Synthesis of all four possible stereoisomers of 5-hydroxylysine

Article abstract of DOI:10.1016/S0957-4166(00)00281-0

A simple protocol for the transformation of L- and D-glutamic acids into the enantiopure forms of all four isomers of 5-hydroxylysine is described. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0957-4166(00)00281-0

Tetrahedron: Asymmetry 11 (2000) 3151±3160
Synthesis of all four possible stereoisomers of 5-hydroxylysine
Pietro Allevi* and Mario Anastasia
Á
Dipartimento di Chimica e Biochimica Medica, Universita di Milano, via Saldini 50, I-20133 Milano, Italy
Received 21 June 2000; accepted 17 July 2000
Abstract
A simple protocol for the transformation of l- and d-glutamic acids into the enantiopure forms of all
four isomers of 5-hydroxylysine is described. # 2000 Elsevier Science Ltd. All rights reserved.
1. Introduction
(2S,5R)-5-Hydroxylysine 1 (l-normal-5-hydroxylysine; Fig. 1) is an amino acid exclusive to
collagen protein. It forms by post-translational hydroxylation of lysine and can be successively
glycosylated with either a b-d-galactopyranosyl- or an a-d-glucopyranosyl-(1-2)-b-d-galacto-
pyranosyl residue.1,2 The extent of glycosylation di€ers for each collagen type but, in all cases,
during collagen degradation, (2S,5R)-5-hydroxylysine is excreted in urine mainly as glycoside.3
Thus, after an acidic hydrolysis, the (2S,5R)-5-hydroxylysine levels in human urine are commonly
Figure 1.
* Corresponding author. Fax: +39 02 2361407; e-mail: pietro.allevi@unimi.it
0957-4166/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(00)00281-0

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measured to evaluate the collagen breakdown.4 Moreover, during the hydrolysis, the (2R,5R)-5-
hydroxylysine 4 (d-allo-5-hydroxylysine) is also formed and any analytical method must detect
this 2-epimer also.4
The hydroxylysine glycosylates are also of relevance for immunological studies concerning the
autoimmune response evidenced in rheumatoid arthritis.5
Surprisingly, while many authors have reported6 8 convenient protections of (2S,5R)-5-hydroxy-
lysine 1 to allow the study of the glycosylation of its hydroxy group, no stereoselective synthesis
of the possible stereoisomers of this amino acid has been reported. All published syntheses9 12
a€ord a mixture of the four possible 5-hydroxylysine stereoisomers and delicate chemical and
enzymatic procedures are then necessary for their separation.13,14 On the other hand, (2R,5R)-5-
hydroxylysine 1, the only pure isomer commercially available, is expensive since it is obtained by
tedious procedures from gelatine acid hydrolysates which also contain the (2R,5R)-epimer 4
which forms by epimerization during the acidic hydrolysis.15
In a very recent paper,16 a strategy for the stereoselective synthesis of 5-hydroxylysine was
proposed which, however, is satisfactory only16 for the preparation of (2S,5S)-hydroxylysine 2.
Thus, we decided to report on the synthesis of both (2S,5R)- and (2S,5S)-5-hydroxylysine 1 and 2
and of their diastereoisomer (2R,5R)- and (2R,5S)-5-hydroxylysine 3 and 4, planned as part of a
larger program concerning various markers of collagen turnover.17,18
The results, here reported, concern the synthesis of (2S,5R)-5-hydroxylysine 1 and of (2S,5S)-5-
hydroxylysine 2 starting with l-glutamic acid. However, they represent a veri®cation of a general
strategy which, starting from commercial d-glutamic acid, also allows the preparation of the
corresponding d-enantiomers 3 and 4.
2. Results and discussion
According to our protocol, the known diazo ketone 5, prepared19 in three steps from l-glutamic
acid, is transformed into the azido ketone 8 which, by reduction with sodium borohydride,
a€ords the diastereoisomeric hydroxy esters 9 and 10 (Scheme 1).
