Novel sugar compounds isolated from high
molecular weight dissolved organic matter
(HMWDOM)
Christos Panagiotopoulos (1), Dan Repeta (2), and J-F Rontani (1)
(1) LMGEM, Center of Oceanology Marseille
(2) Woods Hole Oceanographic Institution
How well do we know the composition of Organic matter ?
DOC
Surface DOC
Deep DOC
15-30% C characterized (AA-C + Lip-C +Sugar-C)
< 10% C characterized (AA-C + Lip-C +Sugar-C)
Most of the DOC remains uncharacterizable at the molecular level
Benner (2002)
Natural Concentrations of compounds in DOM
Concentrations close to the detection limits of the
techniques
-
Pulsed amperometric detection (PAD) ~ 2-10 nmol/L (sugars)
Flame ionization detection (FID/MS) ~ 100-300 nmol/L (lipids)
Fluorescence/UV-visible ~ 200-1000 nmol/L (Amino acids)
+
NMR ~ mg-g/L
Detection limits
Carbohydrates: 100-800 nmol/L for polysaccharides
< 10 nmol/L for monomers
Amino acids: similar range with carbohydrates
Lipids: 200-500 nmol/L
Impossible to characterize the whole DOM with the current
techniques
Get info from concentrated OM [i.e. High Molecular Weight dissolved
Organic matter (HMWDOM)] > 1000Da)
Approaches for the chemical characterization of DOM
(1) Direct analyses of DOM (0.5-1 mg/L DOC)
+
• No contamination and artifacts
• Representative of DOM pool
• Low conc. of compounds (nmol)
• Salts (35-38 g/L)
(2) Analyses of concentrated DOM (HMWDOM > 1kDa)
+
• Almost no salts
• 5-10 g of material
• Large vol. of samples required (>5000L)
• Only 25-30% DOC recovered
NMR; MS-MS; Δ14C on individual comp. etc
HMWDOM composition by 13CNMR
HMWDOM is mostly a mixture of carbohydrate like
molecules [acyl polysaccharides (APS)].
HMWDOM comprize sugars (50-70%; OCO, HCOH
groups), carboxylic + acetate (5%; COOH,
CH3CO), alkyl (5%; CHx), proteins and
humics (20%)
absorbance
HCOH
However, after acid hydrolysis (HCl, TFA,
H2SO4 etc) only 15-20% of HMWDOM
is recovered as neutral sugars.
OCO
CHx
COOH
APS are present in both fresh and
marine waters and their inventory
is large (10-15 GTC at least)
ppm
frequency
Aluwihare et al., (1997); Repeta et al.(2002)
Chemical composition and age of HMWDOM
HMWDOM
Δ14C (‰) = 46 to -381 (<10-3850 yr)
Hydrolysis
desalting (resins)
Elution NH4OH
Elution H2O
Amino acids and
humics
50-70% HMWDOC (APS)
Total Carbohydrates
Rich in –CHOH Hydrophilic component
HP
LC
-R
I
Δ14C (‰) = -416 to -428 (4320-4480 yr)
(3-3600 m)
Rich in –COOH Aliphatic component
(3-3600 m)
Arabinose
Fucose
Galactose
Glucose
Mannose
Rhamnose
Xylose
Δ14C (‰) = 57 to -145 ( <5-1280 yr)
(3-3600 m)
F4
F3
F5
F2
F1
10
20
30
40
Repeta and Aluwihare (2006)
Separation of mono- and oligosaccharides
after HMWDOM hydrolysis using ion chromatography
Unhydrolyzed sugar polymer.
WE TRIED everything
HCl, H2SO4, TFA
glucose
Surface Hawaii sample
HPLC-RI conditions
2 Ag columns at 80C, loop 100 µl
Eluant H2O, 0.5 ml/min, 40 min
Sample
Laminarin
DP-7
DP-5
DP-3
maltose
absorbance
Standard
F4
Neutral sugars
(15-20%)
F3
F5
F2
(75%) F1
10
20
10
30
20
30
40
Time (min)
Chemical composition of the HMWDOM- F1 by NMR
CH3COO
CH3CO
CHOH
CH3
CH3
CHOH
Hydrolysis
Initial HMWDOM
HMWDOM-F1
Unhydrolyzed sugar polymer
most AcOH is gone
Panagiotopoulos et al (2007)
Chemical composition of the HMWDOM- F1 by anion
exchange chromatography
36,00
34,00
Gradiant conditions :
0-25 min 50mM CH
Detector response
32,00
30,00
28,00
26,00
24,00
22,00
mV
20,00
18,00
16,00
14,00
12,00
10,00
8,00
6,00
4,00
F1
2,00
0,00
-2,00
2,00 4,00 6,00 8,00 10,00 12,00 14,00 16,00 18,00 20,00 22,00 24,00 26,00 28,00 30,00 32,00 34,00 36,00 38,00 40,00 42,00 44,00 46,00 48,00 50,00 52,00 54,00
Minutes
10
20
30
40
Time (min)
HO CH3
HO
OH
CH3O
2000000
Detector response
1800000
Confirmation of results by GC-MS
1600000
2-OMe glucose
1400000
1200000
2-OMe fucose
Detector response
GC-MS alditols acetates derivatives in the HMWDOM-F3
F3
1000000
10
800000
2-OMe galactose
600000
20
30
Time (min)
40
3-OMe glucose
400000
3-OMe rhamnose
200000
5.00
10.00
15.00
20.00
25.00
30.00
35.00
Time (min)
MS can not say the difference between epimers (i.e. glucose, galactose, mannose)
Lack of authetic sugar standard (differences only in retention times)
