts were made at this point to separate the diastereomers since our ultimate
goal is to develop a stereospecific synthesis for the vitamers. Further investigations
are in progress dealing with stereospecific reductive amination of KAPA to DAPA.
If this goal is attained, it should be possible to proceed to DTB maintaining complete
stereospecificity using the reactions described in this paper.

SYNTHESIS OF VITAMERS OF BIOTIN
163
SCHEME 8
EXPERIMENTAL
1H NMR spectra at 200 and 300 MHz were obtained on Bruker AC-200 and
AM-300 spectrometers, respectively. When D2O was used as NMR solvent, its own
1
peak was used as a standard for H NMR, while MeOH was used as an internal
standard for 13C NMR. Mass spectra were obtained on a Varian Mat 731 spectrome-
ter (CI ϭchemical ionization). HRMS were obtained on a VG AutoSpec E spec-
trometer. Progress of the reactions was monitored by TLC on silica gel (Merck,
Art. 5554) or alumina (Riedel-de Haen, Art. 37349). Flash chromatography was
carried out on silica gel (Merck, Art. 9385). Most commercially available chemicals
were purchased from Aldrich Co., Sigma Co., and Fluka Co. and were used without
further purification.
Heptanedioic acid monoethyl ester K salt (1). To an ice-cold solution of diethyl
heptanedioate (99.9 g, 0.46 mol) in absolute EtOH (15 ml) was added dropwise an
ice-cold solution of KOH (30.5 g, 0.46 mol, 85%) in absolute EtOH. The mixture
was stirred for 1 h, then ether was added until the sample began to crystallize. The
solid (40 g) was filtered and to the filtrate more ether was added. An additional
1
two crops of white crystals (combined total, 43 g) were collected. H NMR (D2O)
ͳ 1.23 (t, J ϭ 7.2 Hz, 3H, Me), 1.3 (m, 2H, CH2CH2CH2CO), 1.48–1.64 (m, 4H, 2
CH2CH2CO), 2.15 (t, J ϭ 7.7 Hz, 2H, CH2COOK), 2.36 (t, J ϭ 7.4 Hz, 2H,
EtOOCCH2), 4.13 (q, J ϭ 7.2 Hz, 2H, OCH2). The NMR spectrum of the first crop
indicated it was contaminated with about 50% di-K heptanedioate.
Heptanedioic acid monoethyl ester (2). An ice-cold solution of K ethyl heptanedi-
oate (16.5 g, 79 mmol) in H2O (45 ml) was acidified with concd HCl to pH 5 and
was rapidly extracted with ether (2 ϫ 100 ml). The organic phase was dried and
evaporated to give the acid as an oil (11.2 g, 75%). 1H NMR (CDCl3) ͳ 1.25 (t, J ϭ
7.3 Hz, 3H, Me), 1.3–1.5 (m, 2H, CH2CH2CH2CO), 1.6–1.75 (m, 4H, 2 CH2CH2CO),
2.25–2.40 (m, 4H, 2 CH2CO), 4.15 (q, J ϭ 6.9 Hz, 2H, OCH2).
Chlorocarbonylhexanoic acid ethyl ester (3). A solution of monoethyl heptanedi-
oate (40 g, 0.21 mol) in 50 ml of SOCl2 was heated to reflux until no more gas
evolution was evident. The excess SOCl2 was evaporated and the residue was dis-
tilled.