In this sequence of well-established reactions, the easy transformation of the oxazolidinone 5
into the corresponding methyl ester 6, performed by mild treatment with NaHCO3 in methanol,
is new and noteworthy. In previous works20,21 similar transformations were performed in the
presence of sodium methoxide which could put in danger the enantiomeric purity of the amino
acid.
Unfortunately, all attempts to separate the diastereoisomeric hydroxy esters 9 and 10 were
unsuccessful even when the hydroxy group was esteri®ed with achiral or chiral acids (acetates,
tri¯uoroacetates, benzoates, Mosher esters).22 However, the intramolecular transesteri®cation of
the 5-hydroxyl group with the methyl ester of the amino acid a€orded a mixture of
diastereoisomeric cis and trans lactones (2S,5R)-11 and (2S,5S)-12, which showed a di€erent
polarity on normal phase HPLC and were easily separated by rapid chromatography on silica.
The lactonization of the hydroxy esters 9 and 10 was performed with TFA, an acid commonly
used in amino acid chemistry for the regeneration of the amino acid functionalities from tert-
butyl derivatives without a€ecting their stereochemical integrity. In any case, the presence of a
stereogenic center a to the lactonic carbonyls of 11 and 12, suggests a possible epimerization of
the lactones in the acid condition necessary for their formation. This unfavourable event could
transform, at least in part, the initially formed lactones (2S,5R)-11 and (2S,5S)-12 into the

P. Allevi, M. Anastasia / Tetrahedron: Asymmetry 11 (2000) 3151±3160
3153
Scheme 1. Reagents and conditions: (i) NaHCO3, MeOH, re¯ux, 10 min, 85%; (ii) HBr 33% in AcOH, THF, 0ꢀC,
82%; (iii) NaN3, DMF, rt, 1 h, 86%; (iv) NaBH4, MeOH, 0ꢀC, 20 min, 92%; (v) TFA, rt, 40 min; (vi) Cs2CO3,
MeOH:H2O 1:1 v/v, rt, 35 min, 85%; (vii) H2 Pd/C, MeOH:H2O 1:1 v/v, then pH 6.5±7.0, 75%; (viii) a-chymotrypsin,
phosphate bu€er (pH 7.4)/Me2CO, 25ꢀC, 12 h, 96%; (ix) H2 Pd/C, MeOH:H2O 1:1 v/v, then pH 6.5±7.0, 78%
reversed pair of epimers (2R,5R)-12 and (2R,5S)-11 (Table 1), with a consequent reduction of the
enantiomeric purity of the diastereoisomers isolated at the end of the reaction.
In order to check this possibility, we separately tested the stability of lactones 11 and 12 under
various lactonization conditions. The results of this study led to the choice of the conditions
(TFA, rt, 40 min) which caused the complete lactonization of hydroxy methyl esters 9 and 10
without modifying the geometry of the initially formed lactones.
The next step of the synthesis required the transformation of lactones 11 and 12 into the
corresponding hydroxy acids 13 and 14 under basic conditions. Thus, a possible a epimerization
of lactones 11 and 12 was taken in account. In order to exclude this event, the lactone 11 was
hydrolyzed under various basic conditions and the crude product of the reaction (the hydroxy
acid 13) was directly treated with TFA (rt, 10 min) to regenerate the starting lactone group
(Scheme 2). The stereochemical purity of the resulting lactone was then checked by HPLC and it
was possible to select the milder conditions for the opening of the lactone 11 to the hydroxy acid
13 without a-epimerization: a simple treatment with Cs2CO3 of the lactone 11, dissolved in a
mixture of methanol and water (50%, v/v), followed by acidi®cation. In this way, only the
(2S,5R)-hydroxy acid 13 was obtained which, by cyclization, a€orded the stereochemically pure
starting lactone (2S,5R)-11.
In the case of the cis lactone 12 a slower hydrolysis reaction was observed (6 h with Cs2CO3 in
MeOH/H2O) with a longer exposure of cis lactone 12 to basic conditions. This resulted in a
partial equilibration of this lactone with the epimer (2R,5S)-11 and, consequently, the desired
hydroxy acid 14 was accompanied by 12% of inseparable diastereoisomer (2R,5S)-13.
