Enzymatic Synthesis of C- 11 Formaldehyde: Concise Communication

G. Slegers, R.H.D. Lambrecht, 1. Vandewalle, L. Meulewaeter, and C. Vandecasteele

State Universityof Ghent, Ghent, Belgium

We presentan enzymaticsynthesisof C-i i formaldehydefromC-I I methanol,with immobilizedalcoholoxidaseand catalase: a rapid,simpleprocedure,with ahighandreproducibleyield.Carbon-i1 methanolisoxidizedto C-i I formaldehydebypassage over a column on which the enzymes alcohol oxidase and cataiase areimmobilized. The catalase increases reaction velocity by recycling the oxygen,andpreventsdestructionof the alcoholoxidasebyeliminatingthe excessof hydrogenperoxide.Theyieldof the enzyme-catalyzedoxidationwas 80-95 %. A specific activity of 400-450 mCi/j.@molewas obtained at EOB + 20 mm. Various immobilization techniques and the optimal reaction conditions of the Immobilized enzymesare investigated. .

J NucI Med 25: 338—342,1984

Carbon- I 1 formaldehyde is a useful agent for the Iabeling of complex molecules of biomedical interest by

reductive alkylation reactions (1—3). It is usually prepared by catalytic oxidation of C- I I methanol on metalcatalysts at high temperature (4—6).The yield is strongly

dependent on the temperature and the surface activityof the catalyst.

As an alternative, we present an enzyme-catalyzedoxidation of C- I I methanol, with immobilized alcohol

oxidase and catalase. It is a rapid, easy procedure witha high and reproducible yield. The principle of the

method is outlined in Fig. I.

MATERIALS AND METHODS

Setup 9f the C-I I formaldehyde production(Fig. 2).Nitrogen gas (7 bar) is irradiated with l8-MeV protons

at I 5 j.tA for 20 mm. The target gas containing ‘1CO2is pumped to a hot cell where it is trapped at a flow of I

I/mm in 0.5 ml of a 0.5 M solution of LiA1H4 in tetrahydrofurane, cooled to —80°C.The trapping of' ‘CO2takes about 10 mm. Within 1.5 mm the tetrahydrofurane

Received July 18, 1983; revision accepted Oct. 31, 1983.For reprints contact: Guido Slegers, PhD, Laboratory of Analytical

Chemistry, Pharmaceutical Inst., Harelbekestraat 72, B-9000 Ghent,Belgium.

is evaporated to dryness by heating to I60°C.Hydrolysisof the methanolate is achieved by injection of 0.5 ml 6N HCI. By heating to 160°C, the C-I I methanol is

transferred by a N2 flow of I00 ml/min to 0.5 ml phosphate buffer (0. 1 M, pH 7.5, ambient temperature). Tomaintain the pH of this buffer solution, the C- 11 methanol is first sent through 300 mg soda lime to trap theexcess HCI. The soda lime is heated to I 80°C to avoid

condensation of the methanol. In 5 mm, the activity isalmost quantitatively trapped in the buffer solution. Theventilation outlet of the buffer solution was controlledby a wash bottle.

During a few seconds, air is bubbled through thebuffer solution to introduce the substrate oxygen. Thesolution is sucked into a sample loop of0.7 ml (Pump I,Fig. 2). With a flow of I .5 mI/mm, a peristaltic pump(Pump 2, Fig. 2) transfers the loop contents to the enzyme column containing the enzymes alcohol oxidaseand catalase, immobilized on porous glass beads. In 2mm, 3 ml of buffer solution are collected, containing theC- I I formaldehyde. The entire procedure takes about20mm.