7-Imidazol-1-yl-7-oxo-heptanoic acid ethyl ester (4). To a solution of ethyl hep-
tanedioyl chloride (24.5 g, 0.12 mol) in THF (200 ml), cooled in an ice bath, was
added a solution of imidazole (16.1 g, 0.24 mol) in ice-cold THF (100 ml). The

164
NUDELMAN ET AL.
mixture was stirred for 1 h and the solid precipitate was filtered and washed with
THF (150 ml), and the filtrate was evaporated at 60ЊC to give the product as an
1
oil (27.9 g, 98%). H NMR (CDCl3) ͳ 1.25 (t, J ϭ 7.2 Hz, 3H, Me), 1.40–1.50 (m,
2H, CH2CH2CH2CO), 1.69 (quintet, J ϭ 7.6 Hz, 2H, CH2CH2CO), 1.83 (quintet,
J ϭ 7.6 Hz, 2H, CH2CO), 2.33 (t, J ϭ 7.4 Hz, 2H, CH2CON), 2.92 (t, J ϭ 7.3 Hz,
2H, O2CCH2), 4.13 (q, J ϭ 7.1 Hz, 2H, OCH2), 7.08 (m, 1H, Ar), 7.50 (m, 1H, Ar),
8.20 (s, 1H, Ar).
8-Nitro-7-oxononanoic acid ethyl ester (5). To an ice-cold solution of nitroethane
(57 mmol) in DMSO (50 ml) was added tert-BuOK (6.40 g, 57 mmol) followed by
the addition of ethyl 1-imidazolyl heptanedioate (57 mmol). The mixture was stirred
for 72 h, then poured into H2O (300 ml), and washed with ether. The aqueous
phase was acidified to pH 5.8 with concd HCl and was extracted with EtOAc
(2 ϫ 100 ml). The organic phase was dried, mixed with charcoal, filtered, and
1
evaporated to give the ethyl 8-nitro-7-oxononanoate as an oil (76%). H NMR
(CD3OD) ͳ 5.56 (q, J ϭ 7.1 Hz, 1H, CH), 4.06 (q, J ϭ 7.2 Hz, 2H, MeCH2), 2.25–2.35
(m, 4H, 2 CH2CO), 1.65 (d, J ϭ 7.1 Hz, 3H, MeCH), 1.60 (quintet, J ϭ 7.6 Hz, 4H,
2 CH2CH2CO), 1.23–1.40 (m, 2H, CH2CH2CH2CO), 1.24 (t, J ϭ 7.1 Hz, 3H, MeCH2).
MS (EI) C11H19NO5 200 (M–C2H5O), 171 (M–C2H4NO2), 125 (M–C2H4NO2–
C2H6O).
8-Amino-7-oxononanoic acid ethyl ester hydrochloride (6). To solution of ethyl
8-nitro-7-oxononanoate (0.5 g, 2 mmol) in EtOH (5 ml) was added concd HCl (0.2
ml). The solution was hydrogenated over Raney Ni at 35ЊC, 1000 psi for 2 h. After
1 h no further consumption of H2 was detected. The mixture was filtered and to
the filtrate was added a solution of NaHS until no further formation of black NiS
was evident. The mixture was filtered and flash evaporated. 1H NMR (D2O) ͳ 1.24
(t, J ϭ 7.1 Hz, 3H, MeCH2), 1.25–1.45 (m, 2H, CH2CH2CH2CO), 1.55 (d, J ϭ 7.4
Hz, 3H, MeCH), 1.61 (q, J ϭ 7.6 Hz, 4H, 2 CH2CH2CO), 2.30–2.45 (m, 4H, 2
CH2CO), 4.15 (q, J ϭ 7.2 Hz, 2H, MeCH2), 4.28 (q, J ϭ 7.4 Hz, 1H, CH).
