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Carbohydrates
C.D. Jonah and B.S.M. Rao (Editors) Radiation Chemistry: Present Status and Future Trends 9 2001 Elsevier Science B.V. All rights reserved.
481
Carbohydrates C. von Sonntag and H.-P. Schuchmann
Max-Planck-Institut ftir Strahlenchemie P.O. Box 101365, Stiftstrasse 34-36, D-45470 Mtilheim an der Ruhr, Germany
1. I N T R O D U C T I O N The initial impetus for the study of the chemical behaviour of carbohydrates under the influence of ionizing radiation consisted in the hope that the power of the new sources of radioactivity might provide a practical way of transforming abundant natural carbohydrates, especially cellulose which is relatively difficult to deal with chemically or enzymatically, into other useful chemicals [1-3], and in the need to examine the chemical effect of radiation sterilization of carbohydrate-containing foods with respect to its wholesomeness after such treatment [4, 5]; it has been observed that carbohydrate-derived compounds carrying additional keto functions [6] can be mutagenic [7], similar to various aldehydes [8]. A second major incentive was originally derived from radiationprotection considerations. In order to evaluate possible chemical protective strategies it was necessary to obtain mechanistic information on the production of radiation-induced DNA damage such as the formation of strand breaks and the release of nucleobases [9]. The latter processes originate from radicals generated at the deoxyribose moiety of DNA whose backbone is a chain consisting of 2-deoxyribose units bridged by phosphate groups at the positions 3' and 5'. The use of various carbohydrate model compounds, among them functionalized ones, in the detailed mechanistic studies of the radiation-induced damage of its sugar moiety proved to be crucial since DNA offers a variety of reactive sites for flee-radical attack, notably the nucleobases whose reactivity in this situation tends to overshadow the reactivity shown by the deoxyribose moiety as such [10]. Thus, for a better understanding of the mechanistic principles that lead to DNA strand breakage and base release, a large number of low-molecular-weight carbohydrate systems have been investigated [ 11 ]. Ionizing radiation is in fact a convenient means to induce free-radical chemistry such as can be set in motion, for instance, by oxidative processes.
482
Thus, the free-radical-induced degradation of hyaluronic acid, another carbohydrate biopolymer, is a major aspect of rheumatoid arthritis, which has provoked radiation-chemical studies to elucidate the kinetics of its depolymerization (the lubricating property of hyaluronic acid in a joint is rapidly lost when its molecular weight is decreased). Most of such radiation-chemical studies of carbohydrates have been carried out in aqueous solution. Besides, there are crystalline-state studies which in some cases show interesting examples of solid-state chain reactions (cf. [2]). Some of the paths traced by the radical function as the reaction propagates through the crystal can be inferred on the basis of X-ray crystallography (cf. [12, 13]). 2. AQUEOUS S O L U T I O N S Carbohydrates (many of them being at the same time polyhydric alcohols) and those of their derivatives that are of interest in the above context are usually water soluble (notable exceptions, the biopolymers cellulose and chitin). Good water solubility has allowed to study the radiation chemistry of their aqueous solutions without the difficulties that are encountered in the case of poorly soluble substrates. Difficulties are mainly of a product-analytical nature. It is relatively easy to determine the low-molecular weight carbohydrate products that are formed via the unimolecular transformation of the starting radical, while it is very difficult to analyse completely the complex mixtures of the dehydro dimers which arise from the substrates under anoxic conditions as several different primary radicals intercombine and, in addition, different stereochemical options exist for each combination. The formation of the primary radicals in these systems is governed by Habstraction reactions, effected by OH" and H" since the substrates usually exist mainly in the hemiacetal form (or acetal form in the case of disaccharides and polysaccharides). The hydrated electron eaq" is essentially non-reactive in the absence of the free carbonyl function (there are exceptions with certain glycosides when the glycosidic group upon elimination can form a stabilized radical) [14, 15]. In radiation-chemical experimentation eaq is usually transformed into the OH" radical with N20. Due to the much lower bond dissociation energy of the C-H bond compared to the O-H bond, practicality only carbon-centred radicals are formed. For the OH radical, the reaction rate constants are typically near 2 x 10 9 dm 3 mol 1 s-1, and about one to two orders of magnitude slower for the H atom [16]. Generally, there is no pronounced regioselectivity [17], i.e. in D-glucose, radicals 1-6 are formed in comparable
483 yields [ 18]. Higher selectivity is observed with other H-atom abstractors, e.g. the sulfate radical anion [19]. Considering the type of chemistry they undergo, the primary radicals from regular carbohydrates may be divided into several groups (that may partially overlap), e.g., 1,2-diol-type radicals such as 1-4, 2-alkoxyl-hydroxyalkyl radicals such as 4 and 6, and ether-type radicals such as 1 and 5. CH2OH
CH2OH
J--oo.
o~
)--oo.
?.
~o~O~.~ ~o~O~~ I
OH
9
OH
1
2
CH2OH
,L-oo. 9
CH2OH
;--oo.
OH
I
OH 3
CH2OH
-~oo.
~o~O~~
oCHOH
)--ooH
I
OH
I
OH
4
OH
5
6
In disaccharides and polymeric carbohydrates, the ether-type radicals such as 7 and 8 have the radical site proximate to the glycosidic linkage and therefore play a major role in its scission (see below). In glucosamine [20] and Nacetylglucosamine [21] as well as in their related polymers chitin, chitosan and hyaluronic acid, one must further consider radicals of the type 9-11. Thus, one may expect that in addition to the behaviour shown by the prototypical carbohydrate radicals, reactions such as are observed with amino acids (cf. [22]) and peptides (cf. [23-25]), may also play a role. CH2OH
CH2OH
J-:o
)--:oo.
CH2OH
CH2OH
~-:o.
.J-:oo.
~o~~o.~o~~ ~o~O~j~o_~o~~ I
I
OH
OH 7
I
I
OH
OH 8
484 CHeOH J---OOH
CH2OH ~---OOH
_ NH2
Ho~OH.~ , NHCOCH3
-NH
9
CH2OH ~--OOH
10
11
In DNA, five different radicals may be formed at the sugar moiety. The radical site in 12 is proximate to the N-glycosidic linkage and can give rise to base release [26]. Radical 13 appears to behave in the way of an alkyl-type radical. Radicals 14 and 16 have phosphate groups in (x-position, while radical 15 has these groups in a ~-position linked to an oxygen atom which makes heterolytic cleavage possible (see below). ~-Phosphato radicals such as 14 and 16 do not show phosphoester release [27]. In RNA and ribo-polynucleofides which lack the deoxy function one must take radical 17 into consideration which regarding the ease of phophoester release resembles 15.
I
I
O=oHO
ase
I
()H
O=P-O-CH2 ase ()H
O
I o
I o
I
ase
o
i
I
O=P-O~
O=P-O~
O=P-O~
OH
OH
OH
12
13
14
i
I O=PHO
i
i
I
-
ase
I
()n
O
ase
O=P-O-CH2 ()n
ase
I o
I o
I o
O=P-O~
O=P-O~
O=P-O~
i
I
OH 15
i
I
OH 16
I
I OH
I
OH 17
It has been mentioned above that in these systems the radiation chemistry is mainly governed by the free-radical chemistry of the nucleobases, and much of the knowledge about their chemistry has to come from model studies.
485
As discussed below, there are two types of unimolecular reaction pathway open to all of these carbohydrate radicals, i.e. "ionic" elimination, and cleavage of a carbon-oxygen bond [3 to the free-radical site. Depending on whether the substituent is a hydroxyl group, an ester, ether, or other hetero function, preference will lie toward one or the other reaction channel. 2.1. Elimination
of water or alcohol
Many carbohydrates have the structural element -CHOH-CHOH-. Radicals such as 1 - 4, just like 18, generated by H-abstraction from such sites can eliminate water [28]. This reaction occurs at a relatively slow rate spontaneously ("water-catalyzed"), but faster upon acid or base catalysis [reactions (1)-(3)].
H~ w (1)
I_I,O,H OH
OH o -
OH
OH
~H2--~H ___._. - H20
19
H*
(4)
_ H |
,,(3 9C H T - C ,
(2)
2O
18
H
-~
(5)
OH e (3)
OH O 21
Carbinol ROH can be similarly eliminated [reaction (6)] (this reaction has been studied in a variety of model compounds [29, 30]; cf. further [31, 32] and references therein); this reaction as well converts the exocylic glucose-derived radical 6 into a [3-ketoalkyl radical. The elimination of ammonia from ~-aminoc~-hydroxyalkyl radicals has also been reported [33]. --CH-C-I I OR OH 22
(6)
~
--CH-C-II
+
ROH
O
23
While ot-hydroxyalkyl radicals are of a reducing nature [cf. reaction (7)] [17, 34], the resulting [3-ketoalkyl radicals have oxidizing properties. The latter can effect (slow) H-abstraction from the substrate [reaction (8)] which in the case of ethylene glycol leads to a short chain reaction [35].
486
--C--
+
F e ( C N ) 3-
(7)
'
OH
~
--C--
+
Fe(CN)64-
+
H
"
0
O
O
II
II
9C H 2 - - C - H
+
CH2--CH 2 I I OH OH
20
(8)
>
CH3--C-H
+
CH--CH2 I i O H O H 18
Interestingly, the reaction of the (reducing) et-hydroxyalkyl radicals with thiols [reaction (9)] is relatively fast while the (oxidizing) formylmethyl radical does not react with thiols at an appreciable rate [36]. In contrast, in basic solution where the thiolate form predominates, a rapid reduction, by electron transfer, of the formylmethyl radical is observed [reaction (10)] [36].