Preparation of the enzyme reactor. The enzymes areimmobilized by covalent linkage to glutaraldehyde

activated controlled-pore glass (7) (Fig. 3).One gram of controlled-pore glass (CPG 500/80—120

Mesh*) is rinsed in I N HCI and hydrated by boiling in

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BASIC SCIENCESRADIOCHEMISTRY AND RADIOPHARMACEUTICALS

‘4N(p,u)1'C traces02@

4 11C02+ 3 LiA1H4 .—.---..(“CH3O)4A1LI+ 2 A1L102

Production of1‘C-isotope

Reduction

(‘1CH30)4MLI+ 4 H20 HC1 M(OH)3 + LIOH+ 4 11CH30HHydrolysis

EnzymaticOxidation

Recyclingof 02

Immobilized11CH3OH+ 02 Mcoho@ oxidase@ 11CH20+ H202

(E.C.1.1.3.13)

Ininobil izedCatalaseH202@ H20 + 1/2 02

(E.C.1.11.1.6)

FIG.1. SchemeforsynthesisofC-i1formaldehyde.

5% HNO3. Silanization is carried out with 20 ml of a20%-'y-aminopropyItriethoxysilane@ solution in water,adjusted to pH 3.45 with 6 N HCI, by heating to 75°Cfor 2 hr.

Glutaraldehyde is coupled to the silanized glass byadding 25 ml 2.5% glutaraldehyde solution, in 0.05 M

phosphate buffer, pH 7. After I hr of reaction, the glassis exhaustively washed.

To I g glutaraldehyde-activated glass, 30 units of alcohol oxidaset (E.C. I.I.3.13, from Pichia pastoris), and60,000unitsofcatalaset(E.C.1.11.1.6,frombovineliver) are added in I .5 ml phosphate buffer (0. 1 M, pH7.0). One hour of shaking at room temperature completes the reaction. The enzyme-loaded glass beads arepacked in a 0.55- by 10-cm glass column, washed thoroughly with phosphate buffer (0.1 M, pH 7.5) to removeany protein not covalently bound, and stored at 4°C.Under these conditions the enzymes are stable for severalmonths. Blocking of the remaining aldehyde groups canbe done with 1 M glycine, but blocking is not indispensable.

Before use the column is warmed to 26°Cand washedwith phosphate buffer (0. 1 M, pH 7.5). After each C- I Iformaldehyde production or one-half day's storage at

26°C,enzymatic activity decreases by about 5%. Ifproperly handled there is no enzyme leakage.

RESULTS AND DISCUSSION

Yield. Table I shows the distribution of C- I I activity,starting from the conversion of C-I I methanol into C-I 1formaldehyde. Losses of activity in I CO2 and C-I 1methanol synthesis are not included. From the data ofTable 1and the data from radiogaschromatography, wecalculate that 80% to 95% of C-I I methanol is convertedinto C-I I formaldehyde, yielding 200 to 250 mCi atEOB+2Omin.

Specific activity. The specific activity was 400-450mCi/@zmole(EOB + 20 mm), determined as follows.

The total activity of the collection flash was determined with a calibrated gamma spectrometer equippedwith a Ge(Li) detector.

The collected solution was analyzed by radiogaschromatography. The chromatographic system isdescribed in Table 2. The result is given in Fig. 4: 93.5%of the C- I I activity is in the formaldehyde peak.

The carrier amount, determined with Nash reagent(8), wasat most 0.65 zmole.The stock formaldehyde

ViiitilatIoi, (Ne) VIfltll•tlOø (Tif)

:@ob@ (@Zy'@1s

FIG.2. SetupofC-i1formaldehydeproduction.® Pump1.© Pump2.

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SLEGERS. LAMBRECHT. VAN DEWALLE, MEULEWAETER, AND VANDECASTEELE

Support Si1ane Glutaraldehyde Enzyme

( AlcoholoxidaseN-Enzyme@

(.. Catalase

FIG. 3. Structure of immobilized alcohol oxidase and catalase.

or CPG 240/80—120 Mesh)*. Different silanest, spacerarms, and active groups were tested. To compare thedifferent immobilization techniques, we determined thepercentage of bound alcohol oxidase and the alcoholoxidase activity ofthe glass beads (Table 3).

The enzymatic activity is determined by spectrophotometric measurement of formaldehyde with Nash reagent (8).

