Synthesis of novel sugar amino acids by Curtius rearrangement

Article abstract of DOI:10.1002/ejoc.200210682

Sugar amino acids (SAAs) bearing an amine group on the anomeric position are a challenging class of SAAs to synthesize due to the inherent instability of glycosylamines. We developed a novel synthetic strategy towards both furanoid and pyranoid δ-SAAs of

Full text of DOI:10.1002/ejoc.200210682

FULL PAPER
Synthesis of Novel Sugar Amino Acids by Curtius Rearrangement
Renate M. van Well,[a] Herman S. Overkleeft,[a] Jacques H. van Boom,[a] Andrew Coop,[b]
Jia Bei Wang,[b] Hongyan Wang,[b] Gijsbert A. van der Marel,*[a] and Mark Overhand*[a]
Keywords: Amino acids / Carbohydrates / Peptides / Curtius rearrangement / Synthetic methods / Leu-enkephalin
Sugar amino acids (SAAs) bearing an amine group on the
anomeric position are a challenging class of SAAs to syn-
thesize due to the inherent instability of glycosylamines. We
developed a novel synthetic strategy towards both furanoid
and pyranoid δ-SAAs of this type, based on a Curtius re-
arrangement. The latter reaction, which is known to proceed
with retention of configuration, was performed on carboxylic
acids derived from the oxidation of glycosidic primary hy-
droxyls. Leu-enkephalin analogs were prepared by replacing
the Gly-Gly moiety in the parent Leu-enkephalin pentapep-
tide with the furanoid and pyranoid δ-SAA.
( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2003)
Introduction
eric position of the carbohydrate core. The challenge to syn-
thesize this type of SAAs and their application in peptide
synthesis is associated with the inherent lability of glycosyl-
amines, which are prone to mutarotate, hydrolyze or un-
dergo Amadori rearrangement.[5] Several research groups
have disclosed synthetic strategies towards α-, β- and γ-
SAAs having an anomeric amine.[6] One of the most com-
mon methods used for the introduction of an amine at the
anomeric position is the reaction of a reducing sugar with
ammonia or ammonium hydrogen carbonate.[7] This reac-
tion was successfully applied by Danilov et al. in the syn-
thesis of amino-mannuronic acid.[6e] Glycosylamines have
also been prepared by reduction of glycosylazides, obtained
from the glycosyl halide or acetate by nucleophilic displace-
ment. For example, Fleet and Dondoni employed this reac-
tion in the synthesis of furanoid and pyranoid α-SAAs.[6a,6b]
The azide route was also adopted by Kessler and co-work-
ers for the construction of the γ-SAA glucuronosylamine,
which was subsequently incorporated into a somatostatin
analog.[2c] An alternative approach towards glucuronosyl-
amine, based on the acidic ring opening of an α-oxazoline,
was developed in our laboratory.[3e] The γ-SAA obtained
via this approach was used for the preparation of a back-
bone-modified phytoalexin elicitor heptasaccharide analog.
Sugar amino acids (SAAs) Ϫ carbohydrate derivatives
bearing a carboxylic acid and an amino functionality Ϫ
have attracted considerable interest in recent years as pep-
tide and carbohydrate mimetics. The ease of peptide bond
formation together with the potential structural diversity in
the carbohydrate core of SAAs has stimulated the design
and synthesis of an array of novel SAAs as well as their
implementation in different fields of research.[1] With the
objective of stabilizing the secondary structure of pharma-
cologically active peptides SAAs have been utilized as con-
formationally restricted peptide isosters. For instance, sev-
eral research groups have synthesized linear Leu-enkephalin
analogs as well as cyclic somatostatin and RGD peptides
containing a single SAA residue at predesigned positions.[2]
Moreover, oligomers assembled from multiple SAA build-
ing blocks have found interest as oligosaccharide mimetics
with enhanced metabolic and conformational stability.[3] In
line with these efforts, we and others are exploring the de-
velopment of new potential host molecules by the construc-
tion of cyclic oligomers composed of SAAs.[4]
To broaden the scope of SAAs as versatile building
blocks for drug discovery and material sciences the design,
synthesis and application of novel SAAs, varying in the
number, nature and stereochemistry of the functional
groups, is imperative. In this respect our attention was at-
tracted to δ-SAAs having an amino function at the anom-
Here we wish to report the synthesis of the novel fu-
ranoid (I) and pyranoid δ-SAA (II), bearing an amine
group at the anomeric position (see Figure 1). The key steps
in the synthetic protocol involve oxidation of the primary
hydroxyl function of the respective carbohydrate precursors
to the carboxylate, followed by Curtius rearrangement,
which is known to proceed with retention of configura-
tion.[8] The use of the δ-SAAs in peptide synthesis is dem-
onstrated by incorporating SAAs I and II in Leu-enke-
phalin analogs, chosen as representative examples of a short
[a]
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden
University,
P. O. Box 9502, 2300 RA Leiden, The Netherlands
Fax: (internat.) ϩ 31-71/5274307
E-mail: overhand@chem.leidenuniv.nl
[b]
Department of Pharmaceutical Sciences, University of
Maryland School of Pharmacy,
20 N. Pine St., Baltimore, MD 21201, USA
1704
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/ejoc.200200682
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Synthesis of Novel Sugar Amino Acids by Curtius Rearrangement
FULL PAPER
methyl ester under standard basic conditions (1  aq.
NaOH) led to epimerization at the C3 position.[10] To our
satisfaction, deprotection of 3 using LiOOH proceeded
without the undesired epimerization to give furanoid SAA
4 in 63% yield together with 26% of recovered starting ma-
terial.
In a similar sequence of reactions, the pyranoid dipeptide
isoster 8 was obtained starting from the known[4d] methyl
3,7-anhydro-4,5,6-tri-O-benzyl-2-deoxy--gulo--glycero-
octonate (5) as depicted in Scheme 2. In contrast to the
transformation of 1 into 2, TEMPO oxidation of 5 did not
afford the desired acid. Fortunately, treatment of 5 with a
large excess of pyridinium dichromate (PDC) did furnish 6
in 87%, Curtius rearrangement of which under the afore-
mentioned conditions gave the Boc-protected amine 7 in
75%. Ensuing saponification of the methyl ester in 7 in the
presence of NaOH proceeded without racemization to pro-
vide the partially protected SAA 8.
Figure 1. Furanoid δ-SAA I, pyranoid δ-SAA II and Leu-enke-
phalin III
peptide. The flexible Gly-Gly dipeptide in the original Leu-
enkephalin pentapeptide (III in Figure 1) serves as a spacer,
and replacement of this dipeptide by different SAAs has
been previously reported.[2c,2d]
Results and Discussion
The synthesis of furanoid δ-SAA 4 commences with
TEMPO oxidation of the primary alcohol group in 1, which
has been previously synthesized in our laboratory,[4a,4d] to
afford carboxylic acid 2 in good yield (see Scheme 1).
