Synthesis of sugar-lysine chimera with integrated gluco-configured 1,3-hydroxyamine motif

Article abstract of DOI:10.1055/s-2007-968002

The paper describes the synthesis of glucose-configured sugar-lysine chimeras in which the side chain of lysine is conformationally constrained via incorporation into a D-glucose scaffold. Key step in the synthesis is a high-yielding, reductive ring opening of an exocyclic glucose-derived epoxide to form an α-hydroxyester that can be converted into chimeric sugar-lysine analogues. To demonstrate the use of these novel chimeric sugar-lysine building blocks in peptide coupling, we replaced D-lysine in the antimicrobial dipeptide sequence kW by a D-glucose-D-lysine chimera. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-2007-968002

LETTER
239
Synthesis of Sugar–Lysine Chimera with Integrated gluco-Configured
1,3-Hydroxyamine Motif
S
ynthesis of Sugar
a
–Lysine Ch
i
imera dong Zhang, Jialiang Wang, Zhizhi Sun, Dung-Huang Nguyen, Frank Schweizer*
Department of Chemistry, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada
Fax +1(204)4747608; E-mail: schweize@cc.umanitoba.ca
Received 4 July 2006
activity of AMPs.4 In addition, post-translational glyco-
sylation of the antimicrobial peptide drosocin8,9 provided
analogues with increased antibacterial activity. In order to
study the effect of artificial hydroxylation and glyco-
Abstract: The paper describes the synthesis of glucose-configured
sugar–lysine chimeras in which the side chain of lysine is confor-
mationally constrained via incorporation into a D-glucose scaffold.
Key step in the synthesis is a high-yielding, reductive ring opening
of an exocyclic glucose-derived epoxide to form an a-hydroxyester sylation of lysine in antibacterial peptides we describe
that can be converted into chimeric sugar–lysine analogues. To
demonstrate the use of these novel chimeric sugar–lysine building
blocks in peptide coupling, we replaced D-lysine in the antimicro-
natural configurations at the a-carbon. Compound 1
bial dipeptide sequence kW by a D-glucose–D-lysine chimera.
here the synthesis of gluco-configured, conformationally
constrained GlcLysC analogues 1 with natural or un-
contains the gluco-configured RNA 1,3-hydroxyamine
binding motif10 of aminoglycoside antibiotics that has
Key words: sugar–lysine chimera, reductive ring opening, ep-
oxides
been proposed to interact as bidentate RNA hydrogen
bond donor to the phosphodiester backbone or Hoogsteen
face of guanosine (Figure 2). Incorporation of 1 into short
Amino acids containing basic side chains (lysine, orni-
antibacterial peptides may introduce novel and synergistic
thine and arginine) occur frequently in antimicrobial pep-
effects, which could improve the biological, pharmaco-
tides (AMPs). Although the mode of action of AMPs is
logical and chemical properties of antibacterial peptides.
not fully understood, most AMPs appear to manifest their
biological action by enhancing the permeability of lipid
NH2
membranes of pathogenic cells. This typically involves
NH2
NH2
NH2
O
initial electrostatic interactions between the positively
charged basic side chains to the negatively charged lipid
membrane of pathogens, followed by adoption of an
amphipathic a-helical or b-sheet structure.1,2 Although
more potent antibiotics exist, the ability to kill target cells
rapidly, unusually broad activity spectra against some of
the more serious antibiotic resistant pathogens and rela-
tive difficulty with which mutants develop resistance in
vitro make AMPs attractive targets for drug develop-
ment.1,2 However, in vivo studies of many cationic pep-
tide antibiotics have been disappointing most likely due to
the fact that many AMPs exhibit poor bioavailability, sus-
ceptibility to proteolytic cleavage and low antimicrobial
activity.3 In order to overcome some of these drawbacks
novel approaches are in high demand. This paper de-
scribes the synthesis of sugar–lysine chimeras (SlysC, 1,
Figure 1). GlcLysCs are molecules in which a cyclic C-
glycosidic scaffold becomes part of, or mimics, the regu-
lar side chain of lysine. The cyclic nature of the carbohy-
drate constrains the side chain of lysine while the
polyfunctional nature of the scaffold introduces chemical
diversity and artificial post-translational modifications
such as hydroxylation and glycosylation. It is noteworthy
that post-translational hydroxylation of lysine,4 arginine4
and other amino acids5–7 has enhanced the biological
OH
OH
HO
O
O
1
lysine
Figure 1 Structure of a sugar–lysine chimera (SLysC) 1.