Incubation of soluble and immobilized enzyme iscarried out with 20 @tlof methanol in 2.5 ml phosphate

buffer (0.05 M, pH 7.5) at 26°Cfor 10 mm.The method ofchoice is the glutaraldehyde coupling

on y-aminopropylcoated CPG 500 (Fig. 3). It is an easyand gentle immobilization procedure giving the highestloading with active enzyme.

Effect of coimmobilized catalase. To increase theconversion of methanol into formaldehyde, the substrateoxygen can be recycled by addition of catalase to thesystem (Fig. I). When the oxygen-to-methanol ratio isnot too high, addition of hydrogen peroxide to the mixture of enzymes also increases the conversion rate.

TABLEI. DISTRIBUTIONOF C-li ACTIVITY

LlAIH4trap 0.5%Sodalimetrap 5.9%Washbottle(ventilationoutletofbuffersolution) 2.6%Enzymereactor 3.0%Collectionflask 88.0%

. Mean value of five productions

solution was standardized by dimedone precipitation (9)and by iodimetric titration.

The carrier amount originates from the 1‘CO2andC- I I methanol production. The use of the enzyme process makes no significant contribution to the carriercontent.

Overoxidation of formaldehyde into formic acid didnot occur; it is checked by passing the C- I 1 formaldehyde eluate of the enzyme reactor over an AG IX8 anionresin. Any trace of C-I I methanol and the producedC-I I formaldehyde pass through the column, whereasany trace of C- 11 formic acid is retained. No activitycould be detected @nthe anion resin, indicating that therewas no C- I I formic acid present.

Comparison of immobilization techniques. Enzymesare immobilized by covalent linkage to an inert support(7), by means ofan active group, at the end ofa spacerarm, coupled to a silane. The support was controlled

pore glass in all experiments (CPG 500/80—120Mesh

TABLE2. CHROMATOGRAPHYDATA

Column:‘/@“Teflontubing,id. 2.4mm:Part 1 12.5 cm Carbosphere60—80Mesh@,placed in

injection port,Part 2 200 cm Chromosorb 108/60—80Mesht,

placedin ovent°Injection port 170°to oven 146°

to thermal conductivity detector 270°

He flow = 40 mI/mmRadiochemicaldetection:Nal(Tl)Injection volume 2,ul

. Alltech.

t Johns Manville.

L__,/k.Ilk@)k_@.:@ I THERMALCONDUCTIVITY

RETENTION TINE ______________________(MIN)@@@ I

THERMAL CONDUCTIVITY

RETENTION TIME

(MIN)@@ 2@@

FIG. 4. Radiogaschromatography.Peak 1 formaldehyde,Peak2 water, Peak3 methanol.A test mixture; B C-i 1 formaldehydesolution. Peak 1 represents 93.5% of the activity, Peak3, 6.5%.

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TABLE3.COMPARISONOFIMMOBILIZATiONTECHNIOUESGlass

Silane SpacararmActiye groupCoupling% Bound % Active

BASIC SCIENCESRADIOCHEMISTRY AND RADIOPHARMACEUTICALS

CPG240 Al 100 GlutaraldehydeAldehydeShiftbase1009CPG500 Al l20@ GlutaraldehydeAldehydeShiftbase1005CPG500

A l87@—AldehydeShiffbase4015CPG

500 — —CyanogenbromideCarbamate1515CPG500 Al 100ArylamineDiazoniumAzo800.5CPG500

A1100GlutaraldehydeAldehydeShiffbase10030CPG

500 Al 100@—ThiocyanateThiourea909CPG500 Al 100' SuccinateAcidChlorideAmide6015CPG500 Al 100 SuccinateActiveEsterAmide62*

(C2H5O)3-Si@H@H@-CH@-NH2t

(CH3O)a-Si-CH@-CH@-CH@-NH-CH@-CHrNH2t@

0'

EL,2iv

I.7@

we 1500 2000 UnIts C.t.I.s.

