RESOURCE-RELATED RESEARCH

COMPUTERS AND CHEMISTRY

(RR-00612 RENEWAL APPLICATION>

       Submitted to the

BIOTECHNOLOGY RESOURCES BRANCH

        OF THE

NATIONAL INSTITUTES OF HEALTH

December, 1973

School of Medicine
Stanford University


      DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
    PUBLIC HEALTH SERVICE

SECTION I

       Fom AppraMd
      Budget Bureau No. 6ER0249
LEAVE BLANK

GRANT APPLICATION

COUNCIL Ihfonrh, Year)      OATE RECEIVED

                                                  I                         I
TO pE COMPLETED BY PRINCIPAL INVESTIGATOR Ilt8ms 1 thruuolr 78nd 15AI
,1. TITLE OF PROPOSAL (Do notwcwd 53 ty&t@r4==)
Resource Related Research - Computers and Chemistry        (RR-00612 renewal)
          2. PRINCIPAL INVESTIGATOR         13.DATES OF ENTIRE PROPOSED PROJECT PERIOD (Th~aPP~i*tiOn)
2A. NAME (Last Fitzt, Inidel)                IFROM            1 THROUGH

  Djerassi, Carl
26. TITLE OF POSITION

Professor of Ch=mistry

$1,350,795.00  I   $276,197.00

2C. MAI Ll NG Am IStroet, City, SWW, Zip Coda)
Depariment of Chemistry
Stanford University
Stanford, California 94305

B. PERFORMANCE SITE(S) &ernsrructions)
  Department of Genetics,
  Department of Chemistry, and
  Department of Computer Science
   Stanford University

(SW lnsvuctionsl
Department of Chemistry

%f. MAJOR SUBDIVISION (SW /mWuctions~      I

School of Humanities and Sciences                                                          .
I . Rosewch Involving Human Subjects (S@e lnnruCtions~           8. Inventions ~Renaw;rl Applicants Only - S-M hnnrCb0~)
Aa NO 6-a YES Approved:
C.m YES - Pending Review              chte

A.a NO B.0 YES - Not previously reported
C. OYES - Prwio~!t.j rcportM

TO BE COMPLETED BY RESPONSIBLE ADMINISTRATIVE AUTHORITY l/terns8 through 13and 1581
B. APPLICANT ORGANIZATION IS) [SW lmtructions~          TYPE OF ORGANIZATION l&sck app/iciib/e item)
                                  0 FEDERAL  r] STATE  0 LOCAL ?!3 OTtiER (Swifv)
Stanford University                     Private, non-profit University
Stabford, California 94305            , NAME, TITLE, ADDRESS. AND TELEPHONE NUl~6ER OF
  IRS No. 94-1156365                    OFFICIAL IN BUSINESS OFFICE WHO SHOULD ALSO BE
Congressional District No, 17             NOTIFIED IF AN AWARD IS MADE
                               K. D. Creighton

11.
F
12

Deputy Vice Pres.  for Business & Finance
Stanford University
Stanford, California 94305

10. NAME,TITLE, AND TELEPHONE NUMBER OF OFFICIAL(St
SIGNING FOR APPLICANT ORGANIZATION(S)

       T&phone Numter (415 ) 321-2300 ,x2551
13. IDENTlFm '
       ~3s.:slLAtfONAc7NTT~ RECEIVE CREDIT
FOR INSTITUXONAL G,RANT PURPOSES lSt?e lnsouctionsj

I    2p School of Humanities and Sciences

C/O Sponsored Projects Office            14.  ENTITY NUMBER (Formerly PHS Account Number)
  Telap!mneNumbw(c) (415) 321-2300, X2883             458210
16, CERTIFICATION AND ACCEPTANCE. We, the undenigmd, certify thst the statements herein we true and complete to the best of our
Mw nd rcbpt. 8s to uny mnt mrarded, the obllgstlon to &mply with Public Health Sewice terms and conditions in effect at the time of the

(slpnatura muiredon
original copy only.
Lb ink, `?%r" sign8lums   8. &GNATURE(Sj OF PERSONiSi NAME0 IN ITEM IO                     OATE

not wcqowbfd
111~ y$ (FORP~RLY PH~ 398
,Rov. l/73                      /


DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE                LEAVE BLANK
         PUBLIC HEALTH SERVICE                   PROJECT NUMBER

                RESEARCH OBJECTIVES     I
NAME AND 4DDRESS OF APPLICANT ORGANIZATION
Stanford University
Stanford, California   94305
NAME, SOCIAL SECURITY NUMBER, OFFICIAL TITLE, AND DEPARTMENT DF ALL PROFESSIONAL PERSONNEL ENGAGED ON

               Professor of Chemistry, Department of Chemistry; Joshua
              fessor of Genetics, Department of Genetics; Edward Feigenbaurr
               omputer Science, Department of Computer Science; Bruce
               arch Computer Scientist, Department of Computer Science;
                Research Associate, Department of Genetics; Dennis Smith,
                   Department of Genetics;  Nates
           Department of Computer Science; Harold Bro
Associate, Department of Computer Science; Geoff Dromey, SS#
at a later date, Department of Computer Science.
                  .-- _-__- _._ _ - _ .__-
TITLE OF PROJECT

Resource-Related Research -- Computer and Chemistry
USE THIS SPACE TO ABSTRACT YOUR PROPOSED RESEARCH. OUTLINE OBJECTIVES AND METHODS. UNDERSCORE THE KEY WOR'
(NOT TO EXCEED 10) IN YOUR ABSTRACT.

  The ,bjectives of this research program are the development of innovative
computer and biochemical analysis techniques for application in medical research
and closely related aspects of investigative patient care.  We will apply the
unique analytical capabilities of gas chromatography/mass snectrometry ((X/MS)
with the assistance of data interpreting computer programs utilizing artificial
intelligence techniques, to investigate the chemical constituents of human body
fluids in a variety of clinical contexts.  Specific subtasks of-this program include;
1) the application of artificial intelligence (AI) techniques to programs capable of
interpreting mass spectra from basic principles as well as extending mass spectral
theory by analysis of solved spectrum-structure examples, 2) the extension of GC/MS
data systems to provide stand-alone capabilities for collecting low and high resclution
mass spectral and metastable ion data, 3) the application of GC/MS and AI techniques
to analysis of biomolecular structure elucidation problems of a large number of
collaborators,, and 4) the extension of artificial intelligence techniques to an
interactive system for computer assisted structure elucidation based on a variety
of data.

LEAVE BLANK

NIH 398 (FORMERLY PM 398)
Rev. r/73

PAGE 2


SECTION II -PRIVILEGED COMMUNICATION                        FROM               THROUGH

DETAILED BUDGET FOR FIRST 12-MONTH PERIOD  I  5/l/74

           -

                              -

163,935     28.962    192.897

CONSULTANT COSTS                                                                          I    -O-
                                                                           I

EQUIPMENT                                                                           -__
Equipment Purchase (First Year Items Only):
    DEC GT-40 Display Terminal                                     13,400
    PDP 11/20 Upprade                                          --        1
                                                                           -    34~,000
Equipment Maintenance:
    PDP-11 (DEC Contract)                                            4 200
                     --                               I      -L---
MAT-711 (Parts, etc.)                                    6.292
suppLIEsElectronics Supplies                                            4 400
                                                      -                                        -I.--
    GC Supplies -&olumns
    Liquid Nitrogen
---                 stock. etc.
    Data Recordinp Media

PATIENT COSTS (See ins true tionsl                                                     I

I   -o- i

ALTERATIONS AND RENOVATIONS                                                                 -n-

OTHER EXPENSES (Itemize)                                        -~
    Publications,  telephone, office supplies,  p ostage
    Computer Terminal Lease (4)
    Computer Usage - 370/158 (First Year Item Only)

--I        --
      4.000

--- ---UQQ-
      5.OGO

TOTAL DIRECT COST (Enter on Page 1, ltsm 5)

  INDIRECT
   COST
(See Instructions)-

                                                              ( -a",...,
                                                                      v-
           DATE OF DHEW AGREEMENT:

     56 S&W'                     0 WAIVED
                            0 UNDER NEGOTIATION &lTH:
47   %wxHTDC-J une 26. 1973'                           i

        1 `IF TH:S IS A SF'ECIAL RATE le.@ off-site;, SO INDICATE.
`NIH 398 (FORUERLY PHS 398)              PAGE 3
Rev. 1173

.


-PRINCIPAL INVESTIGATORS:
  C. Djerassi
  J. Lederberg
  E. Feigenbaum

RESEARCH ASSOCIATES:
B. Buchanan (1)
  A. Duffield
  D. Smith
  N..Sridharan
  H. Brown
G. Dromey

PROGRAMMERS:
  W. White
  R. Tucker

SENIOR RESEARCH ASSISTANT:
A. Wegmann

ELECTRONICS ENGINEER:
  N. Veizades

GLASS BLOWERIMACHINEST:
  E. Steed

RESEARCH ASSISTANTS:
  L. Masinter
  M. Stefik
To Be Appointed

SECRETARIAL SUPPORT:
  K. Wharton

TOTAL:                  $163,935     $28,962   $192,897

DETAILED SALARY DATA
NIH GRANT #RR-O0612
5/l/74-4/31/75

%
Effort

Salary

Fringe
Benefits

Total

10         -O-         -O-         -O-
10         -O-         -O-         -O-
10       2,910        514     3,424

50       7,000      1,237     8,237
25       6,195      1,094     7,283
100      16,200      2,862    19,062
100      16,050      2,835     18,885
100      16,200      -2,862    19,062
100      15,500      2,738     18,238

100      14,400      2,545    16,945
100      14,100      2,491     16,591

100      15,000      2,650    17,650

60      11,670      2,062    13,732

25.

100
100
100

100

4,410

5,070
4,915
4,915

9,400

779     5,189

895     5,965
868     5,783
868     5,783

1,662

11,062
--

(1)Dr. Buchanan's salary charges do not begin until 9/l/74 at which time
his NIH Research Career Development Award expires.

3a


SECTION II -PRIVILEGED COMMUNICATION
   BUDGET ESTIMATES FOR ALL YEARS OF SUPPORT REQUESTED FROM PUBLIC HEALTH SERVICE
                   DIRECT COSTS ONLY (Omit Cents)

    DESCRIPTION

PERSONNEL
COSTS

`ST PERlO:)
ISAME AS  1 ADDITIONAL YEARS SUPPORT REOUESTED lThisapp/ication only)
TAILED B       2ND YEAR    3RD YEAR    4TH YEAR    5TH YEAR   23XMmds    7TH YEAR
                                        TOTAL

192,897  210,611  225,129  240,630  257,383  1,126,650

CONSULTANT COSTS
(Include fees, travel, etc.)      -O-      -O-      -O-      -O-      -o-       -O-

EQUIPMENT
              58,100   11,770   12,947   14,241   15,665   112,723

SUPPLIES           9,600    6,920   7,612    8,370   9,207    41,709

             DOMESTIC
TRAVEL           1,200    1,320   1,452    1,597   1,757     7,326

             FOREIGN     -O-      -O-     -O-      -O-      -O-       -o-

PATIENT COSTS          -O-      -O-      -O-      -O-     -O-       -O-

ALTERATIONS AND
RENOVATIONS          -O-       -O-      -O-      -O-     -O-       -O-

OTHER EXPENSES      14,400   10,340   11,374   12,511   13,762    62,387

TOTAL DIRECT COSTS   1276,197  l 240,961 ,258,514  1 277,349 1297,774 ,1,350,795(        I

I  TOTAL FOR ENTIRE PROPOSED PROJECT PERIOD (Enteron Page I, /tern 4) - I  $1,350,795     I

REMARKS: Justify all costs for the first year for which the need may not be obvious For future years, justify equipment costs, as we/J as any
significant increases in.any other category. If a recurring annual increase in personnel casts is requested, give percentage. (Use continuation
pege if needed)

See following pages for budget justification.


l?uaqet. Just.i.ficati0n

Tt)t 3vailab%lity of exisiinq equipment - including the mass
sp?ctromet3r and SUMEX computer - svoi3s the need for requesting
f I1 n 3 5 for major laboratory itcns and substantial comp*lting costs.
"bus,  the major expense in the resulting budget is for personnel.
i?e fcol +h3t the personnel listed here are necessary to carry out.
the cesexrzh, as justified hsl3w.  Rec!lrrinq costs are about
?227,0130 par ve3r.  First year expenditures are higher to provide
the tnstrumentation necessary for mass spectromctry service in the
Ei rst ymar.

i?e ace rclqaesting funds for five years to coincide with the
funi!inq of the ATM-SUY??? resource, to which we hope to make
iiqfif icant constribu+ions.

This Surfqet overlaps sliqhtly with the budget for the Genetics
Pnse3rch Canter (J. Lederberq, Principal. Investigator).   Dr.  Alan
9ufFiel+'s 25% salary budqetsfi here is covered by the other budget.
(GtherY 1QOX of his salary is hudqeted).   10% of Ms. Annemarie
$=+qman~*.s salary is covered there (with 10'37 of her salary
h u-! qe t P ,i here) .  These are the only overlapping items.   We have no
officF31 notification of Genetics Center funding;  if the present
pxpos3T! is SUCC@SSfU1,  the Genet~ics Center budget will be
adj31sted accordinqly.

Tn the five-year hudqet, salaries are increased by 6X per year and
staff henpfits are computed at 17% for +h~ period 5/74-81'74, 18%
Fr)r the peti3d ?/74-q/75, and are increased l? per year
thereafter,  based on current l!niversi+y prnjections.  Other budget
zatc~ories are increased by 10" per year to account for inflation.

?ersonn31:

Pf?l!C9 r;.  YJCHANAN
nr.  Brnr:e Buchanan holds an ??I!! Research Career Development Award
to work on applications of artificial intelli~~once to
health-related problews,  including theory Eormation by computer.
3 i.3  work on those aspects of thi.s grant is thus consistent with
+he DeveI39ment Award.  Half-time support is requested after the
thlc3. vear of the Development Award (startinq September, 1974) to
z3ver t-l??? zonticqency that the award wit1 not he extended to the
full  fiv3 years-  These funds will be ret!lrned if the Rward is
ext erlc?e?.

`)ENNIS ff* S'JTTH
?r.  P5nnis 9. Smith has been a member of the DSNDRAL project since
July, lQ71.   He has beer, responsible for +-he ?IS and its computer
Sllpp3f t. *  ani! has been  involved in the application of the AI
proqcrlms to structural studies of biomedically important
23mpaur.?s,  primarily steroids.  These responsibilitj.es will
zontinuo  in the future,  with psrticular emDhasis on providing the
!?lLlSS snPctrometer/AI program link to the usnr community and its
333s sncctrometry and general structure elljcidation needs, and in
nrnvi.?ir:q the necessary chemical knowledge and input for
qevelonment of the cotilputer programs acd user interfaces for the
proposed compatar assis+ed  structure elucidation effort.


?!T,A v 31~PFIELD
3r.  %lan Duffield is the senior sci.nntist in charqr? of the mass
sp?ctrnmetry facilities of the GRZ.  Eocause of his expertise in
t-k=-  analvsis of mass spectra from various fractions of human body
flfli3S,  h? will provide the 'link hetwe~!r:  the s+.ructure elucidation
teshninues of this pronosai  and other scientists with similar
probleas, . The GC/HFMS faciiit.ies are also ~xuecter! to provide
7:3tvi73 &3
        CI    the Sensfics Cent.er for hiah resolution analysis of
zom!a9urifis isolated from hoc?y flujds,

VATFSA SRI3HARAN
nr.  Sridhacan will be responsible for developinq interface
rnlltin?s that allow now researchers to make use of the structure
elncidation proqrams,  We expect these rout.ines to accept
inforaatiol aboot a research problem, in semi-formal terms, and
transl;Lte it into a format the proqram can use.  They should be
zoaple+e enough so that individual researchers do not need to know
ih~?,u% the inner workinqs of the proqrams.  In addition, he will
nnntinu? t3 help Dr. Prnw~! and Fr.  Vasinter with development of
&he cvrlic qenera+or program.  (Within a few days of this writing,
Dr.  Sridharan has decided to take a leave of absence.  During his
3SsPncc we will recruit another Research Fssocia+e to perform his
,?iltias.)