Scheme 2. Synthesis of pyranoid SAA; reagents and conditions: (i)
PDC (10 equiv.), DMF, 16 h; (ii) DPPA, Et3N, tBuOH, 80 °C, 16 h;
(iii) NaOH, dioxane, 3 h
Having both δ-SAAs 4 and 8 in hand, attention was fo-
cused on the solution-phase synthesis of Leu-enkephalin
analogs 11 and 14. Condensation of 4 with HCl·H-Phe-
Leu-OMe in the presence of N-(3-dimethylaminopropyl)-
NЈ-ethylcarbodiimide hydrochloride (EDC·HCl), 1-
hydroxybenzotriazole (HOBt) and N-methylmorpholine
(NMM) provided 9 in 74% yield (see Scheme 3). Acidolysis
(50% TFA/DCM) of the Boc group in 9, followed by con-
densation of the resulting free amine with Boc-Tyr(2BrZ)-
OH (EDC/HOBt/NMM), led to the isolation of two prod-
ucts. The minor product was identified by NMR spec-
troscopy as the expected tetrapeptide 10a, having the 3,6-
cis relationship in the SAA residue. The major product pro-
ved to be the isomer 10b, in which the amine substituent on
the SAA core has anomerized to give the 3,6-trans configu-
Scheme 1. Synthesis of furanoid SAA; reagents and conditions: (i)
cat. TEMPO, NaOCl, NaHCO3, NaCl, KBr, (nBu)4NCl, DCM/
H2O, 30 min, 0 °C; (ii) DPPA, Et3N, tBuOH, 80 °C, 16 h; (iii)
LiOH, H2O2, THF, 0 °C, 2 h
Curtius rearrangement was effected by treatment of 2 ration.
with diphenylphosphorazidate (DPPA) and Et3N in tBuOH
The trans configuration was established from the pres-
at 80 °C under anhydrous conditions.[9] The intermediate ence of an H2/H6 cross peak in the NOESY spectrum in
isocyanate was trapped with tBuOH to give the Boc-pro- combination with the large upfield shift (6 ppm) of C6 in
tected SAA 3 as a single isomer in 64%. The 3,6-cis relation- the 13C NMR spectrum (see Figure 2). Most likely, mutaro-
ship in 3 was ascertained by the medium H3/H6 cross peak tation of the anomeric amine already takes place during the
observed in a NOESY spectrum. Saponification of the acidic Boc-removal step.[5a,7b]
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G. A. van der Marel, M. Overhand et al.
FULL PAPER
Scheme 3. Synthesis of Leu-enkephalin analogs 11 and 14; reagents and conditions: (i) HCl·H-Phe-Leu-OMe, EDC·HCl, HOBt, NMM,
16 h; (ii) a) 50%TFA/DCM, 5 min; b) Boc-Tyr(2BrZ)-OH, EDC·HCl, HOBt, NMM, 16 h; (iii) a) Pd(OH)2/C, MeOH, 5 h; b) 5% H2O/
TFA, 3 h; (iv) a) 50%TFA/DCM, 5 min; b) Z-Tyr-OH, EDC·HCl, HOBt, NMM, 16 h; (v) Pd/C, H2, MeOH, 16 h
during this sequence of reactions judging by the J6,7 of
1
9.2 Hz as observed in the H NMR spectrum. Finally, re-
moval of the Z- and benzyl groups was accomplished by
hydrogenation with palladium on charcoal, yielding H-Tyr-
SAA-Phe-Leu-OMe 14 quantitatively. The structure of 14
was unambiguously established by NMR spectroscopy
and LCMS.
Compounds 11 and 14 were tested for their affinity for
human µ, δ and κ opioid receptors using transfected ham-
ster ovary cells. The SAA-containing compounds were not
able to displace radioligands from their binding sites at con-
centrations as high as 10 µ.[11]
In conclusion, two new SAAs, having an amine group
directly attached to the carbohydrate core, were successfully
synthesized by application of a Curtius rearrangement to
readily available carbohydrate building blocks. Future stud-
ies will focus on the conformational analysis of 11 and 14
in order to determine the structural basis for this loss of
activity. Furthermore, SAAs 4 and 8 can be used for the
incorporation into other biologically active sequences (i.e.
RGD peptides, protein farnesyl transferase inhibitor[12]) as
well as for the construction of cyclodextrin analogs.
Figure 2. Parts of the13C NMR spectra of 10a (a) and 10b (b) re-
corded in CDCl3 at 50 MHz
Deprotection of the 3,6-cis isomer 10a, i.e. PdII catalyzed
hydrogenation of the 2-bromobenzyloxycarbonyl (2BrZ)
group, followed by acidolysis of the Boc and isopropylidene
protective groups, proceeded uneventfully, affording the de-
sired Leu-enkephalin analog 11 in good yield. The structure
of 11 was ascertained by LCMS and NMR spectroscopy.
The synthesis of Leu-enkephalin analog 14, having the
pyranoid δ-SAA II instead of the Gly-Gly dipeptide, was
accomplished in a similar sequence of transformations.
Condensation of 8 with HCl·H-Phe-Leu-OMe (EDC/
HOBt) gave dipeptide 12 in 89% yield. After removal of the
Boc group in 12 (50% TFA/DCM), the resulting free amine
was condensed with Z-Tyr-OH to furnish the protected
Experimental Section
1
General Procedures and Materials: H and 13C NMR spectra were
recorded with a Jeol JNM-FX-200 (200/50.1 MHz), a Bruker WM-
300 (300/75.1 MHz) or a Bruker DMX-600 (600/150 MHz) spec-
trometer. Chemical shifts are given in ppm (δ) relative to tetrameth-
ylsilane as internal standard. All given 13C NMR spectra are pro-
ton decoupled. Mass spectra were recorded with PE/SCIEX API
165 with an electron-spray interface. Column chromatography was
performed on Fluka silica gel 60 (0.04Ϫ0.063 mm). TLC analysis
was conducted on precoated DC sheets (Merck silica gel 60 F254
)
tetrapeptide 13 in 83%. No anomerization was observed with detection by UV absorption (254 nm) where applicable and
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Synthesis of Novel Sugar Amino Acids by Curtius Rearrangement
FULL PAPER
by spraying with 20% H2SO4 in ethanol, a ninhydrin solution or
ammonium molybdate (25 g/L) and ceric ammonium sulfate (10 g/
3,6-Anhydro-6-(tert-butoxycarbonylamino)-2,6-dideoxy-4,5-O-iso-
propylidene-D-allo-hexonic Acid (4): A mixture of compound 3
L), followed by charring at about 150 °C. Reactions were run at (0.90 g, 2.8 mmol) in THF/H2O2 (25 mL, 2:1, v/v) was cooled to 0
ambient temperature, unless stated otherwise. Reactions that re-
quire anhydrous conditions were stirred under an atmosphere of
argon or nitrogen. Acetone (Acros, p.a.), dichloroethane (DCE; Bi-
osolve, HPLC grade), DMF (Baker, p.a.), toluene (Biosolve, p.a.),
1,4-dioxane (Baker, p.a.), pyridine (Baker, p.a.) and dichlorometh-
°C. Aq. LiOH (6.72 mL of a 1.25  solution) was slowly added to
the solution. After stirring the reaction mixture for 3 h it was acidi-
fied to pH 2 with 1  HCl and aq. Na2SO3 (10 mL) was added.
The aqueous phase was extracted with EtOAc (3 ϫ 30 mL), the
combined organic phases were dried (MgSO4) and concentrated.
Purification by silica gel column chromatography (eluent: EtOAc/
˚
ane (DCM; Baker, p.a.) were stored over molecular sieves (4 A).
Acetonitrile (Biosolve, p.a.) and MeOH (Biosolve, p.a.) were stored light petroleum, 1:1 Ǟ 1:0, v/v, containing 0.5% AcOH) furnished
˚
over molecular sieves (3 A). Triethylamine (Acros) was boiled un- compound 4 as a white solid in 63% yield (0.59 g) together with
der reflux for 3 h with CaH2, distilled and stored over KOH. Di-
phenylphosphorazidate (DPPA) was purchased from Aldrich and MeOD): δ ϭ 5.26 (d, J ϭ 1.5 Hz, 1 H, H6), 4.62 (s, 2 H, H4, H5),
stored at ϩ4 °C. Trifluoroacetic acid (TFA, Biosolve), 2,2,6,6-tetra- 4.33 (dt, J ϭ 1.5 Hz, J ϭ 6.9 Hz, 1 H, H3), 2.61 (dd, J ϭ 1.5 Hz,
26% of recovered starting material (0.23 g). 1H NMR (200 MHz,
methyl-4-piperidin-1-oxyl (TEMPO, Acros), 10% palladium on J ϭ 7.0 Hz, 2 H, 2 ϫ H2), 1.49, 1.31 (2 ϫ s, 6 H, 2 ϫ CH3 isopro-
charcoal (Aldrich), 20% palladium() hydroxide on carbon
(Janssen), 1-hydroxybenzotriazole (HOBt, Neosystem), N-[3-(di-
pylidene), 1.45 (s, 9 H, tert-Bu Boc) ppm. 13C NMR (50 MHz,
MeOD): δ ϭ 174.08 (C1), 114.61 (Cq isopropylidene), 89.36, 85.61,
methylamino)propyl]-NЈ-3-ethylcarbodiimide hydrochloride (EDC, 85.20, 82.56 (C3, C4, C5, C6), 39.76 (C2), 28.63 (tert-Bu Boc),
Aldrich) and pyridinium dichromate (PDC, Aldrich) were used as
received.
