Reduced collagen cross links: The first synthesis of all the possible (2S,2′S)-stereoisomers of 5-hydroxylysinonorleucine and of 5,5′-dihydroxylysinonorleucine in enantiomerically pure form

Article abstract of DOI:10.1016/j.tetasy.2005.08.023

The paper reports the first enantioselective synthesis of all the possible collagen reduced cross links as described: (2S,2′S,5R)- and (2S,2′S,5S)-5-hydroxylysinonorleucine 3a and 3b, (2S,2′S,5R, 5′R)-5,5′-dihydroxylysinonorleucine 4a, (2S,2′S,5R,5′S)- 5,

Full text of DOI:10.1016/j.tetasy.2005.08.023

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 16 (2005) 3059–3069
Reduced collagen cross links: the first synthesis of all the
possible (2S,20S)-stereoisomers of 5-hydroxylysinonorleucine and
of 5,50-dihydroxylysinonorleucine in enantiomerically pure form
*
`
Riccardo Cribiu, Pietro Allevi and Mario Anastasia
`
Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Universita di Milano, via Saldini 50, I-20133 Milano, Italy
Received 8 July 2005; accepted 12 August 2005
Abstract—The paper reports the first enantioselective synthesis of all the possible collagen reduced cross links as described:
(2S,20S,5R)- and (2S,20S,5S)-5-hydroxylysinonorleucine 3a and 3b, (2S,20S,5R,50R)-5,50-dihydroxylysinonorleucine 4a, (2S,20S,
5R,50S)-5,50-dihydroxylysinonorleucine 4b and (2S,20S,5S,50S)-5,50-dihydroxylysinonorleucine 4c. The WilliamsÕ glycine template
methodology was used both for the introduction of a stereogenic at the a-position and for an easy protection of the amino acidic
functionalities duringthe synthesis of the dimeric amino acids.
ꢀ 2005 Published by Elsevier Ltd.
1. Introduction
collagen turnover and of bone resorption, useful for the
diagnosis of osteoporosis and other bone diseases.2a,6
Thus, different protocols are available for their synthe-
sis7 and quantification in various tissues.8 Surprisingly,
less attention has been devoted to the immature collagen
cross links. In fact, after the studies of Tanzer9 and of
Bailey,10 which have permitted their localization in var-
ious tissues, and their isolation, after stabilization by
reduction, only the stereoselective synthesis of lysinon-
orleucine 2, which lacks hydroxyls, has been reported.11
The native products lysinonorleucine 2 (Fig. 2; LNL),
5-hydroxylysinonorleucine 3 (HLNL) and 5,50-dihydr-
oxylysinonorleucine 4 (DHLNL) have still not had the
stereochemistry of their hydroxylated centres deter-
mined. While the stereochemistry of the centres binding
the 2-amino groups of the products 2–4 may be reason-
ably considered as (S)-configuration, that of the 5 and 50
hydroxylated carbons could be different from that of
hydroxylysine and should be demonstrated.
Collagen, the most abundant structural protein of the
human body, is responsible for the mechanical proper-
ties of bones, cartilage, skin, tendon, ligament and other
tissues.1 Collagen is secreted as a procollagen molecule
which, duringthe maturation, suffers some post-trans-
criptional modifications endingwith the formation of
intra and intermolecular cross links, which are responsi-
ble for the mechanical strength of collagen fibrils.2 The
extent of the cross links formation starts with the enzy-
matic oxidative deamination of the a-amino group of
the telopeptide lysine and hydroxylysine residues to their
respective aldehydes, allysine and hydroxyallysine,
which spontaneously form some aldimines, known as
Ôimmature collagen cross linksÕ, by reaction with intact
lysine or hydroxylysine (Fig. 1).3
With the aging of the collagen fibrils, some immature
cross links evolve, via an initial rearrangement to keto-
amines, to more complex cross links, called Ômature
cross linksÕ, which, duringbone remodelling, are excised
and excreted unchanged in human urine, as two pyridi-
nium cross links,2d,4 pyridinoline 1a (Py) and deoxypyri-
dinoline 1b (Dpy) (Fig. 1).5 Py 1a and Dpy 1b are now
considered important biochemical markers both of total
Consideringthat the availability of all the (2 S,20S)-iso-
mers in enantiomeric pure form and suitable amounts
could be useful not only for a definitive assessment of
the stereochemistry and of the biological significance
of the native compounds,12 but also to exclude or to give
evidence of their possible presence in biological fluids,
where they could form in small amounts by the enzy-
matic reduction of the parent imines, we herein report
the synthesis of compounds 3a, 3b and 4a–c.
*
Correspondingauthor.
riccardo.cribiu@unimi.it
Fax:
+39
0250316040;
e-mail:
0957-4166/$ - see front matter ꢀ 2005 Published by Elsevier Ltd.
doi:10.1016/j.tetasy.2005.08.023

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R. Cribiu et al. / Tetrahedron: Asymmetry 16 (2005) 3059–3069
3060
R
R
H2NCH2(CH2)3-R
lysine residue
N
O
R =
H
imine
LNL
(CH2)2CHO
R
HN
allysine residue
R
R
H2NCH2CH(CH2)2 R
HO
OH
N
HLNL imine
OH
R
R
R
R
R
R
O
H2NCH2(CH2)3-R
lysine residue
OH
OH
N
N
OH
R'
HLNL imine
R (CH2)CHCHO
hydroxyallysine residue
H2NCH2CH(CH2)2 R
HO
O
R
R
HO
OH
N
N
R'
DHLNL imine
+NH3
hydroxylysine residue
-
+NH3
CO2
-O
R
R
O
-
CO2
OH
N+
N
R'
1a : X = OH
1b : X = H
X
H3N+
-
CO2
Figure 1. Formation of immature and mature collagen cross links.
H3N+ COO-
-OOC +NH3
H
N
2 (2S,2'S)-LNL
H3N+ COO-
OH
-OOC +NH3
H3N+ COO-
OH
-OOC +NH3
H
H
N
N
3b (2S,2'S,5S)-5-HLNL
3a (2S,2'S,5R)-5-HLNL
H3N+ COO-
-OOC +NH3
OH HO
H
N
4a (2S,2'S,5R,5'R)-5,5'-DHLNL
H3N+ COO-
-OOC +NH3
H3N+ COO-
OH
-OOC +NH3
HO
OH HO
H
H
N
N
4c (2S,2'S,5S,5'S)-5,5'-DHLNL
4b (2S,2'S,5R,5'S)-5,5'-DHLNL
Figure 2. Possible reduced collagen cross links.

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R. Cribiu et al. / Tetrahedron: Asymmetry 16 (2005) 3059–3069
3061
2. Results and discussion
tory7b usingthe Williams Õ glycine template methodol-
ogy,13 with lithium bromide (Scheme 1) and obtained
the two bromohydrins 8 and 9, which were separable
by simple column chromatography. The structure of
these bromohydrins was assigned on the basis of their
ability to afford the corresponding( R)- and (S)-hydroxy-
azides under the conditions previously described for the
analogue iodohydrins.7b Moreover, when we treated
each bromohydrin with the protected hydroxylysine 5,
in the presence of various bases (K2CO3 or Cs2CO3),14
necessary as scavengers of HBr, no alkylation occurred.
Even at room temperature, bromohydrins 8 and 9 were
dehydrohalogenated to the parent epoxide 6 or 7,
respectively (Scheme 1).
Herein, we report the first stereoselective synthesis of the
two possible (2S,20S)-isomers of 5-hydroxylysinonorleu-
cine 3a and 3b [(2S,20S,5R)- and (2S,20S,5S)-5-HLNL
Fig. 2], and of the three possible (2S,20S)-isomers of
5,50-dihydroxylysinonorleucine 4a–c [(2S,20S,5R,50R)-,
(2S,20S,5R,50S)- and (2S,20S,5S,50S)-5-50-DHLNL].
