Synthesis of rhodotorulic acid and its 1,4-dimethylated derivative

Article abstract of DOI:10.3987/COM-14-S(K)67

Facile syntheses of rhodotorulic acid, isolated from Rhodotorula pilimanae as a siderophore, and its 1,4-dimethylated derivative have been achieved by microwave-assisted cyclization of the corresponding dipeptide precursors.

Full text of DOI:10.3987/COM-14-S(K)67

HETEROCYCLES, Vol. 90, No. 2, 2015
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HETEROCYCLES, Vol. 90, No. 2, 2015, pp. 1309 - 1316. © 2015 The Japan Institute of Heterocyclic Chemistry
Received, 30th June, 2014, Accepted, 23rd July, 2014, Published online, 5th August, 2014
DOI: 10.3987/COM-14-S(K)67
SYNTHESIS OF RHODOTORULIC ACID AND ITS
1,4-DIMETHYLATED DERIVATIVE
Michiyasu Nakao, Shintaro Fukayama, Syuji Kitaike, and Shigeki Sano*
Graduate School of Pharmaceutical Sciences, The University of Tokushima,
Sho-machi, Tokushima 770-8505, Japan; E-mail: ssano@tokushima-u.ac.jp
Abstract – Facile syntheses of rhodotorulic acid, isolated from Rhodotorula
pilimanae as a siderophore, and its 1,4-dimethylated derivative have been
achieved by microwave-assisted cyclization of the corresponding dipeptide
precursors.
Siderophores are iron-chelating compounds utilized by bacteria and fungi under iron-limiting conditions.1
Rhodotorulic acid [(S,S)-1] is a dihydroxamate siderophore isolated from Rhodotorula pilimanae,2 and its
biological activities3 as well as its iron-chelating ability4 have been investigated. It can be assumed that
the diketopiperazine (DKP) ring of (S,S)-1 is biosynthesized starting with L-ornithine, and that two
N-hydroxyacetamide moieties serve as a tetradentate ligand for Fe(III) coordination. Therefore, (S,S)-1
has been considered to form a 3 : 2 complex with Fe(III) based on CD spectra and potentiometric
titrations, in contrast to hexadentate siderophores such as desferrioxamine B, which forms a 1 : 1 complex
with Fe(III).5 The coordination pattern of (S,S)-1 with Fe(III) has also been suggested by electrospray
ionization mass spectrometry.6 Despite its interesting structural features, there are only a few examples of
(S,S)-1 synthesis.7-10 Herein, we describe a convenient synthesis of (S,S)-1 and its 1,4-dimethylated
derivative [(S,S)-2] through microwave-assisted cyclization of the corresponding dipeptide precursors.
Figure 1. Rhodotorulic acid [(S,S)-1] and its 1,4-dimethylated derivative [(S,S)-2]
Amino acid building blocks, (S)-5-[N-(benzyloxy)acetamido]-2-[(tert-butoxycarbonyl)amino]pentanoic
acid [(S)-9] and methyl (S)-2-amino-5-[N-(benzyloxy)acetamido]pentanoate hydrochloride [(S)-10], for
dipeptide precursors were synthesized as shown in Scheme 1. Esterification of Boc-L-Glu(OBn)-OH

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[(S)-3] with methanol using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC•HCl)
as a coupling reagent in the presence of a catalytic amount of N,N-dimethyl-4-aminopyridine (DMAP)
followed by deprotection of the benzyl ester via catalytic hydrogenolysis under hydrogen with palladium
on carbon (10 wt. % loading) afforded the carboxylic acid (S)-5 in 90% yield (2 steps). Formation of a
mixed anhydride of (S)-5 with ethyl chloroformate in the presence of N-methylmorpholine (NMM)
followed by reduction with sodium borohydride gave the primary alcohol (S)-6 in 89% yield. One-step
transformation of the hydroxy group of (S)-6 into the protected hydroxylamino group was performed
under Mitsunobu conditions. The reaction of (S)-6 with N-[(2,2,2-trichloroethoxy)carbonyl]-
O-benzylhydroxylamine in the presence of diisopropyl azodicarboxylate (DIAD) and triphenylphosphine
in THF provided the Mitsunobu product (S)-7. Without further purification, reductive cleavage of the
2,2,2-trichloroethoxycarbonyl (Troc) group of (S)-7 with zinc powder followed by acetylation of the
resulting hydroxylamine with acetic anhydride furnished (S)-87,9 in 86% yield (2 steps). Finally,
hydrolysis of (S)-8 under aqueous alkaline conditions gave the corresponding N-protected amino acid
(S)-9.7,9 In contrast, amino acid ester hydrochloride (S)-109 was obtained by deprotection of the Boc group
of (S)-8 under acidic conditions.
Scheme 1. Synthesis of amino acid building blocks (S)-9 and (S)-10