27.22, 25.39 (2 ϫ CH3 isopropylidene).
Boc-SAA-(isopropylidene)-Phe-Leu-OMe (9): A solution of Boc-
Phe-Leu-OMe (0.29 g, 0.74 mmol) in DCM/TFA (6 mL, 1:1, v/v)
was stirred for 5 min and subsequently concentrated. The residue
was coevaporated with toluene (3 ϫ 3 mL) and dissolved in DMF
(5 mL), SAA 4 (0.23 g, 0.74 mmol) was added and the resulting
mixture was cooled to 0 °C. HOBt (0.12 g, 0.89 mmol) and
EDC·HCl (0.17 g, 0.89 mmol) were added and the reaction mixture
was neutralized with NMM. After stirring for 16 h at ambient tem-
perature the reaction mixture was concentrated and the residue re-
dissolved in EtOAc (15 mL). The organic phase was washed with
H2O (10 mL), aq. NaHCO3 (2 ϫ 10 mL), H2O (10 mL), KHSO4
(2 ϫ 10 mL) and brine (10 mL), dried (MgSO4) and concentrated.
Purification by silica gel column chromatography (eluent: EtOAc/
light petroleum, 0:1 Ǟ 2:1, v/v) afforded compound 9 as a white
1-Methyl 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-D-allo-heptar-
ate (2): Compound 1 (1.62 g, 6.24 mmol), TEMPO (0.015 g,
0.094 mmol, 0.015 equiv.), KBr (0.059 g, 0.50 mmol) and TBACl
(0.094 g, 0.34 mmol) were dissolved in a mixture of DCM (40 mL)
and aq. NaHCO3 (1 , 12.5 mL). The reaction mixture was cooled
to 0 °C and a solution of NaOCl (13%, 12.5 mL), sat. NaCl
(12.5 mL) and aq. NaHCO3 (6.25 mL) was added dropwise over a
period of 30 min. After stirring the resulting mixture for another
30 min at 0 °C it was washed with DCM (3 ϫ 50 mL). The aqueous
phase was acidified to pH 2 with 1  HCl and extracted with DCM
(3 ϫ 50 mL). The combined organic layers were dried (MgSO4)
and concentrated under reduced pressure to afford crude acid 2 in
94% yield (1.51 g). 1H NMR (200 MHz, CDCl3): δ ϭ 5.01 (dd, J ϭ
2.9 Hz, J ϭ 6.8 Hz, 1 H, H5), 4.63 (dd, J ϭ 2.9 Hz, J ϭ 6.6 Hz,
1 H, H4), 4.60 (d, J ϭ 2.9 Hz, 1 H, H6), 4.50 (dt, J ϭ 2.9 Hz, J ϭ
5.9 Hz, 1 H, H3), 3.73 (s, 3 H, OMe), 2.71 (dd, J ϭ 2.9 Hz, J ϭ
5.9 Hz, 2 H, 2 ϫ H2), 1.56, 1.36 (2 ϫ s, 6 H, 2 ϫ CH3 isopropylid-
ene) ppm. 13C NMR (50 MHz, CDCl3): δ ϭ 173.45 (C6), 171.12
(C1), 114.14 (Cq isopropylidene), 83.91, 83.64, 83.09, 82.31 (C3,
C4, C5, C6), 51.89 (OMe), 37.43 (C2), 26.73, 25.03 (2 ϫ CH3 iso-
propylidene).
1
solid in 74% yield (0.32 g). H NMR, COSY (300 MHz, CDCl3):
δ ϭ 7.33Ϫ7.17 (m, 5 H, Harom Phe), 6.76 (d, J ϭ 7.7 Hz, 1 H, HN-
Phe), 6.12 (d, 1 H, HN-Leu), 5.44 (dd, J ϭ 3.3 Hz, J ϭ 9.5 Hz, 1
H, HN-SAA), 4.64Ϫ4.48 (m, 5 H, Hα-Leu, H3,H4, H5, H6), 4.20
(q, J ϭ 3.7 Hz, 1 H, Hα-Phe,), 3.68 (s, 3 H, OMe), 3.03 (d, J ϭ
7.3 Hz, 2 H, 2 ϫ Hβ-Phe, ), 2.68 (dd, J2a,3 ϭ 4.4 Hz, J2a,2b ϭ 15.0
Hz 1 H, H2a), 2.48 (dd, J2b,3 ϭ 4.0 Hz, J2a,2b ϭ 14.6 Hz, 1 H,
H2b), 1.58Ϫ1.43 (m, 3 H, 2 ϫ Hβ-Leu, Hγ-Leu),1.53, 1.29 (2 ϫ s,
6 H, 2 ϫ CH3 isopropylidene), 1.47 (s, 9 H, tert-Bu Boc), 0.91 (2
ϫ d, J ϭ 5.5 Hz, 6 H, 6 ϫ Hδ-Leu) ppm. 13C NMR (75 MHz,
CDCl3): δ ϭ 172.63, 171.48, 170.06 [C(O)Leu, C(O) Phe, C(O)
SAA), 154.96 [C(O) Boc), 136, 13 (Cq Phe), 129.12, 128.49, 126.91
(Carom Phe), 114.09 (Cq isopropylidene), 87.98, 84.00, 81.76, 80.48
(C3, C4, C5, C6), 80.12 (Cq Boc), 54.74, 52.17, 50.74 (Cα-Phe, Cα-
Leu, OMe), 41.34, 39.19, 38.10 (C2, Cβ-Phe, Cβ-Leu), 28.24 (tert-
Bu Boc), 27.12, 25.26 (2 ϫ CH3 isopropylidene), 24.48 (Cγ-Leu),
22.75, 21.54 (2 ϫ Cδ-Leu).
Methyl 3,6-Anhydro-6-(tert-butoxycarbonylamino)-2,6-dideoxy-4,5-
O-isopropylidene-D-allo-hexonoate (3): Compound 2 (0.894 g,
3.44 mmol) was coevaporated with toluene (3 ϫ 20 mL), and dis-
˚
solved in tBuOH (20 mL). Crushed molecular sieves (4A) were ad-
ded and the reaction mixture was stirred for 30 min under an argon
atmosphere. Et3N (0.46 mL, 3.44 mmol) and DPPA (0.74 mL,
3.44 mmol) were added and the reaction mixture was heated under
reflux. After 16 h the solution was filtered and concentrated, the
residue was applied to a silica gel column. Elution with EtOAc/
light petroleum (0:1 Ǟ 1:1, v/v) gave compound 3 in a 64% yield
(0.89 g) as a white solid. 1H NMR, COSY, NOESY (300 MHz,
Boc-Tyr-(2BrZ)-SAA-(isopropylidene)-Phe-Leu-OMe (10): Com-
pound 9 (0.12 g, 0.21 mmol) was dissolved in TFA/DCM (2 mL,
1:1, v/v) and the resulting mixture was stirred for 5 min. Toluene
CDCl3): δ ϭ 5.59 (br. s, 1 H, HN), 5.34 (s, 1 H, H6), 4.71 (s, 2 H, (10 mL) was added, the solution was concentrated and the residue
H4, H5), 4.37 (t, J ϭ 5.8 Hz, 1 H, H3), 3.71 (s, 3 H, OMe), 2.72
(dd, J ϭ 3.6 Hz, J ϭ 5.8 Hz, 2 H, 2 ϫ H2), 1.53, 1.33 (2 ϫ s, 6 H,
was subsequently coevaporated with toluene (3 ϫ 5 mL). The resi-
due was dissolved in DMF (2 mL), Boc-Tyr -(2BrZ)-OH (0.11 g,
2 ϫ CH3 isopropylidene), 1.45 (s, 9 H, tert-Bu Boc) ppm. 13C NMR 0.23 mmol) was added and the reaction mixture was cooled to 0
(50 MHz, CDCl3): δ ϭ 171.28 (C1), 154.45 [C(O) Boc), 113.69 (Cq °C. HOBt (0.034 g, 0.25 mmol) and EDC·HCl (0.048 g, 0.25 mmol)
isopropylidene), 88.68, 85.01, 83.49, 81.06 (C3, C4, C5, C6), 51.71
were added and the reaction mixture was neutralized with NMM.