We decided to use the WilliamsÕ glycine template meth-
odology13 for the introduction of a stereogenic centre at
the a-position of the desired dimeric amino acids and for
the easy protection of the amino acidic functionalities
duringthe successive synthetic elaborations. In fact, a
simple retrosynthetic disconnection of the structure of
the monohydroxylated cross links 3a and 3b suggested
that they could be obtained (Fig. 3) by simple alkylation
of the 6-amino group of a conveniently protected lysine
5, with an appropriate amino acidic bromohydrin able
to introduce either a natural or unnatural hydroxylysine
side chain.
Consideringthat the regeneration of the epoxides could
be due to the action of the base present for the alkyl-
ation, we decided to take advantage of this undesired
reaction to prepare in diastereomeric pure form, epox-
ides 6 and 7, by separate treatment with Cs2CO3, in
CH3CN at room temperature of each bromohydrin 8
or 9. In our opinion, epoxides 6 and 7 could be useful
to synthesize protected (2S,20S,5R)- and (2S,20S,5S)-5-
HLNL 3a and 3b by a direct attack of the oxirane ring
With this in mind, we treated the inseparable mixture of
diastereomeric epoxides 6 and 7, prepared in our labora-
OtBu
CbzHN
O
Ph
Ph
NH2
H3N+
COO-
OH
-OOC +NH3
O
OtBu
5
BocN
NHCbz
H
O
N
O
OH
+
H
N
3a (2S,2'S,5R)-5-HLNL
3b (2S,2'S,5S)-5-HLNL
Ph
Ph
O
BocN
O
OH
Br
Figure 3. Retrosynthetic disconnection of the structure of the hydroxylated cross links 3a and 3b.
OtBu
5
CbzHN
Ph
Ph
O
Ph
Ph
NH2
O
O
BocN
ii
BocN
O
O
O
OH
Br
6
8
Ph
Ph
i
O
OtBu
O
BocN
O
O
CbzHN
5
Ph
Ph
Ph
Ph
NH2
O
O
6+7
BocN
BocN
ii
O
O
O
OH
Br
7
9
Scheme 1. Reagents and conditions: (i) LiBr, THF, CH3COOH, 4h, rt, 82%; (ii) Cs2CO3, CH3CN, 2 h, rt, 92%.

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R. Cribiu et al. / Tetrahedron: Asymmetry 16 (2005) 3059–3069
Ph
Ph
O
Ph
BocN
H3N+
COO-
OH
Ph
O
O
O
BocN
6
COOR
+NH3
ii
i
O
COOtBu
NHCbz
+
OH
OtBu
O
N
H
CbzHN
N
H
NH2
10a
R = tBu 11a
R = H 3a
5
iii
Ph
Ph
O
Ph
BocN
Ph
BocN
H3N+
O
COO-
OH
O
O
7
COOR
+NH3
i
ii
O
COOtBu
NHCbz
+
OH
OtBu
O
N
H
CbzHN
N
H
R = tBu 11a
R = H 3a
NH2
iii
10b
5
Scheme 2. Reagents and conditions: (i) LiClO4, CH3CN, 12 h, rt, 65%; (ii) Na, NH3 liq, 15 min; (iii) HCl aq 1 M, 2 h, 52%, from 10a or 10b.
with the protected lysine 5 (Scheme 2). In fact, the
reaction of the protected hydroxylysine 5 with the
enantiomerically pure epoxide 6, in the presence of lith-
ium perchlorate,7k which activates the openingof the
epoxide ring, affords the completely protected dimeric
amino acid 10a in satisfactory yield (70%), accompa-
nied only by minor amounts (<5%; TLC, NMR evi-
dences) of the less polar trimeric homologue. Crucial
for a successful reaction were the molar ratio of epox-
ide 6 on amine 5 (1:2), and the reaction temperature
(22–25 ꢁC). A higher reaction temperature or larger
excess of the amine caused the openingof the oxazinone
ringlactone with the formation of the corresponding
amide.
In fact, the crude product of the reaction, purified from
the inorganic salts by short column chromatography on
ion-exchange resin (Dowex 50W-X8, H+ form), was the
amino acid 11a, which lacks the oxazinone ring, the car-
bobenzyloxy group and even the N-t-Boc, a protecting
group which generally survives the opening of the oxaz-
inone ringperformed under chemical reduction condi-
tions.13 The hydrolysis of the N-t-Boc group probably
occurs duringthe chromatographic purification under
acidic conditions of the crude product, to afford the
tert-butyl ester 11a. The crude the tert-butyl ester 11a
was then transformed into the desired dimeric amino
acid 3a by treatment with aqueous HCl (1 M) at room
temperature. The use of this aqueous acid avoids the
concurrent lactonization of the hydroxylysine branch,
which was observed when the hydrolysis of the tert-
butyl ester 11a is carried out with aqueous TFA (80–
90%).15
Next, we attempted the regeneration of the protected
amino acidic functionalities of compound 10a by a
selective removingof its N-t-Boc protectinggroup, by
treatment with aqueous trifluoracetic acid (TFA),15
followed by catalytic hydrogenation under moderate
pressure (PdCl2, /EtOH/THF, 25 ꢁC, 40 psi).13 How-
ever, usingthis procedure, we observed that the expected
compound 11a was accompanied by the corresponding
lactone which, probably formed by inner transesterifica-
tion of the oxazinone lactone 10a with the hydroxyl
group of the hydroxylysine chain. To circumvent this
problem, we decided to attempt deblockingthe pro-
tected amino acidic functionalities of the compound
Similar results were obtained performinga parallel
sequence of reactions with the (5S)-epoxide 7 and amine
5. In this case, (2S,20S,5S)-5-HLNL 3b was obtained
through the diastereomeric intermediates 10b and 11b.
For the synthesis of dihydroxylated reduced cross links
(2S,20S,5R,50R)- and (2S,20S,5S,50S)-5,50-DHLNL 4a
and 4c (Scheme 3), we decided to react each epoxide 6
and 7 with benzylamine, which in our mind, appeared
a good auxiliary molecule not only for the introduction
of the secondary amino group of the target amino acids
4a and 4c, but also for a series of other reasons, includ-
ingits easily alkylation ability and cleavability.
10a usingsodium in liquid NH at À40 ꢁC, the alterna-
3
tive procedure of Williams.13 We were confident that
these conditions would be suitable for cleavingboth
13,16
the oxazinone ringand the carbobenzyloxy group.

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R. Cribiu et al. / Tetrahedron: Asymmetry 16 (2005) 3059–3069
3063
Ph
Ph
Ph
Ph
O
Ph
NR
Ph
RN
O
O
BocN
H3N+ COO-
OH
-OOC +NH3
HO
O
O
O
6
O
OH
HO
H
N
iii
N
i
R = Boc 12a
R = H 13a
4a
ii
NH2
Ph
O
Ph
Ph
Ph
RN
H3N+ COO-
OH
-OOC +NH3
HO
O
iii
ii
i
NR
H
N
O
O
OH
HO
Ph
O
N
Ph
BocN
4c
R = Boc 12b
R = H 13b
O
O
7
Scheme 3. Reagents and conditions: (i) LiClO4, CH3CN, 12 h, 60 ꢁC, 78%; (ii) ZnBr2, CH2Cl2, 2 h, room temp.; (iii) H2, PdCl2, THF–C2H5OH–H2O
(4:4:2, v/v/v), 67% from 12a or 12b.