UnètAICOMIxidsii

FIG.5. Effectofcoimmobilizedcatalaseonactivityofalcoholoxidase.OrdinategivesextinctionbyNashreaction.

column@ 055cmflow 1.25 mI/mmtmp@roture 26 C0.05M phosphate buftr pH 7.50.495 pmol, mithonol

3U Units otcoholosidose

15 cm column length

‘I.cOnversion of mafhoncl

100

90

80

70

60

50

@-5 100

FIG.6. Conversionofmethanolintoformaldehyde,plottedagainstamountofenzymeincolumn.

However, we observed that by addition of hydrogenperoxide to a buffered solution of alcohol oxidase withoutcatalase, the enzymatic activity decreases irreversiblyfrom day to day and more rapidly than the same enzyme

solution without hydrogen peroxide. To obtain an enzyme column with high stability, addition of hydrogenperoxide in the buffered C-I I methanol solution mustbe avoided. Therefore an enzyme column of alcohol

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SLEGERS, LAMBRECHT, VANDEWALLE, MEULEWAETER, AND VANDECASTEELE

oxidase with a sufficient excess of catalase was preferred,so that not oxygen but the substrate methanol limits theconversion rate.

One unit of alcohol oxidase is immobilized withvariable amounts of catalase (0.5—2000units, Fig. 5).For routine preparation of an enzyme reactor, the alcoholoxidase is immobilized with a 2000-fold excess of catalase.

Optimization of enzyme reactor. The amount of immobilized enzyme needed for high-yield conversion ofmethanol into formaldehyde is determined for 0.5 j@tmoleof methanol, which is about the carrier amount presentin the preparation.

The percentage of methanol conversion into formaldehyde is determined with different packed bed volumesin the enzyme column (Fig. 6). A column, containing 2.5

units of alcohol oxidase, that gives a 80—95%conversion

of methanol into formaldehyde, is used for routine production.

FOOTNOTES

* Corning Glass Works.

t Sigma Chemical Company.

xUnionCarbideBenelux.

ACKNOWLEDGMENTS

This research was supported by a grant of the Belgian Government(Geconcerteerde Onderzoeksaktie 80/85.6).

All silaneswerea generousgift of Mr.Tielemansof UnionCarbideBenelux.

REFERENCES

1. STRAATMANMG,WELCHMi: A generalmethodfor Iabeling proteins with 1‘C.J Nuci Med 165:425—428,1975

2. MAZIERE M, BERGERG, COMAR D: Optimization ofI ‘C-labelling of radiopharmaceuticals: I ‘C-clomipramine as

an example. J Label Compd Radiopharm 132:196-197,I977

3. BERGERG, MAZIEREM, MARAZANOC, et al: CarbonI Ilabeling of the psychoactive drug 0-methyl-bufotenine and itsdistribution in the animal organism. Eur J Nuci Med 3:101—104,1978

4. CHRISTMAN D, CRAWFORD J, FRIEDKIN M, et al: Detection of DNA synthesis in intact organisms with positronemitting [methyl-' ‘Cjthymidine.Proc Nail Acad Sci USA69(4):988-992, 1972

5. MARAZANOC, MAZIEREM, BERGERC, Ctal: Synthesisof methyl iodide-' ‘Cand formaldehyde-' ‘C.ml J App! RadialIsot 28:49—52,1977

6. BERGERG, MAZIEREM, SASTREJ, et al: CarrierfreeI‘C-formaldehyde:An approach.J Label Compd Radiopharm17(l):59—71,1980

7. WEETALL HH: Covalent coupling methods for inorganicsupport materials. In !mmobi!ized Enzymes. Methods in Enzymology,Vol44,1976,pp 134-148

8. NASH T: The colorimetric estimation of formaldehyde bymeansof the Hantzschreaction.BiochemJ 55:416—421,I 953

9. HORNINGEC,HORNINGMG: Methonederivativesofaldehydes.JOrgChem 11:95-99, 1946

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1984;25:338-342.J Nucl Med.   G. Slegers, R.H.D. Lambercht, T. Vandewalle, L. Meulewaeter and C. Vandecasteele  Enzymatic Synthesis of C-11 Formaldehyde: Concise Communication

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