Synthesis and inhibitory activity of substrate-analog fructosyl peptide oxidase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2015.07.045

Abstract Fructosyl peptide oxidases (FPOXs) play a crucial role in the diagnosis of diabetes. Their main function is to cleave fructosyl amino acids or fructosyl peptides into glucosone and the corresponding amino acids/dipeptides. In this study, the subs

Full text of DOI:10.1016/j.bmcl.2015.07.045

Bioorganic & Medicinal Chemistry Letters 25 (2015) 3910–3913
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and inhibitory activity of substrate-analog fructosyl
peptide oxidase inhibitors
b
b
b
c
c
Bunta Watanabe a, , Atsushi Ichiyanagi , Kozo Hirokawa , Keiko Gomi , Toru Nakatsu , Hiroaki Kato ,
⇑
Naoki Kajiyama b
a Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan
b Research and Development Division, Kikkoman Corporation, 399 Noda, Noda, Chiba 278-0037, Japan
c Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimo-Adachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Fructosyl peptide oxidases (FPOXs) play a crucial role in the diagnosis of diabetes. Their main function is
to cleave fructosyl amino acids or fructosyl peptides into glucosone and the corresponding amino
acids/dipeptides. In this study, the substrate-analog FPOX inhibitors 1a–c were successfully designed
Received 4 May 2015
Revised 6 July 2015
Accepted 16 July 2015
Available online 23 July 2015
a
a
and synthesized. These inhibitors mimic N -fructosyl-
L-valine (Fru-Val), [N -fructosyl-
L-valyl]-
L-histidine
e
(Fru-ValHis), and N -fructosyl-
L-lysine (
eFru-Lys), respectively. The secondary nitrogen atom in the nat-
ural substrates, linking fructose and amino acid or dipeptide moieties, was substituted in 1a–c with a sul-
fur atom to avoid enzymatic cleavage. Kinetic studies revealed that 1a–c act as competitive inhibitors
Keywords:
Diabetes
Glycation
Hemoglobin A1c
Fructosyl peptide oxidase
Substrate-analog inhibitor
against an FPOX obtained from Coniochaeta sp., and Ki values of 11.1, 66.8, and 782
for 1a–c, respectively.
lM were obtained
Ó 2015 Elsevier Ltd. All rights reserved.
Diabetes is a metabolic disorder, in which the patient suffers
from chronically high concentrations of blood sugar. This condition
inevitably leads to a high level of glycation, a non-enzymatic
reduction between sugars such as glucose and free amino groups
on amino acids or proteins. Clinically, the level of glycated hemo-
globin (HbA1c) represents an important marker for the diagnosis
of diabetes. Typically, the half-life of erythrocytes, a source of
hemoglobin, is 110–120 days,1 and the degree of glycation of
hemoglobin can accordingly be used as a parameter to evaluate
an average blood glucose concentration over the preceding weeks
to months.2 The quantification of HbA1c is therefore a good index
for the therapeutic assessment and monitoring of diabetes
quantification of HbA1c. FPOXs oxidize fructosyl amino acids and
peptides to Schiff bases, which can be hydrolyzed to generate glu-
cosone and free amino acids and peptides (Scheme 1). The quantity
of H2O2 liberated during the catalytic cycle of FPOXs thereby
reflects the level of glycated proteins.
One of the major limitations of the enzymatic method described
above is the degradation step of intact HbA1c. Even though FPOXs
from Coniochaeta sp. (FPOX-C) and Eupenicillium terrenum (FPOX-E)
are active toward fructosyl amino acids and a fructosyl dipeptide
a
a
such as, for example, N -fructosyl-
L
-valine (Fru-Val), [N -fructo-
e
syl- -valyl]-
L
L
-histidine (Fru-ValHis), and N -fructosyl lysine (
eFru-
Lys), they are inactive toward fructosyl polypeptides. However,
recent reports have described the reactivity of FPOX from
Phaeosphaeria nodorum toward fructosyl hexapeptide.5 This result
encouraged us to use protein-engineering approaches to design
and develop novel FPOXs that are able to interact with HbA1c.