In fact, by reactingthe benzylamine with two molecules
of epoxide 6, the tertiary amine 12a, containingthe
masked functions of the dimeric dihydroxylated amino
acid 4a, was obtained in satisfactory yields (78%). The
deblockingof compound 12a was complicated by the
presence of the two lysine hydroxyl groups and of the
benzyl group and required the investigation of different
procedures. The best results were obtained usinga Ôone-
potÕ cleavage of the oxazinone ring and of the benzyl
group, by acidic elimination of the N-t-Boc group, fol-
lowed by catalytic hydrogenation in the presence of
PdCl2. In fact, treatment of compound 12a with ZnBr2
in dichloromethane17 caused the exclusive cleavage of
the N-t-Boc groups and afforded the free dioxazinone
13a, which by catalytic hydrogenation afforded the
desired (2S,20S,5R,50R)-5,50-DHLNL 4a. On the contrary,
when the cleavage of the t-Boc groups was performed
treatingcompound 12a with TFA, an inner transesteri-
fication occurred to afford a mixture of products mainly
12b and 13b and, successively, of the (2S,20S,5S,50S)-
5,50-DHLNL 4c. The use of benzyl amine as a donor
of the nitrogen atom was useful also for the concise
preparation of the third isomeric (2S,20S,5R,50S)-5,50-
DHLNL 4b which has two hydroxylysine branches with
opposite geometries at the hydroxylated carbons
(Scheme 4).
As reported in Scheme 4, an excess of benzyl amine was
reacted with epoxide 6 (in a 2:1 ratio) to afford, in the
first step, the secondary amine 14, which after isolation
and purification, was reacted with the diastereomeric
epoxide 7 (in a 1:1 ratio) to form the completely pro-
tected dimeric amino acid 15a, a key precursor of target
compound 4b. The regeneration of the amino acidic
functionalities of 15a was then performed by a short
treatment of this dioxazinone with ZnBr2 in dichloro-
methane, followed by catalytic hydrogenation. The
Lewis acid cleaves the N-t-Boc groups to afford the free
dioxazinone 15b which was then reduced to the dimeric
amino acid (2S,20S,5R,50S)-5,50-DHLNL 4b.
1
containingthe mono and dilactones (MS and H NMR
evidences), derivingfrom the transesterification of the
oxazinone lactone(s) with the hydroxyls of the chains.
Similar, unsatisfactory results were obtained usingthe
This procedure for the regeneration of the masked func-
tionalities of 4b affords more satisfactory results with
respect to the cleavage of the dioxazinone of 15a by
means of sodium dissolved in liquid ammonia at
À40 ꢁC. In fact, in the last case, a partial survival of
the benzyl group, which was bonded to the secondary
amino group, was observed and the crude product of
the reaction requires a catalytic hydrogenation to
liberate the free amino acid 4b.
dissolvingmetal reduction (sodium in liquid NH
/
3
EtOH/THF) at À40 ꢁC to cleave the oxazinone ring.
This procedure, which was successful in the cleavage
of the oxazinone ringof the protected monohydroxy-
lated diamino acids 10a and 10b, shows in this case
the survival of the benzyl group bonded to the second-
ary amino group, in spite of the reported general cleav-
ability of the N-benzyl groups, under dissolving metal
reduction conditions.16 Thus, a successive catalytic
reduction was necessary to cleave the benzyl group
and to afford the free dimeric amino acid 4a.
The preparation of dimeric amino acid 4b completed the
synthesis of the possible reduced forms of collagen
immature cross links, which are now available in enan-
tiomeric pure form for biological experiments. More-
over, since the (2S)-stereochemistry of the starting
Parallel results, startingwith diastereomeric epoxide 7,
allowed us to obtain the intermediate tertiary amines

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R. Cribiu et al. / Tetrahedron: Asymmetry 16 (2005) 3059–3069
Ph
Ph
Ph
Ph
O
O
7
BocN
Ph
BocN
6
Ph
Ph
O
O
O
O
Ph
Ph
Ph
O
O
O
NBoc
BocN
BocN
ii
O
i
O
OH
HO
O
OH
+
NH2
N
NH
14
15a
Ph
Ph
Ph
NH
Ph
HN
O
O
H3N+ COO-
OH
-OOC +NH3
HO
iv
iii
H
N
O
O
OH
HO
N
15b
4b
Scheme 4. Reagents and conditions: (i) LiClO4, CH3CN, 3 h, 60 ꢁC, 55%; (ii) LiClO4, CH3CN, 72 h, 60 ꢁC, 65%; (iii) ZnBr2, CH2Cl2, 2 h, room
temp.; (iv) H2, PdCl2, THF–C2H5OH–H2O (4:4:2, v/v/v), 68% from 15a.
amino acids used for the synthesis is obtained with one
of the two possible Williams glycine templates, our pro-
cedure is also useful for the synthesis of all unnatural
dimeric amino acids with a (2R)-configuration.
0.25 mm ID, purchased from Macherey-Nagel); carrier
gas was He set at 85 kPa column head pressure and
the column temperature was set at 170 ꢁC. Mass spectra
were obtained usinga Finniagn LCQdeca (Thermo-
Quest) ion trap mass spectrometer fitted with an electro-
spray source (ESI). All reactions were monitored by
thin-layer chromatography (TLC) carried out on
0.25 mm E. Merck silica gel plates (60 F254) usingUV
light, 50% sulfuric acid or 0.2% ninhydrin in ethanol
and heat as developingagent. E. Merck 230–400 mesh
silica gel was used for flash column chromatography.
Usual work-up refers to washingthe organic layer with
3. Experimental
3.1. General
Meltingpoints were measured on a SMP3 mp apparatus
(Stuart Scientific, USA) and are uncorrected. Nuclear
magnetic resonance spectra were recorded at 298 K
on a Bruker AM-500 spectrometer operatingat 500.13
water, dryingover anhydrous Na SO4 and evaporating
2
the organic solvent under reduced pressure. Used Resin
was a DOWEX 50X 8-200 was used as the cation
exchange resin.