"CH--CH2 I
I
OH OH
RSH
(9)
CH2--CH2 "-
I
+
RS
I
OH OH
18
(1)1 - H20 .CH2_c..O 9
RS ~
H
(10)
20
~
CH2--C"O . o
+
RS"
H HG w (11)
CH3_C,"-'O H
In carbohydrates (and polyhydric alcohols), the importance of the waterelimination reaction is reflected in the mix of products. Deoxy compounds dominate among the products (cf. Table 1). A full material balance has been obtained in the case of ethylene glycol, the prototype of this group [35]. However, already in the case of the monomeric carbohydrates
487 Table 1 - y-Radiolysis of D-glucose in N20 [37] and N20/O2-saturated [18] solutions. Products and their G values. Product N20 N20/Oa D-Gluconic acid 0.15 0.90 D-arabino-Hexosulose 0.15 0.90 D-xy/o-Hexos-3-ulose 0.10 0.57 D-xylo-Hexos-4-ulose 0.08 0.50 D-xylo-Hexos-5-ulose 0.18 0.60 D-gluco-Hexodialdose 0.95 1.55 2-Deoxygluconic acid 0.95 absent D-Glucuronic acid absent 0.05* 5-Deoxy-4-keto-glucose 1 2-Deoxy-5-keto-glucose t 0.08 absent 5-Deoxy-xy/o-hexodialdose J 3-Deoxy-4-keto-glucose 4-Deoxy-5-keto-glucose t 0.25 absent 3-Deoxy-glucosone J 6-Deoxy-5-keto-glucose 0.05 absent D-Arabinose 0.01 0.07 D-Xylose < 0.005 0.01 Ribose < 0.005 xy/o-Pentodialdose absent 0.07 2-Deoxy-ribose 0.04 absent 3-Deoxy-pentulose < 0.005 absent D-Arabinonic acid absent 0.03 D-Erythrose 0.01 0.01 L-threo-Tetrodialdose absent 0.20 Butan-2-one- 1,4-diol 0.02 absent D-Erythronic acid absent 0.01 D-Glyceraldehyde absent 0.06 D-Glyceric acid absent 0.07 Dihydroxyacetone 0.03 absent Glyoxal absent O. 11 Glyoxylic/glycolic acids absent 0.4 Formaldehyde absent 0.12 Formic acid absent 0.6 Hydrogen peroxide 3.0 Glucose consumption 5.6 *secondary product
488
Table 2 Approximate values for rate constants of phosphate release from some (~methoxy-~-phosphatoalkyl radicals according.to [38]. Radical
Rate constant /
"CH(OCH3)CH2OPO3H2
106
"CH(OCH3)CH2OPO3H"
103
"CH(OCH3)CH2OPO32"
S "1
0.1-1
"CH(OCH3)CH2OPO3(CH2CH2OCH3)H
107
"CH(OCH3)CH2OPO3 (CH2CHzOCH3)"
103 _ 104
"CH(OCH3)CH2OPO3(CHzCHzOCH3)2
4 x 10 7
such as glucose the number of the products becomes fairly large so that at a given dose the amount of a particular product tends to be small; both reasons contribute to making the analysis of the products difficult. Thus it has not been possible in the case of glucose and other carbohydrates of similar molecular size to determine the dimer fraction, in contrast to the disproportionation products. This leads to a sizeable deficit in the elemental balance (with respect to, say, carbon compared to the amount of OH radicals and H atoms produced which is known from their G value and the applied dose), as can be seen from Table 1. In contrast, the formation of the dimeric products is suppressed in the presence of 02, which permits a better balance to be achieved (cf. Table 1). 2.2. [3-Elimination of anionic leaving groups" phosphate In the case of good anionic leaving groups, such as phosphate cf [38], sulfate [39], or halogen [40], these can be eliminated directly by bond heterolysis. These eliminations can be very fast even in the absence of base catalysis, the rate depending on the state of protonation of the leaving group (Table 2). In ~-hydroxy-[3-phosphatoalkyl radicals, phosphate elimination and hydroxyl proton loss may occur in a concerted manner. In the glycerol phosphates, the elimination occurs within a few microseconds [27, 41, 42]. This type of reaction plays a role in the nucleobase-radical-induccd strand breakage of poly(U) [cf. reaction (17)]. In this reaction, a radical at C(2') 17 is generated. In DNA, strand
489
breakage occurs from the C(4') radical 15 [43]. In this case, however, the rate of strand breakage depends strongly on the pH. This is due to a change in mechanism. As has been shown in model systems (cf. Table 2) the rate of phosphate elimination is very fast when the leaving phosphate group is neutral [reaction (12)]; it drops by three orders of magnitude when it is mono-negatively charged [reaction (14)] and again in about the same proportion upon further deprotonation [reaction (16)]. This is a strong indication that a radical cation must be formed in this reaction which subsequently undergoes hydrolysis.
CH30-Ct-P-CH2-O-~'-OH 24a
OH
(12)
CH30-d~--CHfO--~O ~ 24e
O o
+
O-~x-'OH OH
9
9
CH30_C~H_CH2_O_~_O o 24b OH
~ CH30-CH--CHff 25
CH3O-(14)
(16)
-CH
+
25
CH O-:I -CH3 25
OH
+
6~
The free-radical chemistry of carbohydrate phosphates exhibits the same characteristics [e.g. reaction (17)], with the additional option that if the freeradical site materializes ~ to the carbohydrate-ring oxygen atom, ring cleavage may occur (see below). Ribose-5-phosphate [44-49] provides an example for this kind of behaviour.
490
I
I
o_~oO
O--~nO e
O---(~
?
Ur~acil
~
17 OH
O--- H2
Uracil
(17)
26
+ H|
~e
o=r--o,
o=~o.
O
0---
Further reaction pathways may be opened up in more highly substituted carbohydrates, e.g. nucleosides. Radical cations are strong oxidants, and it has been shown recently that the radical cation 27 formed in reaction (18) can oxidize the neighbouring guanine residue [reaction (19)] [50, 51].
I
I ? ~~---O e
I
o~~oO
?
O--~---O e
O-- ooe
(18) 15
? o=r--o, o---
~174
(19) 27
?~ o=~o. o---
?~
28
O-~-~-OH
O----
2.3 Scission of the glycosidic linkage and ring cleavage In the radiation chemistry of polysaccharides, the scission of the glycosidic linkage is the most conspicuous process as it leads to the reduction of their molecular weight. There are alternative pathways to this process, as well as to ring cleavage (which is observed, of course, already in monosaccharides) which in some cases may precede glycosidic scission: (i) homolytic transfer of the radical site, to end up on the opposite side of the bond to be cleaved (radical 13 cleavage), (ii) hydrolysis of the carbon-alkoxyl bond [3 to the radical site which does not remove. Radical 13 cleavage has been observed in prototypical ~alkoxyalkyl radicals (cf. [52]); the ease of this reaction depends on the degree of stabilization of the ensuing radical (an OH group as in ~-hydroxyalkyl confers
491 some stability). Radical [3 cleavage [reactions (20), (26) and (27); conceivably reaction (23) also occurs, but the products resulting from 34 have not yet be found] can be concurrent with hydrolysis [reactions (22) and (24)] of the radical; these reactions are shown here for the case of cellobiose; similar reactions are observed with other disaccharides [53, 54] (see also [14, 55, 56]).
(20) CH2OH
o~--~o HO
I OH
CH2OH /[----O
CH2OH ,~-OOH
I OH
I OH
~o~O~)~o +~-q~ ~
CH2OH
)--ooH
30
"~ ~ ~ ' 29 OH
31
CHaOH
CH2OH )----O OH
I-I20
+ ~o~O~~
(21)
I OH
I OH 32 CH2OH )---OOH
1
CH2OH
L-o.
CH2OH
CHaOH
~-ooH
OH
I OH 32
OH 33 CHaOH
~-o (23)
.o-~
OH 4
CH20H
~---0OH
~ o~~1?~
OH
OH
34
35
492 CH2OH
CH2OH
CHeOH
CH2OH
rio "HoO l)-~~ H ,~oH )---OOHoH Ho .I~O~ ~H )=K /O (25) I [ I OH OH OH -
36
~-ooH
+
I
OH 33
28
(24)l H20 CHzOH 9; - - : o ~
CH2OH
CH2OH
CH2OH
t=o H ;--:ooH
J-2ooH
(26) I
I
OH
OH
I
OH
38
I
39
OH
(27)1
/~o + ~~--~oHo~H
OH
H
CH2OH
I
I
OH 40
OH 31
The scission of the glycosidic linkage may occur not only on the flee-radical, but also on the product level. When the flee-radical site becomes isolated from the glycosidic function, or removed through a flee-radical termination reaction or oxidation, the ensuing hemiacetal-like or orthocarbonic-acid-like structures undergo hydrolysis relatively fast, e.g. reaction (25). A more complex process leading to this type of scission is conceivable in radicals such as 29, via ring cleavage, acyl migration in the ensuing ester [57], and hydrolysis when the labile C(1) acetal-ester form materializes [58] (though in the case of cellobiose, no product was detected that bears the trace of the corresponding (C4) radical formed upon the ring-opening of 29, therefore reaction not shown). Similarly in the case of DNA and related compounds, oxidation of the (C1 ') radical leads to the formation of the orthoester-amide structure 42 [reaction (28)] which subsequently eliminates the base [reaction (29)] [59]. These reactions may occur
493 in competition with other reactions, including rearrangements that eventually lead to isomerization at (CI') [reactions (30)-(35)] [60, 61].
ox
HO--CH2
HO--CH2
!o ..fo . aso )O:o
(28)
ase
(29)
I
OH 4:1
tO~'~ase
HO--CH2
OH
OH 43
HO--CH2 Red (30)
d/~2 ase r
41
OH 44
HO--CH2 (32) 1-120
Red (31)
v
ase OH 45 Red (34)
HO--CH2
~ ~-o~~ se OH 46
H
OH
-n2o (33)
H[ ~ ~ B a s e
_- ~ o ( 3~ OH 47
48
Base Red (35)
~ :ae OH
49
It has been mentioned that under favourable conditions, e.g. with benzyl glycosides, the glycosidic linkage is readily broken also by the action of the solvated electron whose attachment to the aglycon moiety [15] leads to rapid scission of the glycosidic linkage, in competition with the protonation of the electron adduct by water. The driving force for reaction (36) is the formation of the highly-stabilized benzyl radical 51.