(OMe), 38.21 (C2), 28.15 (tert-Bu Boc), 26.87, 25.20 (2 ϫ CH3 After 16 h TLC analysis showed complete disappearance of the
isopropylidene). ES-MS: m/z ϭ 332.1 [M ϩ H]ϩ, 354.1 [M ϩ Na]ϩ,
685.5 [2M ϩ Na]ϩ.
starting materials and the solvent was removed under reduced
pressure. The residue was taken up in EtOAc (10 mL) and washed
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G. A. van der Marel, M. Overhand et al.
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with H2O (5 mL), aq. NaHCO3 (2 ϫ 5 mL), H2O (2 ϫ 5 mL),
was taken up in a mixture of TFA/p-cresol/H2O (2 mL, 90/5/5,
KHSO4 (2 ϫ 5 mL) and brine (2 ϫ 5 mL), the organic phase was v/v/v) and stirred for 3 h. After addition of toluene (5 mL) the solu-
dried (MgSO4) and concentrated. The crude product was applied
to a silica gel column and eluted with EtOAc/light petroleum (1:9
Ǟ 8:2, v/v) to give a higher running product 10a in 29% yield
tion was concentrated. The crude tetramer was applied on a silica
gel column and eluted with DCM/MeOH (1:0 Ǟ 9:1, v/v) to give
pure 11 in quantitative yield (0.010 g). [α]2D0 ϭ Ϫ0.28 (c ϭ 0.3,
(0.059 g) and a lower running product 10b in 40% yield (0.084 g). MeOH). 1H NMR, COSY, ROESY, HMQC-COSY (600 MHz,
1
10a: H NMR, COSY, NOESY, (600 MHz, CDCl3): δ ϭ 8.42 (d,
[D6]acetone): δ ϭ 7.79 (d, J ϭ 8.0 Hz, 1 H, HN-Leu), 7.63 (d, J ϭ
J ϭ 8.1 Hz, 1 H, HN-SAA), 7.60 (d, J ϭ 8.0 Hz, 1 H, Harom
-
7.9 Hz, 1 H, HN-Phe), 7.25 (m, 4 H, Harom Phe, Tyr), 7.18 (m, 1
2BrZ), 7.49 (d, J ϭ 7.6 Hz, 1 H, Harom-2BrZ), 7.35 (t, J ϭ 7.4 Hz, H, Harom Phe), 7.10 (d, J ϭ 8.4 Hz, 2 H, Harom Phe), 6.76 (d, J ϭ
2 H, Harom-Phe), 7.21 (m, 9 H, Harom-Phe, Harom,-Tyr, Harom-2BrZ), 8.4 Hz, 2 H, Harom Tyr), 4.81 (t, J ϭ 4.6 Hz, 1 H, H5), 4.77 (m, 2
6.98 (d, J ϭ 7.0 Hz, 1 H, HN-Phe), 6.67 (d, J ϭ 6.5 Hz, 1 H, HN-
Leu), 5.59 (d, J ϭ 6.4 Hz, 1 H, H6), 5.46 (d, J ϭ 7.7 Hz, 1 H, HN-
Tyr), 5.35 (s, 2 H, CH2 2BrZ), 4.62 (m, 1 H, Hα-Phe), 4.46 (m, 2
H, H4, Hα-Leu), 4.43 (m, 1 H, Hα-Tyr), 4.28 (br. s, 2 H, H3, H5),
H, H6, Hα-Phe), 4.52 (m, 1Hm Hα-Leu), 4.39 (t, J ϭ 5.5 Hz, 1 H,
H4), 4.09 (dt, J ϭ 5.7 Hz, J ϭ 7.0 Hz, 1 H, H3), 3.66 (s, 3 H,
OMe), 3.65 (m, 1 H, Hα-Tyr), 3.15 (dd, Jα,β ϭ 5.8, Jβ,β ϭ 14.0 Hz,
1 H, Hβ-Phe), 3.01 (dd, Jα,β ϭ 4.0, Jβ,β ϭ 14.4 Hz, 1 H, Hβ-Tyr),
3.68 (s, 3 H, OMe), 3.12Ϫ2.96 (m, 4 H, 2 ϫ Hβ-Phe, 2 ϫ Hβ-Tyr), 2.98 (dd, Jα,β ϭ 7.5, Jβ,β ϭ 13.6 Hz, 1 H, Hβ-Phe), 2.80 (dd, Jα,β ϭ
2.55 (dd, J2a,3 ϭ 4.2 Hz, J2a,2b ϭ 14.0 Hz, 1 H, H2a), 2.34 (dd, 7.7, Jβ,β ϭ 14.3 Hz, 1 H, Hβ-Tyr), 2.69 (dd, J2a,3 ϭ 7.6 Hz, J2a,2b ϭ
J2b,3 ϭ 4.3 Hz, J2a,2b ϭ 14.7 Hz, 1 H, H2b), 1.56Ϫ136 (m, 3 H, 14.6 Hz, 1 H, H2a), 2.55 (dd, J2b,3 ϭ 5.0 Hz, J2a,2b ϭ 14.7 Hz, 1
Hβ-Leu, 2 ϫ Hγ-Leu), 0. 90, 0.89 (2 ϫ s, 6 H, 6 ϫ Hδ-Leu) ppm. H, H2b), 1.68 (m, 1 H, Hγ-Leu), 1.59 (m, 2 H, 2 ϫ Hβ-Leu), 0.90,
13C NMR (150 MHz, CDCl3): δ ϭ 172.62, 172.04, 171. 89, 169.98 0.89 (2 ϫ d, J ϭ 6.7 Hz, J ϭ 6.7 Hz, 6 H, 2 ϫ Hδ-Leu) ppm. 13C
(4 ϫ C(O), Tyr, SAA, Phe, Leu), 155.45 [C(O) Boc), 153.36[C(O)
2BrZ),149.93 (Cζ-Tyr), 136.13, 134.71 (Cγ-Tyr, C1Ϫ2BrZ, Cγ-
Phe), 132.92, 130.49, 130.16, 130.04, 129.19, 128.55, 127.61, 127.00,
120.88 (13 ϫ CH-arom, Tyr, 2BrZ, Phe), 123.37 (C2(q) 2BrZ),
NMR, HMQC-COSY (150 MHz, [D6]acetone): δ ϭ 176.23,
173.53, 171.83, 171.38 (4 ϫ C(O), Tyr, SAA, Phe, Leu), 156.85
(Cq arom, Cζ-Tyr), 138.26, 129.37 (2 ϫ Cq arom, Cγ-Tyr, Cγ-Phe),
131.20, 130.31, 130.26, 130.21, 129.00, 127.25 (7 ϫ CH arom, Phe,
113.88 (Cq isopropylidene), 86.22 (C6), 84.28 (C5), 82.03 (C4), Tyr), 115.92 (2 ϫ CH arom, Tyr), 89.88 (C6), 80.40 (C3), 74.21 (C4),
80.97 (C3), 79.88 (Cq Boc), 69.54 (CH2 2BrZ), 55.86, 55.04, 52.38,
50.86 (Cα-Tyr, Cα-Phe, Cα-Leu, OMe), 41.31, 39.49, 38.52, 38.06
(Cβ-Tyr, Cβ-Phe, Cβ-Leu, C2), 28.21 (tert-Bu Boc), 27.05, 25.24 (2
72.57 (C5), 60.10 (Cα-Tyr), 55.23 (Cα-Phe), 52.35 (OMe), 51.47
(Cα-Leu), 41.38 (Cβ-Leu), 40.06 (C2), 38.50 (Cβ-Phe), 36.88 (Cβ-
Tyr), 25.29 (Cγ-Leu), 23.17, 21.92 (2 ϫ Cδ-Leu). LCMS: Rt 13.44
ϫ CH3 isopropylidene), 24.51 (Cγ-Leu), 22.81, 21.57 (2 ϫ Cδ-Leu). (5 Ǟ 75% ACN/H2O, v/v), m/z ϭ 615.5 [M ϩ H]ϩ. HRMS: calcd.