1
MHz for H and 125.76 MHz for 13C. Chemical shifts
are reported in parts for million (ppm, d units) relative
to the solvent signal.18 Proton and carbon assignments
were established, if necessary, with homonuclear and
heteronuclear 2D J-revolved experiments. 1H NMR
data are tabulated in the followingorder: number of
protons, multiplicity (s, singlet; d, doublet; bs, broad
singlet; m, multiplet), coupling constant(s) in Hertz,
3.2. tert-Butyl(3S,5S,6R)-3-[(3R)-3-hydroxy-4-bromo-
butyl]-2-oxo-5,6-diphenyl-1,4-oxazinane-4-carboxylate 8
and tert-butyl(3S,5S,6R)-3-[(3S)-3-hydroxy-4-bromo-
butyl]-2-oxo-5,6-diphenyl-1,4-oxazinane-4-carboxylate 9
1
assignment of proton(s). H NMR and 13C NMR spec-
A mixture of the previously obtained7b diastereomeric
epoxides 6 and 7 (846 mg, 2 mmol), dissolved in dry
THF (20 mL) containingAcOH (0.34 mL, 6 mmol),
was treated with anhydrous LiBr (280 mg, 3.2 mmol)
and stirred at room temperature for 4 h. The mixture
was then concentrated, diluted with water and extracted
with ethyl acetate. The usual work-up afforded a crude
residue (825 mg; Y = 82%) formed by a stereomeric mix-
ture of the bromohydrins 8 and 9. Rapid chromatogra-
phy (elutingwith dichloromethane–acetone; 100:2, v/v)
gave first the pure tert-butyl(3S,5S,6R)-3-[(3R)-3-hydr-
oxy-4-bromobutyl]-2-oxo-5,6-diphenyl-1,4-oxazinane-4-
carboxylate 8 (397 mg; Y = 41%): mp 157–158 ꢁC, (from
tra of compounds containingoxazinone rin(gs) were
complicated by the presence of distinguishable rota-
mers.13 Optical rotations were taken at 24 ꢁC on a Per-
kin–Elmer 241 polarimeter and [a]D values are given in
10À1 degcm 2 À1. The enantiomeric purity of the final
g
amino acids was ascertained by gas chromatography
of the correspondingtrifluoracetylated methyl esters
obtained as described in previous work on the synthesis
of hydroxylysine.19 Chiral GLC analyses were carried
out on a Hewlett-Packard 5890 gas chromatography
equipped with a octakis(3-O-butyryl-2,6-di-O-pentyl)-
c-cyclodestrin (LipodexE) capillary column (25 m,

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R. Cribiu et al. / Tetrahedron: Asymmetry 16 (2005) 3059–3069
3065
25
D
dichloromethane–benzene); ½aŠ ¼ À46:6 (c 1, CHCl3);
with ethyl acetate. Usual work-up afforded a solid resi-
due, which is crystallized to afford the tert-butyl
(3S,5S,6R)-3-[(3R)-3,4-epoxybutyl]-2-oxo-5,6-diphenyl-
1H NMR (CDCl3): d 7.29–6.97 (8H, aromatics), 6.53
(2H, aromatics), 5.98 (0.2H, d, J = 3 Hz, 6-H; minor
conformer), 5.95 (0.8H, d, J = 3 Hz, 6-H; major
conformer), 5.25 (0.2H, d, J = 3 Hz, 5-H; minor con-
former), 5.03 (1.6H, overlapping, 5-H; major conformer
and 3-H; major conformer), 4.86 (0.2H, dd, J = 10.5,
4.5 Hz, 3-H; minor conformer), 4.14 (0.8H, m, 30-H;
major conformer), 3.96 (0.2H, m, 30-H; minor con-
former), 3.59 (1H, dd, J = 10.5, 3.8 Hz, 40-Ha), 3.46
(1H, dd, J = 10.5, 6.6 Hz, 40-Hb), 3.32 (1H, br s, OH),
2.34 (1H, m, 10-Ha), 2.21 (1H, m, 10-Hb), 1.93 (1H,
m, 20-Ha), 1.81 (1H, m, 20-Hb), 1.41, 1.07 [9H, 2 · s,
2 · C(CH3)3; minor conformer and major conformer];
13C NMR (CDCl3): 168.97, 153.89, 136.12, 134.08,
128.61–126.03, 81.75, 79.14, 71.13, 69.43, 61.43,
55.30, 39.37, 31.16, 30.85, 27.81; ESI-MS (positive)
m/z: 526 (79BrÀM+Na+), 528 (81BrÀM+Na+), 1031
(79BrÀ2M+Na+), 1033 (81BrÀ2M+Na+). Anal. Calcd
for C25H30BrNO5: C, 59.53; H, 5.99; N, 2.78. Found:
C, 59.38; H, 6.12; N, 2.86.
1,4-oxazinane-4-carboxylate 6 as a solid (0.867 mg,
25
D
Y = 92%): mp 129–130 ꢁC (diisopropyl ether); ½aŠ
¼
À48 (c 1, CHCl3); 1H NMR (CDCl3): d 7.28–6.95
(8H, aromatics), 6.54 (2H, aromatics), 5.91 (1H, d, J =
3.00 Hz, 6-H), 5.21 (0.2H, d, J = 3.00 Hz 6-H; minor
conformer), 5.02 (0.8H, dd, J = 10.52, 4.21 Hz, 3-H;
major conformer), 4.99 (0.8H, d, 6-H, J = 3.00 Hz; ma-
jor conformer), 4.84 (0.2H, dd, J = 10.52, 4.21 Hz, 3-H;
minor conformer), 3.03 (1H, m, 30-H), 2.79 (1H, m, 40-
Ha ), 2.57 (1H, m, 40-Hb), 2.32 (1H, m, 20-Ha), 2.12
(1H, m, 20-Hb), 1.96 (1H, m, 1-Ha), 1.70 (1H, m, 1-
Hb), 1.45, 1.08 [9H, s, C(CH3)3; minor and major con-
former]; 13C NMR (CDCl3): 168.91, 153.65, 136.32,
134.25, 128.90–126.19, 81.25, 78.94, 61.56, 56.14,
51.43, 47.35, 31.24, 28.94, 27.76; ESI-MS (positive) m/
z: 446 (M+Na+), 869 (2M+Na+). Anal. Calcd for
C25H29NO5: C, 70.90; H, 6.90; N, 3.31. Found: C,
70.50; H, 6.70; N, 3.20.
Additional elution afforded the diastereomeric tert-butyl
(3S,5S,6R)-3-[(3S)-3-hydroxy-4-bromobutyl]-2-oxo-5,6-
diphenyl-1,4-oxazinane-4-carboxylate 9 (389 mg; Y =
41%): mp 156–158 ꢁC, (from dichloromethane–ben-
Similar treatment of the tert-butyl (3S,5S,6R)-3-[(3S)-3-
hydroxy-4-bromobutyl]-2-oxo-5,6-diphenyl-1,4-oxazin-
ane-4-carboxylate 9 (1.12 g; 2 mmol) afforded the tert-
butyl(3S,5S,6R)-3-[(3S)-3,4-epoxybutyl]-2-oxo-5,6-diph-
25
1
zene); ½aŠ ¼ À45:7 (c 1, CHCl3); H NMR (CDCl3): d
enyl-1,4-oxazinane-4-carboxylate 7 as a solid (0.862 mg;
D
25
7.29–6.58–6.92 (8H, aromatics), 6.58 (2H, aromatics),
5.95 (1H, d, J = 3 Hz, 6-H), 5.26 (0.2H, d, J = 3 Hz,
5-H; minor conformer), 5.16 (0.8H, dd, J = 9.3,
5.4 Hz, 3-H; major conformer), 5.06 (0.8H, d,
J = 3 Hz, 5-H; major conformer), 4.89 (0.2H, dd,
J = 10.5, 4.5 Hz, 3-H; minor conformer), 3.97 (0.8H,
m, 30-H; major conformer), 3.88 (0.2H, m, 30-H; minor
conformer), 3.61 (0.8H, dd, J = 10.5, 3.5 Hz, 40-Ha;
major conformer), 3.58 (0.2H, dd, J = 10.5, 3.5 Hz,
40-Hb; minor conformer), 3.50 (0.8H, dd, J = 10.5,
6.5 Hz, 40-Hb; major conformer), 3.46 (0.2H, dd, J =
10.2, 6.5 Hz, 40-Hb; minor conformer), 2.53 (0.2H, m,
10-Ha; minor conformer), 2.39 (0.8H, m, 10-Ha; major
conformer), 2.11 (0.8H, m, 10-Hb; major conformer),
2.05 (0.2H, m, 10-Hb; minor conformer), 1.93 (1.8H,
m, overlapping, 20-Ha 20-Hb; major conformer and
20-Ha; minor conformer), 1.83 (0.2H, m, 20-Hb; minor
conformer), 1.41, 1.07 [9H, 2 · s, 2 · C(CH3)3; minor
conformer and major conformer]; 13C NMR (CDCl3):
168.97, 153.80, 136.22, 134.23, 129.93–126.27, 81.42, 79.07,
70.61, 61.59, 56.12, 40.05, 31.74, 31.34, 27.82; ESI-MS
(positive) m/z: 526 (79BrÀM+Na+), 528 (81BrÀM+
Na+), 1031 (79BrÀ2M+Na+), 1033 (81BrÀ2M+Na+).