For such manipulations, detailed information about the precise
three-dimensional structure and substrate recognition mechanism
of the parent enzymes are indispensible. Recently, preliminary
results of the crystallographic analysis of FPOXs were reported by
us6 and other research groups.7 However, as the crystals employed
in these studies did not contain any ligands, the substrate recogni-
tion mechanism still remains elusive. The generation of FPOX inhi-
bitors for the co-crystallization with proteins therefore represents
patients. Today,
a combination of enzymatic degradation of
HbA1c and quantification of the resultant fructosyl peptides by
fructosyl peptide oxidases (FPOXs)3,4 is commonly used for the
Abbreviations: HbA1c, hemoglobin A1c; FPOX, fructosyl peptide oxidase; FAD,
flavin adenine dinucleotide; CFP-T7, thermostable fructosyl peptide oxidase
mutant; rt, room temperature; quant, quantitative; aq, aqueous; Tf, trifluo-
romethanesulfonyl; Trt, triphenylmethyl; COMU, N-[1-(cyano-2-ethoxy-2-ox-
oethylideneaminooxy)dimethylamino(morpholino)]uronium
hexafluorophos-
phate; Boc, tert-butoxycarbonyl; DMAP, 4-(dimethylamino)pyridine; BzSCs,
cesium thiobenzoate; RPLC, reverse-phase liquid chromatography; TFA, trifluo-
roacetic acid.
⇑
Corresponding author. Tel.: +81 774 38 3233; fax: +81 774 38 3229.
E-mail address: bunta@scl.kyoto-u.ac.jp (B. Watanabe).
http://dx.doi.org/10.1016/j.bmcl.2015.07.045
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

B. Watanabe et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3910–3913
3911
Scheme 1. FPOX-catalyzed reactions.
a highly desirable research target. Due to their structural similarity
to substrates, substrate-analog inhibitors should be accommo-
dated on the active site of the enzyme but display tolerance toward
enzymatic transformation. In order to successfully design such
inhibitors, minimal structural changes that retain the binding
affinity to target the enzyme are important.
In this study, we designed and synthesized the novel substrate-
analog FPOX inhibitors 1a–c (Fig. 1), which allowed us to unveil the
precise structure and the substrate recognition mechanism of
FPOXs. As shown in Figure 1, 1a–c are analogs of Fru-Val, Fru-
ValHis, and
atom in the natural FPOX substrates (Fru-Val, Fru-ValHis, and
Fru-Lys) with a sulfur atom, as we assumed that this modification
eFru-Lys, respectively. We substituted the nitrogen
e
should avoid a significant loss of binding affinity of 1a–c toward
FPOX while simultaneously preventing the enzymatically-induced
oxidative cleavage described in Scheme 1. A similar approach was
successfully employed for the development of monomeric sar-
cosine oxidase and fructosamine oxidase inhibitors.8,9 We also
determined the inhibitory activity and the inhibition mechanism
of 1a–c against the thermostable FPOX-C mutant CFP-T7.10
The synthesis of the inhibitor 1a is outlined in Scheme 2.
Commercially available (R)-(+)-2-bromo-3-methylbutyric acid (2)
was treated with cesium thiobenzoate (BzSCs), before the resulting
Scheme 2. Synthesis of inhibitor 1a. Reagents and conditions: (a) BzSCs, DMF, rt,
2 d; (b) 5 M NH3 aq, CH2Cl2, rt, 3 h, quant from 2; (c) 5, 1 M KOH aq, acetone, THF,
reflux, 3 d, 72%; (d) TFA, H2O, rt, 1.5 h, 59%.
pyranose form (ca. 75%). Coupling constants of 9.9 and 3.3 Hz were
measured for 3
a-H/4b-H and 4b-H/5b-H, respectively. These val-
ues suggest a 1,2-diaxial coupling between 3
a
-H and 4b-H, and a
1,2-diequatorial coupling between 4b-H and 5b-H. Accordingly, a
chair conformation as shown in Scheme 2 was assigned to the
pyranose ring of inhibitor 1a.
thiobenzoate
3 was hydrolyzed to afford (S)-2-sulfanyl-3-
methylbutanoic acid (4) in quantitative yield.11,12 Introduction of
a fructose moiety was achieved by a coupling reaction between
thiol 4 and triflate 5,13 furnishing protected Fru-Val analog 6 in
72% yield.