ES-MS: m/z ϭ 969.5 [M ϩ H]ϩ, 991.4 [M ϩ Na]ϩ.
for C31H41N4O9 [M ϩ H] 615.3030; found 615.2933.
10b: 1H NMR, COSY, ROESY, HMQC-COSY (600 MHz,
CDCl3): δ ϭ 7.58 (d, 1 H, Harom, J ϭ 8.0 Hz), 7.47 (dd, J ϭ 7.5
Hz, J ϭ 0.8 Hz, 2 H, Harom), 7.32 (t, J ϭ 7.4 Hz, 2 H, Harom),
7.26Ϫ7.09 (m, 9 H, Harom), 7.04 (d, J ϭ 8.3 Hz, 1 H, HN-SAA),
7.00 (dd, J ϭ 8.0 Hz, J ϭ 4.0 Hz, 1 H, HN-Phe), 6.71 (d, J ϭ 7.8
Hz, 1 H, HN-Leu), 5.64 (dd, J ϭ 3.7 Hz, J ϭ 8.7 Hz, 1 H, H6),
5.33 (s, 2 H, CH2 2BrZ), 5.16 (d, J ϭ 8.2 Hz, 1 H, HN-Tyr), 4.66
(q, J ϭ 7.5 Hz, 1 H, Hα-Phe), 4.62 (m, 2 H, H4, H5), 4.53 (m, 1
H, Hα-Leu), 4.40 (m, 1 H, Hα-Tyr), 4.20 (br. s, 1 H, H3), 3.65 (s,
3 H, OMe), 3.08 (dd, Jα,β ϭ 6.6, Jβ,β ϭ 14.0 Hz, 1 H, Hβ-Phe),
1-Methyl 3,7-Anhydro-4,5,6-tri-O-benzyl-2-deoxy- -gulo-D-glycero-
D
octarate (6): Compound 5 (0.72 g, 1.43 mmol) was coevaporated
with toluene (3 ϫ 5 mL) and subsequently dissolved in DMF
˚
(10 mL). Crushed molecular sieves (4 A) were added and the reac-
tion mixture was cooled to 0 °C under an argon atmosphere. PDC
(2.69 g, 7.15 mmol) was added and the reaction mixture was stirred
for 16 h at ambient temperature. EtOAc (25 mL) was added, the
resulting suspension was filtered through hyflo and concentrated
under reduced pressure. Purification by column chromatography
(eluent: EtOAc/light petroleum, 2:8 Ǟ 8:2, v/v, containing 0.5%
AcOH) afforded the acid 6 in 87% yield (0.65 g). 1H NMR
3.01 (dd, Jα,β ϭ 7.2, Jβ,,β 13.9 Hz, 1 H, Hβ-Phe), 2.56 (dd, Jα,β
ϭ
9.9, Jβ,,β 14.9 Hz, 1 H, Hβ-Tyr), 2.48 (dd, Jα,β ϭ 4.5, Jβ,,β 14.9 Hz,
1 H, Hβ-Tyr), 2.43 (dd, J2a,3 ϭ 7.2 Hz, J2a,2b ϭ 15.1 Hz, 1 H, H2a),
2.36 (dd, J2b,3 ϭ 5.8 Hz, J2a,2b ϭ 15.1 Hz, 1 H, H2b), 1.58Ϫ1.49
(m, 2 H, Hβ-Leu), 1.41 (s, 3 H, CH3 isopropylidene), 1.37 (s, 9 H,
tert-Bu Boc), 1.34 (m, 1 H, Hγ-Leu), 1.27 (s, 3 H, CH3 isopropylid-
ene), 0.87 (d, J ϭ 5.8 Hz, 6 H, Hδ-Leu) ppm. 13C NMR (150 MHz,
CDCl3): δ ϭ 172.86, 171.510, 170.711, 169.546 (4 ϫ C(O), Tyr,
SAA, Phe, Leu), 155.15 [C(O) Boc), 153.24 [C(O) 2BrZ), 149.89
(Cζ-Tyr), 136.56, 134.45, 134.13 (Cγ-Tyr, C1 2BrZ, Cγ-Phe,),
132.84, 130.36, 130.34, 130.09, 129.95, 129.29, 128.41, 127.55,
126.80, 120.98 (13 ϫ CH arom, Phe, Tyr, 2BrZ), 126.75 (C2 2BrZ),
113.60 (Cq isopropylidene), 83.65 (C5), 79.56 (C6), 78.96 (C4),
78.53 (C3), 69.51 (CH2 2BrZ), 54.67 (Cα-Phe), 52.22 (OMe), 50.82
(Cα-Leu), 50.72 (Cα-Tyr), 41.09 (Cβ-Phe), 38.01, 37.93 (Cβ-Leu,
C2), 36.06 (Cβ-Tyr), 28.17 (tert-Bu Boc), 26.13, 24.85 (2 ϫ CH3
isopropylidene), 24.59 (Cγ-Leu), 22.65, 21.77 (2 ϫ Cδ-Leu). ES-
MS: m/z ϭ 969.5 [M ϩ H]ϩ, 991.4 [M ϩ Na]ϩ.
(200 MHz, CDCl3) :δ ϭ 7.29 (s, 15 H, H
3 ϫ Bn), 4.92Ϫ4.57
arom
(m, 6 H, 3 ϫ CH2 Bn), 3.99Ϫ3.33 (m,5 H, H3, H4, H5, H6, H7),
3.58 (s, 3 H, OMe), 2.73 (dd, J2a,3 ϭ 3.7 Hz, J2a,2b ϭ 16.1 Hz, 1
H, H2a), 2.49 (dd, J2b,3 ϭ 8.0 Hz, J2a,2b ϭ 15.3 Hz, 1 H, H2b)
ppm. 13C NMR (50 MHz, CDCl3): δ ϭ 172.25 (C8), 171.16 (C1),
138.01, 137.56, 137.44 (3 ϫ Cq Bn), 128.31, 128.04, 127.76, 127.61
(CH
3 ϫ Bn), 85.85, 80.27, 79.82, 77.85, 75.73 (C3, C4, C5,
arom
C6, C7), 75.39Ϫ74.91 (3 ϫ CH2 Bn), 51.74 (OMe), 36.91 (C2).