Anal. Calcd for C25H30BrNO5: C, 59.53; H, 5.99; N,
2.78. Found: C, 59.50; H, 6.02; N, 2.80.
Y = 92%): mp 120–122 ꢁC (diisopropyl ether); ½aŠ ¼
D
1
À71 (c 1, CHCl3); H NMR (CDCl3): d 7.28–6.95 (8H,
aromatics), 6.54 (2H, aromatics), 5.91 (1H, d, J =
3.00 Hz, 6-H), 5.19 (0.8H, d, J = 3.00 Hz, 5-H minor
conformer), 5.02 (0.8H, dd, J = 9.82, 5.61 Hz, 3-H;
major conformer), 4.98 (0.8H, d, J = 3.00 Hz, 5-H
major conformer), 4.80 (0.2H, dd, J = 9.82, 4.82 Hz,
3-H; minor conformer), 3.03 (1H, m, 30-H), 2.79 (1H,
m, 40-Ha ), 2.57 (1H, m, 40-Hb), 2.32 (1H, m, 20-Ha),
2.06 (1H, m, 20-Hb), 1.97 (1H, m, 1-Ha), 1.97 (1H, m,
1-Hb), 1.78 (1H, m, 1-Hb), 1.46, 1.08 [9H, s, C(CH3)3,
minor and major conformer]; 13C NMR (CDCl3):
169.18, 153.73, 136.40, 134.09, 129.98–126.42, 81.29,
78.94, 61.53, 56.16, 51.46, 46.79, 31.47, 28.80, 27.78;
ESI-MS (positive) m/z: 446 (M+Na+), 869 (2M+Na+).
Anal. Calcd for C25H29NO5: C, 70.90; H, 6.90; N,
3.31. Found: C, 70.60; H, 6.80; N, 3.40.
3.4. Alkylation of the 6-amino-(2S,5R)-2-benzyloxy-
carbonylaminohexanoate tert-butyl ester 5 with tert-
butyl(3S,5S,6R)-3-[(3R)-3,4-epoxybutyl]-2-oxo-5,6-
diphenyl-1,4-oxazinane-4-carboxylate 6 and with tert-
butyl(3S,5S,6R)-3-[(3S)-3,4-epoxybutyl]-2-oxo-5,6-di-
phenyl-1,4-oxazinane-4-carboxylate 7
The protected lysine tert-butyl ester 5 (830 mg; 2.5
mmol) dissolved in acetonitrile (5 mL) was treated with
the epoxide 6 (530 mg; 1.25 mmol) in the presence of Li-
ClO4 (130 mg; 1.25 mmol) at room temperature for 12 h
and then diluted with water and extracted with ethyl
acetate. After the usual work-up, the residue was puri-
fied by rapid chromatography, eluting with a solution
formed by CHCl3/MeOH/NH3 (100:2:0.25; v/v/v), to
3.3. tert-Butyl (3S,5S,6R)-3-[(3R)-3,4-epoxybutyl]-2-oxo-
5,6-diphenyl-1,4-oxazinane-4-carboxylate 6 and tert-butyl
(3S,5S,6R)-3-[(3S)-3,4-epoxybutyl]-2-oxo-5,6-diphenyl-
1,4-oxazinane-4-carboxylate 7
tert-Butyl (3S,5S,6R)-3-[(3R)-3-hydroxy-4-bromobutyl]-
2-oxo-5,6-diphenyl-1,4-oxazinane-4-carboxylate 8 (1.12
g; 2 mmol) was dissolved in CH3CN (15 mL) containing
CsCO3 (1.95 g; 6 mmol) and stirred for 2 h. At this
point, the mixture was poured into water and extracted
afford the dialkylated compound 10a as a glass
25
(617 mg; Y = 65% from 6): ½aŠ ¼ À23:5 (c 1, CHCl3);
D

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R. Cribiu et al. / Tetrahedron: Asymmetry 16 (2005) 3059–3069
3066
1H NMR (CDCl3): d 7.37–6.95 (8H, m, aromatics), 6.60
(2H, m, aromatics), 6.01 (0.7H, d, J = 3 Hz,
PhCHOCHPh; major conformer), 5.98 (0.3H, d, J =
3 Hz; PhCHOCHPh; minor conformer), 5.45 (0.7H, d,
J = 8.4 Hz, NH; major conformer), 5.42 (0.3H, d, J =
8.4 Hz, NH; minor conformer), 5.24 (0.3H, d,
J = 3.0 Hz, PhCHNBoc; minor conformer), 5.12 (2H,
s, PhCH2O), 5.02 (0.7H, d, J = 3.0 Hz, PhCHNBoc;
major conformer), 5.01 (0.7H, dd, J = 9.8, 5.9 Hz
COCHN; major conformer), 4.94 (0.3H, dd, J =
9.8 Hz, 4.5 Hz COCHN; minor conformer), 4.26 (1H,
m, COCHNHCbz), 3.90 (0.7H, m, CH2CHOH; major
conformer), 3.77 (0.3H, m, CH2CHOH; minor con-
former), 2.79–2.54 (4H, m, overlapping, NHCH2CHOH
and NHCH2CH2), 2.33–2.20 (2H, m, NCHCH2CH2),
1.87–1.35 (8H, m, overlapping, CH2CH2CH2CH2,
CH2CH2CH2CH2, CH2CH2CH2- CH2, CH2CH2CH2-
CH2), 1.47 [9H, s, COOC(CH3)3], 1.12 [9H, s, NCO-
OC(CH3)3]; 13C NMR (CDCl3): 171.56, 169.14,
155.86, 153,68, 136.41, 134.26, 131.40, 129.07–126.20,
82.03, 81.37, 78.91, 67.92, 66.85, 61.41, 55.93, 55.01,
54.29, 49.08, 32.67, 31.15, 31.21, 28.29, 28.01, 22.68;
ESI-MS (positive) m/z: 760 (M+H+), 782 (M+Na+),
1519 (2M+H+), 1542 (2M+Na+). Anal. Calcd for
C43H57N3O9: C, 67.96; H, 7.56; N, 5.53. Found: C,
67.70; H, 7.70; N, 5.40.
3.5. (2S,5R)-2-Amino-6-[(5S)-5-amino-5-carboxypentyl-
amino]-5-hydroxyhexanoic acid 3a
A flame dried, two-necked, round-bottomed flask
(100 mL), equipped with a magnetic stirring bar was
charged with liquid ammonia (17 mL) and sodium metal
(40 mg; 2 mmol). The resulting solution was stirred at
À40 ꢁC. Meanwhile, a flame-dried round-bottomed
flask, fitted with a rubber stopper and an argon inlet,
was charged with the completely protected dimeric
amino acid 10a (135 mg; 0.18 mmol), anhydrous THF
(1.7 mL) and absolute ethanol (0.100 mL). The resulting
mixture was then transferred to the sodium/liquid
ammonia solution. After 20 min, the reaction was
carefully quenched with solid ammonium chloride and
allowed to warm to room temperature. After evapora-
tion of the remainingammonia, the residue was diluted
with ice cold water and the mixture extracted with ethyl
acetate. Then, the aqueous layer was acidified to pH 1
with HCl (1 M) and the obtained solution was applied
on a DOWEX 50X 8-200 cation exchange resin column
activated with HCl 2 M to afford the amino acid 11a,
elutingwith an aqueous ammonia solution (0.5 M).
