Transformation of glucose into a novel carbasugar amino acid dipeptide isostere

Article abstract of DOI:10.1080/07328300701252623

The synthesis of a novel carbasugar amino acid (15), starting from D-glucose and using the Ferrier rearrangement as a key step, is reported. Compound 15 is implemented as dipeptide isostere in the synthesis of a Leu-enkephalin analog. Copyright Taylor & F

Full text of DOI:10.1080/07328300701252623

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Transformation of Glucose into a Novel
Carbasugar Amino Acid Dipeptide Isostere
Bas Lastdrager a , Mattie S. M. Timmer a , Gijsbert A. van der Marel a ,
Herman S. Overkleeft a & Mark Overhand a
a Leiden Institute of Chemistry, Leiden University , Leiden, The Netherlands
Published online: 07 May 2007.
To cite this article: Bas Lastdrager , Mattie S. M. Timmer , Gijsbert A. van der Marel , Herman S. Overkleeft
& Mark Overhand (2007) Transformation of Glucose into a Novel Carbasugar Amino Acid Dipeptide Isostere,
Journal of Carbohydrate Chemistry, 26:1, 41-59, DOI: 10.1080/07328300701252623
To link to this article: http://dx.doi.org/10.1080/07328300701252623
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Journal of Carbohydrate Chemistry, 26:41–59, 2007
Copyright # Taylor & Francis Group, LLC
ISSN: 0732-8303 print 1532-2327 online
DOI: 10.1080/07328300701252623
Transformation of Glucose
into a Novel Carbasugar
Amino Acid Dipeptide
Isostere
Bas Lastdrager, Mattie S. M. Timmer, Gijsbert A. van der Marel,
Herman S. Overkleeft, and Mark Overhand
Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands
The synthesis of a novel carbasugar amino acid (15), starting from D-glucose and using
the Ferrier rearrangement as a key step, is reported. Compound 15 is implemented as
dipeptide isostere in the synthesis of a Leu-enkephalin analog.
Keywords: Sugar amino acid, Peptidomimetic, Carbasugar, Leu-enkephalin,
Mukaiyama-Michael addition
INTRODUCTION
Sugar amino acids (SAAs) are defined as carbohydrate-based compounds that
feature an amine and a carboxylate.[1] As such, SAAs are widely spread in
nature, and neuraminic acid and N-acetyl muramic acid constitute important
structural elements in many oligo(poly)saccharides and glycoconjugates. Inter-
estingly, SAAs in nature are almost exclusively linked through interglycosidic
bonds, not through amide bonds. The full potential of SAAs[2] as carbohydrate-
peptide hybrids was recognized first by Kessler and coworkers,[3] who disclosed
an efficient synthesis of D-glucose-derived SAAs and their incorporation in a
series of linear and cyclic oligopeptide structures. Inspired by this work,
many researchers have become actively involved in SAA-related research.
SAA homooligomers have been generated with the aim to attain oligosacchar-
ide mimics that have the interglycosidic linkages replaced by amide bonds.[4]
Cyclic SAA homooligomers have been prepared[5,6] with the ultimate goal to
Received August 12, 2006; Accepted January 30, 2007.
Address correspondence to Mark Overhand, Leiden Institute of Chemistry, Gorlaeus
Laboratories, P. O. Box 9502, 2300 RA Leiden, The Netherlands. E-mail: overhand@
chem.leidenuniv.nl
41

B. Lastdrager et al.
42
develop cyclodextrin analogous receptor molecules.[7] The relevance of the
hybrid nature of SAAs comes to the fore in the multitude of reported appli-
cations in which selected amino acid residues are replaced in biologically
relevant oligopeptides. Here the aim is twofold: The nature of the parent carbo-
hydrate (the furan or pyran ring) in combination with the positioning of the
amine and carboxylate may impart a desired secondary structure on the
target oligopeptide, whereas the residual functionalities on the furan/pyran
core may be used to introduce additional desirable properties to the peptide.[8]
In conjunction with the growing interest in the application of SAAs, recent
years have witnessed numerous reports describing synthetic strategies toward
new SAAs.[9] Next to aiming for control over the relative positioning of the
amine- and carboxylate functionalities (as in a, b, g, d, and e SAAs), research
objectives in these studies include the development of constrained SAAs, for
instance, through annulation of a second ring system,[10] but also the prep-
aration of linear SAA derivatives.[11]
Given the extensive research efforts involving SAAs, reported examples of
SAA building blocks in which the ring oxygen is replaced by carbon[12] are
scarce. This is surprising, especially when considering the numerous strategies
available for transforming a carbohydrate into carbasugars.[13] We here
describe the application of the Ferrier rearrangement[14] of a carbohydrate-
derived enopyranoside and its transformation into carbasugar amino acid 15
(CSAA). The applicability of dipeptide isostere CSAA 15 in standard solid
phase peptide synthesis is demonstrated by the synthesis of Leu-enkephalin
derivative 21.
RESULTS AND DISCUSSION
Our synthetic approach commenced with the five-step conversion of commer-
cially available methyl a-D-glucopyranoside 1 into the enopyranoside 2 in
61% yield following a literature procedure (Sch. 1).[15] Submitting compound
2 to Ferrier rearrangement conditions (mercury(II)chloride in aqueous media
at elevated temperature) cleanly provided the known functionalized cyclohex-
anone 3 in 80% yield. Ketone 3 was converted into the intermediate enone 4. At
this stage, it was envisioned that the required carboxymethyl substituent could
be introduced via a Michael addition. In a first attempt, treatment of compound
4 with the diethylmalonate anion, generated under the agency of sodium
hydride or sodium methoxide, did not result in the expected 1,4-addition.
Instead, elimination of benzyl alcohol followed by tautomerization led to the
quantitative formation of phenol derivative 5. Because of the apparent instabil-
ity of enone 4 toward basic conditions, attention was focused on the Lewis
acid-mediated Mukaiyama-Michael addition. Treatment of compound 4 with
1-(tert-butyldimethylsilyloxy)-1-ethoxyethene[16] and titanium(IV)chloride led
to 1,2-addition giving allylic alcohol 6 as a single diastereoisomer in 78%

Novel Carbasugar Amino Acid Dipeptide Isostere
43
Scheme 1: Conditions: i) reference 15 ii) HgCl2 (1.1 equiv.), acetone/H2O (2:1), reflux, 2 h, 80%.
iii) MsCl (2.7 equiv.), DMAP (cat.), pyr., rt, 2 h, 87%. iv) diethylmalonate (1.5 equiv.), NaH or
NaOEt (1.5 equiv.), THF, rt, 45 min, 99%. v) TBSOC(OEt)55CH2 (1.5 equiv.), TiCl4 (1.5 equiv.), DCM,
–788C, 30 min, 78%. vi) TBSOC(OEt)55CH2 (1.5 equiv.), SnCl4 (1.5 equiv.), DCM, –788C, 20 min,
55%. vii) TBSOC(OEt)55CH2 (1.5 equiv.), LiClO4 (5.0 equiv.), Et2O, rt, 48 h, 73%. viii)
.
TBSOC(OMe)55CH2 (3.0 equiv.), LiClO4 (10.0 equiv.), Et2O, rt, 30 min, 98%. ix) HF pyr., THF/pyr.
(4:1), rt, 30 min, 99%. x) p-MeOBnNH2 (2.0 equiv.), Na(OAc)3BH (1.5 equiv.), HOAc (1.0 equiv.),
1,2-DCE, rt, 1 h, 11: 51%, 12: 15% (2 steps). xi) CAN (2.5 equiv.), H2O/MeCN (1:2), rt, 24 h, 56%. xii)
LiOH, H2O/dioxane (1:4), rt, 3 h, quant. xiii) FmocOSu, sat. aq. NaHCO3/dioxane (4:1), 17 h, 92%.
yield. Despite the good yield and selectivity, this approach was rendered inef-
fective as the tertiary allylic hydroxyl functionality proved to be inert toward
further transformations. Upon application of tin(IV)chloride as Lewis acid in
dichloromethane, 1,4-addition was achieved resulting in the formation of

