Branched-chain carbohydrate structures resulting from formaldehyde condensation

CARBOHMRATE

RESEARCH

29

BRANCHED-CHAIN CARBOHYDRATE STRUCTURES FROM FORMALDEHYDE CONDENSATION

RESULTING

R D PARTRIDGE*, A H WEISS*, AND D TODDY Worcester

PoZyrechhnrc Insrrtute,

(Recerved September

Worcester,

Massachusetts

13th, 1971, dccepted in rewed

01609 (U

S

A )

form January 25th, 1972)

ABSTRACT

Sugars prepared by condensation of formaldehyde under cataIysls by calcium hydroxide (the formose reactron) were analyzed by combmed gas-hqmd chromatography and mass spectrometry of the tr~methylsllyl ethers; and when reduced to the correspondrng aldltols, as their O-trlmethylsdyl and O-tnfluoroacetyl derivatives Both branched-cham and straight-cham species are produced by formaldehyde condensatlon, and defu-utlve spectra are provided that characterize the umque branchedcham products INTRODUCTION

The formose reaction, namely the autocatalytlc condensation of formaldehyde to sugars m the presence of alkalme catalysts, was first reported by Butlerowl In 1861, and has been studled intermittently smce then, mamly to ldentlfy the reactlon products The reaction aves a complex rmxture of aldoses and ketoses rangmg from a two-carbon product (glycolaldehyde) through three, four-, five-, SIX-, and sevencarbon, and perhaps even hxgher species, by the general process (n+2)HCHO

Ca@H)z +

HOCH,(CHOH),CHO

The formose reaction IS catalyzed both by divalent metal bases, such as calcrum and barium hydroxides, and lead oxide, and by monovalent bases such as thalhum hydroxide The condensation to sugars occurs In competltlon with the simple Canmzzaro reaction of formaldehyde, the latter course IS the sole n&al reactIon observable when sodium or hthmm hydroxides are used The chemistry of the formose reaction has been revlewed recently by Akerlof and Wtchel12 and by Frankeniield3 m connection with the posslb&ty of usmg the formose reachon to produce edlbie carbohydrates m sustamed space tights Metabohc body wastes such as carbon loxlde would be hydrogenated to methane, and the latter *Department fDepartment

of Chemxal Engmeermg of Chermstry Correspondence

should be addressed to Dr Weiss in this department Carbohyd

Res

,24 (1972) 2944

R D

30

PARTRIDGE,

A

H

WEISS, D

TODD

partially oxidized to formaldehyde and subsequently converted mto formose sugar Akerlof4 and Shapira’ have shown that the complex formose sugar IS toxic, and interest from the mew of feeding during space travel has been dlrected to using the formose reactlon, coupled to a reductive step, to produce glycerol Instead of sugars5 lfj There has aIso been malor Soviet Interest m the utlhzatlon of the formose reaction’ The potential of the formose reactlon for large-scale manufacture of sugars has been conadered’, and the mechamsm and kmetlcs of the reaction have been studled To explain the known products of the formose reactlon, the foIlowmg processes suffice (I) An mltlatmg - and autocatalytlc - condensation of two molecules of I-ICI-IO to @ve OH-CH,CHO (glycolaldehyde), (2) successive aldol condensations mvolvmg HCHO and hydrogen atoms alpha to a carbonyl group, (3) Lobry de Bruynvan Ekenstem mterconverslon, and (4) a crossed-Canmzzaro reaction to produce aldltols and formate ion It IS to be noted that branched cham sugars - rarely found m Nature and not sought by earher mvestlgators of formose would be expected to be major components of formose Furthermore, if the structural features required for processes (2) and (3) were absent m a product, then that product IS a terminal one For example, If glyceraldehyde undergoes aldol condensation with HCHO, the product, ZC-(hydroxymethyl)glyceraldehyde, would be expected to either accumulate or undergo crossedCanmzzaro reduction to the tetrltol CHO HCHO

1 + HCOH

CHO +

I CHIOH

I HOH&-COH I CHZOH

Apparently, the reverse aidol condensation IS not favored under condltlons of the formose synthesis, as large proportlons of such termmal, branched products have now been found m formose The crossed-Canmzzaro reactlon seems to be mmor m formose synthesis, as only a small proportlon of aldltols form Gas-hquld chromatographlc analysis or appropnate denvatives [per(tnmethyblyl = Me,& and per(tnfluoroacety1) =CF,CO] of formose led to very complex patterns Prior reduction of formose by sodium borohydride to convert carboxyl groups mto alcohols, followed by denvatlzation, led to far simpler patterns” Many carbohydrate denvatives have been studled by mass spectrometry”, but only a few of these are amenable to analyas by g 1 c The mass spectra of a number of aldltol per(trlfluoroacetates) have been reported12 Whereas many known sugars have prevlousIy been ldentlfied as components of formose, apparently the findmg of branched-cham sugars In the course of our work 1s new To what degree the toxicity of formose IS due to the L-forms (presumably not easily metabolized) of sugars present, and to what degree it 1s due to the unnatural branched-cham sugars present, remams to be seen Carbohyd