The synthesis of inhibitor 1b is outlined in Scheme 3.
Compound 6 was coupled with the protected histidine derivative
[H-His(Trt)-OMeÁHCl] to afford 7. A hydrolysis of the methyl ester
group under basic conditions, followed by acidic hydrolysis of the
acetonide and triphenylmethyl moieties furnished Fru-ValHis ana-
log inhibitor 1b16 in 67% yield as the trifluoroacetic acid (TFA) free
form after purification by RPLC. The pyranose rings of 7 and 1b
adopted twisted-boat and chair conformations, respectively.
Subsequently, a protected lysine counterpart was prepared for
As illustrated in Scheme 2, the acetonide-protected pyranose
rings of 5 and 6 adopted a twisted-boat conformation in CDCl3.
This is reflected in the 1H NMR coupling constants between the
3a-H and 4b-H (J = 2.4–2.8 Hz), which are considerably smaller
than typical 1,2-diaxial coupling constants for a chair conformation
(J = 8–10 Hz). In contrast, the coupling constants between 4b-H
and 5b-H (J = 7.9 Hz) are considerably larger compared to typical
1,2-diequatorial coupling constants (J = 2–3 Hz); an observation
that is consistent with a report by Maryanoff and co-workers.14
Acidic hydrolysis of 6 resulted in the formation of Fru-Val analog
inhibitor 1a15 in 59% yield after purification by reversed-phase liq-
uid chromatography (RPLC). The 1H NMR spectrum of 1a in D2O
revealed that the sugar moiety adopted predominantly the b-
the synthesis of
eFru-Lys analog inhibitor 1c (Scheme 4). For that
purpose, a combined one-pot protection procedure, using commer-
cially available L-2-aminoadipic acid (8), was applied to the car-
boxy and amino groups,17 resulting in the quantitative formation
of mono-Boc derivative 9. The mono-Boc amino group of 9 was
OH
S
R
O
OH
OH
OH
N
NH
O
O
O
OH
OH
OH
N
H
R:
NH2
O
1a
1b
1c
Scheme 3. Synthesis of inhibitor 1b. Reagents and conditions: (a) [H-His(Trt)-
OMeÁHCl], COMU, 2,4,6-trimethylpyridine, DMF, rt, 2 h, 76%; (b) 0.5 M NaOH aq,
MeOH, rt, 30 min; (c) TFA, H2O, rt, 1 h, 67% from 7.
Figure 1. Chemical structures of FPOX inhibitors 1a–c.

3912
B. Watanabe et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3910–3913
18
CH2Cl2 to furnish mono-Boc alcohol 13.19 The moderate yield of
the reaction (60%) should most likely be ascribed to the formation
of a mixture of the esterified and etherified by-products 14 and 15.
Based on the 1H NMR analysis of the mixture, the yields of 14 and
15 were estimated to be 22% and 11%, respectively. We made no
attempt to separate this mixture, since hydrolysis under mildly
basic conditions converted trifluoroacetate 14 into the more polar
alcohol 13, which allowed us to separate 13 and less polar 15 very
easily. The yield of 13 thus obtained from 14 was 95%, and pure t-
Bu ether 15 was recovered in 87% yield. A subsequent reaction of
13 with methanesulufonyl chloride furnished the protected lysine
counterpart 1620 in quantitative yield.
The synthesis of inhibitor 1c is shown in Scheme 5. Triflate 5
was treated with BzSCs to give thiobenzoate 17 in 93% yield. The
benzoyl group of 17 was hydrolyzed with NaOMe, and the result-
ing thiolate anion was not isolated, but treated with mesylate 16 to
afford the fully protected intermediate 18 in almost quantitative
yield. It should be noted here, that these reaction conditions sup-
press the formation of the oxidative homocoupling by-product,
namely disulfide 19,21 which was only observed in negligible
amounts. The protecting groups of compound 18 were removed
by consecutive basic and acidic hydrolysis. After purification by
Scheme 4. Preparation of protected lysine counterpart 16. Reagents and condi-
tions: (a) (i) TMSCl, MeOH, rt, 2 d, (ii) (Boc)2O, Et3N, rt, 27 h, quant; (b) (Boc)2O,
DMAP, MeCN, 4 d, 95%; (c) (i-Bu)2AlH, Et2O, hexane, À78 °C, 15 min, 66%; (d) NaBH4,
MeOH, 0 °C, 20 min, 94%; (e) TFA, CH2Cl2, rt, 5 h, 60% (13), 22% + 11% (14 + 15); (f)
NaHCO3 aq, MeOH, rt, 30 min, 95%; (g) MsCl, Et3N, CH2Cl2, rt, 1 h, quant.