B. Lastdrager et al.
44
ester 7 in 55% as an inseparable mixture of isomers. The yield of this reaction
could be further optimized by switching to lithium perchlorate in Et2O,[17]
affording after 48 h reaction time ester 7 in 73% yield, again as a mixture of dia-
stereoisomers (4:1). By changing the alkylating species to (tert-butyldimethyl-
silyloxy)-1-methoxyethene and using a larger excess of reagents, a spectacular
change in the outcome of the reaction was observed. Using this reagent, enone
4 was consumed in only 30 min, resulting in Michael adduct 8 as a single dia-
stereoisomer in 98% yield. The newly created stereocenter was unambiguously
assigned using NMR spectroscopy.
In continuation of our synthetic studies, silyl enol ether 8 was transformed
into ketone 9 amenable for ensuing reductive amination. Initially, the conver-
sion of compound 9 from 8 was hampered by the formation of the elimination
product 10 in yields varying from 70% to 80%, under several desilylating
conditions. Fortunately, it was found that elimination could be suppressed
.
completely by switching to HF pyridine as the desilylating agent. Under
these conditions, ketone 9 was isolated as the sole product and in quantitative
.
yield. Importantly, the reaction with HF pyridine was complete within 30 min.
Prolonged reaction times led to the formation of enone 10 through elimination
of benzyl alcohol. It was found that this transformation also occurs, over time,
upon storage of 9, dictating the necessity to proceed with the next step directly.
Thus, after hydrolysis and workup, immediate reductive amination employing
4-methoxybenzylamine, acetic acid, and sodium triacetoxyborohydride
afforded CSAA 11 in 51% over two steps. Rather surprisingly, complete epimer-
ization at C1 occurred under the conditions applied. The absolute configuration
of 11 was firmly established by NOE difference experiments, with key NOEs
between H5b-H1 and H5a-H3. In addition, the trans-disposition of the newly
introduced amine- and carboxymethyl substituent was assigned. The epimeri-
zation of the stereocenter at C1 is most likely the result of tautomerization of
the intermediate imine, resulting in the formation of amine 11 as the more
stable isomer. In a competing process, benzyl alcohol proved to be prone to elim-
ination, leading to the formation of enol ether 12 as major side product, in a
yield of 15% over the last two steps. Removal of the N-methoxybenzyl group
of 11, using ceric ammonium nitrate (CAN), liberated the free amine to give
compound 13. Saponification of the methyl ester under the agency of LiOH
furnished amino acid 14 in 56% yield over two steps. Protection of the amine
function of 14 was accomplished using Fmoc-OSu in saturated aqueous
NaHCO3/dioxane, to give CSAA 15 in a yield of 92%.
CSAA 15 was applied in a standard Fmoc-based solid phase peptide syn-
thesis protocol toward the synthesis of a novel Leu-enkephalin derivative
(Sch. 2). Thus, Wang resin 16 was transformed in a standard way to give
H-Phe-Leu-Wang resin 17. Condensation of acid 15 with 17 using HCTU as
coupling agent proceeded smoothly to give immobilized tripeptide 18. After
removal of the Fmoc group, further elongation with a Boc-protected tyrosine

Novel Carbasugar Amino Acid Dipeptide Isostere
45
Scheme 2: Conditions: i) a) DIC, DMAP, Fmoc-Leu-OH, CH2Cl2; b) 20% piperidine in DMF,
3 ꢀ 10 min; c) Fmoc-Phe-OH, HCTU, DiPEA, DMF, 3 ꢀ 1 h; d) 20% piperidine in DMF, 3 ꢀ 10 min.
ii) 15 (2.0 equiv.), HATU (0.95 equiv.), DiPEA, DMF (2.5 equiv.), 2 ꢀ 10 min. iii) a) 20% piperidine in
DMF, 3 ꢀ 10 min; b) Boc-Tyr(t-Bu)-OH, HCTU, DiPEA, DMF, 2 h. iv) TFA/TIS/H2O (95:2.5:2.5), 15 min;
95% v) H2, 10% Pd/C (cat.), HOAc (1.0 equiv.), t-BuOH/H2O (1:1), 17 h, rt, quant.
(19) and subsequent acidic cleavage from the resin, along with removal of the
acid labile protective groups, gave partially deprotected peptide 20. Finally,
hydrogenolysis of the benzyl ethers followed by reverse-phase HPLC purifi-
cation afforded Leu-enkephalin analog 21 in 95% overall yield.
In summary, a synthetic route to novel CSAA 15 was developed having a high-
yielding and stereoselective lithium perchlorate assisted Mukaiyama-Michael
1,4-addition on enone 4 as a key step. In addition, dipeptide isostere 15 was
successfully applied in a solid phase protocol toward Leu-enkephalin analog 21.
EXPERIMENTAL SECTION
General Methods and Materials
Acetone, 1,2-dichloroethane (1,2 DCE), dichloromethane (DCM), dimethyl
formamide (DMF), 1,4-dioxane, ethanol, n-hexane, pyridine (pyr.), and toluene
˚
(Biosolve) were stored over molecular sieves (4A). Acetonitrile and methanol
˚
(HPLC grade) (Biosolve) were stored over molecular sieves (3A). Diethyl
ether and tetrahydrofuran (THF) (Biosolve) were distilled from LiAlH4 prior
to use. Eluents ethyl acetate, petroleum ether (40–60), and toluene (Riedel-
¨
de Haen) were of technical grade and distilled prior to use. All other chemicals
were used as received. Abbreviations of chemicals used in solid phase peptide