Res ,24

(1972) 29-44

BRANCHED CAFSOHYDRATES

31

FROM FORMALDEHYDE

formose

Trmethylsrlylated

z x

-

Trimethyls~lyloted ; formose

reduced

0

9 x

T(Y)

100

. .

t”“““I!“’ r(mln)

t(mtn)

“““:1”“““:““1~‘1’~

0

c

20

z IO

0

’

’

’

0

’

! 5

’

’

’

’

I IO

40

30

’

’

’

!

(

’

15

Fig 1 Gas-llqmd chromatograms of derwatives of formose and reduced formose produced at complete conversion m a plug-flow reactor

’

’

*

1

20

Formose syrup

RESULTS AND DISCUSSION

The chromatogram m Fig 1 shows the complex product-dlstnbutlon of the Me& ethers of the sugars m formose, together w&h the greatIy slmplrfied chromatoCarbohyd

Res

,24 (1972) 29-44

32

R

D

PARTRIDGE,

A

H

WEISS, D

TODD

grams obtamed after borohydnde reductron of the formose before sllylatlon The branched-chain aldltols are resolved from the straght-cham components as the Me$i ethers Better resolutlon of the straight-cham species was accomplished by usmg the aldltol trlfluoroacetates, but there was incomplete separation of the branched from the straight-cham alltols as the tnfiuoroacetates The mass spectra of the sllylated alditols showed fragmentanon patterns resulting pnmanly from simple cleavage of the molecule w&h a rrummum of rearrangement_ The molecular 1011fragments lmtially by loss of O&Me, (m/e 89), followed by O&Me, molecules (m/e 90) m a manner slmdar to that of the trtiuoroacetates” MaJor fragments at m/e 147 and 191 have been found m all Me,& derlvatlves reported to date, and are explamed by rearrangement 16. A base peak of m/e 73 can be explamed by the loss of formaldehyde from the m/e 103 fragmenti Molecular Ions were observed only with the tnmethylsllylated tetntols The mass spectra (of both the Me,% and CF,CO derlvatlves) of dlastereomerlc straight-cham aldltols comclded, wlthm the lm-nts of error The mass spectra of branched-cham aldItols dlffered from those of straight-cham species m the relative lntenslties of tertiary versus secondary fragments ldentrficatronof tetrltol - Fig 2 presents an analysis of the region correspondmg to four-carbon products m the sdylated reduced formose, and shows possible structures for the aldltols and thelr fragmentation patterns There are three possible four-carbon aldltol structures one branched-cham and two dlastereomenc, stralghtcham forms The chromatogram of the sllylated, reduced formose showed three peaks m tbs reDon The last peak of the chromatogram from formose corresponded m retentron time to the trlmethylsllyl ethers of both erythrltol and threltol, and Its mass spectrum matched that of authentic erythntol or threltol tetrakls(tnmethylaly1) ether, both denvatlves gave identical spectra The 6.rst two peaks thus remamed to be ldent&ed_ The branched-cham tetntol, 2-C-(hydroxymethyl)glycerol*, was synthesized by dlazotlzatlon of tr~s(hydroxymethyl)ammomethane (Trls) The sllylated, total crude-product that resulted from this synthesis gave the three peaks shown m Fig 2 The peak among these rdentied as 2-C-(hydroxymethyl)glycerol was ldentlcal by retention time and mass spectrum (Fig 2) with the peak for that compound m the tetntol reDon of the formose The presence of a molecular ion at nz/e410 suggests that the first product eluted, which cannot be a straight-cham tetntol, IS a branchedchain tetntol The mass spectrum of sllylated 2-C-(hydroxymethyl)glycerol showed a low relative abundance of m/e 205 and a high relative abundance of m/e 307 when compared with the spectra of the straght-cham, sllylated tetntols, mdlcatlve of fragmentation from the branched-cham species The structure of the branched, sllylated tetrltol indicates that there can be no drrect formation of nz/e205 by cleavage Cleavage at C-l does result m a very stable, tertiary fragment at m/e 307 The struc*A preparauon of this compound, characterized by a carbon-hydrogen analysis, has been reported’3 The tnacetate, tns(acetoxymethyl)methanol, has been reported m a patent I4 Ger 874, 774