The crude compound 11a was then dissolved in aqueous
HCl 1 M (5 mL) and the solution stirred for 2 h. Succes-
sive evaporation of the solvent afforded the amino acid
25
3a as a chloridrate salt (36 mg; Y = 52%); ½aŠ ¼ þ4:4
D
1
Similar treatment of the protected lysine tert-butyl ester
5, (830 mg; 2.5 mmol) with the tert-butyl (3S,5S,6R)-
3-[(3S)-3,4-epoxybutyl]-2-oxo-5,6-diphenyl-1,4-oxaz-
inane-4-carboxylate 7 (530 mg; 1.25 mmol) afforded,
after rapid chromatography, eluting with CHCl3/
MeOH/NH3 (100:2.5:0.25; v/v/v), the dialkylated com-
pound 10b as a glass (605 mg Y = 64% from 7);
(c 0.33, H2O); H NMR (D2O): d 4.12 (2H, m, 2-H,
50-H), 3.89 (1H, m, 5-H), 3.23 (1H, dd, 6-Ha,
J = 13.30, 2.45 Hz, 6-Ha), 3.15 (2H, t, J = 8.05 Hz,
1-H), 3.05 (1H, dd, J = 12.60, 2.45 Hz 6-Hb), 2.19
(1H, m, 3-Ha), 2.02 (3H, m, overlapping, 3-Hb, 40-H),
1.81 (2H, m, 20-H), 1.68 (2H, m, 4-H), 1.56 (2H, m,
30-H); 13C NMR (D2O): d 173.19, 172.99, 67.21, 53.83,
53.13, 48.20, 30.55, 30.30, 26.96, 25.96 22.57; ESI-MS
(positive) m/z: 292 (M+H+). Anal. Calcd for
C12H28Cl3N3O5: C, 35.97; H, 7.04; N, 10.49. Found:
C, 36.05; H, 7.15; N, 10.60.
25
D
1
½aŠ ¼ À21:1 (c 1, CHCl3); H NMR (CDCl3): d 7.37–
6.95 (8H, aromatics), 6.95 (2H, aromatics), 6.01 (0.7H,
d, J = 3 Hz, PhCHOCHPh; major conformer), 6.00
(0.3H, d, J = 3 Hz, PhCHOCHPh; minor conformer),
5.44 (0.7H, d, J = 8.4 Hz, NH; major conformer), 5.42
(0.3H, d, J = 8.4 Hz, NH; minor conformer), 5.23
(0.3H, d, J = 3.0 Hz, PhCHNBoc; minor conformer),
5.12 (2H, s, PhCH2O), 5.04 (0.7H, dd, J = 9.4, 5.6 Hz,
COCHN; major conformer), 5.02 (0.3H, d, J = 3.0 Hz,
PhCHNBoc; minor conformer), 4.82 (0.3H, dd,
J = 9.4, 4.2 Hz COCHN; minor conformer), 4.26 (1H,
m, COCHNHCbz), 3.74 (0.7H, m, CH2CHOH; major
conformer), 3.64 (0.3H, m, CH2CHOH; minor con-
former), 2.76–2.60 (3H, m, overlapping, NHCHHa-
CHOH and NHCH2CH2), 2.55 (0.7H, dd, J = 12.2,
9.5 Hz, NHCHbCHOH; major conformer), 2.50 (0.3H,
dd, J = 12.2, 9.5 Hz, NHCHHbCHOH; minor con-
former), 2.39 (1H, m, NCHCHHaCH2), 2.05 (1H,
m, NCHCHHbCH2), 1.85–1.35 (8H, m, overlapping,
CH2CH2CH2CH2, CH2CH2CH2CH2, CH2CH2CH2-
CH2, CH2CH2CH2CH2), 1.47 [9H, s, COOC- (CH3)3],
1.12 [9H, s, NCOOC(CH3)3]; 13C NMR (CDCl3) d
171.62, 169.57, 155.93, 153.72, 136.54, 136.39, 134.36,
128.80–126.34, 82.07, 81.18, 78.82, 68.87, 66.83, 61.51,
56.39, 54.92, 54.21, 49.15, 32.63, 31.67, 31.24, 28.27,
27.99, 22.68; ESI-MS (positive) m/z: 760 (M+H+), 782
(M+Na+), 1519 (2M+H+), 1542 (2M+Na+). Anal.
Calcd for C43H57N3O9: C, 67.96; H, 7.56; N, 5.53.
Found: C, 67.85; H, 7.65; N, 5.60.
3.6. (2S,5S)-2-Amino-6-[(5S)-5-amino-5-carboxypentyl-
amino]-5-hydroxyhexanoic acid 3b
Startingfrom the compound 10b (135 mg; 0.18 mmol),
and usingthe same procedure described above for the
dioxazinone 10a, the amino acid 3b was obtained
25
(37 mg Y = 52%); ½aŠ ¼ þ8:7 (c = 0.33, H2O); 1H
D
NMR (D2O): d 3.98 (1H, m, 5-H), 3.79 (2H, dd,
J = 12.60, 6.30 Hz, 2-H, 50-H), 3.23 (1H, dd,
J = 12.95, 3.15 Hz, 6-Ha), 3.14 (2H, t, J = 8.05, 10-H),
3.07 (1H, dd, J = 12.95, 3.15 Hz, 6-Hb), 1.98 (1H, m,
3-Ha), 1.86 (3H, m, overlapping, 3-Hb, 40-H), 1.71
(2H, m, 20-H), 1.63 (1H, m, 4-Ha), 1.44 (3H, m, overlap-
ping, 4-Hb, 30-H); 13C NMR (D2O): d 175.43, 175.25,
67.22, 63.56, 55.45, 53.05, 49.05, 30.08, 27.48, 25.97,
22.49; ESI-MS (positive) m/z: 292 (M+H+). Anal. Calcd
for C12H28Cl3N3O5: C, 35.97; H, 7.04; N, 10.49. Found:
C, 35.85; H, 9.95; N, 10.55.
3.7. Preparation of the tertiary benzylamines 12a and 12b
To a stirred solution of the epoxide 6 (250 mg,
0.59 mmol) in acetonitrile (0.8 mL), LiClO4 (62.6 mg,
0.59 mmol) and benzylamine (32 ll, 0.29 mmol) were

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R. Cribiu et al. / Tetrahedron: Asymmetry 16 (2005) 3059–3069
3067
added. The resultingsolution was stirred for 12 h at
60 ꢁC. After complete conversion of epoxide 6 (TLC),
the reaction mixture was diluted with ethyl acetate and
worked-up. Evaporation of the solvent under reduced
pressure and purification of the crude product on silica
gel, [eluting with a mixture of CHCl3/solvent A
80:20 v/v, where solvent A is a solution of CHCl3/
MeOH/NH3 (100:4:0.5 v/v/v)], gave the pure trialkyl-
amine 12a as a glassy material which was crystallized
(113 mg; 0.5 mmol). After 3 h, the reaction mixture
was diluted with dichloromethane and washed with
water. Usual work-up afforded the crude compound
13a, which was dissolved in THF/ethanol/water 4:4:2
v/v/v (50 mL) and hydrogenated at 60 psi over PdCl2.
After 48 h, filtration of the catalyst on Celite and re-
moval of the solvent, gave the crude dimeric amino acid
4a, which was then dissolved in HCl 0.1 M and purified
through a cationic exchange resin DOWEX 50X 8-200.
Successive elution with an aqueous ammonia solution
from CH2Cl2/benzine to afford the pure compound
25
12a as a white solid (221 mg, Y = 78%); ½aŠ ¼ À63:5
D
(c 1, CHCl3); mp 120–122 ꢁC; 1H NMR (CDCl3): d
7.42–6.87 (21H, aromatics), 6.55 (4H, aromatics), 5.94
(1H, d, J = 2.80 Hz, PhCHOCHPh), 4.96 (3.4H, m,
overlapping, 2 · PhCHNboc and COCHN major con-
former), 4.79 (0.6H, dd, J = 10.6, 4.7 Hz COCHN;
minor conformer), 3.94 (2.8H, m, overlapping, PhCH2N,
and CH2CHOH, major conformer), 3.80 (0.6H, m,
CH2CHOH; minor conformer), 3.56 (0.6H, d, J =
13.8 Hz, PhCH2N; minor conformer), 2.53 (4H, m,
PhNCH2CHOH), 2.23 (4H, m, NCHCH2CH2CHOH),
1.65–1.47 (4H, m, NCHCH2CH2CHOH), 1.43, 1.09
[2 · 9H, 2 · s, 2 · NCOOC(CH3)3; major and minor
conformer]; 13C NMR (CDCl3): d 169.22, 153.80,
136.44, 134.29, 129.80–125.70, 81.41, 78.87, 67.04,
61.30, 60.41, 59.67, 56.02, 31.17, 30.58, 27.80; ESI-MS
(positive) m/z: 954 (M+H+), 1908 (2M+H+). Anal.