L!RROLP PFOWN
nr. qarold Brown's knowledqe of graph theory and combinatorial
113.t hem3ti.c~ is essential to the development of the cyclic
sfru'3ture generator.  Pany problems with development and
Lmolementation of this program have required sophisticated, new
mathsmaticsl solutions worked out by Dr. Brown.  For example,
qenoratinq the dictionary of cyclic qraphs and assembling
suh.;trr~ct:~~css involve problems irt graph theory that Dr. Brown is
3lirranfly *workinq on.

Dr. 7rown has submitted a proposal to the NSF to cover his salary
for this research.  If that qr3nt is awardc:l, funds requested here
for his salary will not be needed.

P
BS. GEOFF DROMEY
  Senff Dromey is a chemist with strD>nq compu+er science
in+eres+s wh:, has been associat.nd with the project since
T~ptemh?r,  1973. He has become familiar with many aspects of the
D!??DR?` -- performance proqrams a?d will be exnected to help outside
rosearchers  use those proqrams.  Tn addition, he will be
I~veloplnq new proqrams, such as the proqram for molecular ion
?e+ermication from mass spectra.

bJI!.tTA'L: C, #HI'??
Yr. iiillianr Whita provides hiqh-level Droqrnmming support for the
theory formation proqrams, including helpinq to devise new
proqr3tmt: in response to new research problems as well as
iaalamentinq them.  HP wrote almost all of the LISP code for the
TVTS'lY proqram, for example, and is currently responsible for the
3!Jl.FGE?: proqram.

MS .  4EiW??A"IF WRGMAN?r
Yz  4nnqma:ie Wegmanr.
-9. . ..I.           is Che Senior Research Assistant in charge
35  'h2  Y:c/HRP!S system.   She was  fnrmerlv head of Yzwlett-Packard's
Palo Alto qas chromstoqraphy applications laboratory and has been
responsible for the operatior! of the GC/CS system since the

6


?6?1iv?ry to our laboratory of th? MAT-711 (Yovembsr,  1971).   Her
tzzhnical 3hiliSy is absolutely essential. tr, the continued
3Peration and development of the mass spectrometry facility.

`Y 2sc3rs,  Vzizadcs and Steed will assist part time ir! maintaining
tflc  I;:/?5 system. fir.  Veizades is an Electronics Snqineer who is
rssnnnsib2e for the electronic and mechanical systems as well as
pr3viIinq the necessary voltaqe rcaq-out an:! control development
f3r t.he m?tastable analysis data system. Yr, Steed is a Research
?tlqineer r3sponsihI.e for the system glassw2rk 3rd vacuum system
na;nkcr.;tnc2.

R3!?"RT TJJCVFF
Yr.  Pobert Tucker implements and maintains the computer program5
for ,?a+3 azquisition and reduction of MS d3ta.   This includes
translatinq existing pL/ACEE into F3HTRAhT and PDP-11 assembly
lnnsJa7e.   In addition,  he will he resporsi5le for improving these
DrOqrtBS f3r repetitive HRY.S scans,  implement inq the multinlet
r~?snlut;on algorithm 2nd the .sDftware necessary for semi-automated
zollarti9n ?f metastable ion data,

LL;1B?Y H   ?I b.SIKTEF
`r. Lar;v Yasinter.  Descarch Assistant,  will continue to work with
3rs.  Leclerherq and Erown on the developmect of ths cyclic
stfllcturo qcnerator.   His LTSP expertise has been an invaluable
rPsourcF? for nvery member of the research team.

YARK  STF'PIK
P!r. Yark Stefik,  Research Assistant,  combines two years of
?x3erienze on t?ie ACP?E/YS data acquisition system with a long-term
nomni+ment to computer scie!nce.  He has developed interactive
Lihr*rv search capabilities for the mass spectrometer and uill
continue to improve them, tzis knowledge of thP data acquisition.
n3mputzr proqrafls will be very valuable in assistinq initial
tran;la?ion of those programs into FORTRAN  {from PL/AC!lE code) for
t. h 3  ?XtQndea PDP-11/20 system.

P?SPAR:f! I\SSIS!TANT - unnamed
Ye h3ve interviewed +wo prospective Research Assistants, both of
ub3Y h3v9 broad chemical experience and strong computer science
ir,tPros+s.  Ws request funds to hire one of tfiem to provide
additi3n31 links between computer science tpchnigues and structure
~luci32t%on problems.

7n2 E:.llt-time secretary is necessary for the secretarial support
3f this number nf scientists. 5s. Kathlaen iaharton is now with
t hI?  C3mnut3r Science group.

!G 3iszuss?I in thP tax+ (Sxtiop III.%), in the first year we
3lan to auqment our existinq PDF-Il.120 computer (4k memory) to
-tll.?w its operation as a stand-3lonl r?3t3 systea.  We plar to add
15k of memory (S3,OOO) v a floatir?q print arithmetic unit (27,500),
in in-lllstry compatible tape drive ($9,300), a ,disk drive (lO,SOO),
2 low soee2 communications intzrfacp ($l,OOO), and a bootstrap


1->?3?r ann clock ($1, ZOG) .   Th2se devices toqet her with state
saLzs tax total t.0 %3tr,OOO.  The prices nuot?c! ar? representations
-tf the P.ost cost-effective suppliers 0 f the rpsppective devices we
lavcl\ '7c~n 3?>le to locat?.   'rJ? will continue to review the market
S?F3ce Smplementation to maxi.mize technical and cost performance.

1s stitn:l above, we plan to provide irikerf3ce programs to provide
t-h9
.-  ,-i,%CruEication I.ir?k between the users an? the programs.   The
~lr.iversal lanquaqe of molscu3.3r structure is diagrammatic
reDrPs?nt3tion of the structures, drawn usually in two dimensions
(?,r 3s two-~Iimensio~~l represent.3tions of three? dimensional
i r,E 3rn=i+io?) .  ThercJfor?,  WC fsel that a graphics terminal such as
Fhp D9:: <T-U0 is necessarv for effective sharing of the programs
1~3r.q Starfqrd users,  The GT-$0 terFi?al is a good choice for
pPrf3r8i,qq this structural display task, f9r a numhsr of reasons.
Przqr3!ns ars available for input a11d output of structural
information which can he modified to run on a ';T-40 (e.g.,  we have
ius? implem~nte3 on ar expcrim?ntal basis routines made available
to 'is by P. Feldman, NI!I);  Sophisticat:? structural display
programs hxve been written psppcially for a ST-40 which we would
i7one to nolnt; 3nd the ATM-STJMFX res3urcp will specifically
SlIDport 3n? GT-40 for use hy the SIJMEX staff.   This terminal will
he physically located it3  the PC: 1abDratory since all of the llscrs
aill  ir.*-erSct with that laboratory.

e~nrr??YvT !?F!TNT?NANCE:
YaintPn3nc3 is hudqeted for the proposed st.and-alone PDP-11/20
system under DPC cor.fract base;! 31: current prices.   Also included
I; ?I hu;!qet for maintenacce of the FAT-711 system.   This estimate
is h3se;! or! our experience with parts replacements to date. We
will provi?e the necessary maintenance manpower (see personnel
justLffcatY3n) because Varian cannot provide adequate service.

S:JP?LIPS:

TrIpplies are budqeted in various categories haspd on our operating
3xp9rienz-3 to date.  Plectronizs supplies inclu?e parts necessary
f3r m3int~3lninq our electronic; and test eqoipment (Sl,OOO) as
erelZ 3s parts in the fFrst year f3r the metastahle ion data system
1"3,200).  These comprise s~versl D/A 3~3 A/3 converters for
3ccnlzraYinq voltaye, ESA vnltaye, and maqnetic field control as
wnll 3s pacts fo upqrade thp A;111 probe mass marker.  GC supplies
include carrier qases, columns,  phsses, syringes, septa, etc., for
YT/MS npmation.  The liquid r.iCroqen is re:luircd for cold trap
Dner3tion o? the MAT-711.  Chezicals, qlassware, ?tc., include the
various ~rqaric chemicals, glassware, apparatus, glass tubing,
?fC.,  need?? to support t.he SC/ES laboratory operation.   Data
rnrJcr3inq media include special uv sensitive recorling paper for
t!le  BA"r-711, paper for GC an3 instrumentation recorder, and
:~L::ocs~I paD?r apd pens for inn currennt and spectrum plottinq.
"l'iri -computer supplies include paper, magnetic tape, ribbons,
3Dar3 disk zartridqes, Eftc.# fnr ;lata system operation,

?he travel Sudqet covers estim3tod nce?s (2 east coast and 2 west
zoast) tri.ps for attendinq rel3tP:! professiocal meetings and
i~tPrf3ni?q potential prorJram lls:\cs naFionally.   Pomcstic travel
is buclqct.cd for two East Coast trips and two California trips per
yc3r aa9nq a11 personnel.  YO foreign travel is budgeted.

d


!!t;P "nt h??r" hudqet Lncfudes nperating telephone,  of ficc supplies,
p'DS+3q?,  r?productioc, etc.,  support r,ecosssry for this project
base1 on our previous experienz?, The f*computer wageYf allocation
r,r3vidcs a cor.tinued limitcrl usage 3f tf:e 370/158 computer during
the 3uqmentation of the PPP-17/20 system.   This cost does not
anne3r in later years.  Terminal. rental covers four terminals to
be Iistrihated 3mony the F.S 1,.3boratory, the Zompu+er Science
DOD+., and J.  Lederhcrq's Ishor3tory.

?


BIOGRAPHICAL SKETCHES


SECTION II -PRIVILEGED COMMUNICATION           Principal Investigator: Carl Dierassi
                        BIOGRAPHICAL SKETCH
      (Give the following information for all professional personnel listed on page 3, beginning with the Principal Investigator.
                  Use continuation pages apd follow the same Genera; format for each person.1
NAME                                     TITLE                        BIRTHDATE (MO., Day, Yr.1

Carl DJERASSI              Professor of Chemistry       October 29, 1923

PLACE OF BIRTH k3ry, State, Country)           PRESENT NATIONALITY l/f nonUS. citizen,     SEX
                              indicate kind of visa and expiration date)
Vienna, Austria                  U.S.A.                  a Male   0 Female
                  EDUCATION (Regin with baccalaureate training and include postdoctoral)

INSTITUTION AND LOCATION

I    DEGREE  I     YEAR
              CONFERRED I

SCIENTIFIC
FIELD

Kenyon Co1 lege

University of Wisconsin

Chemistry, Biology

"ONoAS Hon. D.Sc., Natl. Univ. of Mexico (1953), Kenyon College (1958), Worcester Polytechnic
Institute (,l972); Hon. Prof., Fed. Univ. Rio de Janeiro (1969). Member U.S. National Academy of
Sciences, American Academy of Arts and Sciences, foreign member, Royal Swedish Academy of Sciences,
German Academy of Natural Scientists (leopoldina), Brazilian Academy of Sciences, (cant, below)
                                                                           --
MAJOR RESEARCH                                ROLE IN PROPOSED PROJECT

                          Principal Investigator

                                        Current   Tota I      % Time
Grant          Title               Period      Year    Budge ted    Effort
                                                                --                 --
NIH AM 04257    Mass Spectrometry in    1 o/1/70 to   $52,3C~6 $316,016     10%
          Organic and Biochemistry  9/30/75
NIH GM AM     Marine Chemistry with
    06840-I 5             l/1/73 to   $75,650   578,180     18%
         special emphasis on steroids 12/31/77
NSF Pending Grant: Application KP3P3689, Magnetic Circular Dichroism in Organic Molecules,
in the amount of $27,640.

RESEARCH AND/OR PROFESSIONAL EXPERIENCE (Starting with present position, &st tra!c/,ng andexpetience relevant to area ofprojec:  List all
or most representative publications,  Do not ex teed 3 pages for each individual. )
Academic Experience:
Protessor ot Chemistry, Stanford University, 1959-present.
Associate Professor ( 1952-l 954) and Professor (1954-1959), Wayne State University.
Industrial Research Experience:
c-b Pharmaceutical Co., Summit, N.J.:
S;nyex Corporation:            Research Chemist, 1942-1943 and 1945-1949.
         Associate Director of Chemical Research (Mexico City) 1949-1952,
Research Vice President (Mexico City) 1957-1960; (Palo Alto, California) 1960-1968,
President, Syntex Research 1968-&en&, Z oecon Corporation (Palo Aito), President, i968--+

Editorial Boards:       rw$z                                                             . . - --

(Current) Journal of the American Chemical Society, Steroids, Tetrahedron, Organic Moss Spectrometry.
                     (continued on next page)

Honors (cont.)
Mexican Academy for Scientific !nvestigation. Hon. Fellow of Phi Lambda Upsilon. Amer. Academy of
Pharmaceuticul Sciences, British Chemical Society and Mexican Chemicai Society, Phi Beta Kappa.
Numerous hon. lectureships including lY64 Centenary Lecturer {The British Chemical Society) and 1969
Annual Chemistry Lecturer, Royal Swedish Academy of Engineering. American Chemical Society Award
in Pure Chemistry (1958), Baekeland Medal (1959): Fritz&e Award (1960): Intra-Science Research
Foundation Award (1969);  F reedman Patent Award of Atne rlcan Institute of Chemists (1971).
Foreign Member, ,Royal Swedish Academy of Sciences (1972). D.Sc. (hon.), Worcester Poiytechnic
Institute (1972). Scheele-Lecturer, Pharmaceutical Society of Sweden (1972); American Chemical
                                                                  5 clence !LgTJ).     --.

Rev. 3-70
nne-398 Society's Award tor Creative~lnventioni~anal Medal a*

I. . .
.- .                                                          .(


BIOGRAPHICAL SKETCH (C. Dierassi)

Continuation page  Principal Investiqator:CarI Dierassi

:ESEARCH AND/OR PROFESSIONAL EXPERIENCE (cont.)

Aisce I laneous:

`hairman of the AAAS Gordon Research Conferences on Steroids and Natural Products (1952-1954);
4
Aember of American Pugwash Committee (1968 to present); Chairman of Latin America Science Board
If National Academy of Sciences (1966-1968); Ch
Ind Technology for International Development.  airman of National Academy's Board on Science

`UBLICATIONS

Author or co-author of six books and approximately 800 publications dealing with
natural products (notably steriods, terpenoids, alkaloids and antibiotics), medicinal
chemistry (primarily antihistamines, oral contraceptives and anti-inflammatory agents)
and applications of physical methods (mass spectrometry, optical rotatory dispersion,
magnetic circular dichroism) to organic and biochemical problems.

PIG-398
Rev. 2-69

Page
6

,


                                                            I
                       .                                     ; .a - ,_. .." _l* w.,p ,ea 2, .- -   ., -v .-

                                                                     -. : -I "I.' 1, :.,
hAh:e,       ! TITLE                          alPThDr\TE ibfo, 15ry. fr.1
                        Professor and Executive Head,
     LEDERBERG, JOSHUA          Departmznt of Genetics          5-23-25             -
PLACE Of 6lRTi-i m:y* Sr3rc: Comvyl             PRESENT NATISNALITY /If mn.U.S or/r~n,   iSEX
                               hdicom kind of visa and erpirarron dare)
   i
     Monte air,  ?:cw Jersey         U.S.A.      I   Q Male   5 CCr".l?
                   ECUCATtC:: :SL+n wrh bxmlaurwre tr31ntn3 Jnd mcludepo~:docrofail

INSTITUTION AN0 LOCATION               DEGREE         YEAQ   i         SC;E ?;TlilC
                             CONFERRED f          FIELD

Columbia College, Sew York              B.A.      1944 .
College of Phys icians & Surgeons,                               .                             .'
                                                                 .
  Columbia University, New York (1944-46
Yale University                        Ph.D.       1547
HONORS
1957 - National Academy of Sciences                                                                  1

1958 - Nobel Prize in Medicine

MAJOR RESEARCH INTEREST                  IROLE IN PROPOSED PROJECX

Molecular Genetics; Artificial Intelligent    PRINCIPAL ISVESTTGATOR

REt&ARCH SUPPORT /.%e insrrucrionrl

SEE ATTACHMENTS:

RESEARCH ANOX)R PRCFEZSl3NAL EXPEF?IENCE (S~niry wth prarentporrtion, i 1 fl rraini,q dnd expencnce relevdnr 10 o re.4 of prc,esC  Lix J/i
ormorc~prsen~ovepubl~w:ionr  Do nor exceed 3 p;cs for min mdwd~al.1
1961-     Stsnforc University
         Director, Kennedy,.Laboratories .for ,Yolec.@ar Mediciqe
"i'is~s'-'.'~'.'.'."Professor, Genetics 'and Biology; and Executive Head,            '.,"... :..;...-'. .-;7 `-'.-': ._..
                         DeiLrtment of

       Genetic:;, Stanford University-                    - _

1957-1959   University of Wisconsin
        Chairman, Department of Medical Genetics
1957    Melbourne University, Australia
        Fullbright Visiting Professor of Bacteriology
1950      University of California, Berkeley                                           .