Under di€erent hydrolysis conditions (Table 2) the hydroxy acid 14 was obtained in nearly
satisfactory diastereoisomeric purity (see entry 3 in Table 2). However, in order to improve its

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P. Allevi, M. Anastasia / Tetrahedron: Asymmetry 11 (2000) 3151±3160
Table 1
Study on a-epimerization of lactones (2S,5R)-11 and (2S,5S)-12
Scheme 2. Reagents and conditions: (i) Cs2CO3, MeOH:H2O 1:1 v/v, rt, 35 min, 85%; (ii) TFA, rt, 10 min; (iii)
CH2N2, Et2O, rt, 10 min; (iv) TFA, rt, 40 min
preparation, the possibility of performing the hydrolysis under neutral conditions mediated by
enzymes was considered.
Initial attempts to use various lipases,23 operating in a phosphate bu€er (pH 7.4), were unsuccessful
and the unchanged starting cis lactone (2S,5S)-12 was recovered. The goal was then achieved
using commercial a-chymotrypsin24 which permits the desired hydroxy acid (2S,5S)-14 to be
obtained quantitatively in pure form.

P. Allevi, M. Anastasia / Tetrahedron: Asymmetry 11 (2000) 3151±3160
3155
Table 2
Hydrolysis of the lactone 12 to the acid 14
With hydroxy acids 13 and 14 in hand it was possible to prove experimentally that no
a-epimerization had occurred during the initial transformation of the methyl esters 9 and 10 into
the corresponding lactones (Schemes 2 and 3). In fact, separate treatment with TFA of the methyl
esters (9 and 10, obtained from the pure acids 13 and 14) give the diastereoisomeric pure lactones
(2S,5R)-11 and (2S,5S)-12 (HPLC).
The synthesis was then accomplished in one step by simple hydrogenolysis of the hydroxy acids
13 and 14. The formed hydroxylysines 1 and 2 were isolated as crystalline monohydrochlorides
which showed physicochemical properties (speci®c rotation, 13C NMR) in agreement with those
reported.13,25 Their stereoisomeric purity (>98%) was more accurately established by means of
GLC using a suitable chiral column.26 This veri®cation, performed on the corresponding tris-tri-
¯uoroacetates methyl esters, allowed retention times to be assigned to each isomer. These data
should permit the detection of the presence of other isomers, now considered unnatural, in
biological materials and solve such various analytical problems.
Scheme 3. Reagents and conditions: (i) a-chymotrypsin, phosphate bu€er (pH 7.4)/Me2CO, 25ꢀC, 12 h; (ii) TFA, rt, 10
min; (iii) CH2N2, Et2O, rt, 10 min; (iv) TFA, rt, 40 min

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P. Allevi, M. Anastasia / Tetrahedron: Asymmetry 11 (2000) 3151±3160
3. Experimental
3.1. General
Nuclear magnetic resonance spectra were recorded at 303 K on a Bruker AM-500 spectrometer
1
operating at 500.13 MHz for H and 125.76 MHz for 13C. Chemical shifts are reported in parts
per million (ppm, ꢀ units) relative to CHCl3 ®xed at 7.24 ppm or to HDO ®xed at 4.54 ppm for
1
1
the H spectra and relative to dioxane ®xed at 67.60 ppm for the 13C spectra. H NMR data are
tabulated in the following order: number of protons, multiplicity (s, singlet; d, doublet; bs, broad
singlet; m, multiplet), coupling constants (J) in hertz, assignment of proton(s). IR spectra were
determined with a Perkin±Elmer 1420 infrared recording spectrophotometer. Optical rotations
were taken at 25ꢀC on a Perkin±Elmer 241 polarimeter. Chiral GLC analyses were carried out on
a Hewlett±Packard 5890 gas chromatography equipped with an octakis(3-O-butyryl-2,6-di-O-
pentyl)-g-cyclodextrin (Lipodex E)26 capillary column (25 m, 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 170ꢀC. HPLC analyses were carried out on a silica direct phase column
(superspher Si-60, 25 cm, 4 mm ID, purchased from Merck); the mobile phase was hexane:2-
propanol, 75:25, v/v; the ¯ow rate was 1 mL/min and the detection was performed at 221 nm. All
reactions were monitored by thin-layer chromatography (TLC) carried out on 0.25 mm E. Merck
silica gel plates (60 F254) using UV light, 50% sulphuric acid or 0.2% ninhydrin in ethanol and heat
as developing agent. E. Merck 230±400 mesh silica gel was used for ¯ash column chromatography.27
Usual work-up refers to washing the organic layer with water, drying over Na2SO4, and evaporating
the solvent under reduced pressure. a-Chymotrypsin (EC 3.4.21.1), type II from Bovine Pancreas
was obtained from Aldrich (Milwaukee, WI; cat. no. C 4129).