Curbohyd Res ,24 (1972) 29-44

34

R

2-C -( h ydroxymechyl)

D. PARTRIDGE,

$H2

2-C-(Hydroxymethyl)

1-glycerol A

-rlfluoracetylared -RIS ‘roducts

from

C,

127

c3

379

CH2

OCOCF

2-C3~ II

100

3

I 0-C-CH2OCOCF

F3COC

(MS) q/e

TODD

c2

253

ca

253

3

I CH 2 ocOCF

Trlfluoracecylated glycerol

3

tHOCOCF

Cd

69(hydroxymethyl)

OCOCF

WEISS, D

I

/

Trlfluoracetylated Reduced Formose

80

H

CHOCOCFj

glycerol

//

100

A

Cl

127

3 c3

379

3

506-393-279

C3

m/e

379-265

Cp

m/e

253-139

Trlfluoracetylated FormXqOse

threltol

69 from

80 393

69Tr~fluoracet~lated 2-G-(hydroxymethyl) glycerol from TRIS

200

300 m/e

Fig

3 Identilicatlon

formose

by g 1 c-m

s of specres m the tetntol region of tnfluoroacetylated,

reduced

and thrertol tiuoroacetates from formose corresponded to those of the authentrc derivattves, and both gave mass spectra essentlaliy rdentrcal wrth one another and those of the pure products Agam, the thud peak of the product from Tns corresponded m retentron trme to the f&t and most abundant peak from the formose, that of the branched-cham tetrltol The mass spectra were rdentrcal and agam indicated the branched-cham specres, having its molecular ran at m/e 506, a Iow relatrve Carbohyd Res ,24 (1972) 2944

BRANCHED CARBOHYDRATES

35

FROM FORMALDEHYDE

abundance of m/e 253 and hrgh abundance of m/e 379 corresponded to the mass numbers of m/e 205 and 307, respectrvely, m the sllylated tetrrtols The longer retentron times and the mass spectra of mmor peaks m the sllylated

tetritol regton and m the trifluoroacatylated tetrrtol region of Frg 2 and 3. respectrvely, suggest the presence of partrally denvatrzed forms of the branched-cham tetntol No molecular Ion correspondmg to the completely denvatrzed specres was present for erther component, and the remammg spectra were very smular to those of the parent species Fig 1 shows peaks that correspond to srmrlar Incomplete derrvatrzatron for ethylene glycol and glycerol m the products from reduced formose y5

c,

103

c4

(Mf)

m/e

512-423-333-243

+, m/e

409-319-229

$3 m/e C, m/e

307-217 205-115

CH20SlMe3

409

CHOSlMe

Me3 SIO-

I

-CH2

3

0SIMe3

c:,

205

Cl

c,

307

c4

409

&HOSIMe

3

c3

307

&HoSIM=

3

C2

205

&HOStMe

3

H20S#Me3

103

CH20S1Me3

c!IHZOStMe3

Trlmethylsllylated

100

80

from

tetrlcol

Trlmerhylsllylated

100

” 2-C-(hydroxymethyl)