        Visiting Professor of Bacteriology
1947-1959   University of Wisconsin
*         Professor of Genetics
1.946-1947   Yale University.  Research Fellow of the Jane Coffin.Childs Fund f.or:       . . .
           Medical Research
1945-1946   Columbia University.  Research Assistant in Zoology

Professional Activities:                              ,

  19670      NIMH:  National Mental' Health Advisory Council
  1961-1962  President (Kennedy)'s Panel on Mental Retardation
  1960-      NASA Committees:
.                      Lunar and Planetary Missions Board
1958--   National Academy of Sciences:  Committees on Space Biology
  1950-.                                                                           *
        President's Science Advisory Committee panels; National Institutes
         of Health, National Science Foundation study sections (genetics)
k HS338                                                                        .
Rev. &70                               f .
                                     
                              t      .
                                        .;      
                                     :      i                13
                                               L


1217173

RESEARCH SUPPORT SUMMARY FOR JOSHUA LEDERBERG

Grant Number

1) NASA:NGR-05-020-004

      Grant Title

Cytochemical Studies of Planetary
Micro-organisms

2) NIH:AI-05160         Genetics of Bacteria

3) NIH:GM

Genetics Research Center

4) NIH:RR-00785

Stanford University Medical
Experimental Computer Facility
(SUMEX) Successor to i/3

                    .
;~a-f-i,ii~~

9  NIH:GM00295

Training Grant in Genetics

Current Year

$150,000

60,000

547,035

571,567

121,172

Total Award     Grant Term    Budgeted
                        % Time

$3,950,000

g/60-8/74       4%
(Future support
dubious)

280,000

9168-8173
(Renewal
Pending)

15%

2,609,383

9173-8178
(Pending)

10%

2,769,262     10/73-7178      20%

321,163    7/l/73-6/30/77 15%


SELECTED LIST OF PUBLICATIONS

Lederberg, J., 1959
    A View of Genetics
   Les Prix Nobel en 1958:  170-89.

Buchs, A., A. B. Delfino, A. M. Duffield, C. Djerassi, B. G. Buchanan,
   E. A. Feigenbaum, and J. Lederberg, 1970.
   Applications of Artificial Intelligence for Chemical Inference.
    VI.  Approach to a general method of interpreting low resolution
   mass spectra with a computer.  Helvitia Chimica Acta 53 (6): 1394-1417.

Feigenbaum, E. A., B. G. Buchanan, J. Lederberg, 1971
   On generality and problem solving: a case study using the DENDRAL
  program in Machine Intelligence 6, (B. Meltzer and D. Michie, eds.),
   Edinburgh University Press, P. 165-190.

Reynolds, W. E., V. A. Bacon, J. C. Bridges, T. C. Coburn, B. Halpern,
   J. Lederberg, E. C. Levinthal, E. Steed, R. B. Tucker, 1970
  . A Computer Operated Mass Spectrometer System.
   Analytical Chem. 42:1122-1129, September 1970.

Lederberg, J.

"Use of Computer to Identify Unknown Compounds: The Automation of
Scientific Inference" in Biochemical Applications of Mass Spectrometry
                                                        --
(G. R. Waller, ed.).  John Wiley & Sons, New York (in press).


SECTION II - PRlVlLEGED COMhWNICATlON
                        BIOGRAPHICAL SKETCH

(Give the following information for all professional personnel listed on page 3, beginning with the Principal Invesrigator.
          Use continuation pages and follow the same ocneral format for each ocrson.1
                                            .

NAME

Feigenbaum, Edward A.
PLACE OF BIRTH (Ciry, Stare, Country)

Weehawken, New Jersey

PRESENT NATIONALITY /If non-US citizen,     SEX
indicate kind of visa and expiration date)
U.S. Citizen               YJ! Male   c Female
                 . . .          .I

EDUCATION (Begin wuh bsccalaurcare rralnrng dnu mciuacposrboctorau

INSTITUTION AND LOCATION               DEGREE         YEAR             SCIENTIFIC
                                            CONFERRED             FIELD

TITLE
Principal Investigator,
DENDRAL Project

BIRTHDATE (MO.. Day, Yr./

l-20-36

Carnegie Institute of Technology
Pittsburgh, Pennsylvania

B.S.     19% - Zlectrical Zcgiceerlrig
Ph.D.      1959     Behavioral Sciences.

.HoNoss and memberships:
    American Psychological Association; Association for Corr;puting Machinery ("ember
   of the National Council 1966-68); American Association for the Advencezent of

    Science, SIGBIO Chairman, 11/73-present.
MAJOR RESEARCH INTEREST                      ROLE IN PROPOSED PROJECT

Artificial Intelligence             Principal Investigator
RESEARCH SUPPORT (See instructions)

-

RESEARCH AND/OR PROFESSIONAL EXPERIENCE (Srarring withpresenrposition,&t rraininpandexperiencerelevanr toarea ofprojer; Lista.`:
or most representau've publicationr  Do not exceed 3 pages for each individual.)

u65-
1965-1968
1963

1962

,lg6o-lg64

1957-196o
19%

Stanford University, Computer Science Departnent Faculty
Stanford University, Director, Computation Center
Summer Research Training Institute in Computer Simulation of Cognitive
Processes (National Science Foundation)
Carnegi,e Corporation.  Smer Research Training Institute in Heuristic
Programming. Faculty member.
University of California, Berkeley
Research-Center for Research in Management Science, 196O-1964
Research-Center for Human Learning, 1961-1964
Assistant and Associate Professor, School of Business Administration, 19%6~
The RAND Corporation, Santa Monica, California
IBM Scientific Computing Center, New York

,    Selected Publications:
      "Applications of Artificial Intelligence for Chemical Inference I. The :'::zber
       of Possible Organic Compounds.  Acyclic Structures Containing C, H, 0 and X1(,
      J. Am. Chem. Sot., 91, 2973 (1969).  (Co-Author).

"Applications of Artificial Intelligence for Chenical Inference II. Icterprezation
of Low Resolution Mass Spectra of Ketones", J. Am. Chem. Sot., 91, 2977 (1969).
(Co-Author).                                                    .
                              .

  R HS-398
  Rev. 3-70                                                                           -

.,                                
                                     1                     .I

",.


Publica.tions of Edward Feigenbautn

"Applications of Artificial Intelligence for Chemical Inference III.
Aliphatic Ethers Diagnosed by their Low Resolution Mass Spectra e.n,d Nuclear
Magnetic Resonance", J. Am. Chem. Sot., 91, 7440 (1969).  (Co-Author).

"Heuristic DENDRAL:  A Program for Generating Explanatory Hnotheses in    -
Organic Chemistry", in Machine Intelligence 4, Edinburgh University Iress,
1969. (Co-Author). '

"Toward an Understanding of Information Processes of Scientific Inference
in the Context of Organic Chemistry", in Machine Intelligence 5, Jdinturgh
University Press, 1970. (Co-Author).

"A Heuristic Program for Solving a Scientific Inference Problem:  SUrr=-,2TJ
of Motivation and Implementation", Stanford Artificial Intelligence Proiect
Memo No. 104, November 1969.  (Co-Author).

"Applications of Artificial Intelligence For Chemical Inference IV.   Sztlxated
Amines Diagnosed by Their Low Resolution Mass Spectra and Zuclear Magnetic
Resonance Spectra", Journal of the -erican Chemical Society, 92, 6831 (1970).
(Co-Author).

"Applications of Artificial Intelligence for Chemical Inference V. An
Approach to the Computer Generation of Cyclic Structures.  DifferentLetlon
Between All the Possible Isomeric Ketones of Composition C6HiOO", Organic
Mass Spectrometry, 4, 493 (1970). (Co-Author).

"Applications of Artificial Intelligence for Chemical Inference VI.  Approach
to a General Method of Interpreting Low Resolution Mass S_oectra with a
Computer", Chem. Acta Helvetica, 53, 1394 (1970).  (Co-Author).

"On Generality and Problem Solving:  A Case Study Using the DEND?&L Program,
in Machine Intelligence 6, Edinburgh University Press (1971). (Co-Auttcr).

"A Heuristic Programming Study of Theory Formation in Science", i.n proceedings
of the Second International Joint Conference on Artificial Intelligence,
Imperial College, London (September 1971). (Co-Author).

"Applications of Artificial Intelligence for Chemical Inference VIII. An
Approach to the Computer InterpreTation of the High Resolution Mass Spectra
of Complex Molecules.  Structure Elucidation of Estrogenic Steroids",
Journal of the American Chemical Society, 94, 5962-5971 (1972). (Co-Author).

"Heuristic Theory Formation:  Data Interpretation and Rule Formation", in
Machine Intelligence 7, Edinburgh University Press (1972).  (Co-AlL;tkor).

"Applications of Artificial Intelligence for Chemical Inference X. Intsum
A Data Interpretation Program 2s Applied to the Collected Mass Spectra of
Estrogeiic Steroids", Tetrahedron, 29, 3117 (1973). (co-author).


SECTION II -PRIVILEGED COM~IMUN    I-ION
                         BIOGRAPHICAL SKETCH
     (Give the following information for ail profcssiond personnel listed on page 3, &ginning with the Principal Jnvestigstor.
                  Use continuation paga Jnd follow the same gcnerJl format for each person.)
NAME                                 TITLE                        ElRTtIDATE (20.. Ddy, Yr./

Buchanan, Bruce G.              Research Computer Scientist     7-7-40

PLACE OF BIRTH (City, Sure, Country)           PRESENT NATIONALITY 1/f non4.S citizen,     SEX
                               indicate kind of visa and expiration date)
St. Louis, Missouri                U.S.Citizen               'a Male   @ Female
                   EDUCATION (B&n with baccalaureate training dnd include postdoctorall

INSTITUTION AN0 LOCATION

Ohio Wesleyan University
Michigan State University

. HONORS

Recipient of National Institutes of Health Career Development Award (lg7i-1976)

DEGREE  I     YEAR  I        SCIENTIFIC
        , CONFERRED            FIELO

B.A.      1961     42thematics
M.A., Ph.D.  1966 i hiiosophjr

.

  MAJOR RESEARCH INTEREST
Artificial Intelligence

  RESEARCH SUPPORT (Seeinstructions)

ROLE IN PROPOSED PROJECT

Associate Investigator

NIH Research Career Development Award, GM-z.9662

RESEARCH AND/OR PROFESSIONAL EXPERIENCE [Starting withpresentposition, list traininqandexperiencerelevafit fo area ofpisjecr LAa!!
or mOSt representative publications.  Do nor exceed 3 pages for eJCh individual.)
1972-present Research Computer Scientist, Stanford University
1966-1971    Research Associate, Stanford Artificial.Intelligence ProJect

Publications:

"On the Design of Inductive Systems:  Some Philosophical Problems'.  British Journal
for the Philosophy of Science 20 (1969), 311-323. (Co-Author).

"Applications of Artificial Intelligence for Chemical Inference II.  Interpretation
of Low Resolution Mass Spectra of Ketones".  Journal of the American Chemical Ssciety,
91, 2977-2981 (1969). (CoyAuthor).

"Applications of Artificial Intelligence for Chemical Inference I. The Number cf
Possible Organic Compounds:  Acyclic Structures Containing C, H, 0 and N".  JourcE.1
of the American Chemical Society, 91, 2973-2976 (1969). (Co-Author).

"Applications of Artificial Intelligence for Chemical Inference III. Aliphatic
Ethers Diagnosed by Their Low Resolution X2ss Spectra and hm Data".   ,Tou~al of the
American Chemical Society, 91, 7440-45 (1969). (Co-Author).

"Heuristic DENDRAL:  A Program for Generating Explanatory Hypotheses in Organic
Chemistry".  Machine Intelligence.4, Edinburgh University Press'(1969). (Co-Author).

RHS-398
Rev. 3-70


Publications of Bruce Buchanan:

'Toward an Understanding of Information Processes of Scientific Inference in the
Context of Organic Chemistry".  Machine Intelligence 5, Edinburgh University
Press (1969). (Co-Author).

'On Generality and Problem Solving:  A Case Study Using the DENDRAL Program".
Machine Intelligence 6, Edinburgh University Press (1969). (Co-Author).

"Some Speculation About Artificial Intelligence and Legal Reasoning". Stmford
Law Review, Vol. 23, No. 1, November 1970.  (Co-Author).              I

'Applications of Artificial Intelligence for Chemical Inference VI. Approach to
a General Method of Interpreting Low Resolution Mass Spectra with a Computer".
Chemica Acta Helvetica, 53, 1394 (1970).  (Co-Author).

"An Application of Artificial Intelligence to the Interpretation of Mass Spectra".
Mass Spectrometry Techniques and Appliances (1970).

"Applications of Artificial Intelligence for Chemical Inference IV. Saturated
Amines Diagnosed by Their Low Resolution bzss Spectra and Nuclear Magnetic Rescnance
Spectra".  Journal of the American Chemical Society, 93, 6831 (1970). (Co-Xcthor).
'The Heuristic DENDRAL Program for Explaining Empirical Data". Proceedings of
IFIP Congress 1971, Ljubljana, Yugoslavia.  (Co-Author).

"A Heuristic Programming Study of Theory Formation in Science".  Proceedings of
Second International Joint Conference on Artificial Intelligence, Imperial College,
London (1971). (Co-Author).

"Applications of Artificial Intelligence for Chemical Inference VIII. An Xpproich
to the Computer Interpretation of the High Resolution Mass Spectra of Complex
Molecules.  Structure Elucidation of Estrogenic Steroids". Journal of the Americsn
Chemical Society, 1972. (Co-Author).

"Heuristic Theory Formation:  Data Interpretation and Rule Formation". Machine
Intelligence 7, Edinburgh University Press (1972). (Co-Author).

"Review of Hubert Dreyf'us' 'What Computers Can't Do: A Critique of Artificial
Reason"', Computing Reviews (January, 1973).

'Applications of Artificial Intelligence for Chemical Inference IX. Analysis of
Mixtures Without Prior Separation as Illustrated for Estrogens". Submitted to i!ie
Journal of the American Chemical Society.  (Co-Author).

'Applications of Artificial Intelligence for Chemical Inference X. Intsum    A 3ata
Interpretation Program as Applied to the Collected Mass Spectra of Estrogenic
Steroids".  Tetrahedron, 29, 3117 (1973). (co-author)

"Rule Formation on Non-Homogeneous Classes of Objects". In proceedings of the Third
International Joint Conference on Artificial Intelligence (Stanford. 1973). (co-author).

"Current Status of the Heuristic DENDRAL Program for Applying Artificial Intelligence to
the Interpretation of Mass SpectFa".  DEhQRAL Project Memo, Augu,st 1973
                                     .


Biographical Sketch of Bruce G. Buchanan

Memberships:

Association for Computing Machinery (ACM)
Philosophy of Science Association
American Association for Advancement of Science (AAAS)


PL,ACE OF BIRTH (City, State, Counvyl         PRESENT NAT13NALITY (lfnon-U.S citizen,     --'
                          indicate kind of viu and expiratton datcJ
Perth, Western Australia       .    Australian, Permanent resloenb
        ~--,:rr,2=f. ~7; -, - A i=' @ dale
                            r; FETE;<
                EDVC;ITIOX lZc;;n :*.+rn D.JCCJ/.IU~C'J~~ rr~rn,r~ Jld I~CIUL'C z~::Js::3rJ/)

INSTITUTION AND LOCATION

I    DEGREE  I   YEA3 '
              CONFERRED I

SCIENTIFIC        7

FIELD

1

University of Western Australia

University of Western Australia

B. Sc(lst Class      I.
       1958 '                             *.
    Hons)        Organic Chemistry .
Ph.D.       1962    Organic Cheasitry

i  MAJOR RESEARCH INTEREST                     AOLr IN PROPOSED PROJECT
  Applications of mas.s spectrometry to   Organic Chemist/mass spectroscop?st
   Biology and Riomedical Problems
RESEARCH SUPPORT (See lnstructionsl
       N/A

`
:  RESEA~~CH r\r;Dfoa ~)RO;ESJ;G~~\L EX?~~~:E:JCE (s:~rr:r.~ wirn prnent position, list fra/r.inqand experience reic'trnt :O dre3 of pr;,c'ri  LG: lil
i  of most rrpmsmtative puoirut~onr  00 NON exceed 3 cages for eszh individual.1                                              .