3.2. Methyl (S)-2-benzyloxycarbonylamino-6-diazo-5-oxohexanoate 6
A mixture of (S)-3-(benzyloxycarbonyl)-4-(4-diazo-3-oxobutyl)-5-oxazolidinone 5 (5.0 g, 15.8
mmol, [ꢁ]D +98.2 (c 1, CHCl3), lit.28 +99.8), NaHCO3 (3.0 g) and MeOH (500 mL) was re¯uxed
for 10 min. After cooling at room temperature and ®ltration on Celite, the solvent was
evaporated, the residue oil was dissolved in AcOEt (80 mL) and worked up to a€ord a crude oil
which, after column chromatography (eluting with hexane:AcOEt, 50:50, v:v), a€orded the pure 6
(4.3 g, 85%): an oil; [ꢁ]D +13.8 (c 1, CHCl3) [lit.29 ^14.4 (c 1, CHCl3)]; [ꢁ]D ^4.2 (c 1, AcOEt)
1
[lit.30 ^7.5 (c 1, AcOEt)]; IR (®lm) 2100, 1730, 1710, 1630 cm^1; H NMR (CDCl3): ꢀ 7.36±7.29
(5H, aromatics-H), 5.45 (1H, d, J=5.6, NH), 5.20 (1H, bs, 6-H), 5.12±5.06 (2H, AB system,
OCH2Ph), 4.35 (1H, m, 2-H), 3.73 (3H, s, OCH3), 2.43±2.32 (2H, overlapping, 4-Ha and 4-Hb),
2.20 (1H, m, 3-Ha), 1.99 (1H, m, 3-Hb). Anal. calcd for C15H17N3O5: C, 56.42; H, 5.37; N, 13.16.
Found: C, 56.87; H, 5.51; N, 13.03.
3.3. Methyl (S)-2-benzyloxycarbonylamino-6-bromo-5-oxohexanoate 7
To a light yellow solution of methyl (S)-2-benzyloxycarbonylamino-6-diazo-5-oxohexanoate 6
(4.0 g, 12.5 mmol) in THF (40 mL) containing Thymol Blue (5 mg), a solution of HBr in AcOH
(33%) was added dropwise under stirring at ^5ꢀC. When the solution colour turned violet, a
saturated solution of NaHCO3 was added to neutralize the acids. Then AcOEt (150 mL) was added
and the mixture was worked up to a€ord a crude product which, after column chromatography

P. Allevi, M. Anastasia / Tetrahedron: Asymmetry 11 (2000) 3151±3160
3157
(eluting with hexane:AcOEt, 70:30, v:v), gave pure 7 (3.8 g, 82%): m.p. 86±87ꢀC (from CH2Cl2/
diisopropyl ether); [ꢁ]D +2.1 (c 1, CHCl3); IR (Nujol) 1735, 1690 cm^1; 1H NMR (CDCl3): ꢀ 7.35±
7.27 (5H, aromatics-H), 5.39 (1H, d, J=6.7, NH), 5.11±5.04 (2H, AB system, OCH2Ph), 4.34
(1H, m, 2-H), 3.83 (2H, s, 6-H2), 3.72 (3H, s, OCH3), 2.78±2.65 (2H, overlapping, 4-Ha and 4-
Hb), 2.20 (1H, m, 3-Ha), 1.91 (1H, m, 3-Hb). Anal. calcd for C15H18BrNO5: C, 48.40; H, 4.87; N,