Methyl
amino)-2-deoxy-
3,7-Anhydro-4,5,6-tri-O-benzyl-7-(tert-butoxycarbonyl-
-gulo- -glycero-heptonate (7): After coevapor-
D
D
ation with toluene (3 ϫ 10 mL) compound 6 (1.11 g, 2.14 mmol)
˚
was dissolved in tBuOH (20 mL). Crushed molecular sieves (4 A)
were added and the reaction mixture was stirred under an argon
atmosphere for 30 min. DPPA (0.46 mL, 2.14 mmol) and Et3N
(0.28 mL, 2.14 mmol) were added and the resulting mixture was
heated under reflux. After 16 h TLC analysis revealed completion
of the reaction and the solution was filtered and concentrated. The
Tyr-SAA-Phe-Leu-OMe (11): Fully protected 10a (0.015 g,
0.016 mmol) was dissolved in MeOH (1 mL), and the solution was crude SAA was purified by silica gel column chromatography (elu-
degassed. Pd(OH)2 (4 mg) was added and the resulting reaction
mixture was stirred under an atmosphere of hydrogen gas. After
ent: EtOAc/light petroleum, 0:1 Ǟ 3:7, v/v) to give the expected
product 7 in a yield of 75% (0.84 g) as a white solid. 1H NMR
5 h the catalyst was removed by filtration through glass fiber (GF/ (200 MHz, CDCl3): δ ϭ 7.31 (s, 15 H, H
3 ϫ Bn), 4.96Ϫ4.59
arom
2A, Whatman). The solvent was removed in vacuo and the residue
(m, 7 H, 3 ϫ CH2 Bn, H7), 3.78 (m, 2 H, H5, H6), 3.59 (s, 3 H,
1708
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2003, 1704Ϫ1710

Synthesis of Novel Sugar Amino Acids by Curtius Rearrangement
FULL PAPER
OMe), 3.30 (m, 2 H, H3, H4), 2.71 (dd, J2a,3 ϭ 4.4 Hz, J2a,2b
15.0 Hz, 1 H, H2a), 2.43 (dd, J2b,3 ϭ 7.3 Hz, J2a,2b ϭ 15.0 Hz, 1
ϭ
170.28 (4 ϫ C(O), Tyr, SAA, Phe, Leu), 155.94, 155.39 [C(O) Z,
Cζ-Tyr), 137.92, 137.56, 137.47, 136.55, 136.23 (3 ϫ Cq Bn, Cq Z,
H, H2b), 1.44 (s, 9 H, tert-Bu Boc) ppm. 13C NMR (50 MHz, Cγ-Phe, Cγ-Tyr), 131.68Ϫ126.07 (5 ϫ CHarom Z, 2 ϫ CHarom Tyr,
CDCl3): δ ϭ 170.67 (C1), 154.48 [C(O) Boc), 138.04, 137.68 (3 ϫ 15 ϫ CHarom Bn, 5 ϫ CHarom Phe),115.42, 115.15 (2 ϫ CHarom
Cq Bn), 128.22, 128.00, 127.58 (CH
3 ϫ Bn), 85.61, 80.70,
Tyr), 85.28, 80.12, 79.21, 72.94 (C3, C4, C5, C6, C7) 75.57, 74.97,
arom
73.06 (C3, C4, C5, C6, C7), 75.42, 74.70 (3 ϫ CH2 Bn), 51.44 74.73 (3 ϫ CH2 Bn), 66.60 (CH2 Z), 54.13, 53.92, 52.04, 50.68 (Cα-
(OMe), 37.06 (C2), 27.99 (tert-Bu Boc).
Tyr, Cα-Phe, Cα-Leu, OMe), 40.61, 37.91, 37.54, 37.03 (Cβ-Tyr,
Cβ-Phe, Cβ-Leu, C2), 24.48 (Cγ-Leu), 22.41, 21.44 (2 ϫ Cδ-Leu).
ESI-MS: m/z ϭ 1049.6 [M ϩ H]ϩ, 1071.8 [M ϩ Na]ϩ, 1087.7 [M
ϩ K]ϩ.
3,7-Anhydro-4,5,6-tri-O-benzyl-7-(tert-butoxycarbonylamino)-2-
deoxy-D-gulo-D-glycero-heptonic Acid (8): Compound 7 (0.95 g,
1.61 mmol) was dissolved in dioxane (10 mL) and aqueous 1 
NaOH (1.77 mL) was added. The resulting mixture was stirred for Tyr-SAA-Phe-Leu-OMe (14): Fully protected 13 (0.45 g,
3 h after which TLC analysis revealed complete consumption of
the starting material. The reaction mixture was acidified with 1 
HCl and extracted with EtOAc (3 ϫ 20 mL). The combined or-
ganic phases were dried (MgSO4), concentrated and the residue
applied to a silica gel column. The pure product 8 was obtained in
0.43 mmol) was dissolved in MeOH and the resulting solution was
degassed. Pd/C (0.01 g) was added and after degassing for a second
time the reaction was stirred under an atmosphere of H2. After
TLC indicated the complete conversion of the starting material to
a lower running product, the solution was filtered through Glass
80% yield (0.74 g) by eluting with EtOAc/PE (2:8 Ǟ 8:2, v/v) con- Fiber (GF/2A, Whatman). The filtrate was concentrated and the
taining 0.5% AcOH. 1H NMR (200 MHz., MeOD): δ ϭ 7.26 (s, residue purified by column chromatography (Eluent: DCM/MeOH,
15 H, H
3 ϫ Bn), 4.88Ϫ4.62 (m, 7 H, 3 ϫ CH2 Bn, H7), 3.67 1:0 Ǟ 9:1, v/v). to afford 14 quantitatively (0.28 g). [α]2D0 ϭ Ϫ0.32
arom
(m, 2 H, H5, H6), 3.46Ϫ3.29 (m, 2 H, H3, H4), 2.69 (dd, J2a,3
3.7 Hz, J2a,2b ϭ 15.3 Hz, 1 H, H2a), 2.38 (dd, J2b,3 ϭ 7.3 Hz,
ϭ
(c ϭ 0.2, MeOH). 1H NMR, COSY, ROESY, HMQC-COSY
(600 MHz, [D6]acetone): δ ϭ 7.71 (d, J ϭ 8.1 Hz, 1 H, HN-Leu),
J2a,2b ϭ 15.0 Hz, 1 H, H2b), 1.44 (s, tert-Bu Boc) ppm. 13C NMR 7.51 (d, J ϭ 8.2 Hz, 1 H, HN-Phe), 7.24 (m, 4 H, Harom Phe), 7.16
(50 MHz, MeOD): δ ϭ 171.32 (C1), 157.20 [C(O) Boc), 139.49, (m, 1 H, Harom Phe, Tyr), 7.12 (d, J ϭ 8.4 Hz, 2 H, Harom Phe),
139, 16 (3 ϫ Cq Bn), 128.22, 128.00, 127.58 (CHarom 3 ϫ Bn),
6.75 (d, 2 H, Harom Tyr), 4.76 (m, 1 H, Hα-Phe), 4.52 (m, 2 H, Hα-
86.73, 82.54, 81.69, 74.50 (C3, C4, C5, C6, C7), 76.41, 75.84 (3 ϫ Leu, H6), 4.40 (d, J ϭ 9.2 Hz, 1 H, H7), 3.70 (m, 1 H, H3), 3.67
CH2 Bn), 38.17 (C2), 28.65 (tert-Bu Boc). ES-MS: m/z ϭ 578.7 [M
ϩ H]ϩ, 600.4 [M ϩ Na]ϩ.
(s, 3 H, OMe), 3.61 (dd, J ϭ 4.1 Hz, J ϭ 8.3 Hz, 1 H, Hα-Tyr),
3.42 (t, J ϭ 8.9 Hz, 1 H, H5), 3.39 (t, J ϭ 8.9 Hz, 1 H, H4), 3.11
(dd, Jα,β ϭ 5.8, Jβ,β ϭ 13.9 Hz, 1 H, Hβ-Phe), 3.08 (dd, Jα,β ϭ 4.0,
Jβ,β ϭ 14.5 Hz, 1 H, Hβ-Tyr), 2.95 (dd, Jα,β ϭ 8.2, Jβ,β ϭ 13.9 Hz,
1 H, Hβ-Phe), 2.78 (dd, Jα,β ϭ 8.4, Jβ,β ϭ 14.4 Hz, 1 H, Hβ-Tyr),
2.63 (dd, J2a,3 ϭ 2.7 Hz, J2a,2b ϭ 15.4 Hz, 1 H, H2a), 2.45 (dd,
J2b,3 ϭ 7.3 Hz, J2a,2b ϭ 15.3 Hz, 1 H, H2b), 1.71 (m, 1 H, Hγ-
Tyr), 1.60 (m, 2 H, 2 ϫ Hβ-Leu), 0.90 (d, J ϭ 6.6 Hz, 3 H, Hδ-
Leu), 0.88 (d, J ϭ 6.5 Hz, 3 H, Hδ-Leu) ppm. 13C NMR, HMQC-
COSY (150 MHz, [D6]acetone): δ ϭ 176.24, 173.80, 171.92, 170.85
[C(O), Tyr, SAA, Phe, Leu), 156.78 (Cζ-Tyr), 138.37, 129.78 (2 ϫ
Cq, Cγ-Tyr, Cγ-Phe), 131.07, 130.26, 128.99, 128.91, 127.20 (7 ϫ
CHarom, Phe, Tyr), 115.90 (2 ϫ CHarom Tyr), 84.79 (C7), 78.89
(C5), 75.70 (C3), 73.60 (C4), 69.56 (C6), 60.24 (Cα-Tyr), 55.18 (Cα-
Phe), 52.41 (OMe), 51.35 (Cα-Leu), 41.44 (Cβ-Leu), 39.13 (C2),
38.53 (Cβ-Phe), 36.87 (Cβ-Tyr), 25.28 (Cγ-Leu), 23.22, 21.90 (2 ϫ
Cδ-Leu). LC-MS: Rt 12.85 (5Ϫ75% ACN/H2O, v/v), m/z ϭ 645.4
[M ϩ H]ϩ. HRMS: calcd. for C32H43N4O10 [M ϩ H] 645.3136;
found 645.3129.