. :.  ..,.... ".._.. ..r: :. . . ..a.. :.;... . . , - . : ,_.C._ . ;: _' * ._.__ &  . . ._.,,) ., , ..`.`, ..,.,.,. a. . . ._.. :. ,.. .   ,        : . . -.-I ,'
      1970 -.  Research Associate,' Department of Genetics,-Stanford iiniversity
                 School of Medicine
        . . 1969 -    %ad of the Xass Spectrometry Laboratory, Chemistry Departmer,:
             Stanford irniversity
      1965 - 69  ?.cscarch Associate, Department of Chemistry, Stanford ScivarsiLy
      1963 - 65   Pos+~octoral r"cllow, Department ofAChemLstry, S'canfo~d University
      1962 - 63   Postdoctoral Fellow, Department of Biochemistry, Slianlord University

.

.     I- -

                          -
School of Medicine.

`.

.__^ _ -I- . __-
._ .
. .

1 .     .1,;
     

* --

1. -.   7*.. .:                    :_:          -_-

         .*.

        ; ; :                                                    .
         , .                               .
    ,.. -I

          ;.

                     *      J .

--s-- -.- -     .      2. * ! L
                     s_- ;                         t '           m -

     :,                            -
       . PUBLICATIONS SINCE 197i

An Application OF Artificial lnte IliS?nce to the Interpretation oi Mass Spectra.
. Mass Spectrometry,  , B.W. G. Milne, Ed., John Wiley and Sons,

New York, 1971, p;, , 121-178
By B . G 3:Jchancnt A. M. Duffield and A. V. Robertson


2.  Mass Spectrometry in Structural and Stereochemical Problems. CCIV. Spectra
  of Hydantoins.11.  Electron Impact Induced Fragmentation of some Substituted
  Hydantoins.

Org. Mass Spectr., 2, 551 (1971)
By R. A. Corral, 0. O.'Orazi, A. M. Duffield and C. Djerassi

3. Electron Impact Induced Hydrogen Scrambling in Cyclohexanol and Isomeric
Methylcyclohexanols.                                                           .-
    Org. Mass Spectr., 5, 383 (1971)                                       _
    By R. H. Shapiro, S. P. Levine and A. M. Duffield

4.. Derivatives of 2-Biphenylcarboxylic Acid.
    Rev. Roumain. Chem., 16, 1095 (1971)
   By A. T. Balaban and CM. Duffield

5. Alkaloide aus Evonymus europaea L.
    Helv. Chim. Acta, 54, 2144 (1971)
    By A. Kldsek, T. Rzchstein, A. M. Duffield and F. Santa*

6. Studies on Indian Medicinal Plarts. XXVIII. Sesquiterpene Lactones of
Enhyira Fluctuans Lour. Structure: of Enhydrin, Fluctuanin and Fluctuadin.
   Tetrahedron, ;28, 3235 (1973).

By E. Ali, P. P. Ghosh Dastidar, S. C. Pakrashi, L. J. Durham
and A. M. Duffield

7. The Electron Impact Promoted Fragmentation of Aurone Epoxides.
     Org. Mass Spectr.  6, 199 C1972j
    By B. A. Brady, WI y. O'Sullivan.and A. M. Duffield

8. The Determination of Cyclohexylanine in Aqueous Solutions of Sodium Cyclamate
by Electron Capture Gas Chromatography.
   Anal. Letters, k, 3Cl (1971)
    -By !!. D. Soloman, W. E. Pereira and A. M. Duffield

9. Computer Recognition of !jetastable  Ions. Nineteenth Annual Conference on
Mass Spectrometry, Atlanta, 1971, p. 63
- : By A. M. Duffield, W. E. Reynolds,.D. A. Anderson, R. A. Stillman, Jr-.
     . and C. E. Carroll

10% Spectrometrie de Masse. VI.  Fragmentation de Dimethyl-2,2-dioxolanes-I,:-
   Insatures.

    Org. Mass Spectr., 5, 1409 (1971)
     By J. Kossanyi, J. Chuche and A. M. Duffield
11% Chlorpromazine Metabolism in Sheep. II. In vitro Metabclism and Preparation
                                                   --.
  of 3H-7-Hydroxychlorpromazine.
     Journees D'Agressologie, 12 , 333 09711
    By L. G. Brooks, M. A. Hol?%s, I. S. Forrest, V. A. Bacon,
      A. M. Duffield and M. D. Solomon

.

12., Mass Spectrometry in Structural and Stereochemical Problems. CCXVII.
. Electron Impact Promoted Fragmentation of 0-Wethyl Oximes of Some
  a&Unsaturated Ketones and Methyl Substituted Cyclohexanones.
    Canadian J. Chem., 50, 2776 (1972)
     By Y. M. Sheikh, R.?. Liedtke, A. Mi Duffield and C. Djerassi

.


r\. >:. ;:-;fit:d
Publications

13.  Thermal Fragmentation of Quinoline and Isoquinoline N-Oxides in the Ion
  Source of a Mass Spectrometer.

Acta Chem. Stand., 26, 2423 (1972).
By A. M. Duffield and 0. Buchardt

.-

14.  -Applications of Artificial Intelligence for Chemical Inference. VII. An
  Approach to the Computer Interpretation of the High Resolution Mass Spectra
  of Complex Molecules.  Structure Elucidation of Estrogenic Steroids.
     J. Amer. Chem. Sot., 94, 5962 (1972)
     By D. H. Smith, B. G. Buchanan, R. S. Englemore, A. M. Duffield,
     A. Yeo, E. A. Feigenbaum, J. Lederberg and C. Djerassi
      ,

15.  Mass Spectrometry in Structural and Stereochemical Problems. CCXIX.
  Identification of a Unidirectional Quadruple Hydrogen Transfer Process
  in 7-Phenyl-hept-3-en-2-one O-Methyl Oxime Ether.
     Org. Mass Spectr., &1?71 (1972).
     By R. J. Liedtke, Y. M. Sheikh, A. M. Duffield and C. Djerassi

16.  &I Automated Gas Chromatographic Analysis of Phenylalanine in Serum.
    Clinical Biochem.,Il 166 (1972) ,
     By E. Steed, TJ. Perelra, B. Halpern, M. D. Solcnon and
       A. M. Duffield

17.  Pyrrolizidine Alkaloids. XIX.  Structure of the Alkaloid Erucifoline.
     .Coll. Czech. Chen. CTmmun.,      (1972)
   By P. Sedmera, A. Klasek, A. M. Duffield and F, Santav;.

18.  Mass Spectrometry in Structural and Stereoch.enical Problems, CCXXII,
  Delineation of Ccnpeting Frapentation Pathxalys of Con?lex !a!olezL.zs
  from a Study of Zetastzble Ion Transitions of Deuterated ZerivatlyJes.
     Org. Mass Spectr.,  1,  (1973) ,
     -By D. H. Smith, A. M. Duffield and C. Djerassi

19.  Chlorination Studies I.  The Reaction of Aqueous-Hypochlorous Acid irith

Cytosine.

Biochem. Biophys. Res. Commun., 48, 880 (1972)
By W. Patton, V. Bacon, A. ?l. Duseid, B. Halpern, Y. Hoyano, :?.
Pereira and J. Lederberg

20.  A Study of the Electron ImDact Fragmentation of Promazine Sulphoxide
  and Promazine using Speci.ficallv Deuterated Analogues.      -
     Austral. J. Chem., 26.,   (1973).
     By M. D. Solomon, R. Summons, 67. Pereira and A. M. Duffield

21.  Spectrometric de Masse. VIII.  Elimination d'eau Induite par Impact
     Electronique dans le Tetrhydro-1,2,3,4-naphtalenediol-1,2.
      Org. Mass. Spectrom., 7   (1973).
     By P. Perros, J. P. Morizui, J. Kossanyi and A. M. Duffield

22.  The Determination of Phenylalanine in Serum by Mass Pragmentography
      Clinical Biochem., submitted for publication (1973).
    By W. E. Pereira, V. A. Bacon, Y. Hoyano, R. Summons and A. M. Duffield


SECTION II -PRIVILEGED COMMUNICATION
                       BIOGRAPHICAL SKETCH

(Give the following information for all professional personnel listed on page 3, beginning with the Principal Investigator.
        Use continuation pages and follow the same general format for each person.1

NAME                    1 TITLE

BIRTHDATE (MO., Day, Yr.l

Research Associate  I  11/12/42

Dennis H. Smith

PLACE OF BIRTH iCity, Stare, Country)

I                                     I
PRESENT NATIONALITY f/f non-U.S citizen,     SEX
indicate kind of vise and expiration date)

New York

              USA
EDUCATION (Begin with baccalaureate training and include postdoctorall

b Male .`d Female

          INSTITUTION AND LOCATION

Massachusetts Inst. of Technology
Cambridge, Mass.

DEGREE

S.B.

  YEAR
CONFERRED

1964

    SCIENTIFIC
       FIELD

Chemistry

University of California, Berkeley
Berkeley, California

Ph.D. I   1967  I  Chemistry

                                                I              I
.!?!?lJ P. Sloan Foundation Scholarship
NASA Predoctoral Traineeship
Phi Lambda Upsilon, Sigma Xi                                                                ----
MAJOR RESEARCH INTEREST                     ROLE IN PROPOSED PROJECT
Mass Spect'ometry and A.I. in Chemistry         Research Associate

RESEARCH SUPPORT (See instructions)

I                                              --

N/A

RESEARCH AND/OR PROFESSIONAL EXPERIENCE (Starting withpresentposition, list traininq and experience reievant to area of projccr.  List ail
or most representative publications.  00 not exceed 3 pages for each individual.)
1971-Present  Research Associate, Stanford University, Stanford,Ca.
1970-1971   Visiting Scientist, University of Bristol, Bristol, England
1967-1970    Assistant Research Chemist, University of Calif.at Berkeley, Berkeley, Ca.
1965-1967    NASA Pre-Doctoral Traineeship, University of Calif.at Berkeley,Berkeley, Ca.

Publications:  See attached list.

RHS-398
Rev. 3-70


PUBLICATIONS: D. H. SMITH

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

H. G. Langer, R. S. Gohlke and D. H. Smith, "Mass Spectrometric
Differential Thermal Analysis," Anal. Chem., 37, 433 (1965).

S. M. Kupchan, J. M. Cassady, J. E. Kelsey, H. K. Schnoes, D. H.
Smith and A. L. Burlingame, "Structural Elucidation and High Resolution
Mass Spectrometry of Gai I lardin, a New Cytotoxic Sesqui terpene Lactone, "
J. Amer. Chem. Sot., 88, 5292 (1966).
                        -

D. H. Smith, Ph.D. Thesis, "High Resolution Mass Spectrometry:
Techniques and Applications to Molecular Structure Problems," Dept. of
Chemistry, University of California, Berkeley, California (1967).

H. K. Schnoes, D. H. Smith, A. L. Burlingame, P. W. Jeffs and W.
DBpke, "Mass Spectra of Amaryl lidaceae Alkaloids:  The Lycorenine
Series, " Tetrahedron, 24, 2825 (1968).
                      -

A. L. Burlingame, D. H. Smith and R. W. Olsen, "High Resolution Mass
Spectrometry in Molecular Structure Studies, XIV. Real-time Data
Acquisition, Processing and Display of High Resolution Mass Spectral
Data," Anal. Chem., 40, 13 (1968).
                       -

A. L. Burlingame and D. H. Smith, "High Resolution Mass Spectrometry
in Molecular Structure Studies II.  Automated Heteroatomic Plotting cs an
Aid to the Presentation and Interpretation of High Resolution Mass
Spectral Data," Tetrahedron, 24, 5749 (1968).
                              -

W. J. Richter, B. R. Simoneit, D. H. Smith ond A. L. Burlingame,
"Detection and Identification of Oxocarboxylic and Dicarboxylic Acids in
Complex Mixtures by Reductive Silylation and Computer-Aided Analysis of
High Resolution Mass Spectral Data," Anal. Chem., 41, 1392 (1969).
                                      -- .w

The Lunar Sample Preliminary Examination Team, "Preliminary Examination
of Lunar Samples from Apollo 11," Science, 765, 1211 (1969).

S. M. Kupchan, W. K. Anderson, P. Bollinger, R. W. Doskotch, R. M.
Smith, J. A. Saenz Renauld, H. K. Schnoes, A. L. Burlingame and D. H.
Smith, "Tumor Inhibitors, XXXIX. Active Principles of Acnistus arborescens.
Isolation and Structural and Spectral Studies of Withaferin A and Withacnistin,"
J. Org. Chem., 34, 3858 (1969).

A. L. Burlingame, D. H. Smith, T. 0. Merren and R. W. Olsen, "Real-
time High Resolution Mass Spectrometry," in Computers in Analytical
Chemistry (vol. 4 in Progress in Analytical Chemistry series ) C. H. 0l.r
and J. Norris, Eds., Plenum Press, New York, 1970, pp. 17-38.


PUBLICATIONS: D. H. SMITH                     Page 2

11.

. 12.

13.

14. '

15.

16.

17.

18.

19.

20.

The Lunar Sample Preliminary Examination Team, "Preliminary Examination
of Lunar Samples from Apollo 12," Science, 167, 1325 (1970).

D. H. Smith, "Mass Spectrometry, " Chapter X in Guide to Modern
Methods of Instrumental Analysis, T. M. GOUW, Ed., Wiley-Interscience,
New York, 1972.

D. H. Smith, R. W. Olsen, F. C. Walls and A. L. Burlingame, "Real-time
Mass Spectrometry:  LOGOS--A Generalized Mass Spectrometry Computer
System for High and Low Resolution, GC/MS and Closed-Loop Applications,"
Anal. Chem., 43-, 1796 (1971).

A. L. Burlingame, J. S . Hauser, B. R. Simonei t, D. H. Smith, K .
Biemann, N. Mancuso, R. Murphy, D. A. Flory and M. A. Reynolds,
"Preliminary Organic Analysis of the Apollo 12 Cores," Proceedings of the
Apollo 12 Lunar Science Conference, E. Levinson, Ed., M.I.T. Press,
Cambridge, Mass., 1971, p. 1891.

D. H. Smith, "A Compound Classifier Based on Computer Analysis of Low
Resolution Mass Spectral Data,`! Anal. Chem., 44, 536 (1972).
                                                -

D. H. Smith and G. Eglinton, "Compound Classi fi cation by Computer
Treatment of Low Resolution Mass Spectra-Application to Geochemical and
Environmental Problems, " Nature, 235, 325 (1972).

D. H. Smith, N. A. B. Gray, C. T. Pillinger, B. J. Kimble and G.
Eglinton, "Complex Mixture Analysis - Geochemical and Environmental
Applications of a Compound Classifier Based on Computer Analysis of Low
Resolution Mass Spectra," Adv. in Org. Geochem., 1971, p. 249.

D. H. Smith, B. G. Buchanan, R. S. Engelmore, A, M. Duffield, A.
Yeo, E. A. Feigenbaum, J. Lederberg and C. Djerassi, "Applications of
Artificial Intelligence for Chemical Inference, VIII. An Approach to the
Computer Interpretation of the High Resolution Mass Spectra of Complex
Molecules.  Structure Elucidation of Estrogenic Steroids,"' J. Amer. Chem.
sot., 94, 5962 (1972).
--

D. H. Smith, A. M. Duffield and C. Djerassi, "Mass Spectrometry in
Structural and Stereochemical Problems, CCXXII. Delineation of
Competing Fragmentation Pathways of Complex Molecules from a Study of
Metastable Ion Transitions of Deuterated Derivatives," Org. Mass.
Spectrom., z, 367 (1973).