B. Lastdrager et al.
46
synthesis: Boc: tert-butyloxycarbonyl; DIC: diisopropylcarbodiimide; DiPEA:
N,N-diisopropylethylamine; DMAP: 4-(dimethylamino)pyridine: Fmoc: 9-fluor-
enylmethyloxycarbonyl; Glu: L-glutamic acid; Gly: glycine; HATU: 2-(7-aza-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HCTU:
2-(6-chloro-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoropho-
sphate; Leu: L-leucine; Phe: L-phenylalanine; Su: succinimide; TFA; trifluoroa-
cetic acid; TIS: triisopropylsilane; Tyr: L-tyrosine. All reactions were performed
under an inert atmosphere and at ambient temperature unless stated other-
wise. Prior to reactions that require anhydrous conditions, traces of water
from starting material and reagents were removed by coevaporation with
toluene or 1,2-dichloroethane. All solvents were removed by evaporation
under reduced pressure. Reactions were monitored by TLC analysis using
DC-fertigfolien (Schleicher & Schuell, F1500, LS254) or HPTLC aluminum
sheets (Merck, silica gel 60, F254). Compounds were visualized by UV absorb-
tion (254 nm) where applicable and by spraying with 20% H2SO4 in ethanol
followed by charring at ꢀ1508C or by spraying with a solution of
.
.
(NH4)6Mo7O24 4H2O (25 g/L) and (NH4)4Ce(SO4)4 2H2O (10 g/L) in 10%
˜
sulfuric acid followed by charring at 1508C. Olefins were visualized by
spraying with a solution of KMnO4 (2%) and K2CO3 (1%) in water. Column
chromatography was performed on silica gel (Merck, 40–60 mm). Optical
rotations ([a]2D0) were measured on a Propol automatic polarimeter (sodium D
line, l ¼ 589 nm). 1H and 13C APT NMR spectra were recorded on a Jeol
JNM-FX-200 (200/50.1 MHz), a Bruker 300 WM-300 (330/75 MHz), a
Bruker AV 400 (400/100 MHz), or a Bruker DMX-600 (600/150 MHz) spec-
trometer. Chemical shifts (d) are given in ppm relative to tetramethylsilane
as internal standard. Coupling constants (J ) are given Hz. Where indicated,
NMR-peak assignments were made using COSY and NOESY experiments.
Infrared spectra were recorded on a Shimadzu FTIR-8300 and data are
reported in cm21. Mass spectra were recorded on a PE/Sciex API 165 instru-
ment with an ion spray interface. High-resolution mass spectra and MS/MS
were recorded on a Finnigan LTQ-FT (Thermo electron). LC-MS analysis
was conducted on a Jasco system (detection simultaneously at 214 nm and
254 nm) equipped with an Alltima C-18 analytical column (Alltech,
4.6 mm ꢀ 150 mm, 5-mm particle size). Preparative HPLC was performed on
a BioCad Vision (Applied Biosystems, Inc.) using a Alltima C-18 column
(Alltech, 10.0 mm ꢀ 250 mm, 5 mm particle size).
(2S,3R,4S,5R/S)-2,3,4-Tris-benzyloxy-5-hydroxycyclo-
hexanone (3)
Compound 2 (5.96 g, 13.36 mmol) was dissolved in a mixture of acetone/
water (2:1). HgCl2 (3.99 g, 14.69 mmol, 1.1 equiv.) was added and the
mixture was heated under reflux. After 2 h the mixture was cooled to rt and

Novel Carbasugar Amino Acid Dipeptide Isostere
47
concentrated. The resulting white solid was dissolved in DCM, washed with
water (2ꢀ) and brine (2ꢀ). The organic layer was separated, dried (MgSO4),
filtered, and concentrated. Purification by silica gel column chromatography(E-
tOAc/PE 1:3 to 2:3) gave 3 as a mixture of hydroxyketones in a combined
overall yield (4.64 g, 10.73 mmol) of 80%. Major isomer 3S: 13C NMR
(50 MHz, CDCl3): d 204.0 (C-1), 138.3, 137.6 (3 ꢀ Cq Bn), 128.6, 128.4, 128.2,
127.9, 127.8, 127.6, 127.5, 127.1 (CHarom), 85.2, 81.6, 81.4 (C-2, C-3, C-4),
75.7, 73.3, 72.9 (3 ꢀ CH2 Bn), 66.3 (C-5), 42.6 (C-6). Minor isomer 3R: 13C
NMR (50 MHz, CDCl3): d 203.2 (C-1), 138.0, 137.9, 137.4 (3 ꢀ Cq Bn), 128.6,
128.4, 128.1, 128.0, 127.9 (CHarom), 85.9, 84.6, 81.9 (C-2, C-3, C-4), 75.6, 75.3,
73.5 (3 ꢀ CH2 Bn), 67.9 (C-5), 44.1 (C-6).
(4S,5R,6S)-4,5,6-Tris-benzyloxycyclohex-2-enone (4)
To a mixture of alcohol 3 (0.432 g, 1.00 mmol) and mesylchloride (0.209 mL,
2.7 mmol, 2.7 equiv.) dissolved in pyridine (10 mL) was added a catalytic
amount of DMAP. After 2 h the reaction was complete according to TLC
analysis (EtOAc/PE 1:1). Ice was added and the mixture was extracted with
Et2O. The organic layer was separated, washed with brine, dried (MgSO4),
and concentrated. After purification by column chromatography (EtOAc/PE
1:3 to 1:1), unsaturated ketone 4 was obtained (0.361 g, 0.87 mmol) in a yield
1
of 87%. H NMR (200 MHz, CDCl3): d 7.46–7.26 (m, 15H, CHarom), 6.81 (dd,
1H, J3,2 ¼ 10.2 Hz, J3,4 ¼ 2.2 Hz, H-3), 6.39 (dd, 1H, J2,3 ¼ 10.2 Hz,
J2,4 ¼ 2.2 Hz, H-2), 5.11–4.70 (m, 6H, 3 ꢀ CH2 Bn), 4.35 (dt, 1H,
J4,2 ¼ J4,3 ¼ 2.2 Hz, J4,5 ¼ 7.3 Hz, H-4), 4.04 (d, 1H, J6,5 ¼ 10.2 Hz, H-6), 3.97
(dd, 1H, J5,4 ¼ 7.3 HZ, J5,6 ¼ 10.2 Hz, H-5). 13C NMR (50 MHz, CDCl3): d
197.3 (C-1), 148.0 (C-3), 138.1, 137.7, 137.5 (3 ꢀ Cq Bn), 128.5 (C-2), 128.3–
127.7 (CHarom), 84.6, 83.7, 78.9 (C-4, C-5, C-6), 75.6, 74.4, 73.5 (3 ꢀ CH2).
2,4-Bis(benzyloxy)phenol (5)
To a mixture of NaH (6 mg, 0.15 mmol 60% dispersion in mineral oil, 1.5
equiv.) or NaOEt (0.15 mL 1.0 M, prepared from 103 mg Na in 45.0 mL EtOH,
1.5 equiv.) in freshly distilled THF (2.5 mL) was added diethylmalonate
(23 mL, 0.15 mmol, 1.5 equiv.) at 08C. After 15 min of stirring a solution of unsa-
turated ketone 4 (42 mg, 0.10 mmol) in THF (0.5 mL) was added dropwise and
the mixture was allowed to reach rt. After 45 min TLC analysis (EtOAc/PE 1:4)
revealed complete consumption of enone 4 and the reaction was quenched by
addition of water and diluted with EtOAc. The aqueous layer was separated
and extracted once more with EtOAc. The combined organic layers were
washed with water and brine, dried (MgSO4), and concentrated. Purification
of the residue by column chromatography (PE to EtOAc/PE 1:19) gave phenol
1
derivative 5 (30 mg, 0.10 mmol) in a quantitative yield. H NMR (200 MHz,
CDCl3): d 7.42–7.31 (m, 10H, CHarom Bn), 6.84 (d, 1H, J6,5 ¼ 8.8 Hz, H-6),