The synthesis started with the readily available D-gluco-
configured lactone 211 (Scheme 1) which reacts with the
enolate of a-bromo acetic acid methylester generated
from lithium bis(trimethylsilyl)amide [LiN(SiMe3)2] in
THF at –78 °C, to produce the exocyclic epoxide 3 in 80%
yield as a single stereoisomer.12 Trimethylsilyltrifluoro-
methanesulfonate (TMSOTf)-promoted reductive ring
opening of epoxide 3 with tributyltin hydride in dichlo-
romethane at 0 °C afforded a mixture containing silylether
4 and alcohol 5 (ratio 4:5 = 3.5:1) in a combined yield of
88%. Compounds 4 and 5 were obtained as a single dia-
stereoisomer. The silylether 4 was hydrolyzed quantita-
tively into alcohol 5 by exposure to trifluoroacetic acid
containing wet THF. The stereochemistry at C-2 of alco-
hol 5 was determined by conversion of 5 into the known
benzylester 6.13 This was achieved in a two-step proce-
dure. At first, methylester 5 was hydrolyzed to the corre-
sponding acid with lithium hydroxide in wet THF.
Subsequently, esterfication of the acid using cesium
carbonate and benzylbromide in DMF afforded benzyl-
ester 6 as a single product in 89% yield. Hydroxyester 5
served as starting material to install an amino function at
C-2. Initially, we activated the hydroxyl group as a tri-
SYNLETT 2007, No. 2, pp 0239–0242
0
1.
0
2.
2
0
0
7
Advanced online publication: 24.01.2007
DOI: 10.1055/s-2007-968002; Art ID: S14206ST
© Georg Thieme Verlag Stuttgart · New York

240
K. Zhang et al.
LETTER
O
N
N
RO
OR
O
H
HN
P
N
H
H
N
H
O
N
O
H
antibacterial
peptide
NH2
O
H
N
NH
H
N
O
H
O
O
HO
antibacterial
peptide
N
NH2
H
NH
OH
O
H
O
O
N
HO
OH
O
Figure 2 Proposed mechanism of an antibacterial peptide containing a 1,3 hydroxyamine binding motif for RNA recognition. Phosphodiesters
and the Hoogsteen face of guanosine represent bidentate RNA hydrogen bond acceptors that may be recognized by hydroxyamines (modified
from ref. 10).
fluoromethanesulfonate ester 7 using trifluoromethane function into the carbohydrate scaffold. This was
sulfonic anhydride in pyridine. Subsequently, we studied achieved by selective tosylation of the primary hydroxy
the displacement reaction of triflate 7 with two nucleo- position using p-toluenesulfonic chloride in pyridine fol-
philes benzylamine and sodium azide. Both reactions pro- lowed by nucleophilic displacement of the tosylate with
vided the corresponding amino ester 8 and azide 9 in 56% sodium azide to produce compound 11 in 65% isolated
and 81% yield, respectively. Catalytic hydrogenation of 8 yield. Exposure of ester 11 to basic conditions (LiOH,
and 9 using Pearlman’s catalyst followed by protection of THF–H2O) resulted in partial epimerization at the C-2
the amino function as tert-butyloxycarbamate provided position, affording an inseparable mixture of acids 12 and
protected amino ester 10 in 90% yield. Compound 10 13. Acids 12 and 13 were converted into the epimeric