P. Longevial le, D. H. Smith, H. M. Fales, R. J. Highet and A. L.
Burlingame, "High Resolution Mass Spectrometry in Molecular Structure
Studies, V.  The Fragmentation of Amaryllis Alkaloids in the Crinine
Set-i-es, I' Org. Mass Spectrom., 7, 401 (1973).
                       --


PUBLICATIONS: D. H. SMITH

21.

22.

23.

24.

B. R. Simoneit, D. H. Smith, G. Eglinton and A. L. Burlingame.
"Applications of Real-time Mass Spectrometric Techniques to Environmental
Organic Geochemistry, I I.  San Francisco Bay Area Waters," Arch. Env.
Contam and Tox., 1, 193 (1973).

D. H. Smith, B. G. Buchanan, R. S. Engelmore, H. Adlercreutz and C.
Djerassi, "Applications of Artificial Intelligence for Chemical inference,
IX. Analysis of Mixtures Without Prior Separation as Illustrated for
Estrogens," J. Amer. Chem. Sot., 95, 6078 (1973).
                                    -

D. H. Smith, B. G. Buchanan, W. C. White, E. A. Feigenbaum, J.
Lederberg and C. Djerassi, "Applications of Artificial Intelligence for
Chemical Inference, X. INTSUM - A Data Interpretation and Summary
Program Applied to the Collected Mass Spectra of Estrogenic Steroids,"
Tetrahedron, 29, 3117 (1973).
             -

G. Loew, M. Chadwick and D. H. Smith, "Applications of Molecular
Orbital Theory to the Interpretation of Mass Spectra. Prediction of
Primary Fragmentation Sites' in Organic Molecules," Org. Mass Spectrom. f
z, 1241 (1973).

.

a7


SECTlON II -PRIVILEGED COMMUNICATION
                      BIOGRAPHICAL SKETCH

(Give the following information for ell professionel personnel listed on pege 3, bqinning with the Primipel Invartiptor.
       Use continuation pegar end follow the seme generel former for each psrronl

NAME                    ITITLE                  IBIRTHDATE ma, OW, rd

Sridharan, Natesa S.               Research Associate

10/2/46

PUCE OF BIRTH (City, Stete, Country)

PRESENT NATIONALITY (II nonUS citizen,    SEX
indicete kind of Vise and expiration data) India .

                                                                           ',
Madras, India              5/73-U.S. permanent residence     (g Male   0 Famals
              EDUCATION (&gin with baccelaurwte training end include postdoctorell
                                                                        SCIENTIFIC
          INSTITUTION AND LOCATION               DEGREE         YEAR
                                                    CONFERRED             FIELD
Indian Institute of Technology, Madras    Bachelor of
India                       Technology     1967    Electrical Engineering
State University of New York, Stony Brook M.S.          1969    Computer Science
                                Ph.D.          1971    Computer Science

HONORS
University Fellow - 1968-1971, SUNY Stony Brook; Graduate Assistant - 1967-1968, SUNY
Stony Brook; Siemens' Award (awarded for top rank in Electrical Engineering) - 1967, ITT
Madras; National Merit Scholarship - 1963-1967, ITT Madras                                   -
MAJOR RESEARCH INTEREST                    ROLE IN PROPOSED PROJECT
Computer Application in Chemistry

and MedicinL

RESEARCH SUPPORT (See instructions)

Research Associate

----

RESEARCH AND,QR PROFESSIONAL EXPERIENCE (Sterling with pfscent posidon,list end experience relevant to eree of p.ro@cl,  L!%t efi
or most representetivepublicetioru.  Do not exceed 3 peges for eech individual.)
1971-present Research Associate, Heuristic Programming Project, Stanford University

1970-1971    Consultant, IAC Computer Corp., Long Island, N.Y.

Sridharan, N.S., "An Application of Artificial Intelligence to Organic Chemical Synti-'esis"
Doctoral Thesis, State University of New York at StonyBrook, 1971.
Sridharan, N.S., "Search Strategies of Organic Chemical Synthesis", Third International
Joint Conference on Artificial Intelligence (3IJCAI), Stanford, 1973
Sridharan, N.S. (co-author), "Heuristic DENDRAL: Analysis of Molecular Structure", Proc.
NATO Advanced Study Institute, Amsterdam, 1973.
Sridharan, N.S. (co-author), "Heuristic Theory Formation", Machine Intelligence, Volume 7,
Edinburgh, 1972.


SECTION II - PRlVlLEGED COMMUNICATION
                        BIOGRAPHICAL SKETCH

NAME

(Give the following Inform&ion for sllprofessional pononnel listed on page 3, brginning w'th the Principal Invertlgator.
         Use continuetion pqm snd follow the snme genaruf format for eech person)
                           TITLE                        BIRTHDATE (Ma, Day, Yr.1

Brown, Harold D.                    Associate Professor        July 12,1934
PLACE OF BIRTH Kiry, Sate, Country)           PRESENT NATIONALITY f/f n0n.U.S citizen,     SEX
                                indicate kind of vis, and expiration date)
South Bend, Indiana                 u.s,               B Male Ofcrnale
                   EDUCATION f8eoin wirh bacce!auraate ttaining and include oostdoctorsll

lNSTlTUTlDN AND LOCATION

University of Notre Dame, Notre Dame,
Indiana

Ohio State University, Columbus, Ohio
       (No Baccalaureate Degree)

HONORS

Summa Cum Laude - Notre Dame

  YEAR            SCIENTIFIC
CONFERRED            FiELD
1963      Mathematics

1966      Mathamatics

MAJOR RESEARCH !NTEREST               %%k IN PROPOSEC) PROJECT
Applied Discrete Mathematics - Computer
   Science                              Research Associate
RESEARCH SUPPORT (See innrvctionrl

Principal Investigator, NSF-GP-16793 (Expires March, 1974)

Pending Proposal NSF (Proposed starting date September, 1974)

RESEARCH AND/OR PROFESSIONAL EXPERIENCE (Starting withprarent position,&J&(&gandexpetien&rel@vant tort-e* i,fproht L.tiz
or most representative publicetions.  Do not exceed 3 pages for each individual.)
Visiting Associate Professor, Computer Science, Stanford University , 1971-72, 1973-present
Associate Professor, Mathematics, Ohio State University, 1966-
Visitin.g Professor, Mathematics, Rhine,Westf. Tech. Hoch., Aachen, 1972 and 1973
Visiting Member, Courant Institute, New York University, 1967-65
Instructor/Assistant Professor, Assistant Chairman, Mathematics, Ohio State U.: 1963-65
Assistant to the Chairman, Mathematics, University of Notre Dame, 1960-63
Director or Associate Director, NSF-SSTP, 1964-70


Vitae   Pace 2

7Ciea.r A`l@'L)ras, Ill. J. Kath, 12(1968), Pg. 215.
Distrjbutor Theory in Rear AlZebras, Comm. Pure App. Eath.
  ??? ?o??O? ??? o ????

An Algorithm for the Determination of Space Groups, Ma& Comp.
  23(w59), Pg. 499.

Some Empirical Observations on Primitive Roots, with H. Zassenhaus,
  J, Number Theory 3(1971), Pg. 306.

A Generalization of Farey Sequences, with K. Eahler, J, Number
  Theory 3(1971), pg.364,

Basic Computations for tiders, Stanford CS Kemo STAN-CS-72-208.
An Application of Zassenhaus' Unit Theorem, Acta Arith. xX(1972),
  Pg, 154.

Integral Groups Tx The Reducible case, with J. Xeub:iser and
    II, Zassenhaus, Numer. Math. 19(i972), Pg. 3%.
Integral Groups II: The Irreducible Case, with J, Neubtiser and
   H. Zassenhaus, Numer. Math. ZO(l972), Pg. 22.
. Integral Groups IIIt Normalizers, with J. NeubGser and
    H, Zassenhaus, l-lath, Conp. 27(1973), PC;. 167.
Constructive Graph Labeling Via Dollble Cosets, with L. Hjelmeland
   and L. Mnsinter, Di&rete Wath, in press and Stanford CS I:e?lo
    STAN-E-72-318.

An Algorithm for the Construction of the Graphs of Organic liioiecljles,
  with I., Kasinter, Discrete Hatll. in press and Stariford CS E:eno
  STAN-G-73-261, )

The Crystallogaphic Groups of &-dimensional Space, with 2, Neubcser,
   H. Wondratschek and H. Zassenhaus, Wiley-Interscience in Fess.

30


SECTION II - PRIVILFtiEO COrulMUNICATlON
               ---         ------
                        8lOGRAPHICAL SKETCH

-II

{Give the following information forsllprofmsionel personnel listed on page 3, brglnning w'th the Princi@ Invartlpgtor.
         Use continuation moos end follow the s?rne aenerrtl former for eech person)

NAME

-
    TITLE                       BIRTHDATE (Ma, Day, Yr.)

DROMEY, Robert Geoffrey
PLACE OF BIRTH (Ciry, Stste, Countryj

Research Associate           11121146
PRESENT NATIONALITY (If non4.S citizen,    SEX
indicete kind of visa end expiration de tel

Castlemaine, Victoria, Australia   ustralian, J-l Visa, Exp. 10/8/74 ~~~~~ o>cms'a
              EDUCATION (Begin with baccelaureate training and include postdoctoralj
                                                                          SCIENTIFIC
         INSTITUTION AND LOCATION              DEGREE        YEAR
                                                      CONFERRED             FIELD
Swinburne College of Technology,       Diploma of     1968   Chemistry
Melbourne, Australia             Appl. Chem.
La Trobe University                     Ph.D.       1973    klolecular Science
Melbourne, Australia

HONORS CSIRO Postdoctoral Studentship
   Commonwealth Postgraduate Research Scholarship
    Walter Lindrum Memorial Scholarship

Artificial Intelligence Techniques to Bio
Medical and Chemical Problems.
RESEARCH SUPPORT (Sseinn~ctionrl

Research Associate

----

RESEARCH ANDjQR PROFESSIONAL EXPERIENCE (Starting withpracent positicn,~~sndexpen'sncerel#ent foor~s ofpmbt Lind
ormort mpnasntetivepublication+  Do not exceed 3pages for sach individual.)
1973   DENDRAL Project, Stanford University, Computer Science Department
1973   Software Development for Graphics Systems, LaTrobe University, Computer Centre
1969-73 Construction, development and applications of an on-line photoelectron spectrometer
     LaTrobe University, Chemistry Department
1569-73 Application of Deconvolution Techniques to the Processing of Experimental Data.

Publications:

"Deconvolution and Its Application to the Processing of Experimental Data", Intl. Journal,of
Mass Spectrometry and Ion Physics, 1970, 5. (co-author).

"Inverse Convolution in Mass Spectrometry", Intl. Jnl. Mass Spec. Ion Phys.,1971, 6. (co-

author).

"A Combined Time Averaging-Deconvolution Technique Applied to Electron Impact Ionization
Efficiency Curves", Internation Journal of Mass Spectrometry & Ion Physics, 1971, 5.
(co-author).

"The Perfect Direction and Velocity Focus at 254034' in a Cylindrical Electrostatic
Field", Reviews of Scientific Instruments, 1373, 44. (co-author).
                                                      -


R. G. Dromey

*'Detection of Spin-Orbit Splitting in the Photoelectron Spectrum of 02+ by Deconvolution",
Chem. Physics Letters (in press), 1973. (co-author)

"The Effect of Finite Line Widths on the Interpretation of Photoelectron Spectra", Journal
of Electron Spectroscopic (accepted for publication). (co-author).

"An On-line Ultraviolet Photoelectron Spectrometer for High-Resolution Studies o-f Molecular
Structure", Australian Journal of Chemistry (accepted for publication). (co-author).

"Photoelectron Spectroscopic Correlation of the Molecular Orbitals of the Alkanes and
Alkyliodides", Journal of Molecular Structure (submitted for publication). (coauthor).

"Comparison of the Photoelectron Spectra and the Photoionization EffFciency Curves for
the Alkyliodides", Transactions of the Faraday Society (submitted for publication).
(co-author).

"A Convolution-Deconvolution Algorithm Using Fast Fourier Transforms), Decuscope,
1973 (in press).


RESEARCH PIAN

33


  I.

rr,

ITT,

?? o

??

? ?? ?

PTOMOLECTJLAR CHARACTPFT7AfI3N:  AFTTFICTAL INTFLLIGENCE
     A Program of Fesource-!?elated 3esearch

A.  3bject.ivcs
n.  Backqround artd Rat ionale
C.  Pelationship to AIY-SUMFX and the Genetics Research Center

SPECIFIC AIFlS

t3FTH3DS

SIZNIFICANCE OF PROPOSED FESEARCE

FnCTLITIES 8 Z;QUIPI?FENT

ETBLIOGRAPHY

SahL~ 1
Fiqurps l-3
Appendix 4:   Letters of Interest
4op enlix R:   1973 Annual Report to the NTii


1.   INTFDDUCTION

Thi 3 r?czec3'L application is intended to susfain and augment the
zapabilities of the mass spectrometry (MS) program which has
;ecv~!il 3s 3 major insTitutional resource at Stanford for some
yp3rs.  Rith previous support from NASA and NSF it has made
oossible a hiqhly interdisciplinary set of research projects
r;tnqiPq ov2r:  artificial intelligence (AI) in biomolecular
~h3r3cterization,  natural product chemistry, clinical biochemical
sttl!ies on steroids,  and the mechanisms of molecular fragment
Formation in mass snectrometry.  While the facility equipment for
mass spectrometry has been funded mostly by other agencies,
cDnne!cted research proqrams embrace several NIY research projects
is wzll,  In addition, this activity was closely coupled with the
?,"FF Ye!ical school computer resource (1966-19731 and will have
sisflnr associations with the  new RI?!-S!JEl?X conputer resource
re:ontly flnded by the BP2 (see Section T.").

Previous support ref1ect.s the diversified facets of this
icperdisciplinacy research,  RASA has supported projects in new
i?:trunent3tion,  inclurlinq the initial mass spectrometer-computer
Ilnk,  gSF has supported chemical research,  and ARPA has supported
our artificial intelliqencc research and initial application to
nass spPctrometry,  Overall cutbacks have forced NASA to reduce
Ftinqinq for this area of research despite their interest.   Under
AFP.4 sunport to Drs. Feigecbaum an3 Lederherg for AI research, the
DENDPAL proqram became recoqnized as one of the most successful AI
3pplic3tions programs.   Eowevec *  ARPA is chartered to fund
Fr3nti~c computer science research and no longer provides funds
fnr the DENDRAL applications programs.   APPA has indicated a
relozt3nz~ to continue funding to this groun for the theory
fncmatior, work in chemistry, although we expect to continue to
receive AfiPA support for more theoretical aspects of OUT research
3r3ffr451  (e*q*,  3utomati.c  proqrarrming).

iJc previously submitted a comprehensive proposal to the NIH
(Rt?-~07SS,  3/25/73) which included an application for the
4fY -SUVEX zr>mputinq resource and a renewal  o f the existing DENDRAL
qrant IPP-00012) o  This proposal was approved for 5 years by the
Pational Advisory Researc+ Fes3urces Council.   Certain
reservations were,  however,  communicate3 to us:  they concerned
especially what we must aqree was an  ambitious effort to close the
control loop for  l'intelliqent. automationfi*  whose costs overreached
t he imme~i3~e utility of the expected result.  During subsequent
3iscussiDn? with the RioTechnDLoqy Resources Branch, takinq into
accnurt the council review aR.d a number of diverse policy issues,
we 37ceoit administratively to seqment the two components of the
3riqinaf proposal.  The ATPI-SU?!EX portion of the original proposal
(exclil3inq DI?F?DRAL) was recently funded for 5 years as a national
resource for artificial intelligence in medicine.  The present
~r~p~ri31 for resource-related research in hiomolecular
characterization and artificial intelliqence is an elaboration of
thz DEN?RAL portion incorporating intensive reexamination and
revision of the previous proposal.

Vith thr? differentiation of priorities represented by AItl-SUEEX,


th?  "tlnetizs  Fesearch Center (ZPC),  and continuinq work on
Irtifizial intelliqecce under Dr.  Feiqanbaum's leadership, the
Dresclt renewal application places more emphasis than heretofore
3n real-world oriented applic2t.ions.  Correspondingly, we have
sqceei that it is now more appropriate thst Dr. Djerassi should be
?ps!qn3ted as Principal Investiqatz)r in this phase of our work.