B. Lastdrager et al.
48
6.63 (d, 1H, J3,5 ¼ 2.9 Hz, H-3), 6.48 (dd, 1H, J5,3 ¼ 2.9 Hz, J5,6 ¼ 8.8 Hz, H-5),
5.28 (s, 1H, OH), 5.04 (s, 2H, CH2 Bn), 4.97 (s, 2H, CH2 Bn). 13C NMR (50 MHz,
CDCl3): d 146.2, 140.2, 140.2, 137.1, 136.1 (5ꢀ Cq), 128.6, 128.5, 128.3, 127.8,
127.5, 126.9 (CH Bn), 114.3, 106.1, 101.7 (C-3, C-5, C-6), 71.0, 70.7 (2 ꢀ CH2
Bn). MS (ESI): m/z ¼ 307.1 [M þ H]þ, 329.2 [M þ Na]þ, 635.4 [2M þ Na]þ.
(4S,5R,6S)-4,5,6-Tris(benzyloxy)-1-ethoxycarbonylmethyl-
cyclohex-2-enol (6)
a,b-Unsatured ketone 4 (0.124 g, 0.30 mmol) was dissolved in freshly dis-
tilled DCM (2.5 mL) under an argon atmosphere. 1-(tert-Butyldimethylsily-
loxy)-1-ethoxyethene[16] (91.0 mg, 0.45 mmol, 1.5 equiv.) was added and the
mixture was cooled to –788C. After dropwise addition of TiCl4 (49.6 mL,
0.45 mmol, 1.5 equiv.), the mixture turned dark red. After 30 min TLC analysis
(EtOAc/PE 1:3) showed complete disappearance of starting material along
with the formation of a lower running spot. The reaction was quenched by
addition of water (1.0 mL) and warmed to rt, after which the red color disap-
peared. The mixture was diluted with Et2O and the separated organic layer
was collected, washed with brine, dried (MgSO4), and concentrated. Purification
of the residue by silica gel column chromatography (EtOAc/PE 1:19 to 1:9) gave
1,2-adduct 6 (0.117 g, 0.23 mmol, 78%). 1H NMR (400 MHz, CDCl3): d 7.35–7.24
(m, 15H, CHarom), 5.80 (2ꢀ dd, 2H, J ¼ 1.8 Hz, J ¼ 10.2 Hz, H-2, H-3), 5.09
(d, 1H, J ¼ 11.2 Hz, CH Bn), 4.91 (d, 1H, J ¼ 11.0 Hz, CH Bn), 4.85 (d, 1H,
J ¼ 11.0 Hz, CH Bn), 4.69 (s, 2H, CH2 Bn), 4.69 (d, 1H, J ¼ 11.2 Hz, CH Bn),
4.16 (dt, 1H, J ¼ 1.8 Hz, J4,5 ¼ 7.8 Hz, H-4), 4.09 (dd, 1H, J5,4 ¼ 7.8 Hz,
J5,6 ¼ 9.8 Hz, H-5), 4.01 (q, 2H, J ¼ 7.1 Hz, CH2 Et), 3.66 (d, 1H, J6,5 ¼ 9.8 Hz,
H-6), 3.36 (s, 1H, OH), 2.57 (dd, 2H, J ¼ 14.3 Hz, CH2CO), 1.18 (t, 3H,
J ¼ 7.1 Hz, CH3 Et). 13C NMR (100 MHz, CDCl3): d 170.5 (C55O), 138.6, 138.4,
138.1 (3 ꢀ Cq Bn), 129.8, 129.7 (C-2, C-3), 128.3–127.5 (CHarom), 81.3 (C-5),
80.4 (C-6), 80.1 (C-4), 75.5, 75.2 (2 ꢀ CH2 Bn), 72.2 (C-1), 71.8 (CH2 Bn), 60.6
(CH2 Et), 43.4 (CH2CO), 14.1 (CH3 Et). IR (thin film): 3030, 1728, 1497, 1454,
1367, 1302, 1209, 1177, 1067, 1026, 734, 696 cm21. MS (ESI): m/z ¼ 503.4
[M þ H]þ, 520.3 [M þ NH4]þ, 1005.7 [2M þ H]þ, 1022.7 [2M þ NH4]þ.
(3R/S,4S,5R,6S)-4,5,6-Tris-benzyloxy-1-(tert-butyldimethyl-
silyloxy)-3- ethoxycarbonylmethylcyclohexene (7)
SnCl4 mediated Mukaiyama-Michael addition: To a solution of unsatu-
rated ketone 4 (57 mg, 0.14 mmol) in freshly distilled DCM (1.0 mL) was
added a solution of silylketen acetal (50 mg, 0.25 mmol, 1.8 equiv.) in DCM
(1.0 mL) under an argon atmosphere. The mixture was cooled to –788C and
two drops of SnCl4 were added. After 20 min TLC analysis (EtOAc/PE 1:4)
revealed complete consumption of starting material, water (0.5 mL) was

Novel Carbasugar Amino Acid Dipeptide Isostere
49
added, and the mixture was warmed to rt. The mixture was diluted with Et2O
and the organic layer was separated. After extraction of the aqueous layer with
Et2O, all organic layers were combined, dried (MgSO4), and concentrated.
Purification of the residue by column chromatography (EtOAc/PE 1:19) gave
ester 7 as a colorless oil as a mixture of diastereoisomers (47 mg,
0.076 mmol, 55%).
LiClO4 mediated Mukaiyama-Michael addition: To a solution of unsatu-
rated ketone 4 (83 mg, 0.20 mmol) in freshly distilled Et2O (2.0 mL) was
added a solution of silylketene acetal (61 mg, 0.30 mmol, 1.5 equiv.) in Et2O
(0.3 mL) at rt. Next, a 1.0 M solution of LiClO4 in Et2O (1.0 mL, 0.106 g, 5.0
equiv.) was added. After 48 h, TLC analysis (EtOAc/PE 1:4) showed all
starting material was converted into a higher running spot. The reaction was
quenched by addition of sat. aq. NaHCO3 and the organic layer was separated,
washed with brine, dried (MgSO4), and concentrated. Purification of the residue
as described above resulted in 1,4-addition product 7 (90 mg, 0.15 mmol, 73%)
as an inseparable mixture of diastereoisomers. 1H NMR did not provide
useful data. 13C NMR (200 MHz, CDCl3): d 173.0, 172.2 (C55O), 149.6, 149.2
(C-1), 128.2, 127.9, 127.8, 127.6, 127.4 (CHarom), 106.3, 106.1 (C-2), 84.7, 82.0,
81.2, 81.0, 80.0, 78.2 (C-4, C-5, C-6), 75.0, 74.8, 74.6, 74.0, 73.7, 72.1 (CH2
Bn), 60.3 (CH2 Et), 37.4, 36.1 (CH2C55O), 37.0, 33.0 (C-1), 25.8 (C-3), 18.2,
16.2 (Cq TBS), 14.2 (CH3 Et). MS (ESI): m/z ¼ 617.5 [M þ H]þ, 639.4 [M þ Na]þ.
(3S,4S,5R,6S)-4,5,6-Tris-benzyloxy-1-(tert-butyldimethyl-
silyloxy)-3- methoxycarbonylmethylcyclohexene (8)
To a 1.0 M solution of LiClO4 in Et2O (110 mL) was added compound 4
(4.575 g, 11.04 mmol) at rt. This mixture was stirred until the ketone dissolved
completely, followed by the addition of (tert-butyldimethylsilyloxy)-1-methox-
yethene (7.23 mL, 33.11 mmol, 3.0 equiv.). Stirring was continued for 30 min
after which TLC analysis (EtOAc/toluene 1:19) indicated complete disappear-
ance of starting material together with the formation of a higher running spot.
After quenching the reaction, by addition of sat. aq. NaHCO3, the organic layer
was separated and the aqueous layer was extracted once more with Et2O. All
ether layers were combined, dried (MgSO4), filtered, and concentrated. Purifi-
cation of the residue by silica gel column chromatography (EtOAc/toluene
1:49) gave methylester 8 (6.53 g, 10.83 mmol, 98%) as a single stereoisomer.
1
[a]2D0 –18.9 (c 1.0, CHCl3). H NMR (400 MHz, CDCl3): d 7.42–7.21 (m, 15H,
CHarom), 4.94 (d, 1H, J2,3 ¼ 5.8 Hz, H-2), 4.85 (d, 1H, J ¼ 11.0 Hz, CH Bn),
4.82 (d, 1H, J ¼ 11.0 Hz, CH Bn), 4.71 (d, 1H, J ¼ 11.0 Hz, CH Bn), 4.70
(d, 1H, J ¼ 11.0 Hz, CH Bn), 4.63 (s, 2H, CH2 Bn), 4.00 (d, 1H, J6,5 ¼ 6.2 Hz,
H-6), 3.83 (dd, 1H, J5,4 ¼ 9.8 Hz, J5,6 ¼ 6.2 Hz, H-5), 3.72 (dd,
1H, J4,3 ¼ 5.6 Hz, J4,5 ¼ 9.8 Hz, H-4), 3.59 (s, 3H, CH3 OMe), 3.10 (ddt,
1H, J3,CHH ¼ J3,4 ¼ 5.6 Hz, J3,CHH ¼ 8.9 Hz, J3,2 ¼ 5.8 Hz, H-3), 2.79 (dd, 1H,