served as starting material to introduce a second amino sugar–lysine hybrids 14 and 15 (ratio 14:15 = 4:1) by
OBn
O
OBn
O
OBn
O
R1
H
BnO
BnO
BnO
BnO
R2
b)
O
BnO
BnO
a)
BnO
OBn
O
ref. 15
O
CO2Me
OBn
4: R1 = OTMS; R2 = OMe
5: R1 = OH; R2 = OMe
6: R1 = OH; R2 = OBn
7: R1 = OTf; R2 = OMe
c)
e)
3
2
d)
f)
N3
OH
OBn
NHBoc
NHBoc
R
H
O
O
i)
HO
HO
O
HO
HO
O
O
O
g)
BnO
BnO
h)
OMe
OH
OH
O
OBn
O
11
n)
10
8: R = NHBn
9: R = N3
j)
14
FmocHN
NHBoc
O
R2
N3
O
R1
FmocHN
HO
H
NHBoc
OH
l)
O
m
HO
HO
R3O
R3O
N
O
R4
O
N
3
HO
H
1
5
OH
OR3
OH
O
O
12: R1 = H; R2 = NHBoc; R3 = H; R4 = OH
13: R1 = NHBoc; R2 = H; R3 = H; R4 = OH
14: R1 = H; R2 = NHBoc; R3 = OAc; R4 = OMe
15: R1 = NHBoc; R2 = H; R3 = OAc; R4 = OMe
BocN
16
17
k)
k)
Scheme 1 Reagents and conditions: (a) LiHMDS, CH2BrCOOMe, THF, –78 °C to r.t., 2 h, 80%; (b) Bu3SnH, TMSOTf, CH2Cl2, 0 °C, 30
min, 88%; (c) THF, TFA (2 equiv) 2 h quant.; (d) (i) LiOH (3.0 equiv), THF, H2O, 12 h; (ii) Cs2CO3, BnBr, DMF, 4 h, 89%; (e) Tf2O, pyridine,
CH2Cl2, 0 °C 1 h, quant.; (f) NaN3, CH2Cl2, 15-crown-5, r.t., 24 h, 81% or BnNH2, CH2ClCH2Cl, 50 °C, 24 h, 56%; (g) Pd(OH)2, MeOH, HCl
(1.3 equiv), quant.; (h) MeOH, Boc2O (2 equiv), Et3NH, 12 h, 90%; (i) TsCl (2.2 equiv), pyridine, r.t., 12 h; then work-up and NaN3, DMF,
80 °C, 12 h, 65%; (j) LiOH (5 equiv), THF, H2O, 8 h; (k) (i) Cs2CO3, DMF, MeI work-up; (ii) Ac2O, pyridine, 12 h, quant.; (l) Pd(OH)2, H2,
MeOH, 30 min then FmocOPfp (2 equiv), NaHCO3 (4 equiv), r.t., acetone–H2O (3:1), 2 h, 63%; (m) TBTU, H-Trp(Boc)-NHBn, DIEA, DMF,
80%; (n) Ac2O–pyridine.
Synlett 2007, No. 2, 239–242 © Thieme Stuttgart · New York

LETTER
Synthesis of Sugar–Lysine Chimera
241
esterfication (Cs2CO3, MeI, DMF) followed by acetyla-
tion of the hydroxyl groups (Ac2O, pyridine). At this stage
it was possible to separate the epimeric diastereomers by
flash chromatography. The major diastereomer was iden-
tical to compound 14 previously obtained by acetylation
of ester 11. Compounds 14 and 15 exhibit characteristic
1H NMR data that confirm their structure. For instance,
compound 14 shows the expected downfield shifts of H-
4, H-5 and H-6 (dH4,H5,H6 > 5.00 ppm) that are characteris-
tic for O-acetylation at C-4, C-5 and C-6. By comparison,
protons H-8a and H-8b in compound 15 appear high field
(dH8a,b = 3.20–3.33 ppm), clearly indicating the installa-
tion of the azido function at C-8. In addition, the observed
vicinal coupling constant between H-3 and H-4 of 9.3 Hz
demonstrates the diaxial relationship of these protons and
proves that no epimerization occurred at C-3 during treat-
ment with LiOH. Analogously, the epimeric sugar–lysine
hybrid 15 exhibits chemical shifts and vicinal diaxial
coupling constants (dH4,H5,H6 > 5.00 ppm; JH3,H4 = 9.9 Hz)
that confirm its C-2 epimeric structure.
References and Notes
(1) Hancock, R. E. W.; Lehrer, R. Trends Biotechnol. 1998, 16,
82.