Fs outline3 in section 3.2, the interests 2nd responsibilities of
?r3f?ssors Djerassi (Chemis+ryl I Feiqenbaum (Computer Science) and
'_e?erberq (Genetics) have? been closely intordigitated.   With their
f!lrther connections with many colleagues,  these programs enjoy a
h irJh acqrs? of university-wide participation.  For example, the
?zRttics rtppartment is also closely affiliated with Biology,
Bi 3zhemis+.ry, Pediatrics, Psychiatry an3 xedicine through joint
lppointrnents or joint research projects or both.   This breadth
would be difficult to obtain except at a few institutions where
t5s medical. schDo3. is both aca4emicslly an3. qeoqraphically
inteqrlted with the university t:, the deqree that characterizes
the Stanford University envir0nmeF.t.

GLOFSARY !IF ABDREVIATIO?JS

AC"F    - Advanced Computer for Yedical Research {Nib-funded computer
           resource,  1968-1973)
91      - artificial intclliqencp
4 It!-S!Tr!??Y- A comprehensive computer resource jntended to serve

the n&iqnal requirement for artificial intelligence
in medicine.  This will be implevsnted at the Stanford
University facLlity called AIM-SVYEX
- Advanced Research Projects 4qency of the Department of
Defense.

- Biot?chngloqy Resources Rranch
- carhDn-l? saqr;etic resonance
- qas chroaatoqraphy or qas chromatoqraph
- Genetics Research Center (Stanford, J. Lederberg,

Principal Investiqator; NIGYS-approved and
awaitinq fundinq.  Grant #PDl-GY 20832-01)
- hiqh resolution mass spectrometry
- infra-red
- Tnstrumentation Research Laboratory

{Stanford Gene%ics Department)
- low resolution mass sppctrometry
- magnetic circular dichroism
- Mass spactronetry or mass sDectrometer
- National Aeronautics G Space Administration
- nuclear maqnetic resonance
- National Science Foilndation
- optinal rotatory dispersion
- a mo-lified version of the PL-1 computer language (for the

Stanford ACME computer fscility)
- Stanford University Eedical Experimental Computer Resource
(WIH funded computer resource, 1973-1978)
- ultra-violet

3&


A.   03JECTTVES:

Core Research.

"he fllnds now applied for would permit

1)  the continued fundinq of the ?!S laboratory as a biomolecular
oh3r37cerization resource;

?)  advancement of laboratory instrumentation capability in
specific areas of GC-HRMS and the exploitation of metastahle peak
linalvsis.

3)  the fucther development of AI computer techniques to match the
instranentation.  This work will emphasize practical utilization
for applications in biomolecular characterization connected with
other on-qoinq biomedical research programs.  It will include, for
3X3UP1, al   the analysis of mixtures by SC/MS; b) metastable peak
3n3lvsis f>r difficult problems of pure compounds and of mixtures
not rcalily separable by CC; c)  optimized data analysis for
zhsr3cterization of MS peaks an3 d) heuristic analysis of spectra
for the molecular ion composition.

?ur project is the only systematic effort, to our knowledge,
cl~rrently underway in this country for computer assisted structure
?licidation,  Subsequent to our early publications, an intensive
proqram has been mounted in Japan in similar areas.   This
situa+.lon may ho contrasted with computer assisted organic
synt.h,+sis, an area receiving considerable attention from several
ccse3rzh qroups,   The%? capabilities can be beneficially provided
t3 a wider community via the ATM-SUP?SX resource.   Research on the
?nulstion of human intellect by computer programs will undoubtedly
Influence the efficiency with which chemical research can be
soplied to ever more complex problems of health, e. q.,
intermediary metabolism and its pathologies; environmental
infl3F?n:a s on hea1t.h; the development and critical validation of
new therapeutic aqents.

"he a:Elevament of these nbjectiQeS depends on the continued
maintenance and development of the DENDP9L AI programming system
{see h313WI .  The advent of the ATM-StMEX facility will remove
some nf the serious computational limits on the exercise of this
system that have delayed recent progress.

Pduc3tion.
rn our university setting, pre-doctoral and post-doctoral
educa+_ion of course constitutes a part of our mission.   As far as
is practically possible, research participation in the DENDRAL
proqram has been couple3 with 3 issertation w3rk by graduate
stu??nts and post-doctoral research experience respectively.
Ex3 mplss of people (and their research area) whose education has
Seen enttan:e d in this way are the followinq:

Srad~late Students: J, Simek, padaqoqical aspects of the structure
qerlerstor;  Rai Lee Tan, synthesis of new estroqen compounds; H.
Fqqcrt,  13ZYYIR of amines and st?roidal ketones; C. Van Antwerp,
13CYR of steroidal alcohols; C. Farrell, theory formation from
~a3s spectral data; L. !!asint.cr, development of the structure
qnnnrator; Y. Stefik,  AI applications to chemistry.

37


F'nst3933rll Fcllous:   r,. Drr)mey,  theory formation f ram analytical
!3t3; P. Sritter, mass spectral  fragmentation of bioloqically
active steroids; R. Carhart, analysis of 13CI?R spectra by
DSNDRAL-like programs; 5, Hammerurn, development of better
fraqmcntation rules for proqesterones.

Fofmll orqlnization.
*his project has been a long-term commitment of Djerassi,
Lederberq and Feiqenbaum functioning in effect as
Jo-investigators,   We coordinate our activities with day-to-day
zDntlc?s i.1 the pursuit of converqent research objectives.   In the
light of the extension of our collaborative activity during the
1.3;t tdo years,  we are now orqanizinq a formal advisory group to
i ~cludp,  in addition to ourselves, H. Cann, J. Barchas, and E. Van
Tamelen,  This qroup will advise the principal investigators on
the direction of the proqram with respect to allocating available
facilities and seekinq out and helping other collaborators,   This
desiqlation simply recoqnizes the fact that many of our colleagues
haIre already been ecqaged in c. alevant collaborative research with

1s.  A '?S resource has recently been funded at the riniversity of
California/Berkeley,  under the direction of Dr. A.I..  Burlingane.
Drs.  Djerassi and Burlingame have recently engaqed in some
zollahorative research which was made more successful by the
sharinq of facilities ard expertise  available at or.e institution
but n?t at the other.  We would hope to maintain and strengthen
t hES2 contacts to avoid unnecessary duplication of effort.

U?  plan to discuss with Dr. A.L.  Burlinqamo the most appropriate
pr3c?Aures for coordinaticq the related activities of our
respect ivn proqrams at the 1Jni.versit.y of California/Berkeley and
!I?_?.   ?hi; may take the form of reciprocal membership in advisory
committees.

fhs
   '*hardware resource w to which this application is pegged has
333~ id entified as the MS facility.   ahile these  instruments alone
represent an investment of over 5`300,000, funded previously by
several aqencies, they do not represent the most important resource.
WC, would use this designation instead for the working team led by
th,e princioal and co-investiqators.  The skills embraced by this
group Fncllde, as mentioned, computer science, structural orqanic
zhemis+rv,  molecular biology,  instrumentation enqincering and a
wide ranqe of other disciplines,  They are represented not only in
tke princinal professors hut in a diversified and accomplished
orofessional research staff (SE?;? Bu3qet Justification).   The
proqrlm for which funds are now requested is the vital means by
which the interests of this qroup can he sustained in a
zn?r3inate? effort that would be very costly both in funds and in
tin4 if  it had to he reconstructed from scratch.   'dithout the
finanziatl support now requested, this line of collaborative
research will have to he abandoned,  with it a unique style of
interdisciplinary collaboration,  and the "1s facility will be
terminated.


9.   BACRGP3JJND AldD RATIOWALE

1,   Phe StrtlctVlre Elucidation Problem

    3)   The General Problem,  Analysis of molecular
structure is a major activity in our program of resource related
research.  For the speci.fic task of efuci3ating molecular
structures, i.e., the topoloqy of atom-to-atom connectivities,
analysts utilize a mixture of information derived from chemical
procedures and spectroscopic techniques.   Each item of
information,  if not red!lndant 3r uninterpretable, contributes to
t4e solution of the problem.  Chemists 3raw upon a tremendous body
7f snecific knowledge about the task area (e.q., clinical
chemistry,  biochemistry), molecular structure, spectroscopic
techniques, etc., in or3er to piece together this information and
l?fer the  structure of aolecules.  These features, and the
relative si!nplicity of the finai concept of a structure, make the
prohl?m particularly well-suited for applications of the
tec!iniqtles of AI to assis t research workers performing the task.

    bl  D-jerassF*s Laboratory.  Professor D jerassi has been concerned
with secncture elucidation problems since the beginning of his
-hamica research.
Y kL,d               His activities at Stanford have been concerned
heavily with the apnlication of particular spectroscopic
techniques to structural studios of biomedically important
~3nP3unas.  These techniques include optical rotatory dispersion
IoR?) and,  more recently,  maqnetic circular dichroism (BCD) (both
3f t.hem supported initially by the NIH),   Since 1961 he and his
?coun have also been concerned with MS because of the power of the
technique, in terms of specificity and sensitivity, as an
an3lyticsl tool for structllre elucidation.   Four books and
anproximately 250 articles on 9s have been published by him and
i i.s  zolIea7ues.

rha technique of E4S does not silffice for all structure
3zt~rminatio~ problems,  but it is a very powerful tool in areas
where thora exists a body of knowledge about the MS behavior of
related molecules,  Bhen sample size is limited MS may well be %he
only technique that tag be utilized.  The recent availability of
hiqh resolution mass spectrometers has madn HFMS the technique of
c: h 3 s z e four ntany applications because under ideal conditions the
exact mass number uniquely specifies the the empirical formula of
3 molecule or fraqraent.  nn a parallel course, the technique of
;:/?pS, routinely available with low resolution mass spectrometers
(r,C,'LRMS),  has revolutionized investigations wherever complex
aixtclrss ace encountered.  All of the above considerations argue
that an extension o f H,S at Stanford to provide routine GC/LRMS and
;C/!fRMS an.alyses would he the next logical step to assist
researcher; depending on this facility for solutions of their
structure elucidation problems.

2.   Aistorical Backqrnund

    91   Y3ss Spectrometry Laboratory.  Prior to the existing DENDHAL
7 r I c t ,  the qroundwork was laid for computerization of the existing
mass spectrometers, an Associated Electrical Industries MS-9 high
resolution mass spectrometer and an Atlas Z'i-4 low resolution mass
spectrometer.  This work, supported primarily by NASA via the


Tnstru~cntation Research Laboratory (T9L) in the Department of
:?r!etiZS, resulted in link-up to the then existinq ACME computer
facility via a PDP-11 mini-comolIter  which acted as a buffer
bctweon tha spectrometers and ACHE.  Initial data acquisition and
ceduztion proqrams were written for thz system and utilized on a
limitz?d basis,  The fundinq of the DENDRAL proposal, NIH qrant
PU-612 [May 7,1Q71-present)  in conjunction  with additional
resources provided by the IRL resulted in a major improvement to
these capabilities. The fruits  of these? efforts are described
un3et section T-B.3 [below).

    h)  Summary of Early DEYDRAL Development.
r? 1364, Lederberq devi.scd a notational algr,rithm for chemical
:):r!lz tllr?s  (termed DENDPAT,,f  that allowed questions of molecular
structure to b? framed in precise qraph-theoretic terms.   (Refs.
1,3-5,12)*  He also showed how to use the DENDPAL algorithm to
qenerate complete and irredundant lists of structural isomers.
(RCfS.  ???? o

Tn lQ65-66 Lederberq and Feiqc?nbaum began exploring the idea of
usin th: isomer qenerator in an artificial intelligence program -
searchinq the space of possible structures for plausible solutions
to a problem much as a chess-playinq program searches the space of
lesal moves for the best moves.   (Refs, 7,12).   This approach
qusrsnt??s that every possible sollfion to a problem is considered
- 2ith9r ianlicitly,  as when whole classes of unstable structures
are rz-jectsd, or explicitly, as when complnto molecules are tested
for plansibility,  In either case,  an investigator easily
?etPrmines the criteria for rejection and acceptance and knows
that no possibilities have been foryottpr.,  l!his approach also
guarantees that structures appear in the list only once - that
3utor3rphi.z representations of the Sam? corrplex molecule have not
h?en included.  In both these respects the computer proqram has an
advantaqe over manual approaches to structure elucidation.

    Cl  Initial collaboration with Djerassi.   (Refs.  14,15,19,
20,21,22,24).
Ledetberq and Feiqonbaum realized that (a) only throuqh
aaplicatio? to real problems could the AT approach be materially
l?v,ancad and critically evaluated, and (b)  MS appeared to be a
fruitful applications area.  MS appeared to be an excellent
orohlem area because of the close relationship between spectral
Tr3qmen+atiop patterns and molecular structure for many classes of
a?leculls*  Djerassigs interest and expertise - an3 daily
intpr3ctio2 between members of his qroup and the AI group - led to
9 seriF5 of joint publications describing the approach and initial
cesrllts of the programs.  The success of these collaborative
?)lf>rts led to the proposa?. to the NIH for initial fundinq to
ex+e?I these efforts.

    1)  Efforts Under NIH Funling for DENDRAL.   (3efs.  25-41).
?h? initial fundinq by NIH provided the opportunity to upgrade the
Fnstrument~tioc and computer pcoqr2ms.  In particular we were able
to !llnun+. a concerted project on both the analysis of mass spectra
of bioTedically important compounds and the mathematical aspects
of m31?cul3r structure.  Proqress reports  to the NIH describe this
research in detail.  The nest recent annual report appears in
4 r?pe!li?x 5.  R series of publications Sirccted to audiences both
in corsputer science and chemistry sre listed in the bibliography.
Ilh? followFnq section [Section 3) summarizes the capabilities for


-tcllcture elucidation which,
3                     in thsmselves, constitute an
impqrtnnt result of past wnrk.

    e)   Related Research.
Fr! important side effect of tha DEWDRAL project is the extent to
vhich additional research was inspired and carried out to fill
q3cIs in existinq knowledqe.  This research,  not supported by the
r)Srt'DRhL qrant, has been beneficial to or-qoicq DENDRAL work, and
vine-versa.  Publications whjch have arisen from this research are
listed in the hiblioqranhy (Pefs, 58-70).   A brief review of these
oublications should indicate the need for precise specification of
the kn7wledqe elicited from chemists and used in computer
Dr3nr3ms.  As an example, consider the description and application
>f an early alqorithm for qeneration of cyclic structural isomers
(311.  This paper considered the problem of spectroscopic
diffar?ntLation of isomers of Z6H100.   Unsaturated ethers fall in
3ne of the classes of isomeric compounds which must be considered,
but th? RS of unsaturated ethers had not been investigated
systematically.  This work WAS subsequently carried out in
Professor Djerassi's laboratory independently of DENDRAL support,
but of hen?Eit to DZ':NDRAL (62).  Other examples will be found in
t h3  Bihlioqraphy (Eefs. 58-70).

3.  Existinq Capabilities

an hlvp worked to develop distinctive capabilities for molecular
structure elucidation, bringing together a hiqh quality HRMS
svstea and AI programs applied to biomolecular characterization.
The feasibility of our analytical approach has been demonstrated
in saveral problem areas, base3 upon the development both of a MS
svst3v ;Init a qeneral se+  of computer proqrams for use in new
areas.

The princioal capabilities are summarized below.   These are now in
3pilq and Here developed primarily under NIH funding to this
pc3 jer=t, with additional support supplied by ARPA and NASA in
snecific areas.  (These agencies have reduced funding levels for
this work because overall cutbacks have forced NASA to cut out
this are3 3,f research despite their interest and ARPA is chartered
to provide funds for frontier coapllter science research but not
f9c 3pplic3tions.  Thus the NLH is the principal of support for
future development of anplications programs in the
interdisciplinary area of artifici31 intelliqence./health related
r:herrical problems.)

      3,  HRYS System and Coupled SC/LRMS System.
We h3ve coupled the PIH-supported Varian -YRT 711 High Resolution
Yass Spectrometer with a Hewlett Packard Gas Cbromatograph and
denonstratad its utility for GC/LR?!S analysis of such difficult
analytic problems as the free -;terols (i.e., not derivatized)
isolated from marine and other sources.   Advanced data reduction
techniques for this instrument Yere written for use with the ACME
conput er svstem  (36Q/50) and cow exist in Stanford's new 370/158
ihI. Ch  :Jnntinues to support the PL/ACNR lanquaqe.   nJC/HRMS scans on
extracts from urine and anniotiz flui demonstrated this system's
zapability to provide hiqh quality mass measurements on complex
nixtures obtained from biological sources.  An example of one
;t/fiRYS run on the amino acid fraction of amniotic fluid is
presented below (Sec. 1II.D).