B. Lastdrager et al.
50
JCHH,CHH ¼ 15.8 Hz, JCHH,3 ¼ 5.6 Hz, CHH), 2.26 (dd, 1H, JCHH,CHH ¼ 15.8
Hz, JCHH,3 ¼ 8.9 Hz, CHH), 0.92 (s, 9H, 3ꢀ CH3 t-Bu), 0.16 (s, 3H, CH3
TBS), 0.15 (s, 3H, CH3 TBS). 13C NMR (100 MHz, CDCl3): d 172.6 (C55O),
149.3 (C-1), 138.4, 138.1 (3 ꢀ Cq Bn), 128.6, 128.2, 128.1, 127.8, 127.7, 127.4,
127.3, 127.2, 127.0 (CHarom), 106.0 (C-2), 80.6 (C-6), 79.4 (C-5), 77.8 (C-4),
74.0, 73.5, 71.1 (3ꢀ CH2 Bn), 50.8 (CH3 OMe), 35.5 (CH2CO), 32.7 (C-3), 25.4
(CH3 t-BuSi), 17.8 (Cq t-BuSi), 24.7, 24.9 (2 ꢀ CH3 SiMe). IR (thin film):
3032, 2928, 2858, 2359, 1734, 1661, 1454, 1205, 1094, 839, 696 cm21. MS
(ESI): m/z ¼ 603.4 [M þ H]þ, 625.4 [M þ Na]þ. HRMS (ESI): calcd for
[C36H46O6Si þ NH4]þ 620.3407. Found 620.3439.
(2S,3R,4S,5R)-2,3,4-Tris-benzyloxy-5-methoxycarbonyl-
methylcyclohexanone (9)
To a solution of enol ether 8 (0.301 g, 0.50 mmol) dissolved in THF (2.0 mL)
.
and pyridine (0.5 mL) was added HF pyridine (70/30 v/v, 0.25 mL). After
30 min water and Et2O were added to the reaction mixture. The organic
phase was separated and the aqueous phase was extracted once more with
Et2O. All organic layers were combined, dried (MgSO4), filtered, and concen-
trated. Purification of the residue by column chromatography (EtOAc/PE 1:9
to 1:4) gave ketone 9 (0.242 g, 0.495 mmol) in a yield of 99%. [a]2D0 –60.6 (c
1.0, CHCl3). 1H NMR (400 MHz, CDCl3): d 7.39–7.7.22 (m, 15H, CHarom),
4.86 (d, 1H, J ¼ 11.8 Hz, CH Bn), 4.75 (d, 1H, J ¼ 11.1 Hz, CH Bn), 4.71
(d, 1H, J ¼ 11.1 Hz, CH Bn), 4.60 (d, 1H, J ¼ 11.4 Hz, CH Bn), 4.50 (d, 1H,
J ¼ 11.8 Hz, CH Bn), 4.49 (d, 1H, J ¼ 11.4 Hz, CH Bn), 4.09 (d, 1H,
J ¼ 6.9 Hz, H-2), 3.80–3.75 (m, 2H, H-3, H-4), 3.59 (s, 3H, CH3 OMe), 2.95–
2.87 (m, 1H, H-5), 2.55 (dd, 1H, J ¼ 5.5 Hz, J ¼ 16.2 Hz, CHH), 2.46 (dd, 1H,
J ¼ 6.0 Hz, J ¼ 15.4 Hz, H-6a), 2.40 (dd, 1H, J ¼ 6.7 Hz, J ¼ 15.4 Hz, H-6b),
2.15 (dd, 1H, J ¼ 8.9 Hz, J ¼ 16.2 Hz, CHH). 13C NMR (100 MHz, CDCl3): d
205.7 (C-1), 172.3 (C55O CO2Me), 137.9, 137.6, 137.5 (3 ꢀ Cq Bn), 128.2–
127.6 (CHarom), 85.0, 81.6, 79.0 (C-2, C-3, C-4), 74.1, 73.0, 71.8 (3 ꢀ CH2 Bn),
51.6 (CH3 OMe), 40.2 (C-6), 33.7 (CH2CO), 32.0 (C-5). IR (thin film): 1730,
1497, 1454, 1437, 1352, 1205, 1092, 1074, 1051, 1026, 908, 731, 696,
611 cm21. MS (ESI): m/z ¼ 489.2 [M þ H]þ, 511.5 [M þ Na]þ. HRMS (ESI):
calcd for [C30H32O6 þ NH4]þ 506.2543. Found 506.2543.
(4S,5R)-2,4-Bis-benzyloxy-5-methoxycarbonylmethylcy-
clohex-2-enone (10)
Ketone 9 degraded into unsaturated ketone 10 upon storage at room temp-
erature: 1HNMR (200 MHz, CDCl3): d 7.38–7.22 (m, 15H, CHarom), 5.86 (d, 1H,
J3,4 ¼ 5.1 Hz, H-3), 4.89 (d, 1H, J ¼ 13.2 Hz, CH Bn), 4.82 (d, 1H, J ¼ 13.2 Hz,
CH Bn), 4.48 (d, 1H, J ¼ 11.7 Hz, CH Bn), 4.41 (d, 1H, J ¼ 11.7 Hz, CH Bn), 4.24