Boc-SAA-(Bn3)-Phe-Leu-OMe (12): The Boc protected SAA 8
(0.58 g, 1.01 mmol) was coupled to HCl·H-Phe-Leu-OMe (0.40 g,
1.01 mmol) as described for 9. The crude product was purified by
column chromatography (eluent: MeOH/DCM, 0:1 Ǟ 5:95, v/v) to
give 12 in 89% yield (0.77 g) as a white solid. 1H NMR, COSY
(300 MHz, CDCl3): δ ϭ 7.31Ϫ7.18 (m, 20 H, Harom, 3 ϫ Bn, Phe),
6.84 (d, J ϭ 7.9 Hz, 1 H, HN-Phe), 6.59 (d, J ϭ 7.9 Hz, 1 H, HN-
Leu), 5.39 (d, J ϭ 8.7 Hz, 1 H, HN-SAA), 4.92, 4.69 (m, 8 H, 3
ϫ CH2Bn, H7, Hα-Phe), 4.60 (m, 1 H, Hα-Leu), 3.67 (m, 4 H,
CH3 OMe, H6), 3.54 (m, 1 H, H3), 3.39 (q, J ϭ 9.5 Hz, 2 H, H4,
H5), 3.18 (dd, Jα,β ϭ 7.5, Jβ,β ϭ 14.0 Hz, 1 H, Hβ-Phe), 3.08 (dd,
Jα,β ϭ 6.1, Jβ,β ϭ 13.8 Hz, 1 H, Hβ-Phe), 2.62 (dd, J2a,3 ϭ 3.0 Hz,
J2a,2b ϭ 15.3 Hz, 1 H, H2a), 2.46 (dd, J2b,3 ϭ 6.5 Hz, J
2a,2b
15.3 Hz, 1 H, H2b), 1.60Ϫ1.48 (m, 3 H, 2 ϫ Hβ-Leu, Hγ-Leu),
1.43 (s, 9 H, tert-Bu Boc), 0.87 (t, J ϭ 6.2 Hz, 6 H, 6 ϫ Hδ-Leu)
ppm. 13C NMR (75 MHz, CDCl3): δ ϭ 173.31, 170.59, 170.22 (3
ϫ C(O), SAA, Phe, Leu), 154.88 [C(O) Boc), 138.20, 137.74, 136.98
(3 ϫ Cq Bn, Cq Phe), 129.37Ϫ126.76 (CHarom 3 ϫ Bn, Phe), 85. Urea 15: Compound 2 (0.16 g, 0.6 mmol) was dissolved in tBuOH
58, 81.46, 79.94, 73.09 (C3, C4, C5, C6, C7), 81.37 (Cq Boc), 75.61,
75.12 (3 ϫ CH2 Bn), 54.16, 52.22, 50.59 (Cα-Phe, Cα-Leu, OMe),
41.58, 37.88, 36.94 (Cβ-Phe, Cβ-Leu, C2), 28.21 (tert-Bu Boc),
24.63 (Cγ-Leu), 22.72, 21.87 (2 ϫ Cδ-Leu).
(5 mL), then Et3N (0.043 mL, 0.6 mmol) and DPPA (0.13 mL,
0.6 mmol) were added and the reaction mixture was heated under
reflux. After 16 h TLC analysis revealed complete disappearance
of the starting material. The solution was filtered and concentrated,
the residue was applied to a silica gel column. Elution with EtOAc/
light petroleum (0:1 Ǟ 1:1, v/v) gave compound 15 in 35% yield
(0.10 g) as a white solid. 13C NMR (50 MHz, CDCl3): δ ϭ 84.31,
83.73, 83.52, 82.49, 81.12 (2 ϫ C3, 2 ϫ C4, 2 ϫ C5, 2 ϫ C6),
51.80 (2 ϫ OMe), 38.27, 37.85 (2 ϫ C2), 26.93, 25.20 (4 ϫ CH3
isopropylidene). ESI-MS: m/z ϭ 489.4 [M ϩ H]ϩ, 511.4 [M ϩ
Na]ϩ.
Z-Tyr-SAA-(Bn3)-Phe-Leu-OMe (13): Trimer 12 (0.44 g,
0.52 mmol) was coupled to Z-Tyr-OH (0.16 g, 0.52 mmol) as de-
scribed for compound 10. Pure 13 was obtained in 83% yield
(0.45 g) by column chromatography using MeOH/DCM (0:1 Ǟ
5:95, v/v) as eluent. 1H NMR, COSY (300 MHz, CDCl3): δ ϭ
7.29Ϫ6.92 (m, 23 H, 15 ϫ Harom Bn, 5 ϫ Harom Phe, 2 ϫ Harom
Tyr, HN-Phe), 6.69 (m, 3 H, 2 ϫ Harom Tyr, HN-Leu), 5.49 (m, 1
H, HN-Tyr), 5.08Ϫ4.51 (m, 9 H, 3 ϫ CH2 Bn, Hα-Phe, Hα-Leu, Biological Assay: Competitive radio-ligand binding assays were
H7), 3.69 (m, 1 H, OMe, H6), 3.63 (s, 3 H, OMe), 3.39 (m, 1 H, performed with Chinese hamster ovary (CHO) cells stably
H3), 3.29 (m, 2 H, H4, H5), 3.09Ϫ2.85 (m, 4 H, 2 ϫ Hβ-Tyr, 2 ϫ transfected with human µ, δ or κ receptors. Cells were harvested
Hβ-Phe), 2.39 (dd, 1 H, H2a), 2.19 (dd, 1 H, H2b), 1.50 (m, 1 H,
Hγ-Leu), 1.25 (m, 2 H, 2 ϫ Hβ-Leu), 0.88 (s, 6 H, 6 ϫ Hδ-Leu)
72 h following plating in 50 m Tris buffer (pH, 7.4) containing
10 m EGTA, 5 m MgCl2 and homogenized using a Dounce
ppm. 13C NMR (75 MHz, CDCl3/MeOD): δ ϭ 172.86, 171.34, homogenizer. The homogenate was then centrifuged (11000 ϫ g)
Eur. J. Org. Chem. 2003, 1704Ϫ1710
www.eurjoc.org
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1709

G. A. van der Marel, M. Overhand et al.
FULL PAPER
Carstenen, G. A. van der Marel, J. H. van Boom, Tetrahedron
for 15 min at 4 °C. The pellet was resuspended in 50 m Tris buffer
(pH 7.4) with 100 m NaCl to yield a protein concentration of 800
µg/mL. Incubations were performed for 90 min at room tempera-
ture with 1 n [3H] DAMGO, [3H] DPDPE or [3H] U69,593 for µ,
δ or κ receptors, respectively. The concentrations of tested drugs
ranged from 10Ϫ10 to 10Ϫ5 . Binding assays were carried out in
mixtures containing 40 µg membrane protein, 10 m EGTA, 5 m
MgCl2 and 0.1% bovine albumin in a final volume of 250 µL. Non-
specific binding was determined in the presence of 1 µ naloxone.