RPLC, e
Fru-Lys analog inhibitor 1c22 was obtained in 49% yield as
the TFA salt.
The inhibitory activity of 1a–c against CFP-T7 was monitored by
a peroxidase-coupled reaction system. The details of the assay are
described in the supporting information. Under the conditions
applied, the Michaelis constants (Km) of CFP-T7 were determined
to be 0.940 0.025, 0.513 0.014, and 25.3 0.5 mM for Fru-Val,
Fru-ValHis, and
e
Fru-Lys, respectively.23 Accordingly, Fru-ValHis
was selected as the substrate for the subsequent enzyme inhibition
assay. Figure 2 shows the Lineweaver–Burk plots for 1a–c, which
clearly demonstrate that these act as competitive inhibitors, and
inhibition constants (Ki) of 11.1 0.9 (1a), 66.8 0.8 (1b), and
782
8 l
M (1c) were obtained from Dixon plots.23 These results
indicate that the affinity of inhibitors 1a and 1b toward CFP-T7 is
about 46 and 7.7 times greater than that of Fru-ValHis, the best
substrate for the enzyme. Even though the affinity of
log inhibitor 1c for CFP-T7 is only 66% of that of Fru-ValHis, it is at
least 30 times higher than that of the parent Fru-Lys, when con-
sidering that the Km value of Fru-Lys (25.3 mM) is nearly 50 times
eFru-Lys ana-
e
Scheme 5. Synthesis of inhibitor 1c. Reagents and conditions: (a) BzSCs, DMF, rt,
17 h, 93%; (b) (i) NaOMe, MeOH, rt, 90 min, (ii) 16, rt, 2 h, 96%; (c) 2.5 M NaOH aq,
MeOH, rt, 1 h, 93%; (d) TFA, H2O, rt, 30 min, 49%.
e
higher than that of Fru-ValHis (0.513 mM).
The difference of inhibitory activity toward CFP-T7 between 1a-
c (Ki: 11.1–782 lM) was higher than that of affinity between the
further protected to afford di-Boc derivative 10 in almost quantita-
natural substrates (Km: 0.940–25.3 mM). This should be attributed
to the presence of the secondary amino group in the natural sub-
strates. Possible formation of hydrogen bonds between the amino
group and the surrounding residue(s) of CFP-T7 should constrain
the substituent at C-1 position of the fructose ring more tightly.
tive yield. The esterified carboxy group at the
x-end of 10 was
selectively reduced with (i-Bu)2AlH,17 affording aldehyde 11 in
66% yield. After the reduction of the formyl group in 11, the di-
Boc protection of alcohol 12 was partially removed with TFA in
(A)
(B)
(C)
[1a] (µM)
0.35
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
25
[1b] (µM)
0.30
0.25
0.20
0.15
0.10
0.05
[1c] (µM)
1250
100
15
60
750
5
0
20
0
250
0
0.00
-2.0 -1.0 0.0 1.0 2.0 3.0 4.0
-2.0 -1.0 0.0 1.0 2.0 3.0 4.0
-2.0 -1.0 0.0 1.0 2.0 3.0 4.0
1 / [Fru-ValHis] (mM-1)
1 / [Fru-ValHis] (mM-1)
1 / [Fru-ValHis] (mM-1)
Figure 2. Lineweaver–Burk plots for various concentrations of the inhibitors 1a (A), 1b (B), and 1c (C).

B. Watanabe et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3910–3913
3913
8. Wagner, M. A.; Trickey, P.; Chen, Z. W.; Mathews, F. S.; Jorns, M. S. Biochemistry
2000, 39, 8813.
9. Collard, F.; Zhang, J.; Nemet, I.; Qanungo, K. R.; Monnier, V. M.; Yee, V. C. J. Biol.
Chem. 2008, 283, 27007.