Calcd for C57H67N3O10: C, 71.75; H, 7.08; N, 4.40.
Found: C, 71.65; H, 7.30; N, 4.30.
(0.5 M) and lyophilization gave the pure compound 4a
25
as a solid (25 mg, Y = 65%); ½aŠ ¼ À3:4 (c 0.5, H2O);
D
1H NMR (D2O): d 3.91 (2H, m, 5-H), 3.66 (2H, t,
J = 6.30 Hz, 2-H), 2.98 (1H, dd, J = 12.95, 3.15 Hz, 6-
Ha), 2.87 (1H, dd, J = 12.95, 3.15 Hz, 6-Hb), 2.01
(2H, m, 3-Ha), 1.86 (2H, m, 3-Hb), 1.59 (4H, m, 4-H);
13C NMR (D2O): d 177.71, 68.77, 55.81, 53.88, 31.00,
28.69; ESI-MS (positive) m/z: 308 (M+H+)+, 330
(M+Na+). Anal. Calcd for C12H25N3O6: C, 46.89; H,
8.20; N, 13.67. Found: C, 47.00; H, 8.30; N, 13.50.
Usingthe same reaction sequence described above for
the synthesis of 4a, startingfrom the compound 12b
(120 mg; 0.125 mmol), the free amino acid 4c was ob-
23
D
1
tained (26 mg Y = 67%); ½aŠ ¼ þ9:1 (c 0.5, H2O); H
NMR (D2O): d 3.84 (2H, m, 5-H), 3.48 (2H, t,
J = 6.30 Hz, 2-H), 2.83 (1H, dd, J = 12.95, 3.15 Hz, 6-
Ha), 2.76 (1H, dd, J = 12.95, 3.15 Hz, 6-Hb), 1.82
(4H, m, 3-H), 1.61 (2H, m, 4-Ha), 1.50 (2H, m, 4-Hb);
13C NMR (D2O): d 181.20, 69.78, 56.32, 54.56, 31.34,
30.37; ESI-MS (m/z): 308 (M+H+). Anal. Calcd for
C12H25N3O6: C, 46.89; H, 8.20; N, 13.67. Found: C,
46.75; H, 8.15; N, 13.70.
Usingthe same procedure for the synthesis of 12a, start-
ingfrom the epoxide 7 (250 mg; 0.59 mmol) the pro-
tected dimeric amino acid 12b was obtained (212 mg
25
Y = 75%): ½aŠ ¼ À21:4 (c 1, CHCl3); mp 125–126 ꢁC,
D
1
CH2Cl2/benzine; H NMR (CDCl3): d 7.43–6.90 (21H,
m, aromatics), 6.55 (4H, m, aromatics), 5.91 (1H, d,
J = 3 Hz, PhCHOCHPh;), 5.18 (0.6H, d, J = 3 Hz,
PhCHNboc; minor conformer), 4.99 (1.4H, dd,
J = 9.3, 5.6 Hz COCHN; major conformer), 4.95
(1.4H, d, J = 3 Hz, PhCHNboc; major conformer),
4.76 (0.6H, dd, J = 10.2, 4.1 Hz COCHN; minor con-
former), 3.88 (1H, dd, J = 13.5, 7.6 Hz PhCHaN), 3.77
(1.4H, m, CH2CHaOH), 3.69 (0.6H, m, RCH2CHbOH),
3.53 (1H, dd, J = 13.5, 4.3 Hz PhCHbN), 2.53 (4H, m,
PhNCH2CHOH), 2.39 (2H, m, NCHCHHaCH2-
CHOH), 2.10 (2H, m, NCHCHHbCH2CHOH), 1.66
(4H, m, NCHCH2CH2CHOH), 1.40, 1.09 [2 · 9H,
2 · s, 2 · NCOOC(CH3)3; major and minor conformer];
13C NMR (CDCl3): d 169.61, 153.80, 136.63, 134.09,
129.80–126.90, 81.14, 78.83, 67.74, 61.55, 60.47, 59.85,
56.33, 31.90, 31.00, 27.82; ESI-MS (positive) m/z: 954
(M+H+), 1908 (2M+H+), 1931 (2M+Na+). Anal. Calcd
for C57H67N3O10: C, 71.75; H, 7.08; N, 4.40. Found: C,
71.85; H, 7.20; N, 4.50%.
3.9. tert-Butyl(3S,5S,6R)-3-[(3R)-4-benzylamino-3-
hydroxybutyl]-2-oxo-5,6-diphenyl-1,4-oxazinane-4-
carboxylate 14
To a stirred solution of epoxide 6 (140 mg, 0.34 mmol)
in 0.5 mL of acetonitrile (0.5 mL), LiClO4 (36 mg
0.34 mmol) and benzylamine (74 lL, 0.68 mmol) were
added. The resultingsolution was stirred for 3 h at
60 ꢁC. After complete conversion of the epoxide 6
(TLC), the reaction mixture was cooled to room temper-
ature, diluted with ethyl acetate and washed with water/
brine. Usual work-up afforded a crude product which,
after purification on silica gel, eluting with CHCl3/
MeOH/NH3; 100:4:0.5 v/v/v, gave the pure dialkyl-
amine 14 as a glassy material, which was crystallized
with CH2Cl2/benzine to afford a solid compound 14
25
(99 mg, Y = 55%); ½aŠ ¼ À44:8 (c = 1, CHCl3); mp
D
1
103–104 ꢁC; H NMR (CDCl3): d 7.36–6.96 (13H, m,
aromatics), 6.56 (2H, m, aromatics), 5.98 (1H, d,
J = 3.0 Hz, PhCHOCHPh), 5.24 (0.2H, d, J = 3.0 Hz,
COCHPh; minor conformer), 5.05 (0.8H, dd, J = 9.1,
5.7 Hz, COCHN; major conformer), 5.01 (0.8H, d,
J = 3.0 Hz, COCHPh; major conformer), 4.82 (0.2H,
dd, J = 9.1, 4.2 Hz, COCHN; minor conformer), 3.87–
3.69 (3H, m, PhCH2N and CH2CHOH), 2.83 (1H, dd,
J = 12.1, 3.0 Hz PhNCHaCHOH), 2.60 (1H, dd,
J = 12.1, 9.2 Hz, PhNCHbCHOH), 2.32–2.13 (2H, m,
NCHCH2CH2CHOH), 1.76–1.56 (2H, m, NCHCH2-
3.8. (2S,5R)-2-Amino-6-[(2R,5S)-5-amino-5-carboxy-2-
hydroxypentylamino]-5-hydroxyhexanoic acid 4a and
(2S,5S)-2-amino-6-[(2S,5S)-5-amino-5-carboxy-2-
hydroxypentylamino]-5-hydroxyhexanoic acid 4c
Tertiary amine 12a (120 mg; 0.125 mmol) was dissolved
in (0.7 mL) of anhydrous dichloromethane and the
resultingsolution was treated with anhydrous ZnBr
2

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R. Cribiu et al. / Tetrahedron: Asymmetry 16 (2005) 3059–3069
3068
CH2CHOH), 1.41, 1.08 [9H, s, NCOOC- (CH3)3, major
and minor conformer]; 13C NMR (CDCl3): d 169.57,
154.01, 136.36, 134.28, 131.47, 128.7–125.37, 81.42,
78.98, 68.05, 61.42, 55.84, 54.51, 53.34, 31.12, 28.44,
27.81; ESI-MS (positive) m/z: 531 (M+H+), 1061
(2M+H+). Anal. Calcd for C32H38- N2O5: C, 72.43; H,
7.22; N, 5.28. Found: C, 72.60; H, 7.30; N, 5.50.