4-tert-Butoxycarbonylamino-3-oxopentanoic acid methyl ester (7). To a solution
of N-Boc-alanine (1 mmol) in dry THF (15 ml) was added, portionwise, CDI
(0.195 g, 1.2 mmol). The solution was stirred at room temperature (RT) under N2
for 0.5 h, followed by the addition of MgCl2 (0.1 g, 1 mmol) and monomethyl
malonate potassium salt (0.16 g, 1 mmol), and the resulting slurry was stirred at
50ЊC overnight. The mixture was filtered and evaporated, and the residue was
partitioned between 1 N HCl and EtOAc. The aqueous layer was extracted with
EtOAc (3 ϫ 15 ml). The combined organic layer was washed with 1 N HCl (15
ml), 5% NaHCO3 (3 ϫ 15 ml), brine (15 ml), dried (MgSO4), and evaporated to
1
give the desired product as white to light yellow crystals (76% yield). H NMR
(CDCl3) ͳ 1.38 (d, J ϭ 7 Hz, 3H, MeCH), 1.45 (s, 9H, Me3C), 3.54 (ABq, JAB
ϭ
17 Hz, 2H, CH2), 3.71 (s, 3H, OMe), 4.55 (quintet, J ϭ 7 Hz, 1H, CH), 5.10 (br d,
1H, NH). 13C NMR (CDCl3) ͳ 15.00 (MeCH), 28.00 (Me3C), 43.75 (CH2), 51.78
(OMe), 53.82 (CH), 80.03 (Me3C), 155.62 (CON), 169.73 (CO2), 200.09
(CHCOCH2). MS (CI, i-Bu) m/e 245 (MHϩ). [Ͱ]D25 50.68Њ (c 0.74, MeOH).
5-Iodopentanoic acid ethyl ester (8). To a solution of NaI (1.1 mmol) in dry
acetone (15 ml) was added ethyl 5-bromovalerate (1 mmol). A precipitance formed
almost immediately and the resulting suspension was stirred at RT overnight. The

SYNTHESIS OF VITAMERS OF BIOTIN
165
mixture was filtered, the salts were washed with acetone, and the filtrate was
evaporated to give a mixture of oily product and unreacted NaI. This mixture was
diluted with CH2Cl2 and filtered. Evaporation of the filtrate gave the product as an
orange liquid (92% yield, ca. 95% conversion of alkyl bromide to the corresponding
1
iodide). H NMR (CDCl3) ͳ 1.26 (t, J ϭ 7 Hz, 3H, CH2Me), 1.65–1.95 (m, 4H,
CH2CH2CH2CH2CO), 2.33 (t, J ϭ 7 Hz, 2H, CH2CO), 3.20 (t, J ϭ 7 Hz, 2H, ICH2),
4.14 (q, J ϭ 7 Hz, 2H, CH2Me). 13C NMR (CDCl3) ͳ 5.70 (CH2I), 14.24 (Me), 25.82
(CH2CH2CO), 32.78 (CH2CO), 33.13 (CH2CH2I), 60.37 (OCH2), 173.01 (CO). MS
(EI) m/e 256 (Mϩ).
2-(2-tert-Butoxycarbonylaminopropionyl]heptanedioic acid 7-ethyl ester 1-methyl
ester (9). To a solution of 7 (1 mmol) in dry acetone (15 ml), was added anhydrous
K2CO3 (4 mmol), followed by the addition of 8 (1 mmol). The resulting suspension
was refluxed under N2 for 6 h. The reaction mixture was cooled and filtered, and
the salts were washed with acetone. The filtrate was concd and the residue was
purified by flash chromatography on a silica gel column using hexane:EtOAc (usu-
ally 2 : 1 or 3 : 1) as eluent, to give the desired C-alkylated product as a light yellow
oil, containing both diastereomers (62% yield). 1H NMR (CDCl3) ͳ 1.25–1.40 (super-
imposed, m, 8H, CH2Me, CHMe, CHCH2), 1.45 (s, 9H, Me3C), 1.60–1.95 (m, 4H,
CHCH2CH2CH2), 2.30 (t, J ϭ 7.5 Hz, 2H, CH2CO2), 3.70–3.80 (superimposed, m,
4H, CHCO2Me), 4.12 (q, J ϭ 7 Hz, 2H, CH2Me), 4.41 (quintet, J ϭ 7 Hz, 1H,
CHMe), 5.22 (br d, J ϭ 7 Hz, 1H, NH). 13C NMR (CDCl3) ͳ 14.23 (CH2Me),
16.95 and 17.45 (2 CHMe), 24.57 and 24.65 (2 CH2CH2COCH), 26.87 and 26.96 (2
CHCH2CH2) 27.64 (CHCH2), 28.33 (Me3C), 33.93 (CH2CO2), 52.35 and 52.48 (2
OMe), 54.60 (CHMe), 55.07 (CHCH2), 60.22 (OCH2), 79.93 (Me3C), 155.07 (CON),
169.60 (CO2Me), 173.30 (CO2Et), 204.48 and 204.63 (2 CHCOCH). MS (CI, i-Bu)
m/e 374 (MHϩ, 13), 318 (MHϩ–C4H8, 22). HRMS (DCI, CH4) calcd for C18H32NO7
(MHϩ): 374.2178. Found: 374.2143.