Novel Carbasugar Amino Acid Dipeptide Isostere
51
(dd, 1H, J4,5 ¼ 2.9 Hz, J4,3 ¼ 5.1 Hz, H-4), 3.65 (s, 3H, CH3 OMe), 2.81-2.29
(m, 5H, H-5, H-6a, H-6b, CHH, CHH). 13C NMR (50 MHz, CDCl3): d 192.4 (C-
1), 172.1 (C55O CO2Me), 150.3 (C-2), 137.6, 135.3 (2 ꢀ Cq Bn), 128.2, 127.9,
127.8, 127.6, 127.3, 126.8, 126.4 (CHarom), 115.1 (C-3), 71.5 (C-4), 70.3, 69.1
(2 ꢀ CH2 Bn), 51.2 (CH3 OMe), 39.3 (C-6), 35.2 (C-5), 34.3 (CH2CO2Me). IR
(thin film): 2363, 2343, 1734, 1697, 1624, 1497, 1456, 1261, 1204, 1140, 1067,
1028, 999, 739, 698, 623 cm21. MS (ESI): m/z ¼ 403.1 [M þ Na]þ.
(1S,2R,3S,4R,6S)-1,2,3-Tris-benzyloxy-6-(para-
methoxybenzylamino)-4- methoxycarbonyl-
methylcyclohexane (11)
.
Enol ether 8 (0.602 g, 1.00 mmol) was cleaved using HF pyridine, following
the procedure described above for the preparation of 9. After workup, without
further purification, intermediate 9 was dissolved in 1,2-DCE (10 mL),
followed by addition of Na(OAc)3BH (0.318 g, 1.50 mmol, 1.5 equiv.). Next,
p-MeOBnNH2 (0.261 mL, 2.0 mmol, 2.0 equiv.) and acetic acid (57.7 mL,
1.0 mmol, 1.0 equiv.) were added. The reaction was stirred for 1 h at rt, after
which TLC analysis (EtOAc/PE 3:7) indicated the complete disappearance of
starting material along with the formation of three lower running spots. The
reaction was quenched by the addition of sat. aq. NaHCO3 and diluted with
DCM. The organic phase was collected, dried (MgSO4), concentrated, and
purified by column chromatography (MeOH/DCM 1:99) resulting in protected
CSAA 11 (0.311 g, 0.51 mmol) in a yield of 51% over two steps. [a]2D0 þ17.0 (c
0.5, CHCl3). 1H NMR (400 MHz, MeOD, T ¼ 333K): d 7.36–7.23 (m, 15H,
CHarom Bn), 7.20–7.15 (m, 2H, CHarom PMB), 6.84–6.80 (m, 2H, CHarom
PMB), 4.68 (s, 2H, CH2 Bn), 4.66 (d, 1H, J ¼ 11.9 Hz, CH Bn), 4.55 (d, 1H,
J ¼ 11.9 Hz, CH Bn), 4.47 (s, 2H, CH2 Bn), 3.89–3.86 (m, 1H, H-1), 3.85–3.82
(m, 1H, H-3), 3.78–3.75 (m, 1H, H-2), 3.75 (s, 3H, CH3 OMe PMB), 3.61
(s, 3H, CH3 OMe), 3.02–2.96 (m, 1H, H-6), 2.69–2.60 (m, 1H, H-4), 2.53 (dd,
1H, J ¼ 7.2 Hz, JCHH,CHH ¼ 15.4 Hz, CHH), 2.20 (dd, 1H, J ¼ 7.4 Hz, JCHH,CH-
H ¼ CHH), 1.72 (ddd, 1H, J ¼ 4.1 Hz, J ¼ 6.8 Hz, J5a,5b ¼ 13.5 Hz, H-5a), 1.58
(ddd, 1H, J ¼ 3.8 Hz, J ¼ 9.0 Hz, J5b,5a ¼ 13.5 Hz, H-5b). IR (thin film): 2872,
1732, 1611, 1510, 1454, 1246, 1099, 737, 698 cm21. MS (ESI): m/z ¼ 610.4
[M þ H]þ, 1220.0 [2M þ H]þ. HRMS (ESI): calcd for [C38H43NO6 þ H]þ
610.3169. Found 610.3213.
(3S,4R,6S)-1,3-Bis-benzyloxy-6-(para-methoxybenzyl-
amino)-4- methoxycarbonylmethylcyclohex-1-ene (12)
Enamine 12 (73.0 mg, 0.15 mmol, 15% over two steps) was formed as a
minor product in the procedure described above, going from compound 8 to
amine 11. 1H NMR (300 MHz, CDCl3): d 7.37–7.22 (m, 10H, CHarom Bn),

B. Lastdrager et al.
52
7.20–7.16 (m, 2H, CHarom PMB), 6.84–6.78 (m, 2H, CHarom PMB), 5.09 (d, 1H,
J2,3 ¼ 5.4 Hz, H-2), 4.75 (s, 2H, CH2 Bn), 4.61 (d, 1H, J ¼ 11.8 Hz, CH Bn), 4.40
(d, 1H, J ¼ 11.8 Hz, CH Bn), 3.97 (dd, 1H, J3,4 ¼ 3.3 Hz, J3,2 ¼ 5.4 Hz, H-3),
3.77–3.67 (m, 2H, CH2 PMB), 3.76 (s, 3H, CH3 OMe PMB), 3.64 (s, 3H, CH3
OMe), 3.47 (dd, 1H, J ¼ 6.4 Hz, J ¼ 10.1 Hz, H-6), 2.65 (dd, 1H, J ¼ 7.4 Hz,
JCHH,CHH ¼ 16.0 Hz, CHH), 2.49 (dd, 1H, J ¼ 7.0 Hz, JCHH,CHH ¼ 16.0 Hz,
CHH), 2.24–2.15 (m, 1H, H-4), 2.00–1.88 (m, 1H, H-5a), 1.86–1.79 (m, 1H,
H-5b). 13C NMR (50 MHz, CDCl3): d 173.5 (C55O), 158.8 (Cq OMe PMB),
158.4 (C-1), 139.1, 136.6 (2 ꢀ Cq Bn), 132.3 (Cq PMB), 129.3 (CHarom PMB),
128.4. 128.1. 127.9, 127.5, 127.2 (CHarom Bn), 113.6 (CHarom PMB), 96.6 (C-3),
72.0 (C-4), 69.8, 69.1 (2ꢀ CH2 Bn), 55.1 (CH3 OMe PMB), 54.9 (C-6), 51.3
(CH3 OMe), 49.3 (CH2 PMB), 36.4 (CH2CO2), 34.9 (C-4), 29.5 (C-5). MS (ESI):
m/z ¼ 502.3 [M þ H]þ, 1003.6 [2M þ H]þ.
(1S,2R,3S,4R,6S)-6-Amino-1,2,3-tris-benzyloxy-4-
methoxycarbonylmethylcyclohexane (13)
To a solution of compound 11 (0.476 g, 0.781 mmol) in a mixture of aceto-
nitrile (6 mL) and water (3 mL) was added CAN (1.07 g, 1.95 mmol, 2.5
equiv.). This orange two-phase system was vigorously stirred for 24 h. After
addition of sat. aq. NaHCO3 followed by dilution with EtOAc, the aqueous
phase was separated and washed twice with EtOAc. All organic layers were
combined, dried (MgSO4), and concentrated. Purification of the residue by
silica gel chromatography (MeOH/DCM 1:19) gave title compound 13
(0.214 g, 0.437 mmol, 56%). 1H NMR (400 MHz, MeOD): d 7.39–7.22
(m, 15H, CHarom), 4.67 (d, 1H, J ¼ 12.3 Hz, CH Bn), 4.62 (d, 1H, J ¼ 12.3 Hz,
CH Bn), 4.59 (s, 2H, CH2 Bn), 4.54 (d, 1H, J ¼ 12.1 Hz, CH Bn), 4.48 (d, 1H,
J ¼ 12.1 Hz, CH Bn), 3.88 (ddd, 1H, J ¼ 1.2 Hz, J ¼ 3.1 Hz, J2,1 ¼ 4.3 Hz,
H-2), 3.78 (dd, 1H, J1,2 ¼ 3.1 Hz, J ¼ 4.4 Hz, H-1), 3.71 (m, 1H, H-3),
3.65–3.55 (m, 1H, H-6), 3.63 (s, 3H, CH3 OMe), 2.52 (m, 1H, H-4), 2.45 (dd,
JCHH,4 ¼ 7.7 Hz, JCHH,CHH ¼ 15.8 Hz, CHH), 2.27 (dd, 1H, JCHH,4 ¼ 6.5 Hz,
JCHH,CHH ¼ 15.8 Hz, CHH), 1.80 (m, 2H, H-5a, H-5b). MS (ESI): m/z ¼ 490.3
[M þ H]þ, 979.7 [2M þ H]þ.
(1S,2R,3S,4R,6S)-6-Amino-1,2,3-tris-benzyloxy-4-
carboxymethylcyclohexane (14)
To a solution of ester 13 (95 mg, 0.194 mmol) in 1,4-dioxane (2.0 mL), was
added an aq. solution of LiOH (0.5 mL, 1.0 M). After 3 h, TLC analysis
(MeOH/DCM 15:85) showed complete conversion of starting material into a
lower running spot. The mixture was neutralized with 1.0 M aq. HCl to pH 7
and extracted thoroughly with EtOAc (three times). The combined organic
phases were dried (MgSO4) and concentrated to give amino acid 14 (92 mg,