      DEWDRAL Structure Generator (Refs.  1-6,14,31,37,38,40,41)
nhe D;iDRAL Structure Generator program accomplishes exhaustive
;   
an? irredundant generation of isomers,  with and without rings.
This pcoqram guarantees consideration of every candidate structure
- either implicitly,  as when whole  classes of structures are
focbi3"en, or explicitly, as when individual compounds in a class
-1ro  spec<,fied,  It corresponds to the "legal move generator" of
c3nnutcriz2d chess playinq and other heuris+ic programs.

      C.  DRNDRAL Planner (Refs. 25,28,33)
de have written a very qeneral set of computer proqrams for
determining structural features from analytical data in
well-defined areas.  Such qeneral planning programs have been
wri+t?n fo: low and hiqh resolution MS,  interpreted proton NJ4R
swectroscopy and 13CMR data,

    3,  INTSUM (ReFs. 26,29,34,35)
TNTS`JM ?s 3 computer proqram that aids in finding interpretive
rales for YS.  The proqraa interprets a larqcl collection of MS
data 1:: orjinq to criteria specified by the investiqator.   Then it
summarizes the data to show which of the possible interpretations
seen most plausible.

      P.  PllLEGEhr (Refs. 26,35)
RITLESEW is the current rule qeneration program that sugqests
various rules of interpretation for th? ES data summarized by
r NTS~JM.  Although not finished,  the program can provide useful
assistance in practical theory formation.

    f.  Ancillary Techniques
1.  The !!S facility provides other types of experiments in MS,
including ultra-high resolution measurements (masses determined
via peak matching),  defocussed metastahle ion determinations
(Barber-Rlliott technique) and low ionizing voltage experiments.
These data are utilized by both scientists and proqrams where
anpropriate.
2.   Additional computer proqraas provide added problem-
  solving assistance.
  3,  Predictor program for predicting major f.. eatures of mass spectra.
h.  Proqcams for drawinq and displayinq chemical structures.
  c.  Subroutines developed in conjunction with or existing as parts of
the Structure Generator for pr2bleol.s of partitioning, construction
>f vertex-graphs, and constructive graph labelling.   These can be
3nnlie? to answer certain questions of isomerism which do not
require the  complete generator.  For example,  the labelling
alqorithm can list alI structlrres resulting from substituting
sites of a carbocyclic skeleton  with stated numbers of different
fuactianal qrouns.

     9*  Other Spectroscopic Techniques
?vailahle to us are the facilities of Professor Djerassi's
Laboratory for work requiring Idditional spectroscopic data.   Also
Ivailahle on a fee for service basis are extensive spectroscopic
facilities (NMR, I.!?.,  3n.d V.V.) of the chemistry department.
These would be utilized for collecting additional data on
particular structure problems and gatherinq data on known
compounds [particularly in the area of 13CMR) as the AI programs
bec?me kn?zledqable about other spectrosconic information.


    h,   Chnaicsl Facilities
l-he  stsff and facilities of thP chemistry department represent
SubstaRt-la1 synthesis capabilities and gerieral chemical know-how.
This r?sollrce can be called upon to pr~vid? assistance i.~
5vnthesi.s of model or labelled cnmpounds, derivatization of
9ixCure9,  3nd so forth,  For example, a graduate student in
shemistry is presently engaqed in tfiesis research dealing with the
lahnratnry synthesis of a now s.strDgen metabolite stronqly
su~~3cte3 to be a component of certain preqnancy urines.   The
prsviously proposed structure 3f this compound was one of the
candidate structures inferred by the nlanner in a study of
?strDqen mixtures (Il-dehydroestradiol-17-alpha, ref. 33).

4.  !Js?r Community
RzDn3rnic utilization of existinq and proposed facilities can be
ce3ilized by sharinq them with 1 community of users.  Lacking
suppfcmentsry funds that would he needed for a comprehensive,
na!nr service facility, this =omaunity will include the following
7 r3'105,  hot will be informally available t.o others.
  A.   Stanford Eommunity
     if  Stanford Chemistry Department (except for Hodgson, all
      3re heavily supported by the hiT!i in their research efforts)
        Letters of interest are sttached to the proposal
       in Appendix A.

?rof. C. Djerassi - Steroids, marine sterols
Prof. W. Johnson - steroi3s
Prof. E.  Van Tamelen - steroids, triterpenoids, other
natural products

Prof. H. Mosher - natural products (e.g., marine toxins)
Prof. K, FIodqson - hiDlogical liqands, ligand-metal complexes
Prof. J. Collmnr. - cytDchrDme P450 models

ii)   Stanford ?ledical School Collaborators

The following research projects in the Stanford
Biomedical ComEunity will furnish samples for mass
eztrometric analysis under the present proposal.
it)tached to this proposal (Appendix A) are copies of
the letters of interest in the proposed facility
r?zzived from the principsl investFqators of these
arants.
    Dr.  James R. Trndell,  Department of Anesthesia,
  Stanford University School of Yedicinz.   Drug
   metabolite identification in humans.

Dr.  Irene S. Forrest, biomedical P,esearch Laboratory,
Veterans Administration i-I3spital, Palo Alto,   Drug
tnetabolite identification in bumar.s.

Dr. T.  Rabinowitz and D.I. Wilkinson, Department
of Dermatoloqy,  Stanford University School of
Medicine.  Prostaqlandins.

PrDf.  Fuqene D.  Eiohin,  Department of Respiratory Medicine,
Stanford Opiversity School of Tedicinc,   Ratio of
NAD+/WADH in cplls by measuring ratio of oxidized
to reduced redox plLrs.

Dr.  Leo E.  Holfister,  Veterans Administration

43


Hospital/Department of Medicine, Stanford 1Jniversity
School of Fedicine,   Mataholism of Marihuana.

Dr.  Hiram ??. Sern,  Pharmacy Deoartment, Stanford
University n0spita.l.  Drug Identification.

Dr.  Sumner p1. dalman,  Department of Pharmacology,
Stanford University School of Medicine.  Drug and
drug metabolitc identification.

Dr.  Jack Rarchas,  Department of Psychiatry, Stanford
University School of Yedicine.   Neurotransmitters
and- related compounds in m?n.

Dr.  Keith A.  Kvecvolden,  Chemical Evolution Branch,
YRSA Ames Research Center, Yountain View, Calif.
Amino acids, acids in geochemical samples, structure
of products formed from electrical discharges in gas
mixtures.

Dr.  William P. Fair,  Department of Urology, Stanford
University School of Medicine,   Identification of
the prostatic antibacterial factor; polyamines
fputrescine, spermnine,  spermidine) in body fluids
of patients with prostatic carcinoma,

R?sil,?s tha user projects just summarized, other major prospects
are in sight,  At the time of writing, the chair of pharmacology
is vacant.  Conversations uith the leadinq candidate have
ina icated a deep-seated interest in GC/HR?IS as the principal
analytical tool for broad ranging studies of drug metabolism in
nal.

     3.   Rxtramural Users
The development of the techniques of OP,D, MS and MCD at Stanford
has beer paralleled with extensive sharing of these resources
natian-  an1 world-wide 513 collaborative research efforts, without
anv ~~l~ditional funding.  Rather than provide routine service,
axneriance has shown th.at discretionary selection of problems
results in better utilization 3f our people an,d instrumentation
resource;.  we would extend this provision of services including
atr2ilablp computer programs,  to a limited number of extramural

IISerT,   vote, for example,  our successful collaboration with
?r?fassor Adlercreutz, Meilahti Hospital, University of Helsinki,
on th? identification of estrogens fron body fluids utilizing the
9X planning proqram {ref. 33).


".  Re?atiDnship to ATE-SrJMEY and the "Jenctics Research Center

I'he present application is strengthened by two research projects
related to,  but not overlapping, the proposed research of this
scant.

1)  RT"P-SUYEX (NIH RR-00785, Oct. 1, 1973, thru July 31, 1978,
prirlcipal Investigator, J. Lederberg).  This is a resource grant
co cst_3tbfi ,sh a national facility for applications of artificial
i?tslliqenzn in medicine (AI%).  Our own use of this facility will
inzlud? SUMEX PDP-10 computer time and file storage necessary to
run the DENDRAL artificial intelligence proyrnms.   This support
vi.11 he furnished without charge to the present proposal. It
represents an annual investmerzt of about R100,OOO in computer time
z?ui.valsnt value.

?he AT?!-SVEX computinq facility is shared equally between a
national user community {AIFP) and a Stanford fledicnl School
community.  The DENDRAL research will he supported out of the
Stanford portion.  The ATM service rill be administered under the
policg control of a national advisory committee and will be
imolemsnted over a national computer network.  AIM-SIIFIFX provides
tt?p W?3?lS for members of thm national user community interested in
?tr!lzt!lre elucidation to access the DSNDRAL programs.

2)  ;enetizs Research Center (NIH POl-SM 20832-01 - approved by
thP  NISMS Zouncil,  awaitinq fundinq,  Principal Investiqator, 3.
i~33tberq).  This research proposal is a comprehensive grant uhich
would support interdepartmentni research at the Stanford Eedical
3ch301 in Yedical Senetics,  Pe3irltrics and other clinical
an3lications.  .9 section of that proposal concerns the use of
SZ/LRMS for screening body fluids for evidence of inborn errors of
metabolism.  (This project qre% out of the initial DENDRAL grant,
one of the research qoals of nhich was the analysis of body fluids
using  ?????? o  This research on inborn metabolic errors will be
n~~?ii!~cted jointly in the Stanford Departments of Genetics and
Pediatrics using existing eguipment {Finniqan 1015 Quadrupole mass
spectrometer, Varian Aerograph GC and a PDP-11/20 based data
??????? o

k?? 3ppr?zisted the value of GC/HPYS analyses of selected extracts
3f ho4y ffjids (i.e., those containinq metaboli tes not identified
bv routine GC/LRMS data) when formulating the Senetics fiescarch
Lleater proposal.  Accordingly,  A small amount of funding was there
reg~lestad for recording selected GZ/HRYS data on the GC/Varian EAT
711 'Rass spectrometer in the Department of Chemistry.   If these
funds are awarded, we will noqotlate with YTH a suitable
elimination of this minor overlap with the present budget.


rr.   Sn'?CIFTC AIMS

1'52 spacj.fic aims enumerated in this section will be pursued in
tfi3 hiqhly inter-disciplinary manner that has characterized the
Ll)7NPRkL project from the start of its NIH support.   The aims are
not Iisjoint,but interactive and inter-dependent.  For example,
the pawor of P!S and, potentially,  other spactroscopic techniques,
z3tn h? enhanced by the use of computer proqrams to perform various
asnez+s of structure elucidation and thaory formation,   From the
star3point of computer science, one measure of the utility of
techniques of artifizi al intelligence is how well they perform in
r??l-v3rld 3Dplications. I+ is necessary in the development of
tFi3s2 proqrams to have a source  of data and informed, involved
teaB-mates able to  criticize methods and results.   The aims are
alaborsted in the methods section.

me have attempt28 tr, keep the proposal to a readable length.
rhF?r?fore,  some detail. has been omitted.   Houever,  many details
can be found in the bilioqraphy an? we are prepare3 to provide
3dditi onal information ;turinq t.he site visit.

1.  Enhance the power of the MS resource.

m he existinq MS resource, toqether with computer programs which
ixist Dr which are proposed (see Aim 2, below), is capable of
-olvinq sane of the structure
3                      zlucidat.ion problems of the user
community given computer supnort for data collection and
re3~lction.  We refer specifically to the areas of GC/LRMS and
ro:lt%ne,  b3tch HRYS samples.  We believe that many of the problems
of the user community require more powerful techniques  (see
YPction III) *  These techniques, specifically fJC/HRZS and
senl-automatic metastable defocussinq,  can be provided with a
minimum of cost and effort,  thus enhancing considerably the
capabilities of the resource.

Dur first aim is to provide the resource with adequate computer
3upp3rt (re placinq the previous ACZ!E system) to enable collection
In3 reduction of mass spectral data including low and high
resolution scans  and data or defocussed mctastable ions.

83 DrnDoSC to develop this computer support in the ways described
30131,  (those aims are writt.en to include the work necessary to
im3lencnt the extended PDP-11/20 computer system.  A description
of the rationale for this choice is provided in Section 1II.A and
tha  specific auqmentations in the Rudqet Justification).

    ,!I 1  Convert Pxistinq,  proven data acquisition and reduction
nc3qra?rs from the PL/ACME langllaqe into Fortran, consistent with
t; n3-:: rikizal assembly lanquaqz proqrams for data acquisition snd
instrument control.  These programs will be written in Fortran to
enhance comnatibility with %hc computer systems of other users of
3uch pzlcka~es*

    3)   Modify these proqrams,  3s requirej,  to handle acquisition and
ce?3u:tion of frequent or repetitive HRYS scans with selected
instLumen~ performance feedhsck to the operator, and to take
aqvantaqe of the cxpandcd canab<ilities of the extended 11/20
svstem.  Prototype GC/RRBS systems have  h eea developed at Stanford
1nd  els.ewhare, but this type :,f facility (in contrast to GC/LRVS)


is not n3ir available to the S+3nford community.  When this system
is d2vclopsd, service will- be available to the Stanford community
and research collaborators and,  if our resources permit, to any
szi.entist r?que!stinq assistance.  In many instances this type of
collaboration will require far more  involvement of convergent
interests,  efforts and skills than merely running samples on
requT?r;t o  me have in mind the chemical and eventually biological
internrotation of the analytical data as a matter of joint
concern,  as appropriate.

We have previously illus&.rated the advantages of high resolution
nass sncctral data in the computer analysis of mass spectra (e.g.,
ref. 29).  Also,  ue have previously shown that the same program
can deal with analyses of mixtures without prior separation
es~ecislly when additional data (e.g.* from selected metastable
lefoc*lssinq ?xporiments) were provided {Pef. 33).   We wish to use
the MS rslsaurce and the computer program in further studies of
nixtures of compounds which are difficult or impossible to
separate by GC. The advent of routine systems for high pressure
liquid chromatography have mado many of these separations
possible,  but the liquid chromatoqraph is not presently interfaced
to the TS.

Hany of +ha problems nf the user community require analysis of
complex mixtures which are amenable to treatment by K/MS
techniques.  We feel that where sample quantities permit,
acquisition of GC/HPMS data is highly desirable.   These data can
ho orovid+? by the resource supplemented width computer support
(abovzf .

Frs prDnose to continue tests of the GC/??S combination, operating
urler moderately high mass resolutions (5000-lOOOO), to define in
IPt3il. the optimum operating conditions of the GC/HRRS
sDmbinstion.   This will provide the necessary information on
maximum practical sensitivity to be expected.   This information
can then he used in collaboration with the user community for
:;9mpln  nrenaration.

r he 5:/H4MS system would normally be operated at reduced mass
sp2ctromzter resolutions to maximize sensitivity.   We have
Fxistinq multiplet resolution programs to increase the resolving
r3oyer of the MS.  We propose to provide the multiplet resolution
nrogram with heuristic quidance based on compositional variations
ir,fe,rred from molecular i0r.s or other singlet peaks,  For example,
a resolving power of 10,000 is barely sufficient. to resolve ions
which differ by CH2 vs. N (delta m = 0.012) for ions of about mass
133.  Alth3uqh it will resolve cH4 vs. 0 doublets (delta m =
3,037) at this mass,  it will c3t resolve closer doublets such as
:3?? vs.  H2o3 (delta m = 0.003).  'nle can prDvi.de exhaustive
tabulation; of multi.plPts by mass separations [based on ref. .?O)
which can be used by the multiplet resolution program.

We hive previously indicated the power of metastable ion
inf9rm3tion in the operation of our proqraas for structure
3117idItion fiefs, 28, 33),  We have extended one of our programs
(the ?IS predictor program)  to propose me%astable defocussing
axperiments in order to avoid collection of unnecessary data [see
Pin 2, bel3w).  Rlthouqh ye can collect these data (Barber-Elliott
technique) manually on our cxis+..ing Varian ??A??-771 YS, this is an


7xcnet4inqly wasteful operation,  both ir, terms of sample
consumption and timp.  we pr3p3s.e to implement som2 automation of
collecti3rt of these data on aetastahle ions.  We also propose to
Benin preliminary ipvestiqation of alternative rrodr?s of metastahle
F3n determination (see MP+hods (Sec. ITT), below).