8-Amino-7-oxononanoic acid · hydrochloride (KAPA · HCl). A suspension of 9
(1 mmol) in 4 N HCl (5 ml) was refluxed at 110ЊC for 2 to 4 h. Gas evolution was
observed. Heating was continued until the mixture became homogeneous. The
resulting dark yellow solution was evaporated and dried under high vacuum. When
the color became darker, the solution was filtered through charcoal, prior to the
evaporation. The residue usually crystallized under high vacuum. Recrystallization
was carried out by dissolving in absolute EtOH and crystallizing upon addition of
dry ether, or ether–HCl, to give a derivative of KAPA · HCl as off-white powder
(55% yield, after recrystallization). 1H NMR (D2O) ͳ 1.24–1.42 (m, 2H,
CH2CH2CH2CO2H), 1.55 (d,
J ϭ 7.5 Hz, 3H, Me), 1.5–1.7 (m, 4H,
CH2CH2CH2CH2CO2H), 2.38 (t, J ϭ 7.5 Hz, 2H, CH2CO2H), 2.68 (ABq of t,
JAB ϭ 18 Hz, JAX ϭ JBX ϭ 7 Hz, 2H, CH2COCH), 4.26 (q, J ϭ 7.5 Hz, 1H, CH).
13C NMR (D2O) ͳ 15.27 (Me), 22.86 (CH2CH2CO2H), 24.57 (CH2CH2COCH),
28.16 (CH2CH2CH2CO2), 34.28 (CH2CO2H), 38.46 (CH2COCH), 55.38 (CH), 179.64
(CO2H), 209.83 (CH2COCH). MS (CI, NH3) m/e 188 (MHϩ–HCl); mp 133–134ЊC
[lit.(2c) 134–136ЊC]; [Ͱ]2D5 Ϫ4.46Њ (c 0.23, MeOH).
8-Amino-7-oxononanoic acid methyl ester hydrochloride (KAPA · HCl methyl es-
ter, 10). To an ice-cold solution of KAPA · HCl (1 mmol) in dry MeOH (10 ml)
was dropwise added acetyl chloride, in order to generate HCl(g) in situ. The resulting

166
NUDELMAN ET AL.
mixture was allowed to warm to RT and was stirred overnight. Evaporation yielded
1
the desired methyl ester as an off-white powder (quantitative yield). H NMR
(D2O) ͳ 1.33 (m, 2H, CH2CH2CH2CO2), 1.58 (superimposed, m and d, Jd ϭ 7.5
Hz, 7H, CHMe, CH2CH2CH2CH2CO2), 2.40 (t, J ϭ 7 Hz, 2H, CH2CO2), 2.68 (ABq
of t, JAB ϭ 18 Hz, JAX ϭ JBX ϭ 7.5 Hz, 2H, CH2COCH), 3.69 (s, 3H, OMe), 4.26
(q, J ϭ 7.5 Hz, 1H, CH). 13C NMR (D2O) ͳ 15.27 (Me), 22.82 (CH2CH2CO2), 24.50
(CH2CH2COCH), 28.16 (CH2CH2CH2CO2), 34.03 (CH2CO2), 38.44 (CH2COCH),
52.66 (OMe), 55.36 (CH), 178.04 (CO2), 209.80 (CH2COCH). MS (CI, i-Bu) m/e
202 (MHϩ–HCl).