3.76. Found: C, 48.13; H, 4.96; N, 5.37.
3.4. Methyl (S)-6-azido-2-benzyloxycarbonylamino-5-oxohexanoate 8
To a solution of methyl (S)-2-benzyloxycarbonylamino-6-bromo-5-oxohexanoate 7 (3.5 g, 9.4
mmol) in DMF (15 mL) cooled at 0ꢀC, NaN3 (0.85 g, 13 mmol) was added under stirring. After
stirring at 0ꢀC for 1 h, the mixture was diluted with AcOEt (100 mL) and worked up to a€ord,
after column chromatography (eluting with hexane:AcOEt, 50:50, v:v), the pure azido ketone 8
(2.7 g, 86%): m.p. 70±72ꢀC (from CH2Cl2/benzene); [ꢁ]D +14.9 (c 1, CHCl3); IR (®lm) 2100, 1780,
1
1520 cm^1; H NMR (CDCl3): ꢀ 7.36±7.29 (5H, aromatics-H), 5.14 (1H, d, J=7.2, NH), 5.10±
5.04 (2H, AB system, OCH2Ph), 4.34 (1H, ddd, J=8.2, 7.2, 5.1, 2-H), 3.91±3.83 (2H, AB system,
6-H2), 3.72 (3H, s, OCH3), 2.55 (1H, m, 4-Ha), 2.45 (1H, m, 4-Hb), 2.23 (1H, m, 3-Ha), 1.91 (1H,
m, 3-Hb). Anal. calcd for C15H18N4O5: C, 53.89; H, 5.43; N, 16.76. Found: C, 53.61; H, 5.27; N,
16.81.
3.5. Methyl (2S,5R)- and (2S,5S)-6-azido-2-benzyloxycarbonylamino-5-hydroxyhexanoates 9 and 10
To a solution of methyl (S)-6-azido-2-benzyloxycarbonylamino-5-oxohexanoate 8 (4 g, 12
mmol) in MeOH (50 mL), NaBH4 (590 mg, 15.5 mmol) was gradually added at ^5ꢀC. The mix-
ture was stirred at ^5ꢀC for 20 min, treated with water (10 mL), acidi®ed with aqueous HCl (2 M)
and extracted with AcOEt (100 mL). Usual work-up a€orded a chromatographically inseparable
1
mixture of diastereoisomers 9 and 10 (3.7 g, 92%, in a 1:1 ratio) which showed in the H NMR
spectrum, diagnostic signal at ꢀ 5.44 and 5.37 ppm corresponding to NH signal, respectively, for 9
and 10.
3.6. (2S,5R)-5-Azidomethyl-2-benzyloxycarbonylamino-ꢀ-valerolactone 11 and (2S,5S)-5-azido-
methyl-2-benzyloxycarbonylamino-ꢀ-valerolactone 12
The mixture of diastereoisomeric azido esters 9 and 10 (4.0 g, 12 mmol) was dissolved in
CF3CO2H (6.0 mL) and the solution was stirred at room temperature for 40 min. The solvent was
then carefully removed under reduced pressure (under 40ꢀC) and the residue (3.2 g) was quickly
chromatographed on column (eluting with CH2Cl2:AcOEt, 100:10, v:v) to a€ord ®rst the lactone
12 (1.37 g, 38%): m.p. 90±92ꢀC (from CH2Cl2/diisopropyl ether); HPLC: Rt=8.9 min; [ꢁ]D +56.2
(c 1, CHCl3); IR (®lm) 2110, 1755, 1715 cm^1; 1H NMR (CDCl3): ꢀ 7.36±7.29 (5H, aromatics-H),
5.62 (1H, d, J=4.2, NH), 5.10 (2H, s, OCH2Ph), 4.51 (1H, m, 6-H), 4.46 (1H, m, 3-H), 3.46 (2H,
d, J=4.4, CH2-N3), 2.63 (1H, m, 4-Ha), 2.00 (1H, m, 5-Ha), 1.87 (1H, m, 5-Hb), 1.62 (1H, m, 4-
Hb). Anal. calcd for C14H16N4O4: C, 55.26; H, 5.30; N, 18.41. Found: C, 55.49; H, 5.41; N, 18.35.