494 ~oOH
~
CHzOH
-CH2--~~) i_IO,~ ( OH
50
eaOq~_.._ H o ~ O ~ (36) 32 OH
51
2.4. Reactions with m o l e c u l a r oxygen The carbon-centered radicals generally react very fast with oxygen [e.g. reaction (37)], so that under conditions of air saturation many of the elimination reactions discussed above are forestalled. This gives rise to the corresponding peroxyl radicals (for reviews on peroxyl radicals in aqueous solution see [62, 63]). Among them, the ~-hydroxyalkylperoxyl radicals stand out, one, because they outnumber the others, and two, because this kind of radical is known relatively easily to eliminate HOz'[e.g. reaction (38)].
CHzOH
CH2OH 02 (37)
1
I OH
.CH2OH
-
H6~
/
52OH
OH
H02"
)--o
?~
(38) 3O
I OH
This spontaneous HO2-elimination is thought to be a concerted process [64]. The rate of the HO2-elimination reaction depends strongly on the nature of neighbouring groups (electronic and steric effects). In carbohydrates the situation becomes rather complex mechanistically [65] as there are several kinds of c~-hydroxyalkylperoxyl radical present. The HO2-elimination reaction is of the type that is also undergone by other compounds that carry the structural element - C ( X ) O H - where X is a good leaving group, such as gem-halohydrins (cf. [66-
68]. In basic solution, deprotonation at the hydroxyl group (typically) leads to a very rapid elimination of the superoxide radical anion [65, 69]. The deprotonation reaction was found to be the rate-limiting step in all of those cases where the kinetics were studied in sufficient detail. The peroxyl radical derived from a carbon-centered radical such as 53 apparently eliminates superoxide in a similar fashion [18]; this probably happens following the pathway (39), (40). In the case of glycosides, this pathway is blocked, and alternatives such as reaction (41) may be envisaged.
495
9 0-0.
CHzOH [ OOH
CH20H
OH ~
9O-O--)--O
H20
CH20H
0 9
_o~.__
(39)
(40)
o OooH~ H
I
53 OH
40 OH
53a OH
CHeOH _.
CHeOH
)--:oo "~ ) - q o .
l_o_O-
I
OH
54
02 ~
H
CHzOH
CHeOH
HooH~o
)--~o.
(41)
I
OH
OH
55 OH
(42)1 1-120
H|
+
CHzOH
CH2OH
oo~Oo.
)--,oH
OH
56 OH
Evidence for this type of superoxide elimination exists in similar "non-ahydroxyl" systems [70], with rates apparently varying across a wide range. Thus, the half-time of reaction (43) is 30 gs [71] whereas that of reaction (44) is at least three orders of magnitude longer [72].
,CH3 O"
,CH3 O"
I
CH3--C-O-O" I
57
(43)
O ~CH3
~
I
CH3--C | !
+
o:
O
58
'CH3
.O. O-O"
Co"t-59
(44)
no superoxide elimination on pulse radiolysis time scale
It seems that these rates are strongly dependent on the flexibility of the acetalic structure. In the present case, the rate is expected to be relatively slow. This means that such peroxyl radicals may competitively undergo bimolecular
496 termination, giving rise to tertiary oxyl radicals that are prone to fragmentation [62, 63], in which the glycosidic linkage may likewise be cleaved. This pathway cannot materialize in monosaccharides which have the faster deprotonationsuperoxide elimination pathway (OH group geminal to peroxyl group, reaction (38)) open to them [65]. For D-glucose, the complete set of reactions (not shown) is not excessively cumbersome to be written out, and the products compiled in Table 1 not only give a complete material balance with respect to the yield of the initiating OH radicals, but also indicates that fragmentation products are formed in very minor yields. The fact that 5-keto-glucose is a major product shows that an HO2"/O2"-elimination must have occurred also in the case of the C(5)-peroxyl radical. This is expected to be much slower than the other HO2"-elimination reactions, and the formation of 5-keto-glucose proceeds via deprotonation at the C(1)-OH group, followed by the elimination of 02"- [reactions (39) and (40)]. Apart from dioxygen, free radicals may also be oxidized by other oxidants, such as transition metal ions in higher valence states, mostly in complexed form [cf. reaction (7)] [34], or nitro compounds (see for instance [72, 73]). These reactions do not necessarily proceed via direct electron transfer but apparently mostly through the intermediacy of an adduct. The finer details remain to be elucidated. 2.5. D e g r a d a t i o n of polymeric carbohydrates From among the natural carbohydrate polymers, we mention here cellulose, chitin and its deacetylated form chitosan, hyaluronic acid (hyaluronan), and heparin. Apart from cellulose, the monomer-unit sequences are not strictly regular, but the structures given below are representative. Chitosan, hyaluronic acid, and heparin are water-soluble because they carry electrically-charged functions. Since cellulose and chitin are insoluble in water, most of their radiation chemistry has been done in the solid state, as discussed below. Yields of molecular-weight reduction have usually been determined by viscosimetry and, more recently, by the laser light-scattering technique.
CH2SO~ C02H
1---o [~O
CHzOH oo-
oH
_
CH2OH
CHeOH o-
N iso
OH
H
NHCOCH3
Hyaluronic acid
i
o
OSO3 Heparin
A Chitosan
I x~ NI COC A: ca. 90%, B: ca. 10%
497 The flee-radical-induced reduction of the molecular weight of polymeric carbohydrates must occur via the scission of a glycosidic linkage, or v i a phosphate release in DNA and related polymers. Mechanistic aspects of these reactions have been discussed above. An additional aspect comes into play on account of the action of shear which tends to enhance the importance of polymer-chain scission by flee-radical transfer [cf. reactions (20) - (27)], versus the other reaction pathways open to the radicals involved, compared with the unstressed situation in, e.g. , disaccharides, because of the drastic weakening of the chemical bonds ~ to the radical site. In polymers, the resulting products are difficult to characterize chemically as each cleavage produces two chemically modified ends per a large number of unmodified units, although this has been achieved for DNA [10]. It is, however, relatively easy to determine the kinetics of the cleavage process by pulse radiolysis, using conductometry (in the case of charged polymers) and low-angle laser light-scattering (charged and noncharged polymers). Where the polymer is charged, a fraction of the counter-ions is condensed onto the polymer due to the high electric field exerted locally by the charged groups along the polymer chain. As the broken ends diffuse apart upon free-radical-induced chain scission, the force of the electric field exerted by the fragments near the cleavage site weakens and some of the counter-ions diffuse into the bulk of the solution. As a consequence of this, the conductance increases above that observed before the irradiation impulse. In contrast to processes where protons (and anions) are released and the conductance increases in the acid pH range but decreases in the basic range as the proton is neutralized and thus both H § and OH- disappear, fragmentation-induced counter-ion release always leads to an increase of conductance, irrespective of pH. Such experiments, originally carried out with poly(U) [74], have been done in the carbohydrate series proper with hyaluronic acid [75, 76] and with chitosan (cf. Figure 1).
498
.....o qD
<1
I
go
,
0
• I
9
I
,
I
0.2 0.4 0.6
time / s I
I
l
i
I
0
10 20 30 time / ms Figure 1. Pulse radio]ysis of chitosan in N20-saturated aqueous solutions. Kinetics of chain scission as detected by changes in conductance [77]. In Fig. 1, the kinetics of counter-ion release are shown for the chitosan system. It is evident that these kinetics cannot be described by a single firstorder process. This also holds for hyaluronic acid [75, 76]. This is in fact not to be expected because here more than one kind of radical may contribute to chain breakage (cf. scission of the glycosidic linkage in disaccharides discussed above). At present, it is not yet possible to correlate a given reaction with a specific time frame of the kinetic experiment. However, the counter-ion release experiment yields quantitative data which can be set in relation to the observed decrease of the molecular weight (chain scission). From such data it is has been calculated that close to 8 counter-ions are released per chain break [78]. Such values vary somewhat with the system, as they depend on the linear density of electrical charge along the polymer molecule. In the presence of oxygen, such conductometry experiments cannot be carried out with confidence because of the copious formation of superoxide in these systems (see above). Its relatively rapid decay (e.g. by reaction of O2"- with polymer-bound peroxyl radicals) itself causes a conductance change. Therefore under those conditions, the kinetics of chain scission must be followed by lowangle laser light-scattering [75]. As it turns out, these reactions are no longer kinetically of first order, but the rates observed depend strongly on the strength of the pulse, i.e. there are important second-order contributions [75]. The chainbreaking reactions that occur in the presence of oxygen are less well understood
499 (for a model system study see [79]) as those in the absence of oxygen, but it is evident that the fixation of radicals by oxygen that in its absence cause chain scission, reduces the yield of chain breakage considerably [73, 75, 76]. This is in contrast to other, non-carbohydrate polymers where a chain scission only becomes noticeable in the presence of oxygen (see for instance [80]). Nevertheless, some polymers such as poly(acrylic acid) and poly(methacrylic acid) undergo effective chain scission (via a ~-fragmentation process) also in the absence of oxygen [81, 82]. Thus, ionizing radiation provides an excellent tool to reduce the molecular weight of a carbohydrate polymer whenever required for medicinal application, e.g. in the case of chitosan [83-85]. On the other hand, an increased stability against free-radical induced depolymerization might be required. This may be achieved by chemically crosslinking the native linear polymer; the radiolytic behaviour of cross-linked hyaluronic acid, called hylan, has been studied [86]. The free-radical-induced degradation of heparin in aqueous solution is reported to proceed without the formation of free sulfate [87] which would suggest that the initial radical attack occurs exclusively at positions C(1) and/or C(4). In puzzling contrast, NMR data regarding the reduced-molecular-weight heparin fragments seem to indicate an increasing loss of sulfate from the fragments as the dose increases and the fragments get smaller [88]. In view of the fact that sulfate is a good ~-elimination leaving group (see above), this is in agreement with expectation.