General procedure for the preparation of KAPA · HCl ketoxime derivatives. To a
solution of a KAPA · HCl derivative (1 mmol) in dry pyridine (1.5 ml) was added
a solution of NH2OH · HCl (3 mmol) in dry pyridine (2.5 ml). The mixture was
stirred at RT overnight and was then evaporated to dryness under high vacuum.
The residue was dissolved in distilled water (2.5 ml) and was made basic by adding
0.5 N NaOH (until pH ϭ8). This solution was extracted with CH2Cl2 (4 ϫ 2.5 ml)
to remove the remaining pyridine. The aqueous phase was evaporated under high
vacuum yielding the desired product as a solid. TLC plates used to monitor the
reaction were sprayed with a solution of 0.5% CuCl2, to give strong blue spots for
oximes. The oximes were obtained as mixtures of two diastereomers (ca. 3 : 1)
derived from syn- and anti-oximes.
8-Amino-7-hydroxyiminononanonic acid hydrochloride (KAPA · HCl oxime, 12).
1
Obtained as a white powder, recrystallized from MeOH (quantitative yield). H
NMR (D2O) ͳ 1.37 (m, 2H, CH2CH2CH2CO2), 1.49 (d, J ϭ 7 Hz, 3H, Me), 1.57
(m, 4H, CH2CH2CH2CH2CO2), 2.21 (superimposed, t and m, Jt ϭ 7 Hz, 2H, CH2CO2,
and 1H, CNCH2), 2.59 (m, 1H, CNCH2), 4.12 (q, J ϭ 7 Hz, 1H, CH). 13C NMR
(D2O)
ͳ
17.09 (Me), 24.92 (CH2CH2CO2), 25.65 (CH2CH2CN), 29.09
(CH2CH2CH2CN), 37.34 (CH2CO2), 49.35 (CH), 159.43 (CN), 183.55 (CO2). MS
(CI, NH3) m/e 203 (MHϩ–HCl).
8-Amino-7-hydroxyiminononanoic acid methyl ester hydrochloride (KAPA · HCl
oxime methyl ester, 13). Obtained as a white powder (quantitative yield). 1H NMR
(D2O) ͳ 1.37 (m, 2H, CH2CH2CH2CO2), 1.48 (d, J ϭ 7 Hz, 3H, Me), 1.60 (m, 4H,
CH2CH2CH2CH2CO2), 2.22 (ddd, J ϭ 14, 9, 5.5 Hz, 1H, CNCH2), 2.40 (t, J ϭ 7.5
Hz, 2H, CH2CO2), 2.56 (ddd, J ϭ 14, 9.5, 6.5 Hz, 1H, CNCH2), 3.69 (s, 3H, OMe),
4.10 (q, J ϭ 7 Hz, 1H, CH). 13C NMR (D2O) ͳ 17.07 (Me), 24.28 (CH2CH2CO2),
24.78 (CH2CH2CN), 25.67 (CH2CN), 28.71 (CH2CH2CH2CN), 33.93 (CH2CO2),
49.38 (CH), 52.54 (OMe), 159.23 (CN), 178.03 (CO2). MS (CI, NH3) m/e 217 (MHϩ–
HCl).
8-Formylamino-7-oxononanoic acid methyl ester (11). Ac2O (0.75 ml) was added
dropwise to an ice-cooled solution of KAPA · HCl methyl ester (0.23 g, 1 mmol)
in HCOOH (2.25 ml). The mixture was stirred at RT for 1 h. A small amount of
ice was added and the mixture was evaporated to dryness to give the desired
1
N-formylated product as a white solid (95% yield). H NMR (CDCl3) ͳ 1.37
(m, 2H, CH2CH2CH2CO2), 1.64 (superimposed, m, 7H, MeCH and
CH2CH2CH2CH2CO2), 2.31 (t, J ϭ 7.4 Hz, 2H, CH2CO2), 2.60 (t, J ϭ 7.3 Hz,
CH2COCH), 3.66 (s, 3H, OMe), 4.42 (m, 1H, CH), 8.29 (m, 2H, NH and
CHO). 13C NMR (CDCl3) ͳ 15.56 (CHMe), 22.66 (CH2CH2CO2), 24.50

SYNTHESIS OF VITAMERS OF BIOTIN
167
(CH2CH2CH2CH2CO2), 28.34 (CH2CH2CH2CO2), 33.66 (CH2CO2), 38.31
(CH2COCH), 51.42 (OMe), 55.23 (CH), 163.23 (COH), 174.00 (CO2), 206.31
(CH2COCH). MS (CI, i-Bu) m/e 230 (MHϩ–HCl).