Further elution gave the lactone 11 (1.12 g, 31%): m.p. 85±86ꢀC (from CH2Cl2/diisopropyl
1
ether); HPLC: Rt=11.7 min; [ꢁ]D ^19.9 (c 1, CHCl3); IR (®lm) 2110, 1755, 1715 cm^1; H NMR
(CDCl3): ꢀ 7.36±7.29 (5H, aromatics-H), 5.52 (1H, bs, NH), 5.10 (2H, s, OCH2Ph), 4.51 (1H, bs,
6-H), 4.14 (1H, m, 3-H), 3.52 (1H, dd, J=13.0, 3.5, CHH-N3), 3.40 (1H, dd, J=13.0, 3.9, CHH-N3),

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P. Allevi, M. Anastasia / Tetrahedron: Asymmetry 11 (2000) 3151±3160
2.47 (1H, m, 4-Ha), 1.98±1.82 (3H, overlapping, 4-Hb, 5-Ha and 5-Hb). Anal. calcd for C14H16N4O4:
C, 55.26; H, 5.30; N, 18.41. Found: C, 55.60; H, 5.25; N, 18.70.
Finally, a washing of the column with CH2Cl2:MeOH (70:30, v/v) a€orded an inseparable
mixture of corresponding acids 13 and 14 (0.57 g, 15%), probably formed by a silica gel opening
of the lactonic ring. These acids could be recovered and subjected again to the lactonization.
3.7. (2S,5R)-6-Azido-2-benzyloxycarbonylamino-5-hydroxyhexanoic acid 13
To a solution of (3S,6R)-3-benzyloxycarbonylamino-6-azidomethyltetrahydro-2H-2-pyranone
11 (1.0 g, 3.29 mmol) in MeOH (25 mL) an aqueous solution of Cs2CO3 (0.27 M, 25 mL) was
added. The resulting light opalescent mixture was stirred at room temperature for 35 min and
then acidi®ed with aqueous HCl (4 M). Addition of AcOEt (100 mL) and the usual work-up
a€orded pure acid 13 (995 mg, 94%): an oil; [ꢁ]D +13.2 (c 1, CHCl3); IR (®lm) 3350, 2100, 1730,
1
1710, 1690 cm^1; H NMR (CDCl3): ꢀ 7.36±7.29 (5H, aromatics-H), 5.68 (1H, d, J=7.0, NH),
5.11±5.05 (2H, AB system, OCH2Ph), 4.39 (1H, m, 2-H), 3.74 (1H, m, 5-H), 3.28 (1H, dd,
J=11.8, 2.4, 6-Ha), 3.21 (1H, dd, J=11.8, 6.6, 6-Hb), 2.02 (1H, m, 3-Ha), 1.75 (1H, m, 3-Hb),
1.54 (2H, m, 4-H2). Anal. calcd for C14H18N4O5: C, 52.17; H, 5.63; N, 17.38. Found: C, 52.04; H,
5.80; N, 17.27.
3.8. (2S,5S)-6-Azido-2-benzyloxycarbonylamino-5-hydroxyhexanoic acid 14
A solution of (3S,6S)-3-benzyloxycarbonylamino-6-azidomethyltetrahydro-2H-2-pyranone 12
(400 mg, 1.32 mmol) in acetone (5.2 mL) was added to a solution of a-chymotrypsin (220 mg) in
0.1 M phosphate bu€er (52 mL, pH 7.4). The suspension was stirred at 25ꢀC for 12 h. When a
solution formed this was acidi®ed with aqueous HCl (4 M) and extracted with AcOEt (60 mL).