(2) Hancock, R. E. W.; Scott, M. G. Proc. Natl. Acad. Sci.
U.S.A. 2000, 97, 8856.
(3) Latham, P. W. Nat. Biotechnol. 1999, 17, 755.
(4) Taylor, S. W.; Craig, A. G.; Fischer, W. H.; Park, M.;
Lehrer, R. I. J. Biol. Chem. 2000, 275, 38417.
(5) Reddy, M. V. R.; Harper, M. K.; Faulkner, D. J. Tetrahedron
1998, 54, 10649.
(6) Baldwin, J. E.; Claridge, T. D. W.; Goh, K. C.; Keeping, J.
W.; Schofield, C. J. Tetrahedron Lett. 1993, 34, 5645.
(7) Postels, H. T.; Koenig, W. A. Tetrahedron Lett. 1994, 35,
535.
(8) Bulet, P.; Dimarcq, J.-L.; Lagueux, M.; Charlet, M.; Hegy,
M.; Van Dorsselaer, A.; Hoffmann, J. A. J. Biol. Chem.
1993, 268, 14893.
(9) Bulet, P.; Urge, L.; Ohresser, S.; Hetru, C.; Otvos, L. Jr. Eur.
J. Biochem. 1996, 238, 64.
(10) Wong, C.-H.; Hendrix, M.; Manning, D. M.; Rosenbohm,
C.; Greenberg, W. A. J. Am. Chem. Soc. 1998, 120, 8319.
(11) Gueyrard, D.; Haddoub, R.; Salem, A.; Bacar, N. S.;
Goekjian, P. G. Synlett 2005, 520.
To demonstrate the use of GlcLysCs in peptide coupling
reactions we decided to convert azido acid 14 into Fmoc-
protected amino acid 16. This was achieved by catalytic
hydrogenation followed by selective protection of the
amino function using 9-fluorenylmethyl pentafluorophen-
yl carbonate (FmocOPfp) to produce 16 in 63% isolated
yield. The lysine analogue 16 is orthogonally protected to
be used in solution-phase peptide coupling. To study the
influence of the constrained sugar moiety and the pres-
ence of the gluco-configured 1,3-hydroxyamine motif on
the bioactivity of small antibacterial peptides, we decided
to incorporate GlcLysC into the amphiphilic antimicrobal
dipeptide sequence kW.14 This was achieved by coupling
of 16 to H-Trp(Boc)-NHBn using 2-(1H-benzotriazole-1-
yl)-1,1,3,3-tetra-methyluronium tetrafluoroborate (TB-
TU) as coupling reagent in DMF to produce dipeptide 17
in 80% isolated yield. During this coupling we did not ob-
serve ester formation as evidenced by MS analysis of the
crude product or exposure to basic conditions (K2CO3,
MeOH) as previously reported by Knorr et al.15
(12) Zhang, K.; Schweizer, F. Synlett 2005, 3111.
(13) Schweizer, F.; Inazu, T. Org. Lett. 2001, 3, 4115.
(14) Strom, M. B.; Haug, B. E.; Skar, M. L.; Stensen, W.; Stiberg,
T.; Svendsen, J. S. J. Med. Chem. 2003, 46, 1567.
(15) Knorr, R.; Treciak, A.; Bannwarth, E.; Gillessen, D.
Tetrahedron Lett. 1989, 30, 1927.
(16) Synthetic Procedures for Compounds 12–17.
Synthesis of Compounds 12–15.
Ester 11 (60 mg, 0.16 mmol) was treated with LiOH (7 mg,
0.31 mmol) for 8 h at r.t. in aq THF (1:1), and then acidified
with formic acid (100 mL). The solution was extracted with
EtOAc (6 × 10 mL) and the combined organic layer solvent
was dried (Na2SO4) and concentrated to afford inseparable
mixture of crude acids 12 and 13 (59 mg, quant.), which was
treated with Cs2CO3 (61 mg, 0.18 mmol) and MeI (30 mL,
0.48 mmol) in DMF. The reaction was worked up with H2O
and extracted with EtOAc (4 × 15 mL); the combined
organic phases were dried (Na2SO4) and concentrated. The
crude was acetylated by dissolving it in a 1:1 mixture
containing Ac2O (0.5 mL) and pyridine (0.5 mL). The crude
mixture was purified by the flash chromatography (EtOAc–
hexane, 1:2) to afford compound 14 (61 mg, 80%) and 15
(15 mg, 20%). Compound 14 was identical to the product
obtained by acetylation of compound 11.
