`
`Review of Possible Boron Speciation
`Relating to Its Essentiality
`William G. Woods*
`
`Office of Environmental Health and Safety, University of California, Riverside,
`Riverside, California
`
`Boron is one of the very few elements known to be essential in plants and is yet to be
`unequivocally proven as essential in animals and humans. Animal and human research on
`essentiality would benefit if the speciation of boron in biological fluids and tissues could be
`determined. This is complicated by the myriad of functional biomolecules with which
`inorganic borates can react and by the exceedingly low concentrations of boron present
`under physiological conditions. This review brings together published literature on the
`interaction of boron with biochemical systems which bear on the question of its essentiality.
`Some fundamentals of boron chemistry that are germane to the issue of speciation in living
`organisms are reviewed. Potential mechanisms of boron action in plants are discussed, with
`a view toward predicting effects in other organisms. Complexation with polyhydroxyl
`compounds, a well-known feature of boron chemistry, and interactions with enzymes,
`cofactors (NAD/NADP), and membranes are proposed as the most likely sites of boron
`involvement. Non-destructive techniques that might be utilized to directly study boron
`speciation in biological systems are discussed.
`© !997 Wiley-Liss. Inc.
`
`Key words: borate ~omplex; enzyme; membrane; NAD; riboflavin
`
`INTRODUCTION
`
`Boron (B) has been known to be essential in plants since I 923 [I] but remains one
`of the few such elements not conclusively proven to be essential in animals [2]. It has
`been theorized that B essentiality arose with the evolution of vascular plants. B also
`has been shown to be essential in diatoms [3] and in nitrogen-fixing cyanobacteria [4],
`with some evidence that it is not essential for green algae [5] or for fungi [5,6]. This
`review will cover aspects of B chemistry relating to the possible interaction of B with
`key biomolecules. The most likely B species to play a role in plant essentiality will
`be reviewed, with implications for essentiality in animals and humans.
`At the low concentration of B found in normal mammalian tissues and fluids
`(0.02-0.5 mg/g, I o-6- I 0-5 M) [7 ,8] and in the absence of interaction with biomol(cid:173)
`ecules, only monomeric boric acid [B(OHh] and tetrahydroxyborate anion [B(OH)4 -1
`are expected to be present. Boric acid is a very weak acid (pKa 9.2). Therefore, at the
`pH of human blood (7.4 ), the B would be >98% in the form of free B(OHh and only
`<2% as B(OH)4 -. Boric acid is known to form esters and complexes with a wide
`variety of mono-, di-, and poly-hydroxy compounds. Such complexes increase the
`
`*Correspondence to: W.G. Woods, Office of Environmental Health & Safety, University of California at
`Riverside, Riverside, CA 92521.
`
`Received 5 October 1996; Accepted 5 November 1996
`
`© 1997 Wiley-Liss, Inc.
`
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`acidity of the boric acid, the classic case being the use of mannitol to increase its
`acidity to a pKa of about 5, making boric acid titrable.
`Besides higher pH, a number of structural factors enhance the stability of such
`complexes. In the case of diols, cis-1,2 are favored over trans or 1,3-diols, and
`five-membered furanosidal are preferred over six-membered pyranosidal 1,2-diols.
`Thus, sugars differ markedly in the stability of their borate complexes [9,10]. The
`presence and nature of a third group in the molecule also can have a stabilizing effect,
`such as a large substituent providing steric protection against hydrolysis, an adjacent
`nitrogen, a group having protons providing H-bonding, or a positively charged sub(cid:173)
`stituent giving electrostatic stabilization.
`Examples of typical biomolecules capable of forming borate complexes are given
`in Figure 1; these are a) Apiose has been found in the cell wall of many plants and
`ribose is a critical component of many biomolecules; both form stable borate com(cid:173)
`plexes, being furanosides with cis-1,2 hydroxyls [ 11 ]. b) As shown by B-affinity
`chromatography [12], the nucleoside adenosine gives a complex more stable than that
`of cytosine, due to the adenine moiety being a more bulky substituent. c) A number
`of neuro-active biomolecules, such as L-dopa and epinephrine, with ortho dihydroxyls
`also are expected to form borate complexes.
`Serine is believed to form a rather stable borate complex~ the amino nitrogen can
`either coordinate or' provide stabilization as a positive charge (Fig. 2). The serine(cid:173)
`borate complex is considered a transition state inhibitor of -y-glutamyl transpeptidase
`and is assumed to form under dilute physiological conditions, since serine or borate
`alone have no effect [ 13, 14]. The borate complex of the coenzyme NAD11 + is 15 times
`stronger than that of NADH due to electrostatic stabilization of the former by the
`positively charged nicotinamide group (Fig. 3) [ 15]. The implications of this for B
`interaction with a number of key biochemical systems is elaborated below.
`
`PLANTS
`
`Some of the functions proposed to account for the essentiality of B in vascular
`plants are listed in Table I [2]. It has been suggested [ l l] that the key role of B is
`cross-linking of the polysaccharide cell wall. Supporting evidence cited was the high
`concentration of B in the cell waH, the presence of apiose for complexation of B as
`well as cross-linking, cell enlargement and wall disorganization with B deficiency
`(-B), and loss of membrane integrity with-B. However, these explanations do not take
`into account the critically specific effects of B on certain enzymes and on membrane
`transport. The pentose phosphate pathway for sugar catabolism is stimulated by -B,
`due to release of inhibition of glucose-6-phosphate and 6-phosphogluconate dehy(cid:173)
`drogenases. This results in the accumulation of phenols, leading to plant damage [ 16].
`These enzymes are held in check by normal levels of B, probably due to the prefer(cid:173)
`ential complexation of cofactor NAO, as shown in Figure 3.
`The glycolytic enzyme phosphoglucomutase has been studied in germination and
`it may also be regulated by B [ 17]. Involvement of borate deficiency in the N AD/
`NADH and NADP/NADPH systems also could alter the energy system of the organ(cid:173)
`ism. In addition, membrane effects of B are very important. Uptake of K 1+, phos(cid:173)
`phate, and glucose by root cells is inhibited by -B and this is reversed by I 0-5 Madded
`B [2]. The trans-membrane proton gradient is decreased by -B. as is ATPase activity
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`155
`
`H
`
`0
`
`OH
`
`OH
`
`0
`
`(a)
`
`OH
`
`OH
`
`Apiose
`
`OH
`
`OH
`
`Ribose
`
`N > N
`
`HOCH2
`
`0
`
`a-i
`
`Cytidine
`
`CH(OH)CH2 NHCHs
`
`1-00--12
`
`0
`
`(b)
`
`a-i
`
`Adenosine
`
`(c)
`
`L-Dopa
`
`a-i
`
`Epinephrine
`
`Fig. 1. Biomolecules capable of forming borate complexes.
`
`[ 18 J. B complexation of membrane glycoproteins and/or glycolipids could be in(cid:173)
`volved in the mechanism of these effects. For example, an adjacent glycoprotein is
`essential for the function of ATPase (see Fig. 4), and the ce11 coat (glycocalyx) has
`many sites for such interactions (see Fig. 5).
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`+ B(OH)3
`
`Fig. 2. Two possible forms of the serine-borate complex. Esterification of a simple primary alcohol in
`dilute, aqueous solution would not be expected unless some special stabilization were available.
`
`OTHER ORGANISMS
`
`In studies of the effects of borate on fungal decay, Lloyd I I 9 I found that the
`non-substrate polyols sorbitol, ribose, and glucose reversed borate dehydrogenase
`inhibition. Similar antidote activity has been seen in the reversal of boric acid inhi(cid:173)
`bition of xanthine oxidase activity by added ribose or sorbitol 1201 and of alcohol
`dehydrogenase by sorbitol, ribose, and mannitol [21 J. The effectiveness of such
`polyols is proportional to the strength of their borate complexes. The most efficient
`diols for reversing ~he inhibition of 6-phosphogluconate were those with cis-adjacent
`hydroxyls in the vicinity of a positive charge, NAO and nicotinamide mononucleotide
`(NMN) both being more efficacious than ribose I 19J.
`Macrocyclic tetrols with ideal geometry for forming tetrahedral borate complexes
`represent the only known B-containing natural products. These are the antibiotics
`boromycin l22], aplasmomycin 123 l, and tartrolon B 1241. Tartrolon A (Fig. 6),
`without the B, extracts B from Pyrex glass to give tartrolon B. The latter inhibits
`RNA, DNA, and protein synthesis in Staph. aureus and in£. coli. Tartrolon Bis better
`than A in removing K+ from S. aureus cell cultures, indicating a membrane transport
`function for the borate complex. Synthetic "cleft" molecules with both a B and a
`crown ether function can transport alkali metal ions and sugars across a synthetic
`membrane (Fig. 7).
`Boric acid rapidly precipitates the rheumatoid factor from serum of human rheu-
`
`NAO-borate
`
`NADH-borate
`
`Fig. 3. Borate complexes of NAD and NADH, showing shifting of concentrations due the greater
`stability of the NAD complex 115].
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`157
`
`TABLE I. Biochemical Systems Involving Boron Potentially Related to Essentiality in Plants*
`
`Biochemical system
`
`Sugar transport
`
`Cell wall synthesis
`
`Lignification
`
`Carbohydrate metabolism
`
`RNA metabolism
`
`Respiration
`
`Phenol metabolism
`
`IAA metabolism
`
`Membrane effects
`
`Method, species used
`- - - - · · - - · · - - - - -
`Movement of sucrose in intact tomato, snap bean; in excised leaves
`of French bean.
`Analysis of tobacco wall; glucose incorporation into field bean wall
`material.
`Lignin determination in sunflower, tobacco: peroxide level and
`distribution in field bean, wheat.
`Effect of borate on in vitro sucrose synthesis, on
`phosphoglucomutase, and on 6-phosphogluconate dehydrogenase.
`Incorporation of labeled precursors. mung bean: -B symptoms by
`base analogs in cotton.
`ATP levels in sunflower; P incorporation into organic phosphates,
`field bean.
`Effect of B on pentose phosphate enzyme; determination of phenol
`levels, compare with -8 effects.
`IAA degradation, compare IAA vs. -B, field bean; IAA levels, oil
`palm: IAA oxidasc, sunflower, squash.
`Effect on mung bean hioelectric fields, on ion transport, membrane
`enzyme, maize; ultrastructure, sunl1ower.
`
`*See ref 121 for original literature citations.
`
`matics, indicating interaction with the sugars on the macroglobulin [25]. Boronate
`affinity chromatography can separate glycosylated hemoglobin from the blood of
`diabetics, pointing to borate complexing of glycoprotein 126}.
`Borate and boronic acids are known to inhibit a variety of serine proteases, in(cid:173)
`cluding a-chymotrypsin [8], Subtilisin Carlsberg [27 j, bovine trypsin [28], and others,
`as well as the manganese-containing allantoate amidohydrolase [291. Borate forms
`a tetrahedral complex at the active site of the enzyme, as shown in Figure 8.
`Baro-amino acids such as boro-leucine [(CH 3)iCHCH2CH(NH2)8(0Hh] are the
`most effective inhibitors known for nasal peptidases [30]. The clotting action of
`thrombin is inhibited by tripeptides terminated by boro-arginine: AA-AA-NH(cid:173)
`CH[B(OHhl-CH2CH2CH2NHC(NH2) = NH 131-331. Leukocyte elastase, which de(cid:173)
`stroys elastin causing emphysema, is inhibited by tetrapeptides terminated by a boro(cid:173)
`amino acid: HOOCCH2CH2CONH-AA-CONH-AA-CONH-AA-CONH-A A-B(OH)z
`[34].
`
`RIBOFLAVIN
`
`Riboflavin (Vitamin B2, 1) is known to form a complex with boric acid which is
`claimed to be 25 times more water soluble than riboflavin itself [35]. A I: I complex
`was isolated and, as judged by effects on optical rotation, some 2: 1 complex also was
`present in solution. Involvement of the hydroxyl on carbon atom 2 of the ribityl side
`chain was indicated. The isoalloxazine ring of 1 is hydrolyzed more slowly in solu(cid:173)
`tions buffered with borate vs. unbuffered solution at the same pH [36]. Excessive
`urinary excretion of 1 has been observed in cases of boric acid ingestion [37,38J.
`Studies in chickens have shown that dietary supplementation with 1 protects against
`boric acid toxicity [39]. It has been proposed [37) that formation of a water-soluble
`borate complex could displace 1 from albumin, which transports it in the plasma.
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`Woods
`
`electrochemical
`
`' sodium
`gradient l •
`
`CYTOSOL
`
`0
`
`D
`
`potassium
`electrochemical
`gradient
`
`~ e
`
`Fig. 4. The Na+-K+ ATPase, showing pumping of Na+ out of and K+ into a cell against their concen(cid:173)
`tration gradients. Also shown is the associated smaller, single-pass glycoprotein. [Reprinted by permission
`from Alberts B, Bray D, Lewis J, Raff M, Robert s K, Watson JD: "Molecular Biology of the Cell," 3rd
`Ed. New York: Garland .Publishing, me. , 1994, p. 514.]
`
`Boric acid also could bind to polyhydroxy flavin nucleotides (FAD, 2), interfering
`with binding affinities of these coenzymes with apoflavoenzymes. Based on the
`above, attempts were made by the author to use proton NMR to investigate the
`structure of the I-borate complex at physiological concentration and pH. Although
`
`adsorbed
`glycoprotein
`
`• = sugar residue
`
`cell coat
`(glycocalyx)
`
`i1,
`
`lipid
`bilayer
`
`CYTOSOL
`
`Fig. 5. Simplified diagram of the cell coat (glycocalyx), showing oligosaccharide side chains (hexagons)
`of glycolipids, glycoproteins, and protoglycans as sites for possible borate complexation. [Reprinted by
`permission from Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD: " Molecular Biology of the
`Cell," 3rd Ed. New York: Garland Publishing, Inc., l994, p. 503 .)
`
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`Boron Essentiality: Speciation Review
`
`159
`
`Tartrolon A
`
`Tartrolon B
`
`Fig. 6. Tartolon A and B I 241. I Reprinted hy permission of the authors and of the Journal of Antibiotics. I
`
`HO
`HO o
`\ /
`-8
`'o
`
`OopNP
`
`Me
`
`Me
`
`Me
`
`-
`
`Me
`
`Fig. 7. Synthesized "cleft" compound with boronic acid and crown ether functionality, shown com(cid:173)
`plexed with sodium ion and p-nitrophenyl (pNP) r3-D-glucopyranoside. [Reprinted with permission from
`Bien JT. Sheng M, Smith BD: Modification of a boronic acid cleft produces a sodium-saccharatc co(cid:173)
`transporter. J Org Chem 60:2147. 1995. Copyright 1995 American Chemical Society.]
`
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`
`Hls-57
`
`/ N---H+ · CH2-CH Ser-195
`'b---H-N
`I
`I
`~ \
`H-0 E) 0
`HN
`,,
`"u'/
`o/ 'o-u"
`:
`I
`H H
`'N
`~
`
`0
`0
`II
`II
`CH2- O-P-O-P-O-CH2
`I
`I
`I
`--o-.....
`o·
`OH
`HOCH
`I
`HOCH
`I
`HOCH
`I
`CH2
`
`H
`
`OH
`
`HOH
`I
`0-P-OH
`I
`0
`
`I N1:;N'r°
`
`NH
`
`#
`N
`
`0
`
`Flavine-Adenine Dinucleotide (FAD)
`
`Fig. 8. Tetrahedral borate complex at the active site of serine protease showing histidine participation
`f8].
`STRUCTURES:
`Riboflavin (Vitamin B2, 1)
`Flavine-Adenine Dinucleotide (FAD, 2)
`
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`
`161
`
`definite peaks due to comp]exation were seen at alkaline pH and 0.0 I M in D20,
`dilution to near physiological levels ( I o-6 M in phosphate buffer, pD 7 .00) gave
`inconclusive results due to interference by impurities in the D20.
`
`NON-DESTRUCTIVE METHODS FOR B SPECIATION
`
`Boron-I I NMR is a non-destructive method which can give useful information
`about the speciation of B. It has been used to study a boronic acid/fj-lactamase
`complex at pH 7.4 and 2.5x 10-4 M concentrations of Band enzyme, showing that the
`B is tetrahedral 140]. However, up to 4 million scans were required to obtain these
`spectra. To get useful information under physiological conditions (i.e., at two orders
`of magnitude lower concentration), the sensitivity of 11 B NMR would have to be
`improved enormously.
`For example, an attempt by the author to detect the 1 1 B signal in a I: I boric acid- I
`solution at I 0-5 M after I million scans was unsuccessful. In plants, however, 11 B
`NMR has been used successfully to directly observe B species in vivo [41]. Three B
`signals were observed from radish root, corresponding to free boric acid and mono
`and di complexes of diols, and three from apple, assigned to free boric acid and mono
`and di complexes of hydroxy acids. Similar peaks to the latter also have been ob(cid:173)
`served in wine [42J. Magnetic resonance imaging (MRI) utilizing II B-free induction
`decay has been applied to a live rat treated with a B neutron capture therapy reagent
`143 I; it can detect 80 mg B/g in rat liver in vivo 144].
`Proton NMR, although capable of sufficient sensitivity to observe protons at physi(cid:173)
`ological concentrations, is largely limited to in vitro studies in D20. Direct observa(cid:173)
`tion of physiolog~cal fluids looking for borate complexes would be difficult due to the
`myriad of proton peaks as well as to the problem of interference from the water peak.
`Secondary ion mass spectrometry with imaging (ion microscopy) can be used to
`locate B in tissue at the ppm level 145]. Electron spectroscopy imaging (ESI) has a
`resolution of 3-5A and a sensitivity of 20--50 atoms~ it has been applied to the study
`of agglutination of acinar cells using boronated protein A [46]. Neutron-capture
`radiography has been used to map B down to 0.03 ppm in histological sections of
`mouse tissue 147}. Although these methods provide useful information on the location
`of the B, they can only give hints as to speciation.
`
`CONCLUSIONS
`This review has pointed out the types of biomolecules or associated functional
`groups that are likely to form complexes with borate. These include glycoproteins,
`glycolipids, enzymes, coenzymes, nucleosides, vitamins, and neuroactive catechols.
`Based on research in plants, B in humans is most likely to be involved in enzyme
`regulation, membrane transport, or energy systems. It is the goal of this article to
`make researchers in the trace element field aware of these potential pathways to aid
`in establishing whether B is an essential element in animals and man.
`
`ACKNOWLEDGMENTS
`
`The author thanks Dr. Kevin Simpson for assistance with the structures and Dr.
`James R. Coughlin, Guest Editor, for useful discussions.
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`Woods
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`
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`22. Budavari S, ed.: "The Merck Index." I Ith ED .• New Jersey: Merck and Co., Inc .. 1989, no. 1344.
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`24. lrschik H, Schummer D, Gerth K. Hc>fle G. Reichenbach H: The tartrolons, new boron-containing
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`25. Badin J, Levesque H: Rapid precipitation of rheumatoid factor in a solution of boric acid and
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`immobilized phenylboronic acid. Biochem J 209:771, 1983.
`
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`163
`
`27. Seufer-Wasserthal P, Martichonok V, Keller TH, Chin B, Martin R, Jones JB: Probing the specificity
`of the S 1 binding site of Subtilisin Carlsberg with boronic acids. Bioorg Med Chem 2:35, 1994.
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