The usual work-up a€orded the pure acid 14 (406 mg, 96%): [ꢁ]D +16.9 (c 1, CHCl3); IR (®lm)
1
3350, 2100, 1730, 1710, 1690 cm^1; H NMR (CDCl3): ꢀ 7.36±7.29 (5H, aromatics-H), 5.63 (1H,
d, J=7.2, NH), 5.11±5.05 (2H, AB system, OCH2Ph), 4.41 (1H, m, 2-H), 3.77 (1H, m, 5-H), 3.29
(1H, dd, J=11.4, 2.5, 6-Ha), 3.21 (1H, dd, J=11.4, 6.8, 6-Hb), 1.95 (1H, m, 3-Ha), 1.88 (1H, m,
3-Hb), 1.52 (2H, m, 4-H2). Anal. calcd for C14H18N4O5: C, 52.17; H, 5.63; N, 17.38. Found: C,
52.35; H, 5.51; N, 17.49.
3.9. Regeneration of the lactonic ring
3.9.1. Directly from hydroxy acid 13 or 14
Each acid 13 or 14 (10 mg) was dissolved in CF3CO2H (0.2 mL) and the solution was kept at
room temperature for 10 min. The solvent was then removed under a nitrogen stream to a€ord
the corresponding diastereoisomeric pure azido lactones 11 or 12 in quantitative yields (diaster-
eoisomeric purity was checked by HPLC).
3.9.2. From methyl esters 9 or 10
Each acid 13 or 14 (30 mg) dissolved in AcOEt (1 mL) was treated with excess CH2N2 in Et2O
at room temperature for 10 min. The solvent was then removed under a nitrogen stream to
a€ord, in quantitative yields, the corresponding methyl ester 9 [1H NMR (CDCl3) ꢀ 7.36±7.29
(5H, aromatics-H), 5.44 (1H, d, J=6.9, NH), 5.12±5.07 (2H, AB system, OCH2Ph), 4.42 (1H, m,
2-H), 3.73 (4H, bs, overlapping, 5-H and OCH3), 3.31 (1H, dd, J=12.2, 3.6, 6-Ha), 3.23 (1H, dd,

P. Allevi, M. Anastasia / Tetrahedron: Asymmetry 11 (2000) 3151±3160
3159
J=12.2, 7.0, 6-Hb), 2.02 (1H, m, 3-Ha), 1.73 (1H, m, 3-Hb), 1.53 (2H, m, 4-H2)] or 10 [1H NMR
(CDCl3) ꢀ 7.36±7.29 (5H, aromatics-H), 5.37 (1H, d, J=7.2, NH), 5.11±5.07 (2H, AB system,
OCH2Ph), 4.40 (1H, m, 2-H), 3.73 (4H, bs, overlapping, 5-H and OCH3), 3.33 (1H, dd, J=12.4,
2.2, 6-Ha), 3.21 (1H, dd, J=12.4, 7.7, 6-Hb), 1.94 (1H, m, 3-Ha), 1.82 (1H, m, 3-Hb), 1.49 (2H,
m, 4-H2)].
The methyl ester 9 or 10 (25 mg) was dissolved in CF3CO2H (0.2 mL) and the solution was
kept at room temperature for 40 min. The solvent was then removed under a nitrogen stream to
a€ord the corresponding diastereoisomeric pure azido lactones 11 or 12 in quantitative yields
(diastereoisomeric purity was checked by HPLC).