In summary, we have developed a synthetic pathway into
suitably protected C-glycosidic glucose–lysine chimeras
with natural and unnatural C(a) configuration that can be
used in peptide coupling reactions without need for hy-
droxyl group protection. The carbohydrate scaffold induc-
es conformational constraint into the side chain of lysine
while, at the same time, introducing artificial post-transla-
tional modifications such as hydroxylation and glycosyla-
tion. Furthermore, the SLysC (1) incorporates the RNA-
recognizing gluco-configured putative 1,3-hydroxyamine
motif which may introduce synergistic effects when in-
corporated into antibacterial peptides. We are currently
studying the lysinemimetic and glycomimetic properties
of 1 in small peptides as well as the antimicrobial proper-
ties of 17.16
Synthesis of Compound 16.
Acid 12 (46 mg, 0.12 mmol) was dissolved in MeOH (4 mL)
and hydrogentated for 20 min using 20 wt% Pd/C. The
solution was filtered and the solvent was evaporated in
vacuo. The solid residue was dissolved in aq acetone (3 mL,
1:1) and treated with 9-fluorenylmethyl pentafluorophenyl
carbonate (91 mg, 0.24 mmol) and NaHCO3 (31 mg, 0.37
mmol) for 4 h at r.t. Then, H2O (10 mL) was added and the
aqueous layer was extracted with EtOAc (6 × 10 mL).
Finally, the solvent was dried (Na2SO4) and concentrated.
The crude product was purified by flash column
chromatography (MeOH–EtOAc, 1:1) to afford compound
16 (45 mg, 63%).
Synthesis of Compound 17.
To the mixture of Fmoc-Trp(Boc)-OH (205 mg, 0.39 mmol)
and benzylamine (165 mL, 1.51 mmol) in DMF (5 mL) was
added TBTU (249 mg, 0.77 mmol) and DIPEA (340 mL,
1.95 mmol). The reaction was stirred for 2 h at r.t. The
solvent was removed in vacuo and the residue was purified
Synlett 2007, No. 2, 239–242 © Thieme Stuttgart · New York

242
K. Zhang et al.
LETTER
Compound 15: 1H NMR (300 MHz, CDCl3, r.t., TMS):
by flash column silica gel chromatography (2:1, hexane–
EtOAc) to yield the Fmoc-Trp(Boc)-NHBn (151 mg, 63%).
The solution of Fmoc-Trp(Boc)-NHBn (151 mg, 0.25
mmol) and piperidine (0.5 mL) in DMF (2 mL) was stirred
for 1 h at r.t. The solvent was removed in vacuo and the
crude product was purified by flash column silica gel
chromatography (from EtOAc to 5% MeOH in EtOAc) to
afford the NH2-Trp(Boc)-NHBn (81 mg, 80%). Compound
16 (23 mg, 0.04 mmol) was dissolved in DMF (2 mL) and
NH2-Trp(Boc)-NHBn (72 mg, 0.18 mmol), TBTU (33 mg,
0.10 mmol), and DIPEA (37 mL, 0.21 mmol). The mixture
was stirred for 4 h at r.t. before removing the solvent under
reduced pressure. The crude product was purified by flash
chromatography using EtOAc as eluent to afford 17 (24 mg,
63%).