3. RADIOLYSIS IN THE SOLID STATE In close analogy to the radiolysis of water which produces the free electron and the water radical cation which in turn gives rise to the OH radical and a proton, the radical cations generated in neat carbohydrates, polyhydroxy compounds, will likewise deprotonate at oxygen yielding oxyl radicals and protons. The latter may react with the electron, forming an H atom which abstracts the carbon-bound hydrogen yielding molecular hydrogen. The oxyl radicals can undergo 13-fragmentation or H-abstraction from neighbouring carbohydrate molecules (the 1,2-H shift of ~-hydrogen-containing oxyl radicals [89-91] seems to be restricted to aqueous solutions). The major part of the radicals remain immobilised in the crystal matrix and only interact with one another when the irradiated sample is dissolved in water for analysis. For instance, the products which are observed after the ~(-radiolysis of D-glucose [92] are compiled in Table 3.
500 Table 3 y-Radiolysis of crystalline D-glucose. Products and their G values [92]. Further (as yet not quantified) products are: glucosone, 3-keto-glucose, 4-keto-glucose, 5-keto-glucose and gluconic acid. Product
G value
Dihydroxyacetone
0.05
3-Deoxytetrose
0.015
1,4-Dideoxy-2-pentulose
0.05
2,4-Dideoxypentose
0.09
Threose / Erythrose / Erythrulose / Erythronic acid
0.04
1 -Deoxy-2-pentulose
0.005
2-Deoxyribose
0.25
3-Deoxypentosulose
0.02
3,5-Dideoxyhexonic acid
0.02
2,3-Dideoxyhexonic acid
0.01
Arabinose
0.25
Ribose / Ribonic acid
0.02
2-Deoxy-2-C-hydroxymethylpentonic acid
0.06
5-Deoxygluconic acid/2-Deoxy-5-keto-glucose
0.02
2-Deoxygluconic acid / 2-Deoxy-5-keto-glucose
0.1
3-Deoxyglucosone / 3-Deoxygluconic acid / 3-Deoxy-4-keto-glucose
0.2
3-Deoxy-4-keto-glucose / 2-Deoxy-3-keto-glucose
0.19
Hydrogen
5.75
501
It can be seen that in contrast to its radiolysis in aqueous solution (see Table 1), in the solid state there is a larger contribution from fragmentation products which we attribute to the participation of oxyl radicals under these conditions. As expected, hydrogen is a major product and the material balance is poor despite the fact that a large number of products have been quantified (Table 3). Again, the missing complement should mainly consist of dimers which would escape analysis. Table 4. y-Radiolysis of crystalline D-fructose. Products and their G values. Further (as yet not quantified) products are: glyceraldehyde, 3-butanone-l,2-diol, 2- and 3deoxyhexodiuloses [92]. Product
G value
2-Deoxytetrose
0.5
Threose / Erytrose / Erythulose
0.65
3-Deoxypentonic acid/3-Deoxypentosulose
0.3
Arabonic acid
0.1
Ribonic acid
0.05
6-Deoxy-D-threo-2,5-hexodiulose Hydrogen
40 4.75
502
Table 5 Radiation-induced chain reactions in some crystalline carbohydrates. The chain products and their G values. .....
Substrate
Product
2-Deoxy-D-ribose c~-Lactobiose 9 H20
D-Fructose
G value
Ref.
2,5-Dideoxypentonic acid
650
[13,93]
2-Deoxylactobionolactone
20
[94, 95]
5-Deoxylactobionic acid
40
4-Deoxy-o-glucose + Galactonolactone
4.5
6-Deoxy-o-threo-hexodiulose
40
[12,92,96]
1.1
[97]
N-Acetylglucosamine 2-Acetylanfmo-2,5-L-threo-dJdeoxyhexodialdose
If a similar situation were always to exist with all crystalline carbohydrates,
i.e. major amounts of hydrogen, a large number of minor products and a considerable unresolved dimer fraction, the attention that this field has found would perhaps have been undeserved. Interestingly, however, radiation-induced chain reactions were observed in the case of several crystalline carbohydrates (see Tables 4 and 5; note also the dominance of the chain product (6-deoxy-Dthreo-2,5-hexodiulose) compared to the fragmentation products, in Table 4). The chain reaction which often leads to an isomer of the substrate molecule or its dehydration product is governed by the crystal structure, as it is no longer observed in crystals of the same compound, but with a different structure. The most effective chain reaction is observed with 2-deoxy-D-ribose in the 2deoxy-~-D-erythro-pentopyranose form [reactions (45) - (47)] [13, 93]. The chain comes to a halt when the propagating radical 62 abstracts an H atom from the product molecule just formed [reaction (48)]. The resulting radical 64 is hence trapped between two product molecules and has been identified by EPR as the most persistent radical in this system [98]. The crystal structure clearly shows the pathway taken through the crystal. In the pristine crystal the distance between the sites of reaction is < 3.3 A [ 13], and upon rearrangement these two centres might come closer to one another in order for reaction (47) to proceed.
503 OOH HOl~- ~
OOH radiation v HO ioniTing ,~ i~ ~ (45)
OH 60
N
o
OH 61 "CH2 0
I (46)
HO•g30 OH
OH 63
Ho~OH OH 62
neighbouring 2-deoxyribose
+
61
~
63
CH30
(47)
OH OH 64
A similar distance has been calculated for the crystal structure of 13-o-fructose for the H-transfer in the propagating step (see below) [12]. The G value drops with increasing dose indicating that termination reactions are favoured by imperfections. There is also a characteristic change of the irradiated 2deoxyribose crystal: it turns highly hygroscopic at moderate doses. The sequence involved in the chain reaction traversing D-fructose is depicted by reactions (49) and (50). In (x-lactose monohydrate, several chain reactions are observed at the same time, yielding 4-deoxy-o-glucose plus galactonolactone, 5-deoxylactobionic acid and 2-deoxylactobionolactone [reactions (51) - (56) [94, 95]. 7-Radiolysis of ~lactobiose monohydrate is the easiest way to prepare 5-deoxylactobionic acid [94], because it is readily isolated from the irradiated material by ion exchange chromatography. N-Acetylglucosamine is listed in Table 5" even though the G value of the corresponding product appears quite small, it is large compared to typical nonchain products (cf. Table 3); this is taken as an indicator for a short chain reaction. Some aspects of the pathway to 2-acetylamino-2,5-L-threodideoxyhexodialdose are fairly well understood: the chain must start from the radical at C(6) with subsequent carbinol elimination [cf. reaction (6)] which here implies ring opening followed by H-abstraction from a neighbouring N-actylglucosamine molecule.
504 r - - - o OH
"CH2 OH
•~ .o~
. o < ~o~oH --
H O ' ~ CH2OH OH 65
(49)
~ CH2OH OH 66
CH30 65
+
0==(
D-Fructose
HO~ [
+ H20
4t
CH2OH OH 67 HO~..~H2~)H
CH2OH
~_~2oOH
68
CH2OH
HO ~---0 [
(51) OH
(50)
OH
~ 0 H 70 OH
69 OH
.CH2OH 68
+
H 71 OH ,CHzOH ,CH2OH HO)---Q ),. H"Jf"'HO
72 +
H OH
75
OH
or-lactose (52)
co-lactose (55) I
CHzOH Ho)--o ~
CHzOH H-~. o
(53)
H OH 73
OH
-
CH2OH 72
OH
Ho)--:o ~
OH
CH2OH
J-Co
(54) CH2OH
Ho~0 ~ 72 +
~
\
CHeOH
OH 74
)--:o
O
~
OH
76
O
~ a-lactose(56) ]
H
505 A considerable amount of work has been devoted to the action of ionizing radiation on polymeric carbohydrates in the solid state, in particular with regard to its effect on the mechanical properties even though some chemical effects have also emerged. For a recent review see [99]. Of these polymers, cellulose has been the most studied (cf. [99, 100]). Native cellulose is locally crystalline. Determination of chain breakage by radiolysis has usually been done after dissolution through complexation, e.g. as the copper-ammine complex (Schweizer' s reagent), or the cadmium-ethylenediamine complex. In contrast to aqueous-dissolved systems where the OH radical and the H atom are the main initiators, radical cations plus electrons and excited states are formed in the solid state. This is also the case with cellulose dispersed in water [101]. The radical cations are transformed by deprotonation [e.g. reactions (57) and (58)]. These suffer fragmentation such as reactions (20) - (23), as discussed above, o r , for example, reaction (59). CH2OH
o
CH2OH
o
I OH
o 77
CH2OH
I 3OH
-
CH2OH
o o,
I OH
I OH
CH2OH
O OH
CH2OH
78
CH2OH
O~
+ H|
OH CH2OH
'O O
OH 79
O I OH
+
- (58) I O.
80
H |
I OH
When cellulose is irradiated in v a c u o G ( H 2 ) = 3, G ( C O 2 ) = 6 and G(CO) = 1 • 10 -7 mol j-a have been observed [99]. The gaseous products CO and CO2 are ascribed to the decay of highly excited states (excited radical cations). It is noted that CO formation is also of some importance in the radiolysis of alcohols in the liquid and frozen state [102, 103]. However, for CO2 and CO, a flee-radical pathway starting with the ~-fragmentation of the oxyl radical 80 [reaction (59)] is also conceivable, as one expects the radicals in the solid to have a long lifetime due to a reduced mobility, which could selectively permit reactions (60) - (64) to occur. Values for G(chain scission) of chitin (1.1 - 1.8 x 10 -v mol Jq) are lower than of (solid) heparin (-- 3 • 10 -7 mol j-l) and cellulose (-- 6 • 10 -7 mol j-l) [99]. Cellulose degradation G values in wood are lower by half or more, presumably
300
because the affinity of the lignin for electrons and holes is greater than of the cellulose fiber [99].
CH2OH
CH2OH
CH2OH
~oHO~o O~~HO O
~
1
~
O.
80
H2OH ~.
O
H
)---O~,
cH OH
O
CH2OH
81
OH
(61)
~o~HOH CH
86
II
O
CH20H
CH
85 OH
82
O
83
(62)1 - CO2
9
(60)
I
CH2OH ~.
x~HcH 84
~oHO
(59)
CH2OH
I
OH
1 (63)
.CH2OH ~, ,,[9. " CH
.