The reactions were terminated by rapid filtration through What-
man GF/B glass fiber filters using a Brandel cell harvester, then
washed with 3 mL 50 m Tris buffer. The filters were presoaked
for at least 2 h in 3% polyethylenimine to decrease nonspecific
binding. The filter disks were then placed in vials with 3 mL of
Econosafe scintillation fluid and counted in Beckman LS 6500
scintillation counter. Ki values were derived from nonlinear re-
gression analysis of competition curves using GraphPad software.
[4b]
Lett. 2000, 41, 9331Ϫ9335.
E. Locardi, M. Stöckle, S.
Gruner, H. Kessler, J. Am. Chem. Soc. 2001, 123, 8189Ϫ8196.
[4c]
M. Stockle, G. Voll, R. Günther, E. Lohof, E. Locardi, S.
[4d]
Gruner, H. Kessler, Org. Lett. 2002, 4, 2501Ϫ2504.
R. M.
van Well, L. Marinelli, K. Erkelens, G. A. van der Marel, H.
S. Overkleeft, H. Kessler, J. H. van Boom, M. Overhand,
manuscript submitted for publication.
[5] [5a]
H. S. Isbell, H. L. Frush, J. Org. Chem. 1958, 23,
1309Ϫ1319. [5b] M. Monsigny, C. Quetard, S. Bourgerie, D. De-
´
lay, C. Pichon, P. Midoux, R. Mayer, A. C. Roche, Biochimie
1998, 80, 99Ϫ108.
[6a] J. C. Estevez, J. W. Burton, R. J. Estevez, H. Ardron, M. R.
[6]
Wormald, R. A. Dwek, D. Brown, G. W. J. Fleet, Tetrahedron
[6b]
Assymetry 1998, 9, 2137Ϫ2154.
A. Dondoni, A. Marra,
Chem. Rev 2000, 100, 4395Ϫ4421, and references cited herein.
[6c]
D. Mostowicz, M. Chmieliwiski, Carbohydr. Res. 1994, 13,
[6d]
137Ϫ143.
B. Drouillat, B. Kellam, G. Dekany, M. S. Starr,
[6e]
I. Toth, Bioorg. Med. Chem. Lett. 1997, 7, 2247Ϫ2250.
L.
G. Revel’skaya, A. N. Anikeeva, S. N. Danilov, Zh. Obshchei
Khimii (Engl. Trans.) 1973, 43, 1606Ϫ1611.
[7]
[8]
[7a] Lobry deM. C. A. Bruyn, Recl. Trav. Chim. Pays-Bas 1895,
Acknowledgments
[7b]
12, 98Ϫ105.
H. Paulsen, K. W. Pflughaupt, The Carbo-
We wish to thank Hans van den Elst and Nico Meeuwenoord for
recording mass spectra and Cees Erkelens for recording the 600-
MHz NMR spectra. The biological studies were supported by a
NIDA grant DA-13583.
hydrates,W. vol. 1B (Pigman, D. Horton, Eds.) p. 881Ϫ927,
[7c]
New York, Academic Press, 1980.
L. M. Likhosherstov, O.
S. Novikova, V. A. Derevitskaja, N. K. Kochetkov, Carbohydr.
Res. 1986, 146, C1ϪC5.
The use of both a Curtius rearrangement and the mechan-
istically similar Hoffman rearrangement have been reported to
afford N-Boc-protected glycosylamines as intermediates in a
fragmentation procedure of glycuronans: [8a] G. O. Aspinall, H.
[1]
[1a]
For reviews on SAAs, see:
S. W. Gruner, E. Locardi, E.
[1b]
[1c]
Lohof, H. Kessler, Chem. Rev. 2002, 102, 491Ϫ514.
Schweizer, F, Angew. Chem. Int. Ed. 2002, 41, 230Ϫ253.
T.
K. Fanous, N. S. Kumar, V. Puvanesarajah, Can. J. Chem.
K. Chakraborty, S. Ghosh, S. Jayaprakash, Curr. Med. Chem.
2002, 9, 421Ϫ435.
[8b]
1983, 61, 1858Ϫ1863.
G. O. Aspinall, M. C. Knebl, Car-
bohydr. Res. 1986, 157, 261Ϫ268.
[2] [2a]
R. M. van Well, H. S. Overkleeft, G. A. van der Marel, D.
Bruss, G. Thibault, P. G. de Groot, J. H. van Boom, M. Over-
[9]
Traces of H2O proved to be capable of partly hydrolyzing the
isocyanate, and the resulting amine reacted readily with the
remaining isocyanate present in the reaction mixture giving
urea 15 as the major product. This side reaction could be pre-
vented by drying the solution of the carboxylic acid in tBuOH
over activated molecular sieves prior to the reaction with
DPPA and Et3N.
[2b]
hand, Bioorg. Med. Chem. Lett. 2003, 13, 331Ϫ334.
E. Lo-
hof, E. Planker, C. Mang, F. Burkhart, M. A. Dechantsreiter,
R. Haubner, H. Wester, M. Schwaiger, G. Hölzemann, S. L.
Goodman, H. Kessler, Angew. Chem. Int. Ed. 2000, 39,
2761Ϫ2764. [2c] Graf vonE. Roedern, E. Lohof, G. Hessler, M.
Hoffman, H. Kessler, J. Am. Chem. Soc. 1996, 118,
10156Ϫ10167. [2d] T. K. Chakraborty, S. Jayaprakash, P. V. Di-
wan, R. Nagaraj, S. R. B. Jampani, A. C. J. Kunwar, J. Am.
Chem. Soc 1998, 120, 12962Ϫ12963.
[3] [3a]
C. Müller, E. Kitas, H. P. Wessel, Chem. Commun. 1995,
23, 2425Ϫ2426. [3b] H. P. Wessel, C. M. Mitchell, C. M. Lobato,
G. Schmid, Angew. Chem. Int. Ed. Engl. 1995, 34, 2712Ϫ2713.
[3c]
Y. Suhara, J. E. K. Hildreth, Y. Ichikawa, Tetrahedron Lett.
[3d]
1996, 37, 1575Ϫ1578.
J. Am. Chem. Soc. 1996, 118, 3818Ϫ3828.
J. P. McDevitt, P. T. Lansburry, Jr. ,
[3e]
[10]
[11]
C. M. Timmers,
H. Ohrui, G. H. Jones, J. G. Moffatt, M. L. Maddox, A. T.
Christensen, S. K. Byram, J. Am. Chem. Soc. 1975, 97,
4602Ϫ4613.
Pyranoid δ-SAA-containing Leu-enkephalin analogs were
shown to be inactive in a guinea pig ileum assay (see ref.[2c]). In
contrast, furanoid δ-SAA-containing analogs showed similar
analgesic activity as the Leu-enkephalin pentapeptide in mouse
hot-plate and tail-clip assays (see ref.[2d]).
H. S. Overkleeft, S. H. L. Verhelst, E. Pieterman, N. J.
Meeuwenoord, M. Overhand, L. H. Cohen, G. A. van der Ma-
rel, J. H. van Boom, Tetrahedron Lett. 1999, 40, 4130.
Received December 5, 2002
J. J. Turner, C. M. Ward, G. A. van der Marel, M. L. C. E.
Kouwijzer, P. D. J. Grootenhuis, J. H. van Boom, Chem. Eur.
J. 1997, 3, 920Ϫ929. [3f] L. Szabo, B. L. Smith, K. D. McReyn-
olds, A. L. Parrill, E. R. Morris, J. Gervay, J. Org. Chem. 1998,
[3g]
63, 1074Ϫ1078.
D. D. Long, N. L. Hungerford, M. D.
Smith, D. E. A. Brittain, D. G. Marquess, T. D. W. Claridge,
[3h]
G. W. J. Fleet, Tetrahedron Lett. 1999, 40, 2195Ϫ2198.
Y.
[12]
Suhara, Y. Yamaguchi, B. Collins, R. Schnaar, M. Yanagishita,
J. E. K. Hildreth, I. Shimada, Y. Ichikawa, Bioorg. Med. Chem.
2002, 10, 1999Ϫ2013.
[4] [4a]
R. M. van Well, H. S. Overkleeft, M. Overhand, E. Vang
1710
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Eur. J. Org. Chem. 2003, 1704Ϫ1710