d = 1.44 (s, 9 H), 1.98 (s, 3 H), 2.03 (s, 3 H), 2.04 (s, 3 H),
3.13–3.29 (m, 2 H, H-8a, H-8b), 3.64–3.71 (m, 1 H, H-7),
3.78 (s, 3 H), 4.16 (dd, 1 H, H-3, J = 9.9, 2.3 Hz), 4.70 (dd,
1 H, H-2, J = 10.5, 2.3 Hz), 5.00 (dd, 1 H, H-6, J = 9.5, 9.5
Hz), 5.06 (dd, 1 H, H-4, J = 9.9, 9.5 Hz), 5.20 (dd, 1 H, H-5,
J = 9.5, 9.5 Hz), 5.13 (d, 1 H, N–H, J = 10.5 Hz). 13C NMR
(100 MHz, CDCl3, r.t.): d = 21.23, 21.34 (2 C), 28.88 (3 C),
51.30, 53.02, 53.46, 68.12, 69.84, 74.59, 78.04, 78.47,
79.82, 167.63, 168.12, 168.84, 169.37, 169.72. MS (ES):
m/z calcd for C20H30N4NaO11 [M + Na]+: 525.18; found:
525.27. Anal. Calcd (%) for C20H30N4O11: C, 47.81; H, 6.02;
N, 11.15. Found: C, 47.98; H, 6.25; N, 11.55.
Compound 16: 1H NMR (300 MHz, CD3OD, r.t., TMS):
d = 1.43 (s, 9 H), 2.98–3.13 (m, 2 H, H-6 and H-8a), 3.18 (m,
1 H, H-7), 3.22–3.44 (m, 2 H, H-3, H-5), 3.62–3.80 (m, 2 H,
H-4, H-8b), 4.23 (t, 1 H, J = 6.8 Hz), 4.32–4.48 (m, 3 H),
7.28–7.87 (m, 8 H). 13C NMR (100 MHz, CDCl3, r.t.):
d = 28.88, 43.42, 48.54, 56.37, 67.78, 72.04, 73.14, 79.03,
80.38, 80.76, 82.97, 120.94–128.78 (arom. C), 142.61–
145.43 (arom. C), 157.71, 159.12, 169.46. MS (ES): m/z
calcd for C28H33N2O10 [M – H]–: 557.21; found: 557.09.
Compound 17: 1H NMR (300 MHz, CDCl3, r.t., TMS):
d = 1.38 (s, 9 H), 1.63 (s, 9 H), 3.04–3.34 (m, 6 H), 3.37–
3.61 (m, 5 H), 3.71–3.83 (m, 1 H), 4.03–4.37 (m, 5 H), 4.45
(dd, 1 H, J = 6.7, 2.2 Hz), 4.75–4.85 (m, 1 H), 5.45–5.56 (br
s, 1 H, NH), 6.12–6.29 (br s, 1 H, NH), 6.29–6.46 (br s, 1 H,
NH), 6.73–6.91 (m, 2 H), 6.96–7.03 (br, 1 H, NH), 7.08–
8.18 (m, 16 H). HRMS (ES): m/z calcd for C51H59N5NaO12
[M + Na]+: 956.40524; found: 956.40537.
Characteristic Spectroscopic Data for Compounds
14–17.
Compound 14: 1H NMR (300 MHz, CDCl3, r.t., TMS):
d = 1.45 (s, 9 H), 2.00 (s, 3 H), 2.02 (s, 3 H), 2.05 (s, 3 H),
3.20–3.33 (m, 2 H), 3.64–3.70 (m, 1 H, H-7), 3.77 (s, 3 H),
3.87 (dd, 1 H, H-3, J = 9.9, 1.7 Hz), 4.66 (dd, 1 H, H-2,
J = 9.2, 1.7 Hz), 5.02 (dd, 1 H, H-6, J = 9.3, 9.7 Hz), 5.16
(dd, 1 H, H-5, J = 9.3, 9.1 Hz), 5.25 (dd, 1 H, H-4, J = 9.9,
9.1 Hz), 5.46 (d, 1 H, N–H, J = 9.2 Hz). 13C NMR (100
MHz, CDCl3, r.t.): d = 20.57, 20.58, 20.66, 28.27 (3 C),
50.83, 52.73, 53.47, 68.77, 69.03, 74.21, 77.77, 78.76,
80.46, 168.78, 168.80, 169.39, 169.51, 170.29. MS (ES):
m/z calcd for C20H30N4NaO11 [M + Na]+: 525.18; found:
525.32. Anal. Calcd (%) for C20H30N4O11: C, 47.81; H, 6.02;
N, 11.15. Found: C, 48.08; H, 6.15; N, 10.77.
Synlett 2007, No. 2, 239–242 © Thieme Stuttgart · New York

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