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Scheme 2. Synthesis of rhodotorulic acid [(S,S)-1] and its 1,4-dimethylated derivative [(S,S)-2]
Benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent) was used in
the condensation of both building blocks (S)-9 and (S)-10 in the presence of N,N-diisopropylethylamine
to furnish the dipeptide (S,S)-11 in 95% yield. In previous reports of the synthesis of (S,S)-1, two steps
have been required to construct the DKP structure via N-terminal deprotection and intramolecular
cyclization of the dipeptide precursor.7-10 In addition, a long reaction time has been needed for
intramolecular cyclization. On the other hand, microwave irradiation has been reported to be efficient for
a one-pot conversion of N-Boc-dipeptide methyl esters into DKPs with a short reaction time.11 We
therefore attempted a one-pot conversion of (S,S)-11 into DKP (S,S)-12 using microwave irradiation. As a
result, removal of the Boc group followed by intramolecular cyclization of (S,S)-11 under microwave
irradiation with a single-mode microwave reactor (InitiatorTM 60; Biotage AB) at 170 °C in a mixed
solvent of water/methanol for 10 min furnished the DKP (S,S)-12 in 63% yield. Finally, catalytic
hydrogenolysis of (S,S)-12 under hydrogen with palladium on carbon (10 wt. % loading) provided
rhodotorulic acid [(S,S)-1] in 80% yield. Furthermore, N-methylation of the DKP ring of (S,S)-12
followed by catalytic hydrogenolysis of the resultant (S,S)-13 afforded 1,4-dimethylated rhodotorulic acid
[(S,S)-2]. The structures of (S,S)-1 and (S,S)-2 were confirmed by spectroscopic methods. In general, DKP
derivatives have poor solubility in various solvents due to their intermolecular hydrogen bonding through

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the amide moiety of the DKP ring.12 Therefore, only a few solvents, including water and
dimethylsulfoxide (DMSO), have been found to be capable of dissolving (S,S)-1. However, (S,S)-2 was
found to be soluble in water, DMSO, methanol, ethanol, chloroform, and ethyl acetate. This enhanced
solubility is likely due to the disappearance of intermolecular hydrogen bonding as a result of
N-methylation.
In conclusion, we have presented the synthesis of rhodotorulic acid [(S,S)-1] and its 1,4-dimethylated
derivative [(S,S)-2] using a microwave-assisted cyclization of the corresponding common dipeptide
precursor (S,S)-11 as a key step. Intriguingly, (S,S)-2 was found to be more soluble in various organic
solvents than (S,S)-1. Derivatization of (S,S)-1 and (S,S)-2 toward the synthesis of novel iron-chelating
compounds is currently underway and will be reported in due course.
EXPERIMENTAL
All melting points were determined on a Yanagimoto micro melting point apparatus and are uncorrected.
1
IR spectra were obtained using a JASCO FT/IR-6200 IR Fourier transform spectrometer. H NMR (500
MHz) and 13C NMR (125 MHz) spectra were recorded on a Bruker AV500 spectrometer. Chemical shifts
are given in ! values (parts per million) using tetramethylsilane (TMS) as an internal standard. Electron
spray ionization mass spectra (ESIMS) were recorded on a Waters LCT Premier spectrometer. Elemental
combustion analyses were performed using a J-SCIENCE LAB JM10. The microwave-assisted reaction
was performed utilizing an automated single-mode microwave synthesizer (InitiatorTM 60; Biotage AB).
All reactions were monitored by TLC employing 0.25-mm silica gel plates (Merck 5715; 60 F254).
Column chromatography was carried out on silica gel [Kanto Chemical 60N (spherical, neutral); 63-210
mm] or [Fuji Silysia Chemical PSQ 60B (spherical)]. Anhydrous THF, CH2Cl2, and DMF were used as
purchased from Kanto Chemical. N-Methylmorpholine (NMM) and N,N-diisopropylethylamine were
distilled prior to use. All other reagents were used as purchased.
Methyl
(S)-5-[N-(Benzyloxy)acetamido]-2-{(S)-5-[N-(benzyloxy)acetamido]-2-[(tert-butoxy-
carbonyl)amino]pentanamide}pentanoate [(S,S)-11]
To a solution of (S)-9 (618 mg, 1.62 mmol) and (S)-10 (537 mg, 1.62 mmol) in anhydrous CH2Cl2 (6 mL)
were added BOP reagent (1.1 g, 2.44 mmol) and N,N-diisopropylethylamine (552 µL, 3.25 mmol) at 0 °C
under argon. The reaction mixture was allowed to warm to rt and stirred for 24 h. The reaction mixture
was treated with 5% citric acid aq (2 mL) and then extracted with CHCl3 (50 mL x 3). The extract was
dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The oily residue was purified by silica
gel column chromatography [Silica Gel PSQ 60B: CHCl3–MeOH (100:0 to 10:1)] to afford (S,S)-11 (1.0
g, 95%). Colorless oil; ["]D19 +5.6 (c 0.51, CHCl3); 1H NMR (500 MHz, CDCl3) ! 1.43 (s, 9H), 1.47–1.88