8-tert-Butoxycarbonylamino-7-oxononanoic acid methyl ester (14). A suspension
of KAPA · HCl methyl ester (1 mmol), di-tert-butyl dicarbonate (0.218 g, 1 mmol),
and NaHCO3 (0.25 g) in dry MeOH (5 ml) was sonicated at RT for 6 h. The
resulting mixture was filtered and the filtrate was evaporated to dryness. The residue
was dissolved in ether, filtered, and evaporated to give the desired product as a
1
light yellow oil (80% yield). H NMR (CDCl3) ͳ 1.31 (d, J ϭ 7.2 Hz, 3H, CHMe),
1.32 (m, 2H, CH2CH2CH2CO2), 1.44 (s, 9H, Me3C), 1.63 (2 m, 4H,
CH2CH2CH2CH2CO2), 2.31 (t, J ϭ 7.4 Hz, 2H, CH2CO2), 2.50 (ABq of t, JAB
ϭ
17.3 Hz, JAX ϭ JBX ϭ 7.3 Hz, 2H, CH2COCH), 3.67 (s, 3H, OMe), 4.30 (quintet,
J ϭ 7.1 Hz, 1H, CHMe), 5.26 (br d, J ϭ 5.3 Hz, 1H, NH). 13C NMR (CDCl3) ͳ
17.86 (CHMe), 23.18 (CH2CH2CO2), 24.67 (CHCOCH2CH2), 28.39 (Me3C), 28.68
(CH2CH2CH2CO2), 33.83 (CHCOCH2), 33.84 (CH2CO2), 51.41 (OMe), 55.13
(CHMe), 79.73 (Me3C), 155.09 (CON), 173.88 (CO2), 209.27 (CHCOCH). MS (CI,
NH3) m/e 319 (MNH4ϩ, 37), 302 (MHϩ, 9), 263 (MNHϩ4 –C4H8, 52), 246 (MHϩ–C4H8,
5), 202 (MHϩ–C5H8O2, 100).
7,8-Diaminononanoic acid methyl ester dihydrochloride (DAPA · 2HCl methyl
ester, 16). Method A. The oxime 12 or 13 (1 mmol) and 4N HCl (2 ml) in absolute
MeOH (10 ml) was hydrogenated at 60 psi/45ЊC/48 h over 10% PtO2 (0.1 g). The
mixture was filtered through celite and washed with MeOH, and the filtrate was
evaporated to dryness to give the product as yellow crystals (88% yield, consisting
of a mixture of two diastereomers). Method B. A solution of N-Boc KAPA methyl
ester 14 (1 mmol), NaBH3CN (63 mg, 0.7 mmol), and NH4OAc (0.77 g, 10 mmol)
in dry MeOH (10 ml) was stirred at RT for 48 h. The resulting mixture was acidified
(HCl conc) to pH 1 and evaporated to dryness. The residue was dissolved in distilled
H2O and brought to pH 10, using solid KOH. The aqueous solution was extracted
with EtOAc (4ϫ) and the combined organic layers were washed with brine, dried
(MgSO4), filtered, and evaporated to give the mono-N-Boc diamine ester. Removal
of the Boc group was carried out under anhydrous conditions, by stirring in ether–
HCl at RT until precipitation of the di-HCl salt. The desired diamine was obtained
as a mixture of two diastereomers (86% yield). 1H-NMR (D2O) ͳ 1.43 (m, 7H, Me,
CH2CH2CH2CH2CO2), 1.70 (m, 4H, CH2CH2CH2CH2CH2CO2), 2.44 (t, J ϭ 7.5
Hz, 2H, CH2CO2), 3.62 (brdt, 1H, CHCH2), 3.71 (s, 3H, OMe), 3.81 (brdq, 1H,