)
CH2OH ~,
co
. .
+
85
(64) 88
87
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481
Carbohydrates C. von Sonntag and H.-P. Schuchmann
Max-Planck-Institut ftir Strahlenchemie P.O. Box 101365, Stiftstrasse 34-36, D-45470 Mtilheim an der Ruhr, Germany
1. I N T R O D U C T I O N The initial impetus for the study of the chemical behaviour of carbohydrates under the influence of ionizing radiation consisted in the hope that the power of the new sources of radioactivity might provide a practical way of transforming abundant natural carbohydrates, especially cellulose which is relatively difficult to deal with chemically or enzymatically, into other useful chemicals [1-3], and in the need to examine the chemical effect of radiation sterilization of carbohydrate-containing foods with respect to its wholesomeness after such treatment [4, 5]; it has been observed that carbohydrate-derived compounds carrying additional keto functions [6] can be mutagenic [7], similar to various aldehydes [8]. A second major incentive was originally derived from radiationprotection considerations. In order to evaluate possible chemical protective strategies it was necessary to obtain mechanistic information on the production of radiation-induced DNA damage such as the formation of strand breaks and the release of nucleobases [9]. The latter processes originate from radicals generated at the deoxyribose moiety of DNA whose backbone is a chain consisting of 2-deoxyribose units bridged by phosphate groups at the positions 3' and 5'. The use of various carbohydrate model compounds, among them functionalized ones, in the detailed mechanistic studies of the radiation-induced damage of its sugar moiety proved to be crucial since DNA offers a variety of reactive sites for flee-radical attack, notably the nucleobases whose reactivity in this situation tends to overshadow the reactivity shown by the deoxyribose moiety as such [10]. Thus, for a better understanding of the mechanistic principles that lead to DNA strand breakage and base release, a large number of low-molecular-weight carbohydrate systems have been investigated [ 11 ]. Ionizing radiation is in fact a convenient means to induce free-radical chemistry such as can be set in motion, for instance, by oxidative processes.
482
Thus, the free-radical-induced degradation of hyaluronic acid, another carbohydrate biopolymer, is a major aspect of rheumatoid arthritis, which has provoked radiation-chemical studies to elucidate the kinetics of its depolymerization (the lubricating property of hyaluronic acid in a joint is rapidly lost when its molecular weight is decreased). Most of such radiation-chemical studies of carbohydrates have been carried out in aqueous solution. Besides, there are crystalline-state studies which in some cases show interesting examples of solid-state chain reactions (cf. [2]). Some of the paths traced by the radical function as the reaction propagates through the crystal can be inferred on the basis of X-ray crystallography (cf. [12, 13]). 2. AQUEOUS S O L U T I O N S Carbohydrates (many of them being at the same time polyhydric alcohols) and those of their derivatives that are of interest in the above context are usually water soluble (notable exceptions, the biopolymers cellulose and chitin). Good water solubility has allowed to study the radiation chemistry of their aqueous solutions without the difficulties that are encountered in the case of poorly soluble substrates. Difficulties are mainly of a product-analytical nature. It is relatively easy to determine the low-molecular weight carbohydrate products that are formed via the unimolecular transformation of the starting radical, while it is very difficult to analyse completely the complex mixtures of the dehydro dimers which arise from the substrates under anoxic conditions as several different primary radicals intercombine and, in addition, different stereochemical options exist for each combination. The formation of the primary radicals in these systems is governed by Habstraction reactions, effected by OH" and H" since the substrates usually exist mainly in the hemiacetal form (or acetal form in the case of disaccharides and polysaccharides). The hydrated electron eaq" is essentially non-reactive in the absence of the free carbonyl function (there are exceptions with certain glycosides when the glycosidic group upon elimination can form a stabilized radical) [14, 15]. In radiation-chemical experimentation eaq is usually transformed into the OH" radical with N20. Due to the much lower bond dissociation energy of the C-H bond compared to the O-H bond, practicality only carbon-centred radicals are formed. For the OH radical, the reaction rate constants are typically near 2 x 10 9 dm 3 mol 1 s-1, and about one to two orders of magnitude slower for the H atom [16]. Generally, there is no pronounced regioselectivity [17], i.e. in D-glucose, radicals 1-6 are formed in comparable
483 yields [ 18]. Higher selectivity is observed with other H-atom abstractors, e.g. the sulfate radical anion [19]. Considering the type of chemistry they undergo, the primary radicals from regular carbohydrates may be divided into several groups (that may partially overlap), e.g., 1,2-diol-type radicals such as 1-4, 2-alkoxyl-hydroxyalkyl radicals such as 4 and 6, and ether-type radicals such as 1 and 5. CH2OH
CH2OH
J--oo.
o~
)--oo.
?.
~o~O~.~ ~o~O~~ I
OH
9
OH
1
2
CH2OH
,L-oo. 9
CH2OH
;--oo.
OH
I
OH 3
CH2OH
-~oo.
~o~O~~
oCHOH
)--ooH
I
OH
I
OH
4
OH
5
6
In disaccharides and polymeric carbohydrates, the ether-type radicals such as 7 and 8 have the radical site proximate to the glycosidic linkage and therefore play a major role in its scission (see below). In glucosamine [20] and Nacetylglucosamine [21] as well as in their related polymers chitin, chitosan and hyaluronic acid, one must further consider radicals of the type 9-11. Thus, one may expect that in addition to the behaviour shown by the prototypical carbohydrate radicals, reactions such as are observed with amino acids (cf. [22]) and peptides (cf. [23-25]), may also play a role. CH2OH
CH2OH
J-:o
)--:oo.
CH2OH
CH2OH
~-:o.
.J-:oo.
~o~~o.~o~~ ~o~O~j~o_~o~~ I
I
OH
OH 7
I
I
OH
OH 8
484 CHeOH J---OOH
CH2OH ~---OOH
_ NH2
Ho~OH.~ , NHCOCH3
-NH
9
CH2OH ~--OOH
10
11
In DNA, five different radicals may be formed at the sugar moiety. The radical site in 12 is proximate to the N-glycosidic linkage and can give rise to base release [26]. Radical 13 appears to behave in the way of an alkyl-type radical. Radicals 14 and 16 have phosphate groups in (x-position, while radical 15 has these groups in a ~-position linked to an oxygen atom which makes heterolytic cleavage possible (see below). ~-Phosphato radicals such as 14 and 16 do not show phosphoester release [27]. In RNA and ribo-polynucleofides which lack the deoxy function one must take radical 17 into consideration which regarding the ease of phophoester release resembles 15.
I
I
O=oHO
ase
I
()H
O=P-O-CH2 ase ()H
O
I o
I o
I
ase
o
i
I
O=P-O~
O=P-O~
O=P-O~
OH
OH
OH
12
13
14
i
I O=PHO
i
i
I
-
ase
I
()n
O
ase
O=P-O-CH2 ()n
ase
I o
I o
I o
O=P-O~
O=P-O~
O=P-O~
i
I
OH 15
i
I
OH 16
I
I OH
I
OH 17
It has been mentioned above that in these systems the radiation chemistry is mainly governed by the free-radical chemistry of the nucleobases, and much of the knowledge about their chemistry has to come from model studies.
485
As discussed below, there are two types of unimolecular reaction pathway open to all of these carbohydrate radicals, i.e. "ionic" elimination, and cleavage of a carbon-oxygen bond [3 to the free-radical site. Depending on whether the substituent is a hydroxyl group, an ester, ether, or other hetero function, preference will lie toward one or the other reaction channel. 2.1. Elimination
of water or alcohol
Many carbohydrates have the structural element -CHOH-CHOH-. Radicals such as 1 - 4, just like 18, generated by H-abstraction from such sites can eliminate water [28]. This reaction occurs at a relatively slow rate spontaneously ("water-catalyzed"), but faster upon acid or base catalysis [reactions (1)-(3)].
H~ w (1)
I_I,O,H OH
OH o -
OH
OH
~H2--~H ___._. - H20
19
H*
(4)
_ H |
,,(3 9C H T - C ,
(2)
2O
18
H
-~
(5)
OH e (3)
OH O 21
Carbinol ROH can be similarly eliminated [reaction (6)] (this reaction has been studied in a variety of model compounds [29, 30]; cf. further [31, 32] and references therein); this reaction as well converts the exocylic glucose-derived radical 6 into a [3-ketoalkyl radical. The elimination of ammonia from ~-aminoc~-hydroxyalkyl radicals has also been reported [33]. --CH-C-I I OR OH 22
(6)
~
--CH-C-II
+
ROH
O
23
While ot-hydroxyalkyl radicals are of a reducing nature [cf. reaction (7)] [17, 34], the resulting [3-ketoalkyl radicals have oxidizing properties. The latter can effect (slow) H-abstraction from the substrate [reaction (8)] which in the case of ethylene glycol leads to a short chain reaction [35].
486
--C--
+
F e ( C N ) 3-
(7)
'
OH
~
--C--
+
Fe(CN)64-
+
H
"
0
O
O
II
II
9C H 2 - - C - H
+
CH2--CH 2 I I OH OH
20
(8)
>
CH3--C-H
+
CH--CH2 I i O H O H 18
Interestingly, the reaction of the (reducing) et-hydroxyalkyl radicals with thiols [reaction (9)] is relatively fast while the (oxidizing) formylmethyl radical does not react with thiols at an appreciable rate [36]. In contrast, in basic solution where the thiolate form predominates, a rapid reduction, by electron transfer, of the formylmethyl radical is observed [reaction (10)] [36].