HETEROCYCLES, Vol. 90, No. 2, 2015
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(m, 8H), 2.09 (s, 3H), 2.11 (s, 3H), 3.41–3.53 (m, 1H), 3.60–3.73 (m, 2H), 3.66 (s, 3H), 4.12–4.27 (m,
1H), 4.32–4.42 (m, 1H), 4.48–4.56 (m, 1H), 4.76–4.88 (m, 4H), 5.22–5.28 (m, 1H), 7.08–7.17 (m, 1H),
13
7.33–7.42 (m, 10H); C NMR (125 MHz, CDCl3) ! 20.4, 20.5, 23.0, 23.2, 28.3, 29.1, 30.6, 43.6, 44.7,
51.9, 52.2, 52.3, 76.31, 76.34, 79.6, 128.72, 128.74, 128.96, 129.01, 129.19, 129.24, 134.3, 134.4, 155.8,
13
172.37, 172.45 (two overlapping singlets), 173.16; C NMR (125 MHz, C6D6) ! 20.5 (two overlapping
singlets), 23.4, 23.7, 28.4, 29.2, 31.1, 43.9, 45.2, 51.7, 52.2, 52.7, 76.1, 76.3, 79.1, 128.77, 128.79, 128.8,
128.9, 129.4, 129.5, 135.1, 135.4, 156.3, 172.1, 172.8, 172.9, 173.1; IR (neat) 3304, 2978, 2935, 1743,
1659, 1499 cm-1; ESI-MS m/z: calcd for C34H48N4NaO9 [M+Na]+, 679.3319; found, 679.3350.
N,N’-{[(2S,5S)-3,6-Dioxopiperazine-2,5-diyl]bis(propane-3,1-diyl)}bis[N-(benzyloxy)acetamide]
[(S,S)-12]
A suspension of (S,S)-11 (611 mg, 0.931 mmol) in a mixed solvent of H2O (15 mL) with MeOH (5 mL)
was irradiated at 170 °C for 10 min utilizing a Biotage Initiator® microwave synthesizer. The reaction
mixture was treated with H2O (20 mL) and then extracted with AcOEt (50 mL x 3). The extract was dried
over anhydrous MgSO4, filtered, and concentrated in vacuo. The oily residue was purified by silica gel
column chromatography [Silica Gel 60N: CHCl3–MeOH (98:2 to 85:15)] to afford (S,S)-12 (305 mg,
63%). Colorless powder (MeOH–Et2O); mp 149–150 °C (lit.7 127–129 °C, lit. 9 129–131 °C, and lit.10
27
25
13
97–99 °C); ["]D -20.5 (c 1.03, MeOH) {lit.7 ["]D -16.5 (c 0.67, MeOH), lit.9 ["]D -16.4 (c 1.01,
EtOH), and lit.10 ["]D20 -16.4 (c 1.01, EtOH)}; 1H NMR (500 MHz, CD3OD) ! 1.67–1.85 (m, 8H), 2.03 (s,
6H), 3.63–3.73 (m, 4H), 3.98 (t, J = 5.2 Hz, 2H), 4.87 (s, 4H), 7.34–7.43 (m, 10H); 13C NMR (125 MHz,
CD3OD) ! 20.5, 23.6, 32.4, 45.6, 55.7, 77.2, 129.8, 130.0, 130.7, 136.1, 170.2, 174.5; IR (KBr) 3193,
3043, 2953, 2886, 1665, 1455 cm-1; ESI-MS m/z: calcd for C28H36N4NaO6 [M+Na]+, 547.2533; found,
547.2525. Anal. Calcd for C28H36N4O6: C, 64.10; H, 6.92; N, 10.68. Found: C, 63.96; H, 6.91; N, 10.53%.
N,N’-{[(2S,5S)-3,6-Dioxopiperazine-2,5-diyl]bis(propane-3,1-diyl)}bis(N-hydroxyacetamide)
[Rhodotorulic Acid, (S,S)-1]
The mixture of (S,S)-12 (100 mg, 0.191 mmol) and 10% Pd–C (20 mg, 0.019 mmol) in MeOH (3 mL)
was stirred at rt for 1 h under hydrogen. The reaction mixture was filtered and concentrated in vacuo to
afford (S,S)-1 (53 mg, 80%). Colorless powder (H2O); mp >217 °C (dec) [lit.7 217–218 °C, lit.8
27
229–232 °C, lit.9 216–218 °C (dec), and lit.10 217–218.5 °C (dec)]; ["]D -30.2 (c 0.16, H2O) {lit.8 ["]D
-30.5 (c 0.67, AcOH), lit.9 ["]D17 -30.4 (c 0.5, H2O), lit.10 ["]D25 -28.8 (c 1.00, H2O)}; 1H NMR (500 MHz,
DMSO-d6) ! 1.50–1.72 (m, 8H), 1.97 (s, 6H), 3.43–3.52 (m, 4H), 3.79–3.86 (m, 2H), 8.16 (brs, 2H), 9.72
13
(brs, 2H); C NMR (125 MHz, DMSO-d6) ! 20.2, 22.0, 30.2, 46.7, 53.7, 167.7, 170.0; IR (KBr) 3187,
3086, 2867, 1686, 1594, 1517, 1473, 1448 cm-1; ESI-MS m/z: calcd for C14H24N4NaO6 [M+Na]+,