CHMe). 13C-NMR (D2O) major diastereomer
ͳ
13.17 (CHMe), 24.31
(CH2CH2CO2), 24.62 (CH2CH2CHN), 27.00 (CH2CHN), 28.23 (CH2CH2CH2CO2),
33.99 (CH2CO2), 48.88 (CHMe), 52.64 (OMe), 53.37 (CHCH2), 177.85 (CO2). Minor
diastereomer ͳ 13.83 (CHMe), 24.31 (CH2CH2CO2), 24.56 (CH2CH2CHN), 28.15
(CH2CH2CH2CO2), 29.24 (CH2CHN), 34.09 (CH2CO2), 48.95 (CHMe), 51.78
(OMe), 54.16 (CHCH2), 179.16 (CO2). MS (CI/NH3) m/e 217 (MHϩ–2HCl).
6-(5-Methyl-2-oxoimidazolidin-4-yl)hexanoic acid (DTB, 17) and methyl ester.
Method A. A mixture of a DAPA · 2HCl methyl ester (1 mmol) and Et3N (0.2 g,
2 mmol) in dry CH2Cl2 (10 ml) was stirred at ca. 0ЊC for 30 min. A solution of CDI
(0.2 g, 1.2 mmol) in dry CH2Cl2 (5 ml) was added and the mixture was stirred at RT
overnight. The mixture was evaporated to dryness and the residue was partitioned

168
NUDELMAN ET AL.
between 1 N HCl and EtOAc. The aqueous layer was extracted with EtOAc (4ϫ)
and the combined organic layers were washed with 5% NaHCO3 (2ϫ) and brine,
dried (MgSO4), filtered, and evaporated to give the desired DTB methyl ester, in
the form of two diastereomers. Method B. A solution of NaOH (0.4 g, 10 mmol)
and a DAPA · 2HCl derivative (1 mmol) in distilled H2O (10 ml) was stirred at RT
for 10 min. A solution of triphosgene (0.3 g, 1 mmol) in dioxane (10 ml) was added.
The mixture became hot and the pH dropped from ȁ10 to ȁ8. The resulting mixture
was stirred at RT for 2 d, after which it was evaporated to dryness. Trituration of
the residue with MeOH, filtration, and evaporation afforded the desired product.
Note. Both the free acid and the ester could be used as substrates. The product
was either a mixture of both or pure acid. In the case of a mixture, the product
was dissolved in 4 N HCl and stirred at RT overnight, to afford, after evaporation,
1
the desired acid as white crystals (quantitative yield, method B). H NMR (D2O)
ͳ 1.09 and 1.19 (2 d, J ϭ 6.2 Hz, 3H, Me), 1.33 (m, 4H, CH2CH2CH2CH2CO2), 1.52
(m, 4H, CH2CH2CH2CH2CH2CO2), 2.40 (t, J ϭ 7 Hz, 2H, CH2CO2), 3.35 and 3.70
(2 m, 1H, CHCH2), 3.53 and 3.85 (2 m, 1H, CHMe). MS (CI, NH3) m/e 215 (MHϩ,
100). HRMS (CI, CH4) calcd for C10H19N2O3 (MHϩ): 215.1396. Found: 215.0874.
(Mϩ): 214.1317. Found: 214.0920.
REFERENCES
1. Izumi, Y., and Tamada, H. (1989) in Biotechnology of Vitamins, Pigments and Growth Factors
(Vandamme, E. J., Ed.), pp. 231–256. Elsevier, London.