arabrnltol,

80

Formose

from

60

and

r bIroi xylltol

Formose 217 ZO? I

40 20 0

‘cc-, 80-

71 Trlmethylsllylared 2-C-(hydroxymethyl)

604o

arablnttol,

101 1.7 J

. ’ 100

100

.?o 2’7 200

300

xylltol

20, ,a7

107 119

rIblEo and

*17

40-

n-e Ag

7~ Trtmethylsllylated

80-

60-

zoo-

1cctetrltol

107

zo400

500

o-

JI

I 100

II 200

I

1 300

400

---zG

m/e

4 Mass spectra of species III the pentitol regron of tnmethylsllylated, redaced formose Carbohyd

Res ,24 (1972) 2944

R D PARTRIDGE, A H WEISS, D. TODD

36

Frg I shows two peaks m the pentrtol regron of the srlylated, reduced formose The first peak corresponded m retention time to a branched-cham, C, polyol, a 2-C-(hydroxymethyl)tetrrtol, as the per(tnmethylsdy1) ether The second peak corresponded m retentron trme to the possrble strarght-cham spectes as then- Me.& ethers* Frg 4 compares the mass spectra obtamed from the formose peaks and from the authentrc pentrtol specres Agam, the drastereomenc strarght-cham pentrtol tnfluoroacetates gave rdenticaI spectra, as drd the tnmethylsriyl ethersl’ The mass spectrum of the branched-chain sdylated species IS drstmct from those of the stratght-chain srlylated pentttols, showmg a lower relative abundance of m/e 205 and 217 and a greater abundance of m/e 307 and 3 19, since the tertiary m/e 307 fragment from the branched-chain species IS more stable The mass spectrum of. the srlylated 2-C-(hydroxymethyl)tetntol from formose corresponded rdentrcally wrth that of the known branched pentrtol (made by borohydnde reduction of aprose) and the mass spectrum of the group of srlylated strarght-cham pentitols was rdentrcal with the spectra of the mdrvrdual analogs The pentrtol trrfiuoroacetate regron of the chromatogram from reduced formose (Frg I) indicates by retention trmes the presence of all three straight-chain pentrtols Overlap of the straight-cham and branched-chain species m thrs and higher carbonnumber aldrtol regrons Interferes wrth the rdentrficatron of structures by the mass spectra of them tnfiuoroacetates Identzfcatzon of hexztols Fig 1 indicates the straight-chain specres identified m the hexltol regton of the stlylated and tnfluoroacetylated reduced formose All SIX of the strarght-chain hexrtoIs were rdentrfied by their retention trmes The tist two mayor peaks m the trrmethylsrlylated hexrtol regron remained umdenttied by retention time and are branched-chain hexltols Frg 5 shows the fragmentation patterns of the possrble sdylated hexrtol structures resultmg from the formose reactton, and a comparrson of one mass spectrum of a strarght-cham product taken m the formose hexrtol regton wtth that for the pure straight-chain species Agam the drastereomenc strarght-cham specres gave essentrally identrcal spectra Scans of straight-cham, tnmethylsrlylated hexttol peaks from reduced formose, as shown m Frg 1, gave xdentrcal spectra correspondmg to spectra of the pure specres The mass spectra from scans of the tnmethylsrlylated branched-chain hexrtols gave fragments indrcatrve of the srlylated hexltols, but which were dlstmctly different from the fragments from the strarght-chain species The shorter retention-times Identzficatron

of

pezztztols -

*It should be noted that (consldenng only open cham structures) there are 8 possible straxght-cham aldo- and keto-pentoses m forrnose that, followmg borohydrlde reductxon, gve three straight-cham penutols Furthermore, the a- and fl- anorners of sugars and aIso the pyranose and furanose tautomers generally show different retention times when subjected, as standard denvatlves, to g I c It 1s thus possible to have 22 g 1 c -separable aldo- and keto-pentose denvatives from the ongmal formose rmrture Cmbohjd

Res ,24

(1972) 29-44

BRANCHED CARBOHYDRATES

37

FROM FORMALDEHYDE

observed, combmed wrth the spectral interpretatrons. mdrcate that the two peaks corresponded to branched-cham hexrtol structures Both spectra, reproduced m Frg 5, show a lower relatrve abundance of nz/e205 and a greater abundance of m/e 307 when compared with the abundance of these fragments m the strarght-cham tnmethylstlylated specres The relative abundance of m/e 307 m the peak desrgnated 2-C-(hydroxymethyl)pentxtol IS about twrce that of m/e 307 m the peak assrgned the 3-C-(hydroxymethyl)pentltol structure (as cleaFH2OSiMe3

Cl

c6

103

c5

5’1

c,

307

C4

c;

Cl c,

103 511

Me3S10-y-CH20S1Me

3

m/e

m/e

409-3

C3

m/e

307-217

C2

m/e

205-l

19-229

15

c2

205

c5

103 51 I

$HOSIMe3

c;,

205

c4

409

c3

307

CHOSlMe3

c4

409

C3

307

vHoSIMe3

CH20SIMe3

YH20SIMe3

FHOSlMe3 CHOSlMe !~2

100

Trlmechyis~lylaced

2-c-

(hydroxymechyl) Formose _-

6 I4-525-435-345-255

51 I-421-331-241

Cl

FH20SIMe3 cjHOSIMe3

(MT)

Cg m/o

pentltol T

from

”

OSIM~

3

Trlmerhylsllylated GalactIcol

a0

3

G luc~tol from

Formose

60

II

Trlmethylsllylated

Stralghc-chain

Hexlcols

mle Fig 5 Mass

J9

ml=

spectra of species m the hexltol regxon of tnmethylsdylated,

reduced formose

Carbohyd

Res ,24

(1972) 29#

and

R

38

D PARTRIDGE,

A H

‘WEISS, D

TODD

vage at C-3 posltlon of the 2-substituted species gwes two fragments of m/e 307 with one bemg the stable tertiary fragment, and as there IS no direct cleavage of the 3substituted species to give m/e 307) Probably there are dlastereomers of the two mono-branched structures present that are not resolved under the chromatographlc condltlons employed C7