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367.1594; found, 367.1588. Anal. Calcd for C14H24N4O6: C, 48.83; H, 7.02; N, 16.27. Found: C, 48.53; H,
6.99; N, 16.14%.
N,N’-{[(2S,5S)-1,4-Dimethyl-3,6-dioxopiperazine-2,5-diyl]bis(propane-3,1-diyl)}bis[N-(benzyloxy)-
acetamide] [(S,S)-13]
NaH (50–72%, 13.6 mg, 0.284 mmol) was added to a solution of (S,S)-12 (49.7 mg, 0.0947 mmol) in
anhydrous DMF (2 mL) and stirred at 0 °C for 5 min under argon. After adding MeI (17.7 µL, 0.284
mmol), the mixture was stirred at 0 °C for 30 min under argon. The reaction mixture was treated with 1N
HCl aq (1 mL) and then extracted with AcOEt (20 mL x 3). The extract was washed with sat. Na2S2O3 aq
(5 mL) and H2O (5 mL x 3). The organic layer was dried over anhydrous MgSO4, filtered, and
concentrated in vacuo. The oily residue was purified by silica gel column chromatography [Silica Gel
PSQ 60B: CHCl3–MeOH (20:1 to 10:1)] to afford (S,S)-13 (43.2 mg, 83%). Colorless oil; ["]D20 +11.4 (c
0.90, CHCl3); 1H NMR (500 MHz, CDCl3) ! 1.60–1.92 (m, 8H), 2.09 (s, 6H), 2.89 (s, 6H), 3.58–3.68 (m,
13
2H), 3.74–3.87 (m, 4H), 4.81 (dd, J = 10.5, 13.7 Hz, 4H), 7.34–7.43 (m, 10H); C NMR (125 MHz,
CDCl3) ! 20.5, 23.5, 30.5, 32.6, 44.1, 61.7, 76.4, 128.8, 129.1, 129.2, 134.3, 165.7, 172.3; IR (neat) 2937,
2878, 1660, 1454, 1403 cm-1; ESI-MS m/z: calcd for C30H40N4NaO6 [M+Na]+, 575.2846; found, 575.2815.
N,N’-{[(2S,5S)-1,4-Dimethyl-3,6-dioxopiperazine-2,5-diyl]bis(propane-3,1-diyl)}bis(N-hydroxyacet-a
mide) [1,4-Dimethylated Rhodotorulic Acid, (S,S)-2]
The mixture of (S,S)-13 (24.2 mg, 0.0438 mmol) and 10% Pd–C (2.3 mg, 0.00219 mmol) in MeOH (1
mL) was stirred at rt for 2 h under hydrogen. The reaction mixture was filtered and concentrated in vacuo
28
to afford (S,S)-2 (12 mg, 80%). Colorless prism (CHCl3–Et2O); mp 134–135.5 °C; ["]D +31.0 (c 0.45,
CHCl3); 1H NMR (500 MHz, CDCl3) ! 1.67–1.89 (m, 6H), 2.05–2.18 (m, 2H), 2.16 (s, 6H), 2.98 (s, 6H),
13
3.57–3.67 (m, 2H), 3.77–3.92 (m, 4H), 9.35 (brs, 2H); C NMR (125 MHz, CDCl3) ! 20.6, 22.2, 30.9,
32.9, 47.3, 61.9, 166.7, 172.7; IR (KBr) 3351, 3115, 2939, 2868, 1663, 1636, 1600 cm-1; ESI-MS m/z:
calcd for C16H28N4NaO6 [M+Na]+, 395.1907; found, 395.1920. Anal. Calcd for C16H28N4O6: C, 51.60; H,
7.58; N, 15.04. Found: C, 51.30; H, 7.51; N, 14.91%.
ACKNOWLEDGEMENTS
This work was supported in part by a Grant for the Regional Innovation Cluster Program (Global Type)
promoted by MEXT.
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