10. Hirokawa, K.; Ichiyanagi, A.; Kajiyama, N. Appl. Microbiol. Biotechnol. 2008, 78,
775.
11. Strijtveen, B.; Kellogg, R. M. J. Org. Chem. 1986, 51, 3664.
12. Perrine, S. P. US2008/0075692.
Since the inhibitors used in this study are unable to form such
hydrogen bonds with CFP-T7, the corresponding inhibitor moiety
seems to exhibit more degrees of freedom relative to the amino
acid/dipeptide moiety of the natural compounds. The latitude of
the inhibitors should be a drawback for the binding affinity toward
the enzyme, and sterically less demanding substituents should be
preferable. This effect probably affects the inhibitory activity of
1a-c toward CFP-T7 strongly, and the inhibitory activity of Fru-
Val analog 1a, which contains the smallest substituent at the C-1
position of the fructose ring, was higher than that of Fru-ValHis
analog 1b. Regardless, natural Fru-ValHis proved to be the best
substrate for CFP-T7.
13. Maryanoff, B. E.; McComsey, D. F.; Costanzo, M. J.; Hochman, C.; Smith-
Swintosky, V.; Shank, R. P. J. Med. Chem. 1941, 2005, 48.
14. Maryanoff, B. E.; Nortey, Samuel O.; Gardocki, J. F.; Shank, R. P.; Dodgson, S. P. J.
Med. Chem. 1987, 30, 880.
15. Compound 1a: colorless, hygroscopic solid. 1H NMR (600 MHz, D2O) dH: 1.02
(3H, d, J = 6.8 Hz, Val-
m, Val-bH), 2.95 (1H, d, J = 14.1 Hz, Fru-1Ha), 3.01 (1H, d, J = 14.1 Hz, Fru-1Hb),
3.35 (1H, d, J = 8.4 Hz, Val- H), 3.65 (1H, dd, J = 12.9, 1.3 Hz, Fru-6Ha), 3.82 (1H,
cH3-2), 1.07 (3H, d, J = 6.8 Hz, Val-cH3-1), 1.99–2.07 (1H,
a
In conclusion, we successfully designed and synthesized sub-
strate-analog inhibitors for CFP-T7 and quantitatively analyzed
their inhibition properties. Co-crystallization of the enzyme with
inhibitors 1a–c in order to determine the three-dimensional struc-
ture of CFP-T7 by X-ray crystallography is currently undertaken in
our laboratories and results will be disseminated in due course.
d, J = 9.9 Hz, Fru-3H), 3.85 (1H, dd, J = 9.9, 3.3 Hz, Fru-4H), 3.96–3.99 (1H, m,
Fru-5H), 3.97 (1H, dd, J = 12.9, 1.3 Hz, Fru-6Hb). 13C NMR (151 MHz, D2O) dC:
22.07 (Val-c-1), 22.58 (Val-c-2), 32.94 (Val-b), 41.40 (Fru-1), 59.17 (Val-a),
66.26 (Fru-6), 71.92 (Fru-5), 72.26 (Fru-3), 72.56 (Fru-4), 101.40 (Fru-2),
180.24 (Val-carbonyl). HRMS-FAB (m/z): [M+Na]+ calcd. for C11H20NaO7S:
319.0827; found: 319.0826.
16. Compound 1b: colorless solid. 1H NMR (600 MHz, D2O) dH: 0.90 (3H, d,
J = 6.7 Hz, Val-
b), 2.69 (1H, d, J = 13.8 Hz, Fru-1Ha), 2.83 (1H, d, J = 13.8 Hz, Fru-1Hb), 3.13 (1H,
dd, J = 15.6, 9.3 Hz, His-bHa), 3.30 (1H, d, J = 8.9 Hz, Val- H), 3.32 (1H, ddd,
cH3-1), 1.01 (3H, d, J = 6.7 Hz, Val-cH3-2), 1.87–1.97 (1H, m, Val-
a
Acknowledgements
J = 15.6, 5.0, 1.0 Hz, His-bHb), 3.66 (1H, dd, J = 12.8, 1.8 Hz, Fru-6Ha), 3.72 (1H,
d, J = 9.9 Hz, Fru-3H), 3.86 (1H, dd, J = 9.9, 3.5 Hz, Fru-4H), 3.97 (1H, dd, J = 12.8,
1.3 Hz, Fru-6Hb), 3.98–3.99 (1H, m, Fru-5H), 4.60 (1H, dd, J = 9.3, 5.0 Hz, His-
Parts of the experimental measurements were carried out using
the Bruker and JEOL 600 MHz NMR spectrometers, as well as the
JOEL JMS-700 mass spectrometer in the Joint Usage/Research
Center (JURC) at the Institute for Chemical Research, Kyoto
University.