1.76 (2H, m, 3-H, 30-H), 1.65, 1.39 (2H, m, 4-H, 40-H);
13C NMR (D2O) d 183.66, 67,71, 67.63, 55.49, 55.43,
53.44, 30.88, 30.73, 27.72; ESI-MS (positive) m/z: 308
(M+H+). Anal. Calcd for C57H67N3O10: C, 71.75; H,
7.08; N, 4.40. Found: C, 71.80; H, 7.20; N, 4.50.
References
3.10. Preparation of the tertiary benzylamine 15a
1. For a comprehensive review on biology of bones, see:
Principles of Bone Biology; Bilezikan, J. P., Raicz, L. G.,
Rogan, G., Eds.; Academic Press: New York, 2001.
2. For recent reviews on the collagen cross links, see: (a)
Gineyts, E.; Garnero, P.; Delmas, P. Rheumatology 2001,
40, 315–323; (b) Watts, N. B. Clin. Chem. 1999, 45, 1359–
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(d) James, I. T.; Walne, A. J.; Perret, D. Ann. Clin.
Biochem. 1996, 33, 397–420, and references cited therein;
(e) Yamauchi, M.; Mechanic, G. L. Cross-linkingof
Collagen. In Collagen; Nimni, M. E., Ed.; Biochemistry;
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3. (a) Banse, X.; Devogelaer, J. P.; Lafosse, A.; Sims, T. J.;
Grynpas, M.; Bailey, A. J. Bone 2002, 31, 70–76; (b)
Bailey, A. J.; Paul, R. G.; Knott, L. Bone 1998, 106, 1–56.
4. (a) Garnero, P.; Delmas, P. D. Curr. Opinion Rheumatol.
2003, 15, 641–646; (b) Garnero, P.; Gineyts, E.; Christgau,
S.; Finck, B.; Delmas, P. D. Arthritis Rheumatol. 2002, 46,
21–30.
To epoxide 7 (92 mg, 0.17 mmol) dissolved in acetoni-
trile (0.6 mL), LiClO4 (0.17 mmol, 19 mg) and trialkyl-
amine 14 were added. The resultingsolution was then
stirred for 72 h at 60 ꢁC. After complete conversion of
the epoxide 7 (TLC), the reaction mixture was cooled
to room temperature, diluted with ethyl acetate and
washed with water. Usual work-up afforded a crude
product, which was purified on silica gel, [eluting with
a mixture of CHCl3/solvent A 80:20 v/v where solvent
A is a solution of CHCl3/MeOH/NH3 (100:4:0.5 v/v/
v)] and gave the pure dialkylamine as a glassy material,
which was crystallized from CH2Cl2/benzine to afford
compound 15a as a white solid (105 mg, Y = 65%);
25
1
½aŠ ¼ À44:0 (c 1, CHCl3); mp 123–124 ꢁC; H NMR
D
(CDCl3): d 7.37–6.89 (21H, m, aromatics), 6.52 (4H,
m, aromatics), 5.90 (1H, d, J = 3 Hz, PhCHOCHPh;
in oxazinone of the S branch), 5.87 (1H, d, J = 3 Hz,
PhCHOCHPh; in oxazinone of the R branch), 5.16
(0.3 · 2H, d, J = 3 Hz, COCHPh; minor conformer),
4.99 (0.7H, dd, J = 8.99, 6.14 Hz, COCHN; in oxazi-
none of the S branch; major conformer), 4.94 (2.1H,
m, overlapping, 2 · PhCHNBoc and COCHN of the R
branch; major conformer), 4.76 (2 · 0.3H, dd,
J = 8.99, 6.14 Hz, 5-H; COCHN; minor conformer),
3.95 (1, 2H, m, overlapping; PhCH2N and CH2CHOH;
minor conformer), 3.74 (2.8H, m, overlapping; PhCH2N
and CH2CHOH; major conformer), 2.72–2.51 (4H, m,
PhNCH2CHOH), 2.31 (1H, m, NCHCHaCH2CHOH,
in S branch), 2.24–2.09 (2H, m, NCHCH2CH2CHOH,
in R branch), 1.98 (1H, m, NCHCHbCH2CHOH, in S
branch), 1.70–1.47 (4H, m, NCHCH2CH2CHOH, in S
and R branch), 1.40, 1.07 [2 · 9H, 2 · s, 2 · NCO-
OC(CH3)3; major and minor conformer]; 13C NMR
(CDCl3): d 169.57, 169.03, 153.80, 136.58, 136.29,
134.33, 134.26, 129.46, 126.20, 81.48, 81.14, 78.98,
78.80, 68.98, 67.97, 61.55, 61.30, 60.28, 60.13, 56.32,
55.52, 31.81, 31.02, 30.73; ESI-MS (positive) m/z:
954 (M+H+), 1908 (2M+H+). Anal. Calcd for
C57H67N3O10: C, 71.75; H, 7.08; N, 4.40. Found: C,
71.55; H, 7.10; N, 4.20.
5. For isolation of Pyd 1a see: Fujimoto, D.; Moriguchi, T.;
Ishida, T.; Hayashi, H. Biochem. Biophys. Res. Commun.
1978, 84, 52–57; For that of Dpy 1b see: Ogawa, T.; Ono,
T.; Tsuda, M.; Kawanishi, Y. Biochem. Biophys. Res.
Commun. 1982, 107, 1252–1257.
6. (a) Garnero, P.; Delmas, P. D. Biochemical Markers of
Bone Turnover. In Osteoporosis; Marcus, R., Feldman,
D., Kelsey, J., Eds.; San Diego: Academic Press, 1996, pp
1075–1085; (b) Delmas, P. D.; Takahashi, M.; Suzuki, M.;
Naitou, K.; Miyamoto, S.; Kushida, K. Rheumatology
1999, 38, 133–138; (c) Garnero, P.; Hausherr, E.; Chapuy,
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7. For work on total synthesis of pyridinoline cross links see:
`
(a) Allevi, P.; Cribiu, R.; Giannini, E.; Anastasia, M.
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bc050107h, 2005.; (b) Allevi, P.; Anastasia, M. Tetra-
hedron: Asymmetry 2004, 15, 2091–2096; (c) Allevi, P.;
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2012; (d) Allevi, P.; Galligani, M.; Anastasia, M. Tetra-
hedron: Asymmetry 2002, 13, 1901–1910; (e) Anastasia, L.;
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11, 5017; (g) Adamczyk, M.; Johnson, D. D.; Reddy, R. E.
Tetrahedron Lett. 1999, 40, 8993–8994, and Corrigendum:
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3.11. (2S,5R)-2-Amino-6-[(2S,5S)-5-amino-5-carboxy-2-
hydroxypentylamino]-5 hydroxyhexanoic acid 4b
The protected functions of compound 15a (95 mg;
0.10 mmol) were deblocked adoptingthe procedure de-
scribed for compounds 12a and 12b, startingfrom 15a.
The compound 15b was obtained and directly hydroge-
nated to afford the dimeric amino acid 4b (meso form):
(21 mg, Y = 68%); 1H NMR (D2O): d 3.85 (2H, m,
5-H, 50-H), 3.66 (2H, m, 2-H, 20H), 3.08 (2H, dd, 6-Ha,
J = 12.95, 2.80 Hz), 2.92–2.86 (2H, dd, 6-Hb, J =
12.95, 2.80 Hz,), 2.02–1.89 (2H, m, 3-H, 30-H), 1.88–

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R. Cribiu et al. / Tetrahedron: Asymmetry 16 (2005) 3059–3069
3069
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