"CH--CH2 I
I
OH OH
RSH
(9)
CH2--CH2 "-
I
+
RS
I
OH OH
18
(1)1 - H20 .CH2_c..O 9
RS ~
H
(10)
20
~
CH2--C"O . o
+
RS"
H HG w (11)
CH3_C,"-'O H
In carbohydrates (and polyhydric alcohols), the importance of the waterelimination reaction is reflected in the mix of products. Deoxy compounds dominate among the products (cf. Table 1). A full material balance has been obtained in the case of ethylene glycol, the prototype of this group [35]. However, already in the case of the monomeric carbohydrates
487 Table 1 - y-Radiolysis of D-glucose in N20 [37] and N20/O2-saturated [18] solutions. Products and their G values. Product N20 N20/Oa D-Gluconic acid 0.15 0.90 D-arabino-Hexosulose 0.15 0.90 D-xy/o-Hexos-3-ulose 0.10 0.57 D-xylo-Hexos-4-ulose 0.08 0.50 D-xylo-Hexos-5-ulose 0.18 0.60 D-gluco-Hexodialdose 0.95 1.55 2-Deoxygluconic acid 0.95 absent D-Glucuronic acid absent 0.05* 5-Deoxy-4-keto-glucose 1 2-Deoxy-5-keto-glucose t 0.08 absent 5-Deoxy-xy/o-hexodialdose J 3-Deoxy-4-keto-glucose 4-Deoxy-5-keto-glucose t 0.25 absent 3-Deoxy-glucosone J 6-Deoxy-5-keto-glucose 0.05 absent D-Arabinose 0.01 0.07 D-Xylose < 0.005 0.01 Ribose < 0.005 xy/o-Pentodialdose absent 0.07 2-Deoxy-ribose 0.04 absent 3-Deoxy-pentulose < 0.005 absent D-Arabinonic acid absent 0.03 D-Erythrose 0.01 0.01 L-threo-Tetrodialdose absent 0.20 Butan-2-one- 1,4-diol 0.02 absent D-Erythronic acid absent 0.01 D-Glyceraldehyde absent 0.06 D-Glyceric acid absent 0.07 Dihydroxyacetone 0.03 absent Glyoxal absent O. 11 Glyoxylic/glycolic acids absent 0.4 Formaldehyde absent 0.12 Formic acid absent 0.6 Hydrogen peroxide 3.0 Glucose consumption 5.6 *secondary product
488
Table 2 Approximate values for rate constants of phosphate release from some (~methoxy-~-phosphatoalkyl radicals according.to [38]. Radical
Rate constant /
"CH(OCH3)CH2OPO3H2
106
"CH(OCH3)CH2OPO3H"
103
"CH(OCH3)CH2OPO32"
S "1
0.1-1
"CH(OCH3)CH2OPO3(CH2CH2OCH3)H
107
"CH(OCH3)CH2OPO3 (CH2CHzOCH3)"
103 _ 104
"CH(OCH3)CH2OPO3(CHzCHzOCH3)2
4 x 10 7
such as glucose the number of the products becomes fairly large so that at a given dose the amount of a particular product tends to be small; both reasons contribute to making the analysis of the products difficult. Thus it has not been possible in the case of glucose and other carbohydrates of similar molecular size to determine the dimer fraction, in contrast to the disproportionation products. This leads to a sizeable deficit in the elemental balance (with respect to, say, carbon compared to the amount of OH radicals and H atoms produced which is known from their G value and the applied dose), as can be seen from Table 1. In contrast, the formation of the dimeric products is suppressed in the presence of 02, which permits a better balance to be achieved (cf. Table 1). 2.2. [3-Elimination of anionic leaving groups" phosphate In the case of good anionic leaving groups, such as phosphate cf [38], sulfate [39], or halogen [40], these can be eliminated directly by bond heterolysis. These eliminations can be very fast even in the absence of base catalysis, the rate depending on the state of protonation of the leaving group (Table 2). In ~-hydroxy-[3-phosphatoalkyl radicals, phosphate elimination and hydroxyl proton loss may occur in a concerted manner. In the glycerol phosphates, the elimination occurs within a few microseconds [27, 41, 42]. This type of reaction plays a role in the nucleobase-radical-induccd strand breakage of poly(U) [cf. reaction (17)]. In this reaction, a radical at C(2') 17 is generated. In DNA, strand
489
breakage occurs from the C(4') radical 15 [43]. In this case, however, the rate of strand breakage depends strongly on the pH. This is due to a change in mechanism. As has been shown in model systems (cf. Table 2) the rate of phosphate elimination is very fast when the leaving phosphate group is neutral [reaction (12)]; it drops by three orders of magnitude when it is mono-negatively charged [reaction (14)] and again in about the same proportion upon further deprotonation [reaction (16)]. This is a strong indication that a radical cation must be formed in this reaction which subsequently undergoes hydrolysis.
CH30-Ct-P-CH2-O-~'-OH 24a
OH
(12)
CH30-d~--CHfO--~O ~ 24e
O o
+
O-~x-'OH OH
9
9
CH30_C~H_CH2_O_~_O o 24b OH
~ CH30-CH--CHff 25
CH3O-(14)
(16)
-CH
+
25
CH O-:I -CH3 25
OH
+
6~
The free-radical chemistry of carbohydrate phosphates exhibits the same characteristics [e.g. reaction (17)], with the additional option that if the freeradical site materializes ~ to the carbohydrate-ring oxygen atom, ring cleavage may occur (see below). Ribose-5-phosphate [44-49] provides an example for this kind of behaviour.
490
I
I
o_~oO
O--~nO e
O---(~
?
Ur~acil
~
17 OH
O--- H2
Uracil
(17)
26
+ H|
~e
o=r--o,
o=~o.
O
0---
Further reaction pathways may be opened up in more highly substituted carbohydrates, e.g. nucleosides. Radical cations are strong oxidants, and it has been shown recently that the radical cation 27 formed in reaction (18) can oxidize the neighbouring guanine residue [reaction (19)] [50, 51].
I
I ? ~~---O e
I
o~~oO
?
O--~---O e
O-- ooe
(18) 15
? o=r--o, o---
~174
(19) 27
?~ o=~o. o---
?~
28
O-~-~-OH
O----
2.3 Scission of the glycosidic linkage and ring cleavage In the radiation chemistry of polysaccharides, the scission of the glycosidic linkage is the most conspicuous process as it leads to the reduction of their molecular weight. There are alternative pathways to this process, as well as to ring cleavage (which is observed, of course, already in monosaccharides) which in some cases may precede glycosidic scission: (i) homolytic transfer of the radical site, to end up on the opposite side of the bond to be cleaved (radical 13 cleavage), (ii) hydrolysis of the carbon-alkoxyl bond [3 to the radical site which does not remove. Radical 13 cleavage has been observed in prototypical ~alkoxyalkyl radicals (cf. [52]); the ease of this reaction depends on the degree of stabilization of the ensuing radical (an OH group as in ~-hydroxyalkyl confers
491 some stability). Radical [3 cleavage [reactions (20), (26) and (27); conceivably reaction (23) also occurs, but the products resulting from 34 have not yet be found] can be concurrent with hydrolysis [reactions (22) and (24)] of the radical; these reactions are shown here for the case of cellobiose; similar reactions are observed with other disaccharides [53, 54] (see also [14, 55, 56]).
(20) CH2OH
o~--~o HO
I OH
CH2OH /[----O
CH2OH ,~-OOH
I OH
I OH
~o~O~)~o +~-q~ ~
CH2OH
)--ooH
30
"~ ~ ~ ' 29 OH
31
CHaOH
CH2OH )----O OH
I-I20
+ ~o~O~~
(21)
I OH
I OH 32 CH2OH )---OOH
1
CH2OH
L-o.
CH2OH
CHaOH
~-ooH
OH
I OH 32
OH 33 CHaOH
~-o (23)
.o-~
OH 4
CH20H
~---0OH
~ o~~1?~
OH
OH
34
35
492 CH2OH
CH2OH
CHeOH
CH2OH
rio "HoO l)-~~ H ,~oH )---OOHoH Ho .I~O~ ~H )=K /O (25) I [ I OH OH OH -
36
~-ooH
+
I
OH 33
28
(24)l H20 CHzOH 9; - - : o ~
CH2OH
CH2OH
CH2OH
t=o H ;--:ooH
J-2ooH
(26) I
I
OH
OH
I
OH
38
I
39
OH
(27)1
/~o + ~~--~oHo~H
OH
H
CH2OH
I
I
OH 40
OH 31
The scission of the glycosidic linkage may occur not only on the flee-radical, but also on the product level. When the flee-radical site becomes isolated from the glycosidic function, or removed through a flee-radical termination reaction or oxidation, the ensuing hemiacetal-like or orthocarbonic-acid-like structures undergo hydrolysis relatively fast, e.g. reaction (25). A more complex process leading to this type of scission is conceivable in radicals such as 29, via ring cleavage, acyl migration in the ensuing ester [57], and hydrolysis when the labile C(1) acetal-ester form materializes [58] (though in the case of cellobiose, no product was detected that bears the trace of the corresponding (C4) radical formed upon the ring-opening of 29, therefore reaction not shown). Similarly in the case of DNA and related compounds, oxidation of the (C1 ') radical leads to the formation of the orthoester-amide structure 42 [reaction (28)] which subsequently eliminates the base [reaction (29)] [59]. These reactions may occur
493 in competition with other reactions, including rearrangements that eventually lead to isomerization at (CI') [reactions (30)-(35)] [60, 61].
ox
HO--CH2
HO--CH2
!o ..fo . aso )O:o
(28)
ase
(29)
I
OH 4:1
tO~'~ase
HO--CH2
OH
OH 43
HO--CH2 Red (30)
d/~2 ase r
41
OH 44
HO--CH2 (32) 1-120
Red (31)
v
ase OH 45 Red (34)
HO--CH2
~ ~-o~~ se OH 46
H
OH
-n2o (33)
H[ ~ ~ B a s e
_- ~ o ( 3~ OH 47
48
Base Red (35)
~ :ae OH
49
It has been mentioned that under favourable conditions, e.g. with benzyl glycosides, the glycosidic linkage is readily broken also by the action of the solvated electron whose attachment to the aglycon moiety [15] leads to rapid scission of the glycosidic linkage, in competition with the protonation of the electron adduct by water. The driving force for reaction (36) is the formation of the highly-stabilized benzyl radical 51.