(M+) m/e

SH2051Me

7 16-627-537-447-357-267 Me3

3

SIO-C-CH20StMe3

Cgm/e

6 13-523-433-343-253

c3

307

C5mle

51 I-421-331-241

c4

409

$HOStMe

3

Cd

409

C4m/e

409-3

19-229

c3

307

GHOStMe

~

I7

c6

613

cc, -C2

511

307-2 205-l

I5

Cl

103

C3de

Q-f&

Cl

103

&H20SiMe3

C,

613

GHOStMe,

f~2

205

cx -I C4

307

?HOStMei

c5

511

409

$HOStMe3

CA

205

c,

307

C2

205

c5

511

3

Me3

SIO-F-CH20S1Me3 YHOStMe

c5

I_

1 I

80

Trlmechylsllylated from

103

c,

613

C,

409

c3

307

kH2051Me3

SCAN-7-I

SCAN-7-3 heptttol

I

60

3

c,

CHOSlMe3

511

c,

100

205

branched-chain

”

Trtmechyls~lylaced heprttols

Formose

from

Scralgh[-chaln Formose

j

4020

1 I

OL

SCAN-7-2 !”

Trtmerhylsllyla~ed

D-gulo-heptltoi

mle Fig 6

Mass

Carbohyd

D-g/ycero

Trtmethylsllylaced

I

heptitols

from

m/e

spectra of specvzs m the hepbtol remon of tnmethylsllylated,

Res ) 24 (1972) 29-44

Straight-chain

Formose

reduced formose

BRANCHED

CARBOHYDRA-IES

FROM FORMALDEHYDE

39

The relative quantities and mass spectra of the hexltols m the reduced formose tnfluoroacetate mdlcate overlap of the branched-cham and straight-cham species, and preclude comparison of the mass-spectral data Unfortunately, no known reference branched-cham hexlto1 was avaIlable Identzfcatron

of heptrtols

-

The chromatograms

of the trrmethyls~lylated and

tnfluoroacetyIated denvatlves of the reduced formose shown m Fig 1 mdlcate the presence m major proportlons of a number of species havmg more than SIX carbon atoms The sllylated, reduced formose gives a dlstmct band of peaks m the heptltol re@on OF the many possible straght-chain species possible, only D-gljxerO-D-p/Oheptltol and D-g&cero-D-g&cto-heptitol have so far been ldentdied by retention times Mass-spectral scans were taken m the O-trlmethylsllylated heptltol region of the reduced formose Fig 6 shows the spectra obtamed and the possible fragmentatlons of a number of the heptitol structures The mass spectra of scans 7-1 and 7-2 compare favorably with one another and with that of the pure, straight-cham heptaO-Qlmethylsdyl-D-g&eyrero-D-gztlo-heptitol of nz/e 205 and a greater relative abundance

Scan 7-3 shows a lower relative abundance of m/e 307 compared

with the other spec-

tra, suggestmg that this peak contained a branched-chain species The longer retentlon time does not necessarily mdlcate that the product Bvmg the peak IS not branched The trend noted for the earller retention times of smgly branched species with respect to their straight-cham isomers might Imply that the species shown by scan 7-3 may have multiple branchmg This suggestlon IS not ruled out for the seven-carbon species by the reaction mechamsm postulated for formose 13, but xt IS only speculative, all that can be stated with any degree of certamty IS that the region IS composed of heptltols havmg both straight- and branched-cham structures The mass spectra of the tnfluoroacetylated, reduced formose m the reaons greater than SIX carbon atoms also Indicate the presence of C, species Identzfcatron of aldoses and ketoses - Although the aldltols of formose give a good mdlcation of carbon number and dlstrlbutlon of branched-and straght-cham specres, they also slmphfy the actual formose reactlon mixture. Attempts have been made to Identify the species present m the ongmal formose mixture by g 1 c of the (unreduced) sllylated derlvatlves A number of peaks are ldentlfied by retention time m Fig 1 (see ref 15), but there IS overlap m a number of Instances Not only IS there overlap w&m a given carbon-number dlstnbutlon, but also, for example, an overlap In the C, region by dlmers of 1,3-dlhydroxy-2-propanone and glyceraldehyde” l5 A number of peaks could not be Ident&ed by comparison of retention hmes with those of known species Fig 7 shows an attempt to slmphfy the mterpretatlons of the mass spectra taken The top chromatogram of the figure IS the normal flame-lomzatron chromatogram of sllylated formose Simultaneously, a “mass” chromatogram was obtamed by settmg the mass spectrometer to momtor a smgle Ion and recordmg the mtenslty of this ron versus time Two masses, m/e 204 and 217, were exammed m tls manner The ratlo of the two mass numbers chosen has been shown to be an effective measure Carbohyd

Res ,24

(1972) 29-44

40

R

D. PARTRIDGE,

A

H

WEISS, D

TODD

of the rmg srze of a number of carbohydratesi6; the fragments grvmg rise to these peaks are shown m FIN 7

CH-CH + .

CH=CH

Fig 7 Gas and “mass” mJe 217 were momtored

chromatogramsof tnmethylsrlylated formose, massnumbersm/e 204 and

The maJor peak m the 2-C-(hydroxymethyl)glyceraldehyde regron of the chromatogram of srlylated formose desxguated m Fig 1 has not been previously Identified As the major component of thus partrcular formose m the tetntol regron of the chromatograms of srlylated and of tnfluoroacetylated, reduced formosers the branched cham tetrrtol, 2-C-(hydroxymethyl)glycerol, we were led to suspect that the major component of the four-carbon regron of the srlylated formose mrght be 2-C-(hydroxymethyl)glyceraldehyde (Frg 1) There IS no possible keto-analog of thus sugar and thus rt IS unhkely to dmrenze or form any rmg structures Frg 7 mdrcates that thrs peak contams very httle component m/e 204 as compared wrth nz/b 217 Generally, for the higher sugars’ ’ 16, thrs observatron would rmpiy a furanord ring-form, but when the formatron of rmg forms IS ruled out, a branched specres is indicated The complete mass spectrum of this peak 1s shown m Frg 8 and 1s compared wrth the mass spectrum of trlmethylsllylated erythrose The scan for 2-C-(hydroxymethyl)glyceraldehyde in Ftg 8 agam shows the small relatrve amount of m/e 204 compared wrth m/e 217 The large relative abundance of m/e 307 mdrcates a stable, tertrary C, fragment, charactenstrc of the branched specres, as prevrously noted The mass spectrum of the erythrose as the Me,% derrvatrve, CUfWIYdKes.,24 (1972) 29-44

BRANCHED

CARBOHYDRATES

41

FROM FORMALDEHYDE

CHO c2

131

C2

205

CH2 CHO I

c,

Me 3 SIO-C-CH20SlMe I CH 2 OSlMe

3 3

29 307

cs

c-2

233

Cl

103

Trlmethylszlylared

100

Fig

8 Identlficatlon

from

OSlMe

3

k=o

131

c,

c3

llo) 2-C-(hydroxymethyl)

I giyceraldehyde

Cl

Formose

by g 1 c -m s of branched-cham

1

Trlmethylsllylated

29

c3

307

cs

233

Cl

103

Cl

103

c3

233

C3

233

Cl

103

erythrose

*’

tetrose

In tnmethyls~lylated

formose

however, shows much greater relative abundance of m/e 204 and 205, mdxcatmg the expected cleavage at C-Z The low relative abundance of m/e 307 and the appearance of ??z/e319 IS characterlstlc for erythrose, an aldose No molecular Ion at m/e 336 was observed Therefore, It IS concluded 2-C-(hydroxymethyl)glyceraldehyde

that the species

IS the branched

tetrose,

EXPERIMENTAL.

General methods - Many of the carbohydrates and aldltols used m thrs study were commercmlly avalable preparations 2-C-(Hydroxymethyl)tetntol was prepared by borohytide reduction of aplose obtamed by hydrolysis of dl-O-lsopropyhdeneaplose The Me,& derlvatlve gave a smgle peak by g 1 c 2-C-(Hydroxymethyl)gIycero1, (HOCH,),COH, was synthesized by deammatlon of tns(hqdroxymethyl)ammomethane (Tns) to a solution of 12 1 g (0 10 mole) of Trrs m 17 1 ml of acetlc acid and 100 ml of water at 5” was slowly added, with external coolmg, a solution of 6 9 g of sodmm mtnte m 25 ml of water After bemg kept cold for 0 5 h the solution Carbohyd

Res ,24

(1972) 29-44

42

R

D

PARTRIDGE,

A

H

WEISS, D

TODD

was warmed to room temperature and then evaporated to dryness zn vacua The residue was dissolved m water, passed through a column of Dowex SOW-X8 resin (H’ form), and the eluate evaporated zn vaczzo A sample of the syrupy resrdue was converted into the Me& derivative and subjected to g 1 c About 98% of the Injected material emerges as one peak This result was confirmed by Ingh-pressure hqmdliqmd chromatography (Waters ALC-201 equipment) The Me,% derivatives were prepared accordm g to the method of Sweeley, et I9 by usmg Trf Sil (Pierce Chemical Company) premixed reagent The sample al, (IO-20 mg) was drssolved in 1 ml of the reagent, wnh shght warmmg if necessary, and allowed to react overmght at room temperature Prior to gas chromatography, the sample was extracted rnto chloroform20 The trifluoroacetates of the aldnols were prepared and analyzed accordmgly to the method of Shapna lo by usmg a 200 1 mixture of trlfluoroacetrc anhydnde and pyridme Generally, 1 ml of reagent was used for a 10 mg sample However, much more sample could be tolerated Reduction of the formose sugars was accomphshed by adding approximately 10 mg of sodium borohydride m 1 ml of water to a neutrahzed .soIutron of formose (l-2 ml) contarnrng the equivalent of 10-20 mg of sugar This mixture was then shaken and allowed to react overmght at room temperature Acetone (1 ml) was then added and the acidity of the solutron adjusted to pH 3-4 with HCI (about 10 drops) The solution was then evaporated to near dryness by a stream of dry nitrogen, treated with 10 ml of methanol, boiled to about 5 ml, and then evaporated to dryness under mtrogen The methanol treatment was repeated twice and the derrvatlzmg reagents added A Perkm-Elmer Model 900 gas chromatograph having a capillary inlet-system and dual flame-iomzatron detectors was mterfaced to a DuPont (CEC) 21-491 double-focusmg, mass spectrometer by means of a heated capillary hne and a jet separator G 1 c of the trrfluoroacetates was accomplished on a 50 ft x 0 020 m I D FS 1265 SCOT column (Perkm-Elmer) operated from 100” to 225” at 4”/mm, with a hehum flow of 4 0 ml/mm The mlector and detector temperatures were mamtamed at 225” and 250”, respectively Combined gas chromatography-mass spectrometry of the Me& and CF&O derivatives was accomphshed with the SCOT coIumns under the same operating condrtrons Hehum make-up gas (10 ml/mm) was added to the effluent of the SCOT columns to perrrnt optimum Interfaced operation An electron energy of 82 eV was employed for a11mass spectra The source temperature was mamtamed at 180” for the aIdno trifluoroacetates and from 200° to 250” for the Me,‘& derivatrves The scanning time over the mass range )n/e 20 to 600 was varied from about 3 to 7 seconds, depending on the peak width and resolution required The Interface Imes and separator temperatures were mamtamed at 180” to 200” for the aIdno trifluoroacetates and from 225” to 250” for the Me,& derlva: tives Too high a temperature m the case of the trlfluoroacetates resulted m decomposCarbohqd

Res _ 23 (1972)

29-44

BRANCHED

CARBOHYDRATES

43

FROM FORMALDEHYDE

ItIon of the sample, @vmg decreased resolution and poor, or m some cases useless,

spectra Identticatlons were made on the basis of a comparison of retention time and mass-spectraI data with that of known sugar and aldltol denvatlves Retention data were obtalneci by notmg an Increase m a peak height when the sample was mlxed with some known reference material The formose sugar preparatlon$ was prepared as follows A plug-flow umt capable of producing 100 g/h of deionized concentrated formose has been described prevlousIy2’ The unit consIsted of a stlrred feed tank contammg a mixture of 8% formaldehyde (prepared from 37%) CP formaldehyde solution, 0 1 moles of Ca(OH), per mole or formaldehyde and 0 01 moles of CaO per mole of formaldehyde, which was metered at a rate of 2000 ml/h through a co11 of l/4 m Eastman Polyffow tubmg with a total volume of 100 ml When the reactor was kept at 82” m a water bath, conversion mto a pale-yellow product occurred m the last few turns of the co11 The effluent was cooled, neutrahzed with gaseous carbon dloxlde, filtered to remove preclpitated calcium carbonate, and delomzed with a 12 x 45 cm co!umn of mlxed anxonand cation exchange resins The almost colorless product was concentrated m a rotary evaporator at 50” and 30 torr to a thick syrup of variable color e CONCLUSIONS

Although tests of the product of condensation of formaldehyde catalyzed by calcium hydroxide show the presence of small proportions of many known sugars, the present study rndlcates that the maJor Cq, Cs, and C6 products are branched-cham aldoses and ketoses Slgmficant proportlons of higher molecular-weight sugars are also suspected to be branched-chain The mass spectral fragmentation pattern of tr~methylsllylated 2-C-(hydroxymethyl)glyceraldehyde provides conclusive evidence of the formatIon of 2-C-(hydroxymethyl)glycera!dehyde m the formose reactlon, and spectra of previously unreported, branched-cham aldltols derived from formose pernut inference of the skeletal branched-cham structure of the parent aldoses and ketoses The formose reactron can be consldered as a umque method for producmg branched-chain carbohydrates ACKNOWLEDGMENT

The research was pursued under NASA Grant NGR 22-170-008 “Study of the Technrques Feasible for Food Synthesis m Sustamed Space Fhght” The authors appreciate experlmental assistawe by MISS Sandra Wilson

*The Cahf

formose 94035

sugar

used was prepared

b> 3 Shaplra

NASA

Ames

Research

Curbohvd

Center,

Moffett

Field,

Res , 24 (1972)

29-44

R

44

D

PARTRIDGE,

A

H

WJBS,

D

TODD

REFERENCES IAB 2 G

IJTLFZOW, Ann Chem , 120 (1861) 295, Compr Rend, 53 (1861) 145 C AICERLOFAND P W. D MITCHELL, A Study of the Feaslbdrty of the Regeneration of Carbohydrates IR a Closed Cvcurt Respwatory System, Final Report, Natlonal Aeronautics and Space Admmstra~on NASr-88, March 1, 1963 ~JWFRANKEN FIELD, Srudy of Methods,cor Synthesis of Fatty Acids and Lrprds from Metabohc Wastes Aboard A Space Craft, Final Techmcal Report, National Aeronautics and Space AdamIstratlon, Contract NAS 2-3708, 1967 4 G C AKERLOF, J Spacecraft, 1 (1964) 303. 5 J S-IRA, Cereal SCI , 13 (1968), J Agrx Food Chem , 18 (1970) 992, Proc Wesr Pharmacot Sot, 13 (1970) 57, Chem Abs;r 74 (1971) 138734b 6 V. JOHNSON, A J CARLSON, AND A JOHNSON, Amer J Physlol, 103 (1933) 517. 7 Y Y SINYAK, Space Btol Med (USSR), 2 (1968) 9-16 8 A H WEISS AND J SHAPIRA, CEPSymp Ser ,67,No 108 (1971) 137-147, Hydr Proc ,49, NO 2, (Feb 1970) 119-26, Abstr Papers Amer Chem Sot Meetang, 155 (1968) CO65 9 A H WEISS, R LAPIERRE, AND J SHAPIRA, J Catal, 16 (1970) 332 10 J SHAPIRA, Nature (London), 222 (1969) 792 11 D C DEJONGH, Anal Chem , 42 (1970) 169R 12 0 S CHIZHOV, B A DMURIEV, B M ZOLOTAREV, A Y CHERNYAK, AND N K KOCHRXOV, Org Mass Spectrom , 2 (1969) 947 13 G HEARNE AND H W DEJONG, Ind Eng Chem , 33 (1941) 940 14 0 WULFF, Ger Pai 874,774 (1953, to Farbwerke Hoechst, A G ), Chenr Abstr 52 (1958) 11113g 15 A H WEISS, R B LAPIERRE AND J SHAPIRA, J Catal, 16 (1970) 332, A H WEISS, H TAXIBAWALA, R D PARTRIDGE, AND J SWAPIRA, De&emu Monographlen 68, Neue Verfahren der Chenuschen Technrk, Verfag Chemre, GMbH, Wernhean/Bergstrasse, 1971 p 239 16 D C DFJONGH, T RADFORD, J D HRIBAR, S HANESSIAN, M BIEBUR, G DA~VSON, AND C C SWEELEY, J Amer Chem Sot, 91 (1969) 1728 17 B ARREQUIN AND J TAROADA, J Chromntogr Scl , 8 (1970) 187 18 H C CURTIUS, M MULLER AND J A V~LL~IIN, .T Chromatogr , 37 (1968) 216 19 C C SWEELEY, R BENTLEY, M MAKKA AND W W WELLS, I Amer Chem Sue, 85 (1963) 2497 20 R D PARTFCIDGEANDA H WEISS, J Chromatogr Scz ,8 (1970) 553 21 J SHAPIRA m The Closed tzfe Support System, NASA SP-134, 1967, pp 175-88

Carbohyd

Res ,24

(1972) 2944