    2
in thi*  Develop performance and theory formation programs to assist
     solution of structure elucidation problems in biomedicine.

TonDutzr programs have already been written for analysis of low
2ilnd hiqb resolution mass spectra,  for generation of acyclic and
zyz lit  molecular structures, f3r lsbellinq structural skeletons
with atoms,  for acalyzinq 13CMR spectra Df nmines and for
interpretation and summary of large volumes of da%a gathered on
!ilO1&?1 compounds (see Pxisting Capabilities above, for references).
Gr'e wish to increase the utility of these programs by providing
Lnt,eractive facilities +hat allow easier access to them, by
incrcn5inq their qenerallty an3 p3wer,  an? by supplementing them
with nBw rnasonlnq pr3qrams.

Parf9cmance Proqrams:

   The current structure qenerator proqrsm will be subjected to
further flitailed tests before usinq it for structure determination
nr7hlens.

   A n?w alqorithm for qencratinq cyclic skeletons (with
no multiple bonds) will be programBed and checked,  The algorithm
is written and informally prnv>d.   A formal proof will be devised
=ls W?ll.  This s'lqorithm represents one very powerful approach to
t hn problem of implementation 3f constraints, 3s discussed in the
f~3 ?.ovinq paraqraph.

   The qencratinq proqrams will be modified to allow isomer
qpnQr3tion within constraints,   Different kinds of constraints can
h? inferred from different kinds of spectroscopic data.   !?e intend
t7 qive thz  proqram knowledqe of 3 variety of these.

   Thp Planner proqrams that infer constraints from mass
sDectron?try data will be hrnadened to include additional
kn?wlerIqe shout the spectral behavior of classes of compounds of
r"levan~e to thcz NIH-sponsored research of the user community. In
3??i.i+i.on,  83 will add the capability for utilization of
izf3rma+t3n about chemical isolation procedures (e.q., one expects
scijic and neutral compounds in  solvent extraction of acidified
bo3v fluins) and relative GC retlntior! times (e.g., to admit the
possibility of homaloqous series).

     w F!  ~~303s~ to implement a more qeneral method for
ir,fsrrirq the i33ntity of thp mol?sular ion whether or not this
1!3pe1cs  explicitly in the spectrum,   This  information is important
f3r the Tuccessful operation of the structure qenerator and the
n?3rlrlf?r.  'de want the program to use whatever information is
3v3il3bl2  -Ind not depend,  as it ctlrrently does, on having
knowledqe 3f the structural slass together with inference rules
for that class.

   Tnt?rfsce routines will he written to mak? it easier for other
;nicntis?s to use tF.ese proqrans,   We have to wait for an


inker3ctivc system before startLng this:   AI!?-SI!MEX will he ideal.
rr.p'lf/output routines will he crucial to ~ssy use of the system.
u~u3vcc, WC als3 want t3 give users the facility to understand the
system's reasoning steps so they can t3k~ 34vantage of it.

Tn a4ditirn to makinq the compilter programs available through
4T"-TTJYEX, we w0da like to translate parts of the LISP code into
another 13nquaqe - for reasons of both efficiency and
?xp3rtability.  We have talked with computer professionals at IBM
Pesaaroh Canter about usinq the APL lanquaqe.   FORTRAN,  ALGOL and
PL/l arc other lanquages whose merits for our purposes we will
explor?.

   W? wish to continue a low-level of effort on computer
proqrams that interpret other kinds of spectroscopic data.
Plannicq proqrams similar to the ?IS Planner could be written for
automatic analysis of data fro3 3,ther spectroscopic
tc:hniques(e.q., IF, IJV) #  as we have illustrated for 13CMP (ref.
??? o

   Th? structure qenerator's view of chemical strlictlre is
topDloqizal and is presentlv unconstrained by bond lengths and
angles.  3ecauss stereochemjcal considerations are frequently
important in structure el.ucidation, we propose to begin
corsidsration of stereochemistry in the structure generation and
2vaLuation processes.

   !! nrolram with detailed knowledge about information
?htain?Sle from various spectroscopic techniques could be written
t3  C?X3Zli!l? a list Df candidate solutions and propose experiments
necessary and sufficient to distinquish among them.  The program
would represet?? an extended Predictor (e.q., ref. 27).   Be have a
Eir- ,t version of a program that suggests "crucial" metastable
pz3ks t.0 he snuqht in order to distinguish among car,didate
stru~tutz?s.  Work on this proqram will con%inuc at a fow level of
3ct!vity,  possibly expandinq  into areas other than ES.  One topic
we will continue to pursue is our collaborative effort uith Dr.
;iLd3 Loew,  Geneti.cs Department, on the potential application of
molez:rllar orbital theory to prediction of mass spectra (ref. 71).

Th33ry 3rnation Proqrams::

  The rile formation proqran (RtJLEGF:N) will he extended so that
i + car? search a larqer space of rules.  Present a priori
zo3str3int; on the rule qeneration qive us a search reduction from
te?s of millions to a thousand possihl? rules,  Even though search
heuristics now 3110~ efficient search of these possibilities, we
want to be able to deal with much larger spaces efficiently, as
wh?? the number of primitive predicates is drastically increased,

   The PULFGEN proqram will. be modified so th-at complex
Eraqmectation and rearranqement processes are manipulated nearly
3s easily 3s simple fraqmentations.  The program currently finds
frlqmentstion rules invnlvinq 3ne 3r two bonds, possibly followed
bv hydroqen miqration,   Ir. the case of cyclic systems such as
=S~CD~ZCS, however, the proqram must be able to work with sets of
. .
three or more bonds in some cleavages,

   Interactive proqrams will. be provided 3~ AIM-SUP!:EX for the
inv3stiqat3r to query the rule qeneration program.  For example,


msnv questions now arise about the proqram steps by which the
proqram Tnfer; the rules it suggests as explanations of the
re7l~larltins.  Why,  for example,  was some particular rule not
considered plausible?

   New d2it.a will have to be selected in order to test the rules
3nd to differentiate amnnq competinq rules.   Be will mite a
nroqram that suqqests new experiments (i.e., new data to obtain),
deosniinq on the nature of the existing rules,

   The t?st phase of the theory formation program will be
written as an evaluation function of each rule against new data.
Tn3;7f3r 3s 3ny new experiments are **crucialtl experiments,  the
evslu-ation function may merely reject a proposed rule.  Mostly,
13wevor , riles will have to be evaluated against new data along
3apv dimensions:  frequency,  strenqth of evidence,  uniqueness,
simplicity,  and the like.

   We wish to experiment with the whole theory formation program
to ,lztermi3e the critical aspects of our design.  For example, 11)
how sensitive is the proqram to discrepancies, inconsistencies and
ecr:>r5  in the data?   (2)  how well can the proqram find rules
within a sliqhtly different model of chemistry?  (3) how well can
the proqrsm perform w?%h one pass through the data, or several
passes? and (4) how critical are the principles of theory
Eormati~on?

    3.  Apply the structure plucidati3n techniques - both
i?;tcumsnt3tion and computer programs -  to hFomedically relevant
compounds.

3ur own interests are in elucidatinq the structures of, and
SnIerstandFnq the MS of, marine sterols, hormonal steroids, and
compounds isolated from human bofly fluids that can be associated
with qenatic disorders (from research in the GRC).  In addition,
W? riff be workinq closely with members of the Stanford Medical
School 3nd Chemistry Department  - in particular those mentioned
aih3ve fSect?.on 1.3.4) - on their structure elucidation problems in
which YS will be used.  Althouqh most users expect to require HRMS
3na  SZ/HRYS data,  some of their problems will be attacked
utilizicq SC/LRMS techniques and library search through (usually)
restricted libraries  of mass slnectral data,   We propose to
investiaate some extensions to the technique of library search
(SIP Methods) to complement our existinq and planned DENDRAL
prcqrams.  WE! plan to continue 3ur exchanqe of mass spectral data
an? library search information as we have previously done with Dr.
5, Uarkey (University of Colorado Medizsl School) and Dr. F. W.
YcLaffnrtv (Cornell Universi.tvI *

P.s !.n the Dast,  attention to new biomedFcnL research problems will
lead t-o increased capabilities in  the computer proqrams. We
rejuir? close communication with the people engaged in the
research so that the proqrams actually assist the researcher while
iycreasinq in power,   Collahor3 tive proposals have come out of
sjxzh past DENDRAL sponsored work, for exampl?, larqe portions of
t ?-lP ;9C Droposal apd a proposal. for 13CMH research.

k'3 2~visi.92  the interaction and collaboration with the user
zoamunity to involve the followinq:


    a)   In all casts, WC plar close cooperation with the users in
311 3SD?Cf';  of the problem,  Rlthouqh the basic isolation
procedures are the problem of each invcstiqator, his knowledge of
th? 3va:lable facil ities ar.? their limitations can be an important
3i3 t3 ssmple preparation and Inalysis of the results.   This is
gartizularty true for collaborst3rs who arc unfamiliar with the
tczhniques of HRFS (e*q*, sample size and resolving power
I en 2 :s s I r y t-, separate the mass doublets that can be realistically
nxpez+oT in different contexts).

    b)  The needs of the user comnunity will be varied.   Drs.  Duffield
3n? Smith sill, in collaboration with the users, determine the
kir.3~ Qf MS experiments which will he most useful, considering
33Klpl2 complexity, stability, guantity, and so forth.   We wish to
lltilize fully the existlnq resource and our proposed extensions,
5ricqinq +:, bear on a problem any techriqu2 which is appropriat-e
snd  z3n h? provided.  This will include the full scope of
available experimental techniques in MS (LRPS, RRMS, GC/LRNS,
;C/HRYS,  mztastable defocussinq,  ultra-high resolution mass
asas.Jrlments) and available computer proqr2ms (see below).

    cl  Y3ny problems will be amenable to treatment by computer
proqrams  which exist or which uill be 3evelope3, fDr example,
3tru:tur31 isomer problf?ms or HEMS interpretation on compounds in
3 w-11-understood class.   We will take the responsibility for
utilizinq t.hese proqfams where appropriate to assist in structure
?J.nci3?+ion problems,  Ide will instruct members of the community
irl else of the proqrams when pr3qrams are used routinely by
c3l.labor3t3rs.


Wolezu13 r structure elucidati31  entails the intelligent and
~2tieri+ application of a large body of krouledqc to each specific
Dr?hlen.  The importance and relative difficulty of the problem
;.~pel us to seek the powerful assistance of computer programs to
helo chemists in their analyses.   It is unlikely that such
prnqraas will ever replace chemists,  especially because computer
pcoqrams are readily written only to focus on rather narrow
3s~ects of problems.  Nevertheless,  our past research is
roasonahly forwarded as a demonstration of the computer's ability
tn Issist in practica, 1 biomolecular characterization although this
was 3 spinoff from theoretically oriented research.

Tn order t-, meet the major objectives of this pronosal we will
fnzus our Itter.tior! primarily 3n structure elucidation of
b13merIiz sllfy important compounds throuqh YS and AI.   However, many
of t.ho cDmoute!r proqrams can already use information from other
analytical techniques.  Sr, we want to be able to think of
structure alucifiation in  the context. of an ensemble of analytical
caoabi? ities.

A.  Snbancing the Power of the Yyi.lss Sptctrometry F?esource

Wo have developed a siqnificant resource consisting of
instrunentation  (the Varian CAT'-711 and ancillary equipment) and
conou+.rr programs for instrument zvalu%tion, data acquisition and
reduction.   Pouti ne reduction of high resolution mass spectra to
elsEsnt31 compositions and ion  abundances without human
intervcrtion provides the capability for efficient handling of
13cqe volumes of hiqh resolution mass spectra (such as will result
Er3m  SZ/HF??IS runs).  The development of the GC and of the SC/MS
combination is in the excellent hands of Ys. Annemarie Wegmann,
vh3 is responsible for operation  of the complete system.   We now
!L~VP mpr? than two years  o f operational experience with the WS,
I- ha
. .I ._  :;C and related equipmsr-t. under a wide variety of experimental
conditions.

U?ne of the resource-related research discussed in this proposal
cd2 ba carried out without siqnifisant quantities of mass spectral
33ta.  The ?xistencc and extensions of the HS resource, the
d~velopmert of computer techniques and the applications to
biomedical problems demand an efficient atecAar!isn! for acquisition
3nd ro?Iuction of p4S data,  and eventual transmission of the data to
the S:JFJ?X resource.  Th!lS, operation of the MS requires
s;rrbstar t Lal computer sunport t9 deal with the large volumes of
data produzcd by the system at hiqh data rates.   WP feel that a
properly confiqured system of hardware an3 software should
orovide,  at. a minimum, the followinq clpahiliti6s:

1)  nptailed evaluation of the condition an4 performance of t_he
PS pri3r to recDrdinq data on valuable samples, with feedback to
the 9P?ra+or.

2) A coordinated system of hardware and software for signal
~nn3itiotlinq, peak detection and peak analysis.


31  D3ta reduction techniques based on a compute3 (not theoretical)
a3?el 3,f the MS,  j.ncluding peak shaoes, mass/time function, and
resolving power as a function of mass.

4)  Pe3k profile analysis for aultiplet <otsction and resolution.

5)  Conpllter control cf scan rates, clock rates and optimum
Tn3loJ and diqital filterinq parameters.

3)  some on-line feedback, t? the operator, assessing the
D?rform2ncz of the svstem Burir,q an experimcn+.

7)  The system must cleal with frequent DC repetitive HRMS
SZITS, req:~li.rinq the capability for rapid st.orage and analysis of
laras volumes of data,

Pr?v.ious support of our research by the NIH an? NASA has given US
a firm foundation of pr9qrams and experience.  WC have,  up until
thp termiRltion of the ACHE computer facility (July 31, 1973) ,
demonstrated canabilities 1-5 above.  we ueco precluded from
o,rrsuinq canabilities h and 7 d!le to the configuration of the ACME
fazilitv.

Fhe ??mi.sc of the ACME computer facility an4 thp subsequent
i?:ornorati.on of the Pf./ACFT;` 13nguaqe into a new XR?! 370/158
factlity under S%anford auspices has forced a reevaluation of the
means for providinq HFP!S laboratory computinq support.   We had
3reviousl.y depended exclusively on AC#E for data reduction
pro:zssinq.  The AZMR transition poses both technical and fiscal
~?JiSiOTlS in that the real-time support capabilities of the new
f3r~ilicy will he different from  ACME9s and the fee for service
basis of the facility requires an explicit budqet allocation for
its use.  Previously we had received ACME computing support
Y i. t. h 3 II t sbsrqe as Dart of the core research effort.   Since we were
thus required to revise our computinq plans, we have explored a
number ot: options for near-term as well as longer term solutions,

r!s outline? in the attached annual report, we haoe chosen an
ir.t?rim approach (throuqh the end of the current grant, 4/30/74)
whish mS.nin\izes near-term costs, includinq hardware and software
:3lv2rs%3n3 as well as operatinq expenses.  This approach entails
zonnectinq the WA I v-711 spcctrome+er to the 370/158 computer
throunb an IBM 1300 interface.  It allows use of the existing
?L/A:ME nroqrams but will have real-time response limitations at
least 3s severe as ACME had (which is inaflequate for either
;?//!?P'S or SC,'HRPlS).  Our existing cornouting budget provides for
3nly a very low level of instrument utilization in this mode.

For a lonqar term solution these constraints are unacceptable.
:urre?t estinates are that ror,tinued use of the inadequate
19~0-37~/1SS connection and PIJACYE interactive programs under
full instrument productivity would cost up +o $4,100 per month.
Three alternatives have he!er. investiqated for improving technical
nerf7rmanc.2  and reducing cost.  This review has resulted in our
zurr?ntr pr3oosal to augment t hz existiI?q mini-computer system
(PDP-17/20) with local storaqe and arithmetic capabilities.   This
stand-31 on3 system would not support real-time, on-line data
reAuztion but woulif allow routine 4ata acquisition and instrument
Dcrform3nc3 evaluation, followed by off-line data reduction.


Alternatives consi.dPre:l include:

1)  Yