PRODUCT AND PROCESS DEVELOPMENT
Controlled Enzymatic Synthesis of Dextran CONDITIONS FOR PRODUCING CLINICALLY SUITABLE MOLECULAR WEIGHT N. N. HELLMAN, H. M. TSUCHIYA, S. P. ROGOVIN, B. L. LAMBERTS, ROBERT TOBIN, C. A. GLASS, C. S. STRINGER, R. W. JACKSON, AND F. R. SENTI N o r t h e r n Ufilizafion Research Branch, Agricultural Research Service,
D
EXTRAN, a polymer of glucose joined chiefly through cr-1,6-glucosidic linkages, has found major application as a blood plasma volume expander. To be suitable for clinical use, according t o present military specifications, the molecular weight of dextran must be 75,000 f 25,000 ( 2 0 ) . Dextran produced by the customary bacterial fermentation ( 8 )or enzymatic method ( 6 , 9 ) , however, is,much too high in molecular weight and must be depolymerized b y some means and fractionated t o yield the desired clinical dextran preparation ( 1 , 3, ?',id, 81). Previous work a t this laboratory (10,li, i7, 18) and by Hehre ( 5 ) has shown that the molecular weight of dextran synthesized by moans of cell-free enzyme liquors can be controlled by adding to reaction mixtures appropriate concentration of primer, which may be either certain oligosaccharides or dextran of low molecular weight (10,11, 17, 18). Nadel and others (IS) also have shown t h a t addition of these substances to a dextran-yielding bacterial fermentation similarly affects molecular weight of dextran produced. T h e present authors have shown t h a t in enzymatic synthesis of dextran the following factors influence molecularweight distribution: primer type, primer concentration, enzyme concentration, sucrose concentration, and temperature at which synthesis is conducted. This paper discusses the above factors particularly as they influence the yield of dextran having clinically useful molecular weight. Experimental materials and methods
Dextran-sucrase. Culture liquors rich in dextran-sucrase were obtained from Leuconostoc inesenteroides N R R L B-512, cultivated in a medium containing sucrose, Basamin-Busch yeast hydrolyzate, and salts (9,18,19).Basamin-Busch yeast hydrolyzate was substituted for corn steep liquor previously employed because its use yielded liquors of higher and more uniform enzyme potency. Upon completion of fermentations, cultures were super-centrifuged to remove most of the bacterial cells. The technique for removal of cells which involved adjustment of p H followed by filtration, found useful with corn steep liquor media, did not result in adequate clarification with this yeast hydrolyzate medium. Culture liquors with activities of 50 t o 70 dextransucrase units per ml. were routinely obtained. One dextransucrase unit is defined as the amount which, under ideal conditions, will convert 1 mg. of sucrose t o dextran in 1 hour at 30" C. and p H 5.0, as determined by increase in reducing power calculated as fructose (19). Culture liquors from which most of the bacterial cells had been removed were stored a t 4" and 16" C. a t pH 5.0 t o 5.3 with a small amount of toluene added t o inhibit contamination. Under these conditions the enzyme solutions were stable for several months. Primers. Dextran of low molecular weight for use as primer was obtained from three sources. 1. From maltose-primed syntheses of dextran, either ( A ) as a fraction obtained by precipitation of dextran between 50 and August 1955
U. S.
Deparfmenf of Agricolfore, Peoria, 111.
60% volume methanol (all methanol concentrations are expressed on a volume-volume basis), or ( B ) a8 the entire unfractionated mixture. 2. From syntheses primed with dextran of low molecular weight, either ( A ) by precipitation of dextran of low molecular weight remaining after recovery of clinical product, or ( B ) by hydrolysis of the fraction too high in molecular weight for clinical product. Primers of Type 2A were derived from reactions primed with unfractionated dext,ran of Type 1B or with dextran fractions of low molecular weight, T y p e 2A. Although t.he upper methanol precipitation limit for all samples of Type 2A was consistently GO%, the lower limit varied from 48 t o 51%. The molecular weight of such fractions generally lay in the range of 15,000 t o 25,000, and the int,rinsic viscosity determined in water a t 25" C. was in the range 0.14 t,o 0.18. The dextran of high molecular weight hydrolyzed for primers of Type 2B was derived from a reaction conducted at 10% sucrose concentration and primed with an unfractionated dextran synthesis mixture (1R). The fraction of high molecular weight was precipitated from the reaction mixture by 42% methanol and was obtained in 18% yield based on the weight of reaction sucrose. Characterization of the hydrolyzates is described below. 3. From a n unprimeci synthesis, by hydrolysis of crude dextran of high molecular weight without subsequent fractionation. The synthesis was conducted with 10% initial sucrose concentration a t 27" C., and crude dextran was recovered by precipitat,ion from the fermentation solution at 50% methanol. Dextran solutions, 5y0 in concentration, were hydrolyzed for primer production at SO" C. in stainless-steel vessels ueing sulfuric acid a t p H 1.0. Hydrolysis required from 1 t o 5 hours, depending on the degree of degradation sought. Characterization of the hydrolyzates is described below.
Enzymatic Syntheses. To sufficient enzyme liquor a t 16" C. to bring the complete reaction mixture to 40 dextran-sucrase units per ml. (9, 19) were added, first', the primer solution, then water t o the desired volume, and finally solid sucrose Reaction mixtures were allowed to stand a t 16" C. for 16 t o 24 hours. Reducing values ( 1 4 )slightly in excess of theory were consistenD1y found a t the end of the reaction period. At, termination of the synthesis, dextran was precipitated by addition of methanol t o 60% concentration. Fractionation. Dextran was first precipitated from the reaction mixture by addition of methanol tJo60% concentration in order t80 remove partially the large amount, of fructose, buffer salts, and culture medium nutrients, and to permit subsequent fractionations t o be performed at predetermined uniform dextran concentrations. All fractionations n-ere performed a t 25' C. by addition of methanol t o solutions containing 5% dextran. Depending on primers and synthesis conditions used, precipitates obtained from reaction mixture a t 60% methanol varied in physical character from dense cakes t o soft flocculent sediments and also in solubility. Depending on the difficulty of securing relatively clear solutions, solutions for fractionation were prepared by dissolving precipitat.es with strong agitation either a t room temperature or a t 90" C., or by autoclaving a t 15 pounds' pressure for 30 minutes. Methanol -concentrations for fractionation are given as volume-volume concentrations calculated from methanol addition without allowance for volume changes on mixing or for
INDUSTRIAL AND ENGINEERING CHEMISTRY
1593
PRODUCT AND PROCESS DEVELOPMENT Table 1. Influence of Dextran-Primer Concentration and Fractionation Conditions on Yield and Molecular Weighj of Fractions Products from Synthesesa Conducted a t Primerb Concentration of: 1%
YieldC,
%
Over-all precipitation 4 4 . 5 of dextran at 60% methanol Methanol 0-40% 40-4870 48-60%
9.8 23.1 10.7
2%
Mol. wt.
YieldC,
%
4%
Mol. wt.
57.8
Yieldc,
%
Mol. wt.
7.0 26.1 23.3
COSR.
69.8
Fractionation I
126;OOO
The middle fraction separated from dextran precipitated from reaction mixtures a t 60% methanol concentration contained approximately 0.03% nitrogen and 0.6% ash. Two more methanol precipitations lowered the nitrogen to the limit of detection, and ash to approximately 0.07%. For the once-fractionated products, [CY]: was 200' i 3 O , and the yield of formic acid in periodate oxidation analysis was 0.95 mole per mole of anhydroglu-
....
76,000 27,800
25.0 7.4 34.5
....
Primer type influences enzymatic synthesis of dextran
82.600 18,400
Generally, dextran produced in primed syntheses appeared in two widely separated molecular-weight ranges, the molecular.O-41% 13.2 8.0 . . . . 27.0 .... weight distribution exhibiting two maxima (I?', 18). One com41-49% 21.6 9i:400 29.6 57,800 7.4 56,000 ponent having a weight-average molecular weight of approxi49-60% 9.1 22,300 17.5 28,000 3 0 . 4 19,400 mately 100 X lo6was equivalent in this respect to the customary Fractionation I11 Methanol product derived from unprimed enzymatic synthesis. The 0-42 % 15.1 8.9 . , .. 2 8 . 5 20.0 4240% 76:300 31.7 65,000 10.6 39:700 other component was much lower in average molecular weight, 8.3 24,400 16 5 50-60% 17,200 26.3 19,100 generally less than 1 X 106, and exhibited varying breadth in a Reaction conditions. Sucrose concentration, 10%; molecular weight of its distribution of molecular weights depending on the primers primer, 13,000; enzyme concentration, 40 dextran-sucrase units per ml.; temperature, 16O C. and reaction conditions employed. Objectives in this investigab Primer, 50-60% methanol fraction from reaction using 20% sucrose and 0.33% maltose: yield, 10.5% reaction-sucrose weight; molecular weight. tion were to determine practical conditions for obtaining maxi13,100,. mum yield of a fraction meeting military mole,cular-weight speciYield expressed as percentage of reaction sucrose weight. fications for clinical dextran (20). Maltose a s a Primer. Of the auxiliary sugars which were tested and showed capability of altering the molecular weight of methanol carried over in the precipitates. As a consequence of the synthesized dextran ( I O ) , maltose seemed most readily variation in treatment of solution, varying amounts of alcohol available in large quantity for process purposes. Laboratory were transferred in the initial precipitate from the reaction mixexperiments, however, showed t h a t maltose had little promise for ture to fractionation solutions. Generally, dextran in the deproduction of dextran of desired molecular weight. Characterissired molecular weight range could be obtained with fractionatically, dextran from maltose-primed enzymatic syntheses was tion limits of 42 and 50% total methanol content. low in molecular weight, and its distribution of molecular weights Analysis. Yields of dextran were based on concentrations was very wide. Yield of clinical-sized dextran was small, as determined polarimetrically, using [a]: = 200' in water or exemplified by the fractionation of R synthesis initiated a t 20% methanol mixtures. Yields of dextran are reported as percentsucrose and 0.33% maltose concentrations. The yield of fraction age of weight of sucrose added to the reaction mixture. Theoof useful molecular weight was only 1.270 of the reaction sucrose. retical maximum yield of dextran in the absence of primer is Greater concentration of maltose favored production of dextran 47.4%. I n reactions employing dextran primers, yields in excess of lower molecular weight, and lower maltose concentration of this figure occurred because the primer dextran contributed to allowed extensive production of the component of very high over-all yield. The efficiency of sucrose conversion was determolecular weight. These results on the use of maltose in enmined by subtracting the weight of primer from the total weight zymatic reactions parallel those observed in whole culture ferof recovered dextran and calculating the theoretical yieltl from mentations ( 1 3 ) . sucrose. Low-Molecular-Weight Dextran Fraction a s a Primer. MalMolecular weights were determined by the light-scattering tose can be used, however, to synthesize dextran of low molecular method using a modified Brice-Speiser light-scattering photomweight which, in turn, may be used more successfully as a primer. eter (2'). Solutions were clarified by means of ultracentrifugaUnfractionated reaction mixture from the maltose-primed syntion and/or filtration through Selas porous clay filters of 01 t o thesis may be added directly to a fresh reaction mixture, in 05 porosity. Usually, centrifugation for 3 hours in a field 42,000 which i t will act as primer. If all sucrose and maltose in a times gravity, followed by filtration through a 02-porosity Selas solution containing 20% sucrose and 0.33% maltose concentrafilter, resulted in adequate clarification of the solutions. Retions were converted to dextran, the concentration of the latter liable analyses of solutions containing dextran of low molecular would be approximately 9.8%. Assuming this dextran content weight could not be performed unless the dissymmetry (146/1135) and adding t h e entire mixture as primer a t 1.5'% theoretical dexwas less than 1.05. Many of the molecular-weight determinatran content to a fresh reaction mixture with 10% sucrose contions were performed by measuring the turbidities of solutions centration resulted in the synthesis of dextran having the proper a t a single concentration in the range 0.25 to 0.50%, and extramolecular weight. By fractionation of the above-described polating the reciprocal specific turbidities to infinite dilution by reaction mixture between 42 and 50% methanol, dextran, 73,000 means of coefficients for the concentration dependence which in molecular weight, was obtained in 11.3% yield based on had been previously determined. A complete description of sucrose used in synthesis of primer plus product dextran. this procedure will be given elsewhere (16). A practical process would be one which would provide not only Viscosities were determined a t 25" C. in a jacketed capillary dextran suitable as an expander of blood plasma volume but flow viscometer which had a flow time for water of 58 seconds. also, as a by-product, the requisite amount of primer for continuaInherent viscosities (4)were calculated from the flow time for tion of the process. First experiments were designed t o teat solutions which exhibited a relative viscosity of 1.10 to 1.50. the feasibility of controlling the primer concentration in the reacAs the coefficient of the first power concentration term of the tion, so that the resulting distribution of molecular weight power series expansion of intrinsic viscosity ( 1 6 ) for dextran in of the synthesized dextran would yield, upon suitable fracthe molecular-weight range of 10,000 to 200,000 is approximately tionation, maximal amounts of clinically useful dextran together 0.5, inherent viscosity - in dilute solution is very nearly with a dextran fraction of low molecular weight adequate in equal t o intrinsic viscosity. amount and molecular weight t o replace the primer consumed. Methanol
31,100
Fractionation I1
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INDUSTRIAL AND ENGINEERING CHEMISTRY
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PRODUCT AND PROCESS DEVELOPMENT
Table 11.
Influence of Primer Molecular Weight on Yield and Molecular Weight of Fractions Methanol Fractionation Limits. %, V./V.
Primer Concn.,
%
Products from Syntheses Conducted with Primer of Molecular Weight 13,100 17,600 Yield, 70 Mol. &. Yield, % Mol. wt. 30.4 13.2 3.0
62,800 24,000
....
13.0 33.2 11.8
86,800 22,300
0-42 42-50 50-60
15.5 20.1 8.3
65,100 19,600
2
0-40 40-48 48-60
6.2 28.9 21.7
66,800 17,500
Table 111.
....
....
1
....
Characterizations of Acid-Hydrolyzed Dextrans Used as Primers
Parent Dextran High-mol.-wt. fraction Crude “native” dextran I Crude “native” dextran I1
Sample No. 15
14 16 22-4 22-8 L-28A L-28B L-2% L-32c 41
Relative Viscosity of 5% Solution
Molecular Weight
Inherent Viscosity
2.69 2.35 2.16 1.92 1.73 2.75 2.33 2.06 2.59 2.69
38,400 32,500 27,400 17,800 13,000 28,700 17,600 10,600 31,000 41 ,400
0.206 0.188 0.180 0.116 0.104 0.194 0.168 0.140 0.185 0.195
trans of molecular weight 13,100 and 17,200 were compared as primers a t 1 and 2% concentrations (Table 11). Fractionation of dextran synthesized a t primer concentrationa of either 1 or 2% showed t h a t the molectilar-weight distribution was shifted markedly t o higher molecular weights by the larger primer. Again, it was noted t h a t the molecular weight fo the fraction of low molecular weight was greater than the primer employed, even though less dextran was isolated in this fraction than was originally added as primer. These findings confirmed t h a t a process for synthesis of clinical dextran would not be selfsustaining if it employed as primer only the fraction too low in molecular weight for clinical use. Dextran Hydrolyzates as Primers. Additional primer was obtained by using unfractionated hydrolyzed dextran of high molecular weight. T h e extent of hydrolysis controls the distribution and average molecular weight of the hydrolyzed dextran which, in turn, controls its effectiveness in synthesis reactions. Samples of a dextran fraction of high molecular weight from a primed synthesis and two crude dextrans from unprimed synthesis were degraded with sulfuric acid to provide a series of primers of varying molecular weight. Characterizations of the samples used in the investigations reported here are presented in Table 111. Samples with the prefix L were small scale laboratory hydrolyses prepared in a glass apparatus, all other hydrolyses were carried out on a larger scale in the pilot plant. The hydrolyzates from crude native dextran were used in 1 and 2% concentrations as primers for reactions a t 10% sucrose concentration. The relation between molecular weight of hydrolyzed-dextran primers and t h a t of synthesized product is shown in Table IV. T o characterize the entire component of low molecular weight, dextran precipitated from the primed reaction mixtures b y 60% methanol was prepared for analysis by ultracentrifugation in a field of 34,000 times gravity for 3 hours. Ultracentrifugation removes the component of very high molecular weight without seriously altering the molecular-weight distribution of t h e component of low molecular weight. Losses in centrifugation ranged from 9.5 t o %a/, of the total dextran synthesized. LOSSwas usually greater for dextran synthesized with 1 % primer than with 2% primer. I n Table IV, the samples are arranged in the order of decreasing molecular weight of t h e product obtained in reaction mixtures a t 2% primer concentration. Although light-scattering molecular meightv of primers do not precisely define their effectiveness, approximate specifications can be established. For use a t 2% primer concentration and synthesis of a product of molecular weight 75,000 f 25,000, the molecular weight of primer produced by acid hydrolysis probably should be between 20,000 and 40,000. Primers of lower molecular weight at 2y0 concentration depress the molecular weight of dextran synthesized. For example, fractionation of dextran synthesized at 10% sucrose concentration and 2% concentration of primers No. 22-4 (molecular weight 17,800) and No. 22-8
Syntheses were conducted a t 10% sucrose and 1, 2, or 4% primer concentrations (Table I). Precipitates obtained a t 60% methanol from each reaction mixture were dissolved to yield 5% aqueous solutions. These solutions were divided into three portions, and one portion of each was fractionated at one of the following series of methanol concentrations: (1) 0 t o 40, 40 t o 48, 48 to 60; (2) 0 t o 41, 41 t o 49, 49 t o 60; and (3) 0 t o 42,42 t o 50, 50 to 60 volume %. Yields of dextran found by precipitation of reaction mixtures a t 6070 methanol and expressed as percentage of reaction-sucrose weight increased with increasing primer concentration. Corrected for added primer a t 1, 2, and 4% primer concentration, 74; S1, and 64%, respectively, of the theoretical yield from sucrose were achieved. Depressed yield a t high primer concentration may arise from side reactions leading to formation of oligosaccharides or dextran of very low molecular weight. Highest yields of fractions with average molecular weights of 75,000 Z!C 25,000 were achieved at 2% primer concentration. Average molecular weights of the middle fractions tended to be high from reactions a t 1% primer concentration, and yield was low from the reaction occurring a t 4% primer concentration. Only in two instances (fractionation I, 1 and 2% primer concentration) was sufficient dextran recovered in the fraction of low molecular weight to replace primer supplied t o the reaction. I n all cases, average molecular weight of the fracTable IV. Relation Between Average Molecular Weight of Hydrolyzed-Dextran tion of low molecular weight Primers and of Low-Molecular-Weight Component of Dextran Synthesized increased substantially over t h a t of the original primer. Increasing the molecular weight of primer at constant concentration in the reaction mixture increased the molecular weight of synthesized dextran. This effect is a joint consequence of the decreasing molarity of t h e primer and the increased initial molecular weight preceding synthesis. I n the following experiments, dex-
August 1955
Component from Synthesisa Using: Ratio of Molecular Weights 1% Primer 2% Primer A l % Prjmero 1% Primer 2% Primer Mol. wt. Yieldb, % Mol. wt. Yield, % A2% Primerc Primer Primer .... 2.4 99,500 .... .... 226:ooo 76.0 Si.6 3.3 7.9 88,400 3.1 223,000 79.9 83.4 7.2 81,400 2.6 3.8 4 0 80.6 3.4 198,000 87.6 11.3 71,300 113,000 3.2 83.6 90.5 2.5 6.3 56.500 5.1 80.6 2.8 131,000 12.4 53,200 86 2 3.2 87.3 41,800 89.5 2.9 95,400 7.3 Av. 3 . 1 8.7 3.4 a Reaction conditions. Sucrose concentration, 10%; enzyme concentration, 40 dextran-sucrase units per ml.; temperature, 16’ C. 6 Yield expressed as percentage recovered after centrifugation, based on total dextran present in precipitate froin reaction mixture a t 60% methanol concentration (v./v.). C A primer signifies increase in molecular weight of dextran synthesized over t h a t of primer used. Primer No. Mol. wt. PP-41 41,400 L-28.4 28,700 L-32D 31,000 L-28B 17,600 22-4 17,800 L-28C 10,500 22-8 13,000
INDUSTRIAL AND ENGINEERING CHEMISTRY
1595
PRODUCT AND PROCESS DEVELOPMENT proximate allowance for methanol carry-over in the precipitates, SO t h a t methanol fracFractionation of Middle Fractionb tionation limits corresponded Yield, % of Reaction Sucrose Total High Middle Low Mol. Low Mol. Wt. High Mol. Wt. t o true 42 and 50% methanol. dextran mol. wt. mol. wt. mol. wt. Wt. Yield, % Mol. wt. Yield, Vo Mol. wt. It can be seen t h a t the second Fractionation I fractionation procedure yielded Com osite 59.0 30.8 18.1 10.0 44,090 .... .... .... .... a middle fraction of more suitAB Bd 56.0 23.3 23.8 9.0 43.000 .8...8. .... able molecular weight, even Cd 59.2 11.7 32.1 11.2 76,000 22,500 7.5 24i;ooo though t h e yields in both fracFractionation I1 c tionations were about equal. Composite Ad 63.2 27.1 19.2 15.3 60,000 8.1 25,600 11.9 84,700 For composite C, both fracBd 59.2 20.3 23.2 15.3 59,500 7.9 25,000 5.7 109,000 Cd 63.5 17.4 28.7 16.9 73,500 6.7 24,300 6.6 146,000 tionation Drocedures vielded middle fractions of t h e same Reaction conditions. Sucrose concentration, 10% ; primer, 27" : enzyme concentration, 40 dextran-sucrase units per ml.; temperature, 16' C. molecular weight. Separation b Separation of dextran into fractions of low and high molecular weight according t o procedures of (19). e For fractionation I, total dextran precipitated a t 60% methanol from reaction mixture was rogressively preof the subfractions of highest cipitated a t 42, 50 and 60% methanol. For fractionation 11, total dextran precipitated a t 60g0 methanol from and lowest molecular weight reaction mixture ;as first Droeressivelv orecioitated a t 40. 48. and 60% methanol and then 40-48% methanol from the middle fraction, however, revealed t h a t the twostep-fractionation procedure resulted in a narrower molecular-weight distribution. Onlv rarely did a single fractionation (molecular weight 13,000) yielded 40 to 48% methanol fractions after the 60% methanol precipitation yield a fraction of sharpness required b y military specifications for clinical dextran ( d o ) , having molecular weights of 35,000 and 29,100, respectively. which limit subfractions of highest and lowest molecular If monomer were deposited uniformly over all primer molecules, weight, separated in yield of 5 to lo%, t o be no higher i n t h e ratio of the increase of molecular weights of dextran synthesized over t h e primer molecular weight using 1 and 2% primer molecular weight than 200,000, or lower than 25,000, respectively. Generally, the concentration of dextran below 25,000 concentrations would be 2; t h e average value found for this ratio molecular weight was too high a t t h e completion of the reaction was 3.1. T h e ratio of molecular weights of dextran synthesized t o yield a once-fractionated product conforming t o present speciusing 1 and 2% primer concentrations t o primer molecular weight fications. should be 5.7 and 3.4, respectively; the averages found for this I n these fractionations, the effect of the molecular weight of ratio were 8.7and 3.4. T h e experimental values of these ratios the primer cannot be adequately evaluated from t h e molecular tend t o be higher than the predicted ratios and this would result weights of the middle fractions. T h e results must be interpreted if monomer were deposited unequally on different primer molerather from a combined consideration of the yields and molecular cules. So great an uncertainty, however, must be assigned t o the determination of molecular-weight values. representative of the weights of t h e fractions. It is probable t h a t a greater degree of polydispersity in t h e synthesized dextran accompanies t h e lower entire low-molecular-weight component, t h a t theoretical conclueffectiveness of the primers of higher molecular weight. This sions at this stage are questionable and these ratios are presented greater polydispersity complicates the fractionation because a only as approximate correlations of molecular weights. high yield of polydisperse fraction of high molecular weight may remove major amounts of useful material and result in depression Composite of dextran hydrolyzate and of the molecular weight of the middle fraction. With optimum fractions has value as primer fractionation procedures, these reaction mixtures probably would yield only slightly different amounts of clinicai molecular-weight For experimental purposes it was assumed that in a continuous fractions. The effect of variation in the molecular weights of the process one might obtain approximately equal quantities of dexhydrolyzates is minimized in composites b y the uniform priming tran greater and less in molecular weight than the clinical fraction. effect of t h e dextran fraction. I n such a process t h e primer would then be a composite of equal All fractionations shown in Table V resulted in a total yield of weights of the unfractionated hydrolyzed dextran of high molecfractions of high and low molecular weight more than sufficient to ular weight and t h e dextran of low molecular weight. Such replace the original primer. Although the molecular weight of composites were prepared from the three hydrolyzates of a the fraction of low molecular weight was greater than t h a t of dextran fraction of high molecular weight shown in Table I11 and a the fraction originally composited, this can be compensated in dextran fraction of low molecular weight (molecular weight composite primer for a succeeding reaction by more extensive 16,500) derived from a 50 t o 60% methanol precipitate of a hydrolysis of the fraction of high molecular weight. previous synthesis. As given in Table V, the calculated weightaverage molecular weights of the composite primers ranged from 27,450 t o 21,950. The composite primers were used in Sucrose concentration affects yield and molecular dextran syntheses a t 2% primer concentration and 10% sucrose weight of synthesized dextran concentration (Table V). All three composite primers yielded Previously reported investigations ( 28) showed t h a t increasing fractions (fractionation 11) of molecular weight suitable for clinical purposes aa an expander of blood plasma volume ( 2 0 ) . sucrose concentration from 5 to 20% causes decreasing yield and molecular weight of synthesized dextran. Increasing the suTwo fractionation procedures were applied t o the dextran precrose concentration results in synthesis a t increased fructose cipitate obtained with 60% methanol from the reaction mixture. concentration and thus increases total primer concentration, I n the first procedure dextran was progressively precipitated a t since fructose also can function as a primer (10,18). The denominal 42, 50, and 60% methanol. I n the second procedure dextran was first precipitated a t nominal 40,48, and 60% methpression in yield at high sucrose concentrations might be due to anol, followed by refractionation of t h e 40 t o 48% methanol formation of products of very low molecular weight from the action of fructose primer. fraction at nominal 41, 49, and 60% methanol. Nominal alcohol T h e reduction in yield and moleculnr weight of synthesized concentrations of t h e second fractionation procedure made ap-
Table V.
Utilization of Composite of Dextran Hydrolyzate and Low-Molecular-Weight Fraction as Primer for Dextran Synthesisa
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PRODUCT AND PROCESS DEVELOPMENT ~~~~
dextran associated with increased sucrose concentration is shown by reactions conducted using 10 or 20% sucrose concentration (Table VI). So great was the reduction in yield with elevated sucrose concentrations that in a process for dextran synthesis i t would be necessary t o adjust sucrose concentrations so as t o compromise between output from a given volume of solution and efficiency in sucrose utilization.
Table VI. Influence of Sucrose Concentration on Dextran-Primed Enzymatic Synthesis of Dextran Yield, % of Reaction Sucrose Total Methanol Precipitate, % dextran 0-42 42-50 50-60
42-50% Methanol ti^^ Mol. Wt:
Reactantsa 1% primer 3.0 62,800 30.4 13.2 10% sucrose 47.7 64,200 10.3 5.0 20% sucrose 37.0 19.4 2% primer 29.9 11.2 68,000 16.5 10% sucrose 58.1 5.4 62,500 11.3 15.8 TOYo aucrose 34.4 a Reaction condition. Sucrose and primer concentrations as indicated: primer, low-mol.-wt.-dextran fraction of mol. wt. 17.500; enzyme conrentration, 40 dextran-sucrase units per rnl.; temperature, 16' C .
Table VII. Influence of Reaction Temperature on Dextran-Primed Enzymatic Synthesis. Temp.,
Total h dextran
30 15 4
45.9 47.4 47.2
c.
Yield, % of Reaction Sucrose Low-mol.-wt. High-niol.-wt. range b range 21.2 24 7
43.4 45 9
4 0 1 3
Reaction conditions. Sucrose concentration, 10% ; primer-dextran fraction of mol. wt.,21,000 at 1.0% concentration; enzyme concentiation, 40 dextran-sucrase units per mi. 6 Yields determined from plateau or inflection in function of dextran precipitated versus amount of methanol added t o reaction mixture ( 1 . 9 ) .
Increasing enzyme concentration favors production of low-molecular-weight dextran
At temperatures near 30" C., extenyive synthesis of dextran component of very high molecular weight occurs in primed reactions (18). I n this temperature range, increasing enzyme conrentration favors production of the dextran components of low molecular weight. At temperatures of approximately 15" C. or less, however, production of dextran component of high molecular weight is minimized, and the molecular-weight diatribution of the synthesized d e x h n i.; less sensitive t o variation of enzyme concentration. Al1owanc.e must, be made in reactions conducted at lower teinperatures for the influence of temperature on reaction rate. Dropping the reaction temperature from 30" t o 15' C. results in nearly a twofold decrease in reaction rate. Although specification of enzyme concentration in terms of dextran-sucrase units (19) would define the rate of synthesis a t 30" C. and sucrose concentration greater than 5%, it was found experimentally that reaction time must be increased approximately 50 to 75% over theoretical t o assure completion of the reaction Thus, conversion t o dextran of a sucrose solution 10% in concentration (100 mg. per ml.) b y enzyme of 40 units per ml. concentration, requires for completion somewhat less than 8 hours a t 15" C. Reaction temperature affects molecularweight distribution of products
As in a11 enzymatic reactions, the temperature of the dextransucrase-cataly~ed reaction affects reaction rate and stability of enzyme. For this reaction, however, the temperature also affects molecular-weight distribution of the products (18). T h e predominant effect of temperature is t o control the relative proportion of dextran in the two molecular-weight ranges (Table
August 1955
VII). At 30" C. more than half of the product may appear in the range of very high molecular weight, whereas a t 4" C only 1.3y0 of the product appears in this size range. At 15" C. the yield of dextran of low molecular weight is nearly as high as a t 4" C. Accompanying this effect, sucrose is used for preferential synthesis of dextran of low molecular weight and the average molecular weight. of the product in the range of low molecular weight increases. Summary
Synthesis of dextran from sucrose in the presence of dextransucrase can be controlled so t h a t the molecular weights of the dextran products primarily occur in the range 25,000 t o 200,000, suitable for clinical use as an expander of blood plasma volume. Enzyme, sucrose, and primer concentrations, primer type, and reaction temperature will affect the molecular weight of synthesized dextran. Dextran of low molecular weight is preferable t o maltose as a primer. Dextran primer of low molecular weight may be obtained from synthesis reactions as a by-product of recovery of dextran of clinical molecular weight either by using the dextran below the "clinical" fraction in molecular weight, or b y hydrolyzing the dextran higher in molecular weight than the clinical fraction. Increasing the molecular weight of the primer increases t h e molecular weight of dextran synthesized. For production of dextran of molecular weight 75,000 f 25,000, suitable weight-average primer molecular weights are in the range of 15,000 to 20,000 for dextran fractions and 20,000 t o 40,000 for dextran hydrolysates. For a primer recycling process in which primer utilization is balanced by primer production, it probably would be necessary to use composite primers of fractions of low molecular weight and hydrolyzed fractionsof high molecular weight. Investigations are being conducted t o determine the practicability of such a recycling operation. High yieIds of products suitable in molecular weight for clinical use have been found b y performing the reaction a t approximately 15" C. or less, using 10% sucrose concentration, enzyme concentrations of 20 to 40 dextran-sucrase units per ml., and dextran of low molecular weight as primer. The synthetic product is adjusted t o the average molecular weight desired primarily by varying primer molecular weight and concentration and, secondarily, b y adjusting fractionation conditions. Products of molecular weight 75,000 i 25,000 have been obtained in yields greater than 25% of reaction sucrose by using 2% concentration of primer dextran, having molecular weight in the vicinity of 20,000, and b y fractionating the reaction mixture between the limits of 42 and 50% actual methanol concentration. Two fractionation cycles may be necessary to produce fractions of sufficiently narrow molecular-weight distribution to meet present military specifications for clinical dextran. Acknowledgment
The authors are grateful to i\. E. Johnston, Mary 0. Bogard, and Bettye L. Wilson for their analytical determinations, and to V. E. Sohns for valuable discussion. literature cited (1) Rixler, G. H., Hines, G. E., McGhee, E. RI., and Shurter, R. .I., IND.ENQ.CHEM.,45, 692-705 (1953). (2) Brice, B.A., Halwer, M., and Speiser, R., J. Opt. Soc. Arne?., 40,
76s-78 (1950). (3) Chem. Eng., 59,240-3 (December 1952). (4) Cragg. L. H., J . Colloid Sci., 1, 261-9 (1946).
(5) Hehre. E.J., J . Am. Chem. Boc., 7 5 , 4866 (1953). (6) Hehre, E.J., J. Bdol. Chem., 163,221-33 (1946). (7) Ingelman, B., and Halling, M. S., Arkiw Kemi, 1, 61-SO (1950). (8) Jeanes, A., Wilham, C. A , , and Miers, J. C . , J . B i d . Chem., 176, 603-15 (1948). (9) Xoepsell, H. J., and Tsuchiya, H. M., J. Bacterid., 63, 293-6 (1952).
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Koepsell, H. J., Tsuchiya, H. M., Hellman, N. N., Kazenko, A., Hoffman, C. A,, Sharpe, E. S., and Jackson, R. W., J . Biol. Chem., 200, 793-801 (1953).
Koepsell, H. J., Tsuchiya, H. M., Hellman, N. N., Kazenko, A., Sharpe, E. S., Hoffman, C. A,, and Jackson, R. W., Bacteriol. Proc., 52nd mtg., p. 23 (1952). M f g . C h m i s t , 23, 49-55
2 952).
Nadel, H., Randlea, C. i s and Stahly. G. L., A p p l . Microbial., 1, 217--24 (1953).
Nelson, N., J . Biol. Chenz., 153, 375-80 (1944). Senti, F. R., Hellman, N. N., Ludwig, N. H., Babcock, G . E., Tobin, Robert, Glass, C. A,, and Lamberts, B. L., J . Polymer Sci., in press. Simha, R., Research NatL B u r . Standardst 42, 409-18 (1949). Tsuchiya, H. M., Hellman, N. N., and Koepsell, H. J., J . Am. Chem. Soc., 75, 757-8 (1953). Tsuchiya, H. M., Hellman, N. N., Koepsell, H. J., Corman, J., Stringer, C . S., Rogovin, S. P., Bogard, M. O., Bryant, G.,
Vol. 47, No. 8
Feger, V. H., Hoffman, C. A., Senti, F. R., and Jackson, R. W., Ibid., 77, 2412-19 (1956). (19) Tsuchiya, H. M., Koepsell, H. J., Corman, J., Bryant, G., Bogard, M. O., Feger, V. H., and Jackson, R. W., J . Eacteriol., 64, 521-6 (1952). (30) U. S. Military Medical Purchase Description, No. 4, Sept. 19, 1952, Stock NO. 1-161-890, Dextran Injection, M-1 S%, 500 cc., Armed Services Medical Procurement Agency, Brooklyn 1, N. Y . (21) Wolff, I. A., Illehltretter, C . R., Mellies, R. L., Watson, P. R., Hofreiter, R. T., Patrick, P. L., and Rist, C. E., IND.ENG. CHEM.,46, 370-7 (1954). RECEIVED for review May 10, 1954. ACCEPTED January 20, 1955. Presented before the Division of Carbohydrate Chemistry, Symposium on Dextran, at the 125th Meeting of the AMERICAN CHEMICAL SOCIETY,Kansas City, Mo.. March 1954. The mention of firm names or commercial products does not constitute an endorsement of such firms or products by the U. 5. Department of Agriculture.
END OF PRODUCT AND PROCESS DEVELOPMENT SECTION
New Polymeric Dispersants for Hydrocarbon Systems C. B. BISWELL, W. E. CATLIN, J. F. FRONING, AND G. B. ROBBINS E. I. du Pont de Nemours & Co., Wilmington, Del.
L
I T T L E fundamental work has been done on t h e behavior of surface-active agents in nonaqueous media. However, a variety of additives of this type has been used empirically in connection with painta, printing inks, dry cleaning and numerous other applications. I n general, these surface-active materials have been solvent-soluble modifications of t h e anionic, cationic, or nonionic types which have proved effective in aqueous systems. A rapidly growing demand for such additives has been found in t h e lubricating oil and fuel oil industries. Lubricating oil detergents have been principally oil-soluble sulfonates of various types although alkaline earth phenates have also been employed. Such additives have been extremely effective in promoting engine cleanliness under t h e high temperature conditions which exist in Diesel and spark-ignited engines during heavy-duty performance. Under t h e low temperature conditions t h a t exist for most passenger cars during city driving conditions and for stop-and-go delivery service, these detergents are effective only when used in high concentrations. This fact has spurred t h e search for novel additive types t h a t are more effective under this critical, b u t widespread, type of service. T h e broad class of oil-soluble surface-active agents described here is a result of t h a t search. These compositions can be broadly characterized as polymers containing basic nitrogen substituents. They are actually copolymers of two monomer types which have distinct and separate functions. One type of monomer is predominately nonpolar. It has t h e function of contributing t h e property of solubility in oil to t h e polymer and is referred t o as a n oleophilic monomer. T h e other type of monomer contains basic nitrogen. Its function is t o contribute surface activity t o t h e polymer through nonionic or cationic mechaniems and to act as t h e point of attraction for polar materials such as t h e sludge in fuel oil or lubricants. I n certain cases i t is possible t o add other relatively nonpolar mpnomers which d o not contribute t o either t h e oleophilic nature or to basicity b u t act as innocuous extenders of t h e polymer chain. T h e ratio in which these different func-
tional types of monomers exist in the copolymer has a great influence on its properties. A variety of monomer structures can fulfill these different functions. Esters or amides of methacrylic and acrylic acids or polymerizable polycarboxylic acids, vinyl esters of carboxylic acids, vinyl ethers, or vinyl-substituted aromatic compounds are among the structuraI classes of monoiners t h a t can be employed. Such a polymer might be represented for a simple vinyl system b y a general formula:
-CH-CH2-CH-CH2-CH-CHz-CH--CH2-CH-CHS I
1
I
I
where 0 is a n oleophilic group, B is a basic group, and L is a group of atoms linking an 0 or B group t o t h e polymer chain Different radicals which may function in these different relations are as follows : 0 --CnHzn
L
B
+1
-(
CHz),--NRz
( m = 2 or more)
(n
=
8 or more)
-C-NHII
a -0(direct bond for aryl compounds -e.g., styrene)