2. (a) Suyama, T., and Kanao, S. (1962) Nippon Kagaku Zasshi 83, 1058–1060; (b) Suyama, T., Tsugame,
R., Okamura, S., and Kanao, S. (1968) Yakugaku Zasshi 88, 223–226; (c) Suyama, T., and Kanao,
S. (Ajinomoto Co., Inc.) (1963) Japan Patent 19,715; (1964) Chem. Abstr. 60, 4013f.
3. (a) Ohrui, H., and Emoto, S. (1975) Tetrahedron Lett. 32, 2765–2766; (b) Confalone, P. N., Pizzolato,
G., Baggiolini, E. G., Lollar, D., and Uskokovic, M. R. (1977) J. Am. Chem. Soc. 99, 7020–7026;
(c) Baggiolini, E. G., Lee, H. L., Pizzolato, G., and Uskokovic, M. R. (1982) J. Am. Chem. Soc.
104, 6460–6462; (d) Corey, E. J., and Mehrotra, M. M. (1988) Tetrahedron Lett. 29, 57–60; (e)
Fujisawa, T., Nagai, M., Koike, Y., and Shimizu, M. (1994) J. Org. Chem. 59, 5865–5867; (f) Deroose,
F. D., and De Clercq, P. J. (1995) J. Org. Chem. 60, 321–330. [and references cited therein]
4. (a) Fujisawa, A., Abe, T., Ohsawa, I., Kamogawa, K., and Izumi, Y. (1993) FEMS Microbiol. Lett.
110, 1–4; (b) Sanyal, I., Cohen, G., and Flint, D. H. (1994) Biochemistry 33, 3625–31; (c) Ohshiro,
T., Yamamoto, M., Izumi, Y., Tse Sum Bui, B., Florentin, D., and Marquet, A. (1994) Biosci.
Biotechnol. Biochem. 58, 1738–41; (d) Birch, O. M., Furhmann, M., and Shaw, N. M. (1995) J. Biol.
Chem. 270, 19,158–19,165; (e) Sanyal, I., Gibson, K. J., and Flint, D. H. (1996) Arch. Biochem.
Biophys. 326, 48–56.
5. (a) Eisenberg, M. (1987) in Escherichia coli and Salmonella typhimurium Cellular and Molecular
Biology (Neidhardt, F. C., Ingraham, J. L., Low, K. B., Magasanik, B., Schaechter, M., and Umbarger,
H. E., Eds.), Vol. 1, pp. 544–550, Am. Soc. Microbiol., Washington, DC.; (b) Parry, R. J., and Naidu,
M. V. (1980) Tetrahedron Lett. 21, 4783–4786; (c) Parry, R. J. (1983) Tetrahedron 39, 1215–1238;
(d) DeMoll, E., and Shive, W. (1983) Biochem. Biophys. Res. Commun. 110, 243–249; (e) DeMoll,
E., White, R. H., and Shive, W. (1984) Biochemistry 23, 558–562; (f) Baxter, R. L., Camp, D. J.,
Coutts, A., and Shaw, N. (1992) J. Chem. Soc. Perkin Trans. I, 255–258.
6. (a) Mansour, T. S., and Evans, C. A. (1990) Syn. Commun. 20, 773–781; (b) Koga, T., Inoe, K.,
and Takahashi, S. (1994) Japan Kokai Tokkyo Koho JP 06,145,107. May 1994; (1994) Chem. Abstr.
121, 179569j.
7. Nudelman, A., Marcovici, D., and Nachum, A. (1994) Heterocycles 38, 751–758.
8. Bringmann, G., and Geisler J.-P. (1989) Synthesis 608.
9. Sheehan, J. C., and Young, D.-D. H. (1958) J. Am. Chem. Soc. 80, 1154–1158.
10. Nudelman, A., Bechor, Y., Falb, E., Fischer, B., Wexler, A. B., and Nudelman, A. (1998) Syn.
Commun. 28, 471–474.