Novel Carbasugar Amino Acid Dipeptide Isostere
53
1
0.194 mmol) in quantitative yield. H NMR (400 MHz, MeOD): d 7.33–7.26
(m, 15H, CHarom), 4.63 (d, 1H, J ¼12.0 Hz, CH Bn), 4.57 (d, 1H, J ¼ 12.0 Hz,
CH Bn), 4.53 (d, 1H, J ¼ 11.9 Hz, CH Bn), 4.43 (d, 1H, J ¼ 11.9 Hz, CH Bn),
4.40 (d, 1H, J ¼ 11.8 Hz, CH Bn), 4.27 (d, 1H, J ¼ 11.8 Hz, CH Bn),
3.85–3.82 (ddd, 1H, J ¼ 1.3 Hz, J ¼ 2.9 Hz, J2,1 ¼ 4.3 Hz, H-2), 3.76 (dd, 1H,
J ¼ 3.1 Hz, J1,2 ¼ 4.3 Hz, H-1), 3.76 (m, 1H, H-3), 3.70 (dd, 1H, J ¼ 3.6 Hz,
J ¼ 7.3 Hz, H-6), 2.51 (m, 1H, H-4), 2.42 (dd, 1H, JCHH,4 ¼ 7.2 Hz,
JCHH,CHH ¼ 16.0 Hz, CHH), 2.29 (1H, dd, JCHH,4 ¼ 6.6 Hz, JCHH,CHH ¼ 16.0
Hz, CHH), 1.99-1.92 (dddd, 1H, J ¼ 1.2 Hz, J ¼ 3.1 Hz, J ¼ 4.1 Hz,
J5a,5b ¼ 14.8 Hz, H-5a), 1.86–1.77 (dd, 1H, J ¼ 3.8 Hz, J5b,5a ¼ 14.8 Hz,
H-5b). MS (ESI): m/z ¼ 476.2 [M þ H]þ.
(1S,2R,3S,4R,6S)-N-(9-Fluorenylmethoxycarbonyl)-
6-amino-1,2,3-tris-benzyloxy-4-carboxymethyl-
cyclohexane (15)
To a suspension of amino acid 14 (0.194 mmol) in dioxane (0.5 mL) and sat.
aq. NaHCO3 (2.0 mL) was added Fmoc-OSu (85 mg, 0.252 mmol, 1.3 equiv.).
After stirring for 17 h, TLC analysis (MeOH/DCM 1:19) revealed complete con-
sumption of starting material into a higher running spot. Water and dioxane
were added to the suspension and the resulting solution was acidified with
1.0 M aq. HCl to pH 5. The mixture was diluted with EtOAc, the organic
layer was separated, and the aqueous layer was extracted twice with EtOAc.
All organic layers were combined, dried (MgSO4), filtered, and concentrated.
After purification by silica gel column chromatography (EtOAc/PE
1:1 þ 1.0% HOAc), carbamate 15 was obtained (0.124 g, 0.178 mmol, 92%).
[a]2D0 þ 25.5 (c 1.0, CHCl3). H NMR (600 MHz, C6D6): d 7.56–7.54 (m, 4H,
1
CHarom Fmoc), 7.37–7.30 (m, 4H, CHarom Fmoc), 7.22–7.06 (m, 15H, CHarom
Bn), 6.79 (bs, 1H, NH), 4.64 (d, 1H, J ¼ 11.8 Hz, CH Bn), 4.60 (d, 1H,
J ¼ 11.8 Hz, CH Bn), 4.58 (m, 1H, H-6), 4.54 (d, 1H, J ¼ 11.8 Hz, CH Bn),
4.50 (dd, 1H, J ¼ 10.6 Hz, J ¼ 7.3 Hz, CHH, CH2Fmoc), 4.27 (dd, 1H,
J ¼ 10.6 Hz, J ¼ 7.3 Hz, CHH CH2Fmoc), 4.22 (m, 1H, CH Bn), 4.16 (m, 3H,
CH2 Bn, CH Fmoc), 3.84 (m, 1H, H-2), 3.74 (m, 1H, H-3), 3.71 (m, 1H, H-1),
2.69 (m, 1H, H-4), 2.40 (m, 1H, CHH CH2CO2H), 2.11 (m, 1H, CHH
CH2CO2H), 1.92 (m, 1H, H-5a), 1.60 (m, 1H, H-5b). 13C-NMR (100 MHz,
CDCl3): d 177.6 (C55O CO2H), 156.5 (C55O Fmoc), 144.1, 141.2 (2 ꢀ Cq
Fmoc), 138.3, 137.9, 137.8 (3 ꢀ Cq Bn), 129.0, 128.5, 128.4, 128.4, 127.9,
127.8, 127.6, 127.5, 127.0 (CHarom Bn), 125.3, 119.8 (CHarom Fmoc), 77.3 77.0,
76.7 (C-1, C-2, C-3), 73.8, 72.6, 70.4 (3 ꢀ CH2 Bn), 66.8 (CH2 Fmoc), 47.8
(C-6), 47.2 (CH Fmoc), 30.7 (CH2CO2), 29.7 (C-4), 28.2 (C-5). IR (thin film):
3032, 2924, 2870, 2363, 2341, 1705, 1514, 1452, 1248, 1055, 739, 698 cm21
.
MS (ESI): m/z ¼ 698.5 [M þ H]þ, 1395.6 [2M þ H]þ. HRMS (ESI): calcd for
[C44H43NO7 þ NH4]þ 715.3383. Found 715.3370.