Determine the absolute configuration of sugars (D or L). Other derivatization procedures
(e.g. trimethylsilylated dithioacetals)
Detector response
GC-MS alditols acetates derivarives in the HMWDOM-F3
2000000
Detector response
1800000
1600000
F3
1400000
10
1200000
20
30
Time (min)
40
1000000
800000
600000
HO
HO
400000
7
CH2CH2OH
O
OH
CH3O
200000
5.00
10.00
15.00
20.00
25.00
30.00
35.00
Time (min)
3,6-dideoxyhexose; 2,3-di-O-methylrhmnose; 2,4-di-O-methylrhamnose; 2-O-methyl-pentose
2,6-di-O-methylhexose; 3,6-di-O-methylhexose; 2,3-di-O-methylhexose; 6-O-methylhexose
Glucosamine, 2-O-methylheptoses
Detector response
GC-MS alditols acetates derivarives in the HMWDOM-F3
2000000
1600000
F3
1400000
10
1200000
1000000
A lot of mono and dimethylated sugars
800000
600000
HO
HO
400000
20
30
Time (min)
40
7
CH2CH2OH
O
OH
CH3O
200000
5.00
10.00
15.00
20.00
25.00
30.00
35.00
Time (min)
3,6-dideoxyhexose; 2,3-di-O-methylrhmnose; 2,4-di-O-methylrhamnose; 2-O-methyl-pentose
2,6-di-O-methylhexose; 3,6-di-O-methylhexose; 2,3-di-O-methylhexose; 6-O-methylhexose
Glucosamine, 2-O-methylheptoses
Detector response
GC-MS alditols acetates derivarives in the HMWDOM-F5
95000
Detector response
Detector response
1800000
85000
75000
65000
F5
55000
10
45000
35000
20
30
Time (min)
25000
15000
5000
10
12
14
16
18
20
22
24
26
28
30
32
34
36
Time (min)
1,6 anhydro hexoses (i.e levoglucosan, galactoglucosan, mannoglucosan etc)
Hexoses, Pentoses (leftovers from the F4 neutral sugar fraction)
Heptoses
38
40
Levoglucosan mass spectra
No NaBH4 treatment
NaBH4 treatment
81
Abundance
Relative Abundance
data.ms (-773) (-)
7500
81
4500
data.ms (-767) (-)
7000
6500
4000
6000
3500
98
5500
5000
3000
98
4500
157
2500
157
4000
3500
2000
97
115
3000
102
69
1500
115
69
2500
1000
112
127
55
1500
140
144
1000
158
188
50
60
70
80
127
229
500
0
186
140
2000
186
228 232
243 245
229
53
500
168
243
0
90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250
50
m/z-->
60
70
80
90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240
m/z
Data from Somkovic et al. 2003
Levoglucosan
Hexose ring does not open
Biogeochemical importance
A. Methylated, dimethylated hexoses have been found in bacterial and algal cell
walls as part of structural polysaccharides, however their chemical structure and
function is poorly understood.
Are these compounds contributors to the refractory DOM ?
B. 3-6-dideoxysugars and heptoses have been as antigenic polysaccharides in
Gram-negative bacteria as well as in antibiotics.
It is fundamental to get more info about bacteria structures. Do bacteria
assimilate metylated or deoxysugars ?
C. Levoglucosan is a component of atmospheric smoke particles derived from wood
burning (cellulose degradation product) and this is the FIRST time that has been
found in DOM in tiny amounts.
Does black carbon (part of the uncharacterizable DOM carbon) enter the ocean via
atmospheric deposition ? (this was assumed but not proven by molecular level
analysis by Masiello & Druffel, 1998).
Measurements of d13C of pure levoglucosan in surface and deeper DOM samples
Beyond Biochemistry
Erythromycin
Azythromycin
Beyond Biochemistry
Sugar moiety
Sugar moiety
Methylated 2,6 or 3,6 dideoxysugars
Erythromycin
Azythromycin
ANTIOBIOTICS: Antibacterial agents
The sugars moieties in antiobiotics actively contribute as recognition elements to the mechanism of
action of the respective drug and their removal often results in the loss of all biological activity.
IDENTIFY new sugar compounds in the HMWDOM may help discovering new antiobiotic
analogs (pharmaceutical chemistry etc..)
Much more to do….
A. Technological development of the HPAEC-MS/MS for structural characterization
of the unhydrolyzed HMWDOM-F1 fraction.
B. Chemical characterization of the F2 fraction
C. Dig out more sugars from the F3 fraction and get someway authentic sugar
standards (buy them or synthetize them). Perform Δ14C measurements on
individual methyl, deoxy sugars.
D. Start to identify all sugar components in marine bacteria and algae
E. Isotopic analysis of levoglucosan in surface and deep samples (info about black
carbon)