3.10. (2S,5R)-5-Hydroxylysine monohydrochloride 1
A solution of (2S,5R)-6-azido-2-benzyloxycarbonylamino-5-hydroxyhexanoic acid 13 (800 mg,
2.48 mmol) in MeOH:H2O (80 mL, 1:1, v/v) was hydrogenated for 15 h, in the presence of
palladium on charcoal at room temperature and atmospheric pressure. Filtration of the catalyst,
evaporation of the MeOH and lyophilization a€orded a residue which was dissolved in water (1.5
mL) and the pH adjusted to 6.5±7.0 with HCl (4 M). The addition of EtOH (2.5 mL) and the
storage at 0ꢀC e€ected the precipitation of (2S,5R)-5-hydroxylysine (normal-l-hydroxylysine)
1
monohydrochloride 1 (368 mg, 75%): [ꢁ]D +15.2 (HCl 6 M, c 1) [lit.13 +14.5 (c 2)]; H NMR
(D2O): ꢀ 3.69 (1H, m, 5-H), 3.59 (1H, dd, J=6.1, 6.1, 2-H), 2.97 (1H, dd, J=13.3, 3.1, 6-Ha), 2.73
(1H, dd, J=13.3, 9.6, 6-Hb), 1.87 (1H, m, 3-Ha), 1.72 (1H, m, 3-Hb), 1.45±1.38 (2H, overlapping,
4-H2); 13C NMR (D2O): ꢀ 175.37 (C-1), 68.27 (C-5), 55.43 (C-2), 45.35 (C-6), 30.56 (C-4), 27.59
(C-3).25 Anal. calcd for C6H15ClN2O3: C, 36.28; H, 7.61; N, 14.10. Found: C, 36.35; H, 7.69; N,
14.08.
3.11. (2S,5S)-5-Hydroxylysine monohydrochloride 2
The (2S,5S)-6-azido-2-benzyloxycarbonylamino-5-hydroxyhexanoic acid 14 (600 mg, 1.86
mmol) in MeOH:water (60 mL, 1:1, v/v) was hydrogenated for 15 h, in the presence of palladium
on charcoal at room temperature and atmospheric pressure. Filtration of the catalyst, evaporation
of the MeOH and lyophilization a€orded a residue which was dissolved in water (1.0 mL) and the
pH adjusted to 6.5±7.0 with HCl (4 M). The addition of EtOH (2.0 mL) and the storage at 0ꢀC
e€ected the precipitation of (2S,5S)-5-hydroxylysine (allo-l-hydroxylysine) monohydrochloride 2
1
(288 mg, 78%): [ꢁ]D +25.5 (HCl 6 M, c 1) [lit.13 +25.8 (c 2)]; H NMR (D2O): ꢀ 3.68 (1H, m, 5-
H), 3.56 (1H, dd, J=5.7, 5.7, 2-H), 2.96 (1H, dd, J=13.1, 1.7, 6-Ha), 2.73 (1H, dd, J=13.1,
10.0, 6-Hb), 1.84 (1H, m, 3-Ha), 1.73 (1H, m, 3-Hb), 1.49 (1H, m, 4-Ha), 1.33 (1H, m, 4-Hb); 13C
NMR (D2O): ꢀ 175.38 (C-1), 68.16 (C-5), 55.47 (C-2), 45.35 (C-6), 30.64 (C-4), 27.59 (C-3).25
Anal. calcd for C6H15ClN2O3: C, 36.28; H, 7.61; N, 14.10. Found: C, 36.18; H, 7.45; N,
14.23.
3.12. GLC derivation of 5-hydroxylysines
Hydroxylysines (1±2 mg) were esteri®ed with methanolic HCl (1 M, 0.2 mL, 25ꢀC, 12 h). The
solvent was removed under a nitrogen stream and the residue was treated with a 50% mixture of
(CF3CO)2O in CF3CO2H (0.2 mL, 25ꢀC, 2 h). After removal of the solvent, the residue (tris-tri-
¯uoroacetates of 5-hydroxylysine methyl ester) was dissolved in AcOEt and injected.

3160
P. Allevi, M. Anastasia / Tetrahedron: Asymmetry 11 (2000) 3151±3160
In the used analyses conditions (see General, Section 3.1), the derivatives showed the following
retention times: 23.35 min for (2R,5R)-5-hydroxylysine 4; 24.26 min for (2S,5R)-5-hydroxylysine
1, 24.59 min for (2S,5S)-5-hydroxylysine 2 and 28.67 min for (2R,5S)-5-hydroxylysine 3.
Acknowledgements
This work was supported ®nancially by MURST COFIN progetto di ricerca `Nuove metodologie
e strategie di Sintesi di Composti di Interesse Biologico'. The authors are grateful to the student
Vincenza Vau for technical assistance. This paper is dedicated to the memory of Professor Giorgio
Traverso.
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