494 ~oOH
~
CHzOH
-CH2--~~) i_IO,~ ( OH
50
eaOq~_.._ H o ~ O ~ (36) 32 OH
51
2.4. Reactions with m o l e c u l a r oxygen The carbon-centered radicals generally react very fast with oxygen [e.g. reaction (37)], so that under conditions of air saturation many of the elimination reactions discussed above are forestalled. This gives rise to the corresponding peroxyl radicals (for reviews on peroxyl radicals in aqueous solution see [62, 63]). Among them, the ~-hydroxyalkylperoxyl radicals stand out, one, because they outnumber the others, and two, because this kind of radical is known relatively easily to eliminate HOz'[e.g. reaction (38)].
CHzOH
CH2OH 02 (37)
1
I OH
.CH2OH
-
H6~
/
52OH
OH
H02"
)--o
?~
(38) 3O
I OH
This spontaneous HO2-elimination is thought to be a concerted process [64]. The rate of the HO2-elimination reaction depends strongly on the nature of neighbouring groups (electronic and steric effects). In carbohydrates the situation becomes rather complex mechanistically [65] as there are several kinds of c~-hydroxyalkylperoxyl radical present. The HO2-elimination reaction is of the type that is also undergone by other compounds that carry the structural element - C ( X ) O H - where X is a good leaving group, such as gem-halohydrins (cf. [66-
68]. In basic solution, deprotonation at the hydroxyl group (typically) leads to a very rapid elimination of the superoxide radical anion [65, 69]. The deprotonation reaction was found to be the rate-limiting step in all of those cases where the kinetics were studied in sufficient detail. The peroxyl radical derived from a carbon-centered radical such as 53 apparently eliminates superoxide in a similar fashion [18]; this probably happens following the pathway (39), (40). In the case of glycosides, this pathway is blocked, and alternatives such as reaction (41) may be envisaged.
495
9 0-0.
CHzOH [ OOH
CH20H
OH ~
9O-O--)--O
H20
CH20H
0 9
_o~.__
(39)
(40)
o OooH~ H
I
53 OH
40 OH
53a OH
CHeOH _.
CHeOH
)--:oo "~ ) - q o .
l_o_O-
I
OH
54
02 ~
H
CHzOH
CHeOH
HooH~o
)--~o.
(41)
I
OH
OH
55 OH
(42)1 1-120
H|
+
CHzOH
CH2OH
oo~Oo.
)--,oH
OH
56 OH
Evidence for this type of superoxide elimination exists in similar "non-ahydroxyl" systems [70], with rates apparently varying across a wide range. Thus, the half-time of reaction (43) is 30 gs [71] whereas that of reaction (44) is at least three orders of magnitude longer [72].
,CH3 O"
,CH3 O"
I
CH3--C-O-O" I
57
(43)
O ~CH3
~
I
CH3--C | !
+
o:
O
58
'CH3
.O. O-O"
Co"t-59
(44)
no superoxide elimination on pulse radiolysis time scale
It seems that these rates are strongly dependent on the flexibility of the acetalic structure. In the present case, the rate is expected to be relatively slow. This means that such peroxyl radicals may competitively undergo bimolecular
496 termination, giving rise to tertiary oxyl radicals that are prone to fragmentation [62, 63], in which the glycosidic linkage may likewise be cleaved. This pathway cannot materialize in monosaccharides which have the faster deprotonationsuperoxide elimination pathway (OH group geminal to peroxyl group, reaction (38)) open to them [65]. For D-glucose, the complete set of reactions (not shown) is not excessively cumbersome to be written out, and the products compiled in Table 1 not only give a complete material balance with respect to the yield of the initiating OH radicals, but also indicates that fragmentation products are formed in very minor yields. The fact that 5-keto-glucose is a major product shows that an HO2"/O2"-elimination must have occurred also in the case of the C(5)-peroxyl radical. This is expected to be much slower than the other HO2"-elimination reactions, and the formation of 5-keto-glucose proceeds via deprotonation at the C(1)-OH group, followed by the elimination of 02"- [reactions (39) and (40)]. Apart from dioxygen, free radicals may also be oxidized by other oxidants, such as transition metal ions in higher valence states, mostly in complexed form [cf. reaction (7)] [34], or nitro compounds (see for instance [72, 73]). These reactions do not necessarily proceed via direct electron transfer but apparently mostly through the intermediacy of an adduct. The finer details remain to be elucidated. 2.5. D e g r a d a t i o n of polymeric carbohydrates From among the natural carbohydrate polymers, we mention here cellulose, chitin and its deacetylated form chitosan, hyaluronic acid (hyaluronan), and heparin. Apart from cellulose, the monomer-unit sequences are not strictly regular, but the structures given below are representative. Chitosan, hyaluronic acid, and heparin are water-soluble because they carry electrically-charged functions. Since cellulose and chitin are insoluble in water, most of their radiation chemistry has been done in the solid state, as discussed below. Yields of molecular-weight reduction have usually been determined by viscosimetry and, more recently, by the laser light-scattering technique.
CH2SO~ C02H
1---o [~O
CHzOH oo-
oH
_
CH2OH
CHeOH o-
N iso
OH
H
NHCOCH3
Hyaluronic acid
i
o
OSO3 Heparin
A Chitosan
I x~ NI COC A: ca. 90%, B: ca. 10%
497 The flee-radical-induced reduction of the molecular weight of polymeric carbohydrates must occur via the scission of a glycosidic linkage, or v i a phosphate release in DNA and related polymers. Mechanistic aspects of these reactions have been discussed above. An additional aspect comes into play on account of the action of shear which tends to enhance the importance of polymer-chain scission by flee-radical transfer [cf. reactions (20) - (27)], versus the other reaction pathways open to the radicals involved, compared with the unstressed situation in, e.g. , disaccharides, because of the drastic weakening of the chemical bonds ~ to the radical site. In polymers, the resulting products are difficult to characterize chemically as each cleavage produces two chemically modified ends per a large number of unmodified units, although this has been achieved for DNA [10]. It is, however, relatively easy to determine the kinetics of the cleavage process by pulse radiolysis, using conductometry (in the case of charged polymers) and low-angle laser light-scattering (charged and noncharged polymers). Where the polymer is charged, a fraction of the counter-ions is condensed onto the polymer due to the high electric field exerted locally by the charged groups along the polymer chain. As the broken ends diffuse apart upon free-radical-induced chain scission, the force of the electric field exerted by the fragments near the cleavage site weakens and some of the counter-ions diffuse into the bulk of the solution. As a consequence of this, the conductance increases above that observed before the irradiation impulse. In contrast to processes where protons (and anions) are released and the conductance increases in the acid pH range but decreases in the basic range as the proton is neutralized and thus both H § and OH- disappear, fragmentation-induced counter-ion release always leads to an increase of conductance, irrespective of pH. Such experiments, originally carried out with poly(U) [74], have been done in the carbohydrate series proper with hyaluronic acid [75, 76] and with chitosan (cf. Figure 1).
498
.....o qD
<1
I
go
,
0
• I
9
I
,
I
0.2 0.4 0.6
time / s I
I
l
i
I
0
10 20 30 time / ms Figure 1. Pulse radio]ysis of chitosan in N20-saturated aqueous solutions. Kinetics of chain scission as detected by changes in conductance [77]. In Fig. 1, the kinetics of counter-ion release are shown for the chitosan system. It is evident that these kinetics cannot be described by a single firstorder process. This also holds for hyaluronic acid [75, 76]. This is in fact not to be expected because here more than one kind of radical may contribute to chain breakage (cf. scission of the glycosidic linkage in disaccharides discussed above). At present, it is not yet possible to correlate a given reaction with a specific time frame of the kinetic experiment. However, the counter-ion release experiment yields quantitative data which can be set in relation to the observed decrease of the molecular weight (chain scission). From such data it is has been calculated that close to 8 counter-ions are released per chain break [78]. Such values vary somewhat with the system, as they depend on the linear density of electrical charge along the polymer molecule. In the presence of oxygen, such conductometry experiments cannot be carried out with confidence because of the copious formation of superoxide in these systems (see above). Its relatively rapid decay (e.g. by reaction of O2"- with polymer-bound peroxyl radicals) itself causes a conductance change. Therefore under those conditions, the kinetics of chain scission must be followed by lowangle laser light-scattering [75]. As it turns out, these reactions are no longer kinetically of first order, but the rates observed depend strongly on the strength of the pulse, i.e. there are important second-order contributions [75]. The chainbreaking reactions that occur in the presence of oxygen are less well understood
499 (for a model system study see [79]) as those in the absence of oxygen, but it is evident that the fixation of radicals by oxygen that in its absence cause chain scission, reduces the yield of chain breakage considerably [73, 75, 76]. This is in contrast to other, non-carbohydrate polymers where a chain scission only becomes noticeable in the presence of oxygen (see for instance [80]). Nevertheless, some polymers such as poly(acrylic acid) and poly(methacrylic acid) undergo effective chain scission (via a ~-fragmentation process) also in the absence of oxygen [81, 82]. Thus, ionizing radiation provides an excellent tool to reduce the molecular weight of a carbohydrate polymer whenever required for medicinal application, e.g. in the case of chitosan [83-85]. On the other hand, an increased stability against free-radical induced depolymerization might be required. This may be achieved by chemically crosslinking the native linear polymer; the radiolytic behaviour of cross-linked hyaluronic acid, called hylan, has been studied [86]. The free-radical-induced degradation of heparin in aqueous solution is reported to proceed without the formation of free sulfate [87] which would suggest that the initial radical attack occurs exclusively at positions C(1) and/or C(4). In puzzling contrast, NMR data regarding the reduced-molecular-weight heparin fragments seem to indicate an increasing loss of sulfate from the fragments as the dose increases and the fragments get smaller [88]. In view of the fact that sulfate is a good ~-elimination leaving group (see above), this is in agreement with expectation.