B. Lastdrager et al.
54
H-Phe-Leu-Wang resin (17)
Loading of resin: Commercially available Wang resin 16 (0.96 mmol/g,
1.79 g) was allowed to swell in DCM (40 mL). A solution was prepared of DIC
(1.07 mL, 0.87 g, 6.88 mmol, 4.0 equiv.), Fmoc-Leu-OH (2.43 g, 6.88 mmol, 4.0
equiv.), and DMAP (cat.) in DCM. The mixture was left for 16 h with occasional
shaking. The resin was filtered, subsequently washed with DMF and DCM,
and dried (air). The loading was determined by treatment of the dried resin
(2.3 mg) with a solution of 20% piperidine/DMF (1.0 mL). After stirring for
10 min followed by dilution to 10.00 mL with EtOH, the absorption of the
solution was measured at 300 nm. The loading was calculated to be
0.49 mmol/g, using the formula: Loading (mmol/g) ¼ [A300]ꢁ10/[7.8ꢁm].
Peptide coupling: Fmoc-Leu-Wang-resin (1.00 g, 0.49 mmol) was treated with
10 mL 20% piperidine/DMF (3 ꢀ 10 min) to effect Fmoc cleavage. The resin was
filtered, washed (DMF and DCM), and swollen in DMF. The resin was treated
with a solution of Fmoc-Phe-OH (0.75 g, 1.94 mmol, 4.0 equiv.), HCTU (0.80 g,
1.94 mmol, 4.0 equiv.), and DiPEA (0.64 mL, 3.88 mmol, 8.0 equiv.) in DMF
(5.0 mL). After shaking the mixture for 1 h, the resin was filtered and rinsed
with DMF. This procedure was repeated twice to ensure complete coupling indi-
cated by a negative Kaiser test. Any unreacted amines were capped using a
solution of 0.5 M Ac2O and 0.125 M DiPEA in DMF (50 mL, 5 min). After filtration
and washing with DMF and DCM, the resin was filtered and dried by an air flow.
The Fmoc was removed with 20% piperidine/DMF solution as described above.
Fmoc-CSAA-(OBn)3-Phe-Leu-Wang resin (18)
Resin 17 (70.0 mg, 34.3 mmol) was swollen in DMF. CSAA 15 (47.9 mg,
68.7 mmol, 2.0 equiv.) in DMF (700 mL) was preactivated for 30 sec with
HATU (12.0 mg, 32.3 mmol, 0.95 equiv.) and DiPEA (15.0 mL, 84.9 mmol, 2.5
equiv) and the mixture was subsequently added to the resin and shaken for
10 min. After filtration of the resin, followed by rinsing with DMF, this
coupling procedure was repeated once more. After a negative Kaiser test
revealed complete consumption of free amines, the resin was washed with
DMF and DCM and dried (air flow).
Boc-Tyr(t-Bu)-CSAA-(OBn)3-Phe-Leu-Wang resin (19)
Resin 18 was swollen in DMF followed by Fmoc cleavage using a 20% piper-
idine/DMF (3 ꢀ 10 min), washed with DMF and DCM, filtered, and dried (air).
Boc-Tyr(t-OBu)-OH (50.6 mg, 0.15 mmol, 4.4 equiv.) was coupled using HCTU
(62.1 mg, 0.15 mmol, 4.4 equiv.) and DiPEA (49.6 mL, 0.300 mmol, 8.8 equiv.) in
DMF and shaken for 2 h. A negative Kaiser test indicated a complete coupling
of the free amines. The resin was filtered, washed with DMF and DCM, and
air dried.

Novel Carbasugar Amino Acid Dipeptide Isostere
55
H-Tyr-CSAA-Phe-Leu-OH (21)
Cleavage from the resin along with the removal of the t-Bu and Boc group
was effected transferring immobilized peptide 19 into a glass tube followed by
the addition of a mixture of TFA/TIS/water (95:2.5:2.5, 1.0 mL). After
shaking for 15 min, the mixture was filtered, washed with DMF and DCM,
and dried. Purification by silica gel chromatography (MeOH/DCM 1:9) gave
compound 20 (29.4 mg, 32.7 mmol, 95%). LC/MS (50% to 90% acetonitrile/
water), Rt 10.02 min. MS (ESI): m/z ¼ 899.9 [M þ H]þ, 1799.4 [2M þ H]þ.
Compound 20 (16.2 mg, 18.0 mmol) was dissolved in t-BuOH (1.0 mL) and
water (1.0 mL), and the resulting solution was degassed. A catalytic
amount of Pd/C was added and after degassing the solution was stirred
under a hydrogen atmosphere for a second time. After 15 h, TLC analysis
(MeOH/DCM 15:85) indicated complete conversion of starting material into
a lower running spot. The reaction mixture was filtered over Celite and the
filtrate was concentrated to give Leu-enkephalin analog 21 in quantitative
yield (11.3 mg, 18.0 mmol). After HPLC purification (18% to 30% aceto-
nitrile/water), an analytical sample was obtained. LC/MS (10–40% aceto-
nitrile/water), Rt 14.02 min. MS (ESI): m/z ¼ 629.5 [M þ H]þ, 651.4
[M þ Na]þ, 1257.8 [2M þ H]þ. 1H NMR (600 MHz, DMSO d6, T ¼ 313K): d
9.25 (s, 1H), 8.09 (d, 1H, J ¼ 7.2 Hz), 7.99 (bs, 1H), 7.84 (d, 1H, J ¼ 7.2 Hz),
7.32–6.91 (m, 9H), 6.71–6.68 (m, 2H), 4.62 (m, 1H), 4.48 (m, 1H), 4.20
(m, 1H), 4.04–3.93 (m, 2H), 3.69 (m, 1H), 3.59 (m, 1H), 3.35–3.21 (m, 2H),
3.08–3.00 (m, 2H), 2.81 (m, 1H), 2.78 (m, 1H), 2.64 (m, 1H), 2.31 (m, 1H),
2.23 (m, 1H), 1.95 (m, 1H), 1.77–1.74 (m, 2H), 1.75 (m, 1H), 1.71–1.66
(m, 2H), 0.89 (m, 6H). IR (thin film): 3275, 2963, 2361, 2341, 1678,
1015 cm21. HRMS (ESI): calcd for [C32H44N4O9 þ H]þ 629.3181. Found
629.3178. The following sequence ions were detected after recording a MS/
MS spectrum:

B. Lastdrager et al.
56
ACKNOWLEDGEMENTS
We thank the Dutch Science Foundation (NWO-CW) for financial support,
Kees Erkelens and Fons Lefeber for their assistance with NMR measurements,
and Hans van den Elst for recording mass spectra.
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