a
H), 7.338–7.340 (1H, m, His-Im-5H), 8.63 (1H, d, J = 1.4 Hz, His-Im-2H). 13C
NMR (151 MHz, D2O) dC: 22.32 (Val-
c
-1), 22.65 (Val-
c-2), 30.02 (His-b), 33.36
(Val-b), 41.06 (Fru-1), 56.55 (His- ), 59.98 (Val-
a
a
), 66.32 (Fru-6), 71.85 (Fru-5),
72.54 (Fru-4), 72.67 (Fru-3), 101.31 (Fru-2), 119.76 (His-Im-5), 132.40 (His-Im-
4), 136.11 (His-Im-2), 177.39 (Val-carbonyl), 178.68 (His-carbonyl). HRMS-FAB
(m/z): [M+H]+ calcd. for C17H28N3O8S: 434.1597; found: 434.1594.
17. Padrón, J. M.; Kokotos, G.; Martín, T.; Markidis, T.; Gibbons, W. A.; Martín, V. S.
Tetrahedron: Asymmetry 1998, 9, 3381.
Supplementary data
18. Koseki, Y.; Yamada, H.; Usuki, T. Tetrahedron: Asymmetry 2011, 22, 580.
19. Compound 13 has previously been synthesized via an independent route; see:
Rodriguez, A.; Miller, D. D.; Jackson, R. F. W. Org. Biomol. Chem. 2003, 1, 973.
20. Compound 16 has been previously synthesized by an independent route, but
NMR and MS data were not reported; see: Fanning, K. N.; Sutherland, A.
Tetrahedron Lett. 2007, 48, 8479.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.07.
045.
21. Goodwin, J. C. Carbohydr. Res. 1989, 195, 150.
22. Compound 1c: pale yellow, hygroscopic solid. 1H NMR (600 MHz, D2O) dH:
References and notes
1.45–1.58 (2H, m, Lys-
(2H, m, Lys-bH2), 2.65 (1H, dt, J = 12.9, 7.3 Hz, Lys-
7.3 Hz, Lys- Hb), 2.91 (1H, d, J = 14.0 Hz, Fru-1Ha), 2.94 (1H, d, J = 14.0 Hz, Fru-
c
H2), 1.66 (2H, dddd, J = 7.3 Hz each, Lys-dH2), 1.89–2.02
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eHa), 2.68 (1H, dt, J = 12.9,
e
1Hb), 3.64 (1H, dd, J = 12.9, 2.1 Hz, Fru-6Ha), 3.80 (1H, d, J = 10.0 Hz, Fru-3H),
3.86 (1H, dd, J = 10.0, 3.4 Hz, Fru-4H), 3.96–3.98 (1H, m, Fru-5H), 3.97 (1H, dd,
J = 12.9, 1.4 Hz, Fru-6Hb), 4.06 (1H, dd, J = 7.0, 5.6 Hz, Lys-
(151 MHz, D2O) dC: 25.98 (Lys- ), 30.97 (Lys-d), 32.08 (Lys-b), 35.15 (Lys-
41.61 (Fru-1), 55.68 (Lys- ), 66.32 (Fru-6), 71.87 (Fru-5), 72.45 (Fru-3), 72.56
a
H). 13C NMR
),
c
e
a
(Fru-4), 101.50 (Fru-2), 119.16 (q, JC–F = 291 Hz, TFA-CF3), 165.79 (q, JC–
F = 35.6 Hz, TFA-carbonyl), 175.11 (Lys-carbonyl). 19F NMR (565 MHz, D2O)
dF: À75.5. HRMS-FAB (m/z): [M–TFA+H]+ calcd. for C12H24NO7S: 326.1273;
found: 326.1275.
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23. Data given represent the mean results standard deviations from three
independent experiments.