3. RADIOLYSIS IN THE SOLID STATE In close analogy to the radiolysis of water which produces the free electron and the water radical cation which in turn gives rise to the OH radical and a proton, the radical cations generated in neat carbohydrates, polyhydroxy compounds, will likewise deprotonate at oxygen yielding oxyl radicals and protons. The latter may react with the electron, forming an H atom which abstracts the carbon-bound hydrogen yielding molecular hydrogen. The oxyl radicals can undergo 13-fragmentation or H-abstraction from neighbouring carbohydrate molecules (the 1,2-H shift of ~-hydrogen-containing oxyl radicals [89-91] seems to be restricted to aqueous solutions). The major part of the radicals remain immobilised in the crystal matrix and only interact with one another when the irradiated sample is dissolved in water for analysis. For instance, the products which are observed after the ~(-radiolysis of D-glucose [92] are compiled in Table 3.
500 Table 3 y-Radiolysis of crystalline D-glucose. Products and their G values [92]. Further (as yet not quantified) products are: glucosone, 3-keto-glucose, 4-keto-glucose, 5-keto-glucose and gluconic acid. Product
G value
Dihydroxyacetone
0.05
3-Deoxytetrose
0.015
1,4-Dideoxy-2-pentulose
0.05
2,4-Dideoxypentose
0.09
Threose / Erythrose / Erythrulose / Erythronic acid
0.04
1 -Deoxy-2-pentulose
0.005
2-Deoxyribose
0.25
3-Deoxypentosulose
0.02
3,5-Dideoxyhexonic acid
0.02
2,3-Dideoxyhexonic acid
0.01
Arabinose
0.25
Ribose / Ribonic acid
0.02
2-Deoxy-2-C-hydroxymethylpentonic acid
0.06
5-Deoxygluconic acid/2-Deoxy-5-keto-glucose
0.02
2-Deoxygluconic acid / 2-Deoxy-5-keto-glucose
0.1
3-Deoxyglucosone / 3-Deoxygluconic acid / 3-Deoxy-4-keto-glucose
0.2
3-Deoxy-4-keto-glucose / 2-Deoxy-3-keto-glucose
0.19
Hydrogen
5.75
501
It can be seen that in contrast to its radiolysis in aqueous solution (see Table 1), in the solid state there is a larger contribution from fragmentation products which we attribute to the participation of oxyl radicals under these conditions. As expected, hydrogen is a major product and the material balance is poor despite the fact that a large number of products have been quantified (Table 3). Again, the missing complement should mainly consist of dimers which would escape analysis. Table 4. y-Radiolysis of crystalline D-fructose. Products and their G values. Further (as yet not quantified) products are: glyceraldehyde, 3-butanone-l,2-diol, 2- and 3deoxyhexodiuloses [92]. Product
G value
2-Deoxytetrose
0.5
Threose / Erytrose / Erythulose
0.65
3-Deoxypentonic acid/3-Deoxypentosulose
0.3
Arabonic acid
0.1
Ribonic acid
0.05
6-Deoxy-D-threo-2,5-hexodiulose Hydrogen
40 4.75
502
Table 5 Radiation-induced chain reactions in some crystalline carbohydrates. The chain products and their G values. .....
Substrate
Product
2-Deoxy-D-ribose c~-Lactobiose 9 H20
D-Fructose
G value
Ref.
2,5-Dideoxypentonic acid
650
[13,93]
2-Deoxylactobionolactone
20
[94, 95]
5-Deoxylactobionic acid
40
4-Deoxy-o-glucose + Galactonolactone
4.5
6-Deoxy-o-threo-hexodiulose
40
[12,92,96]
1.1
[97]
N-Acetylglucosamine 2-Acetylanfmo-2,5-L-threo-dJdeoxyhexodialdose
If a similar situation were always to exist with all crystalline carbohydrates,
i.e. major amounts of hydrogen, a large number of minor products and a considerable unresolved dimer fraction, the attention that this field has found would perhaps have been undeserved. Interestingly, however, radiation-induced chain reactions were observed in the case of several crystalline carbohydrates (see Tables 4 and 5; note also the dominance of the chain product (6-deoxy-Dthreo-2,5-hexodiulose) compared to the fragmentation products, in Table 4). The chain reaction which often leads to an isomer of the substrate molecule or its dehydration product is governed by the crystal structure, as it is no longer observed in crystals of the same compound, but with a different structure. The most effective chain reaction is observed with 2-deoxy-D-ribose in the 2deoxy-~-D-erythro-pentopyranose form [reactions (45) - (47)] [13, 93]. The chain comes to a halt when the propagating radical 62 abstracts an H atom from the product molecule just formed [reaction (48)]. The resulting radical 64 is hence trapped between two product molecules and has been identified by EPR as the most persistent radical in this system [98]. The crystal structure clearly shows the pathway taken through the crystal. In the pristine crystal the distance between the sites of reaction is < 3.3 A [ 13], and upon rearrangement these two centres might come closer to one another in order for reaction (47) to proceed.
503 OOH HOl~- ~
OOH radiation v HO ioniTing ,~ i~ ~ (45)
OH 60
N
o
OH 61 "CH2 0
I (46)
HO•g30 OH
OH 63
Ho~OH OH 62
neighbouring 2-deoxyribose
+
61
~
63
CH30
(47)
OH OH 64
A similar distance has been calculated for the crystal structure of 13-o-fructose for the H-transfer in the propagating step (see below) [12]. The G value drops with increasing dose indicating that termination reactions are favoured by imperfections. There is also a characteristic change of the irradiated 2deoxyribose crystal: it turns highly hygroscopic at moderate doses. The sequence involved in the chain reaction traversing D-fructose is depicted by reactions (49) and (50). In (x-lactose monohydrate, several chain reactions are observed at the same time, yielding 4-deoxy-o-glucose plus galactonolactone, 5-deoxylactobionic acid and 2-deoxylactobionolactone [reactions (51) - (56) [94, 95]. 7-Radiolysis of ~lactobiose monohydrate is the easiest way to prepare 5-deoxylactobionic acid [94], because it is readily isolated from the irradiated material by ion exchange chromatography. N-Acetylglucosamine is listed in Table 5" even though the G value of the corresponding product appears quite small, it is large compared to typical nonchain products (cf. Table 3); this is taken as an indicator for a short chain reaction. Some aspects of the pathway to 2-acetylamino-2,5-L-threodideoxyhexodialdose are fairly well understood: the chain must start from the radical at C(6) with subsequent carbinol elimination [cf. reaction (6)] which here implies ring opening followed by H-abstraction from a neighbouring N-actylglucosamine molecule.
504 r - - - o OH
"CH2 OH
•~ .o~
. o < ~o~oH --
H O ' ~ CH2OH OH 65
(49)
~ CH2OH OH 66
CH30 65
+
0==(
D-Fructose
HO~ [
+ H20
4t
CH2OH OH 67 HO~..~H2~)H
CH2OH
~_~2oOH
68
CH2OH
HO ~---0 [
(51) OH
(50)
OH
~ 0 H 70 OH
69 OH
.CH2OH 68
+
H 71 OH ,CHzOH ,CH2OH HO)---Q ),. H"Jf"'HO
72 +
H OH
75
OH
or-lactose (52)
co-lactose (55) I
CHzOH Ho)--o ~
CHzOH H-~. o
(53)
H OH 73
OH
-
CH2OH 72
OH
Ho)--:o ~
OH
CH2OH
J-Co
(54) CH2OH
Ho~0 ~ 72 +
~
\
CHeOH
OH 74
)--:o
O
~
OH
76
O
~ a-lactose(56) ]
H
505 A considerable amount of work has been devoted to the action of ionizing radiation on polymeric carbohydrates in the solid state, in particular with regard to its effect on the mechanical properties even though some chemical effects have also emerged. For a recent review see [99]. Of these polymers, cellulose has been the most studied (cf. [99, 100]). Native cellulose is locally crystalline. Determination of chain breakage by radiolysis has usually been done after dissolution through complexation, e.g. as the copper-ammine complex (Schweizer' s reagent), or the cadmium-ethylenediamine complex. In contrast to aqueous-dissolved systems where the OH radical and the H atom are the main initiators, radical cations plus electrons and excited states are formed in the solid state. This is also the case with cellulose dispersed in water [101]. The radical cations are transformed by deprotonation [e.g. reactions (57) and (58)]. These suffer fragmentation such as reactions (20) - (23), as discussed above, o r , for example, reaction (59). CH2OH
o
CH2OH
o
I OH
o 77
CH2OH
I 3OH
-
CH2OH
o o,
I OH
I OH
CH2OH
O OH
CH2OH
78
CH2OH
O~
+ H|
OH CH2OH
'O O
OH 79
O I OH
+
- (58) I O.
80
H |
I OH
When cellulose is irradiated in v a c u o G ( H 2 ) = 3, G ( C O 2 ) = 6 and G(CO) = 1 • 10 -7 mol j-a have been observed [99]. The gaseous products CO and CO2 are ascribed to the decay of highly excited states (excited radical cations). It is noted that CO formation is also of some importance in the radiolysis of alcohols in the liquid and frozen state [102, 103]. However, for CO2 and CO, a flee-radical pathway starting with the ~-fragmentation of the oxyl radical 80 [reaction (59)] is also conceivable, as one expects the radicals in the solid to have a long lifetime due to a reduced mobility, which could selectively permit reactions (60) - (64) to occur. Values for G(chain scission) of chitin (1.1 - 1.8 x 10 -v mol Jq) are lower than of (solid) heparin (-- 3 • 10 -7 mol j-l) and cellulose (-- 6 • 10 -7 mol j-l) [99]. Cellulose degradation G values in wood are lower by half or more, presumably
300
because the affinity of the lignin for electrons and holes is greater than of the cellulose fiber [99].
CH2OH
CH2OH
CH2OH
~oHO~o O~~HO O
~
1
~
O.
80
H2OH ~.
O
H
)---O~,
cH OH
O
CH2OH
81
OH
(61)
~o~HOH CH
86
II
O
CH20H
CH
85 OH
82
O
83
(62)1 - CO2
9
(60)
I
CH2OH ~.
x~HcH 84
~oHO
(59)
CH2OH
I
OH
1 (63)
.CH2OH ~, ,,[9. " CH
.
)
CH2OH ~,
co
. .
+
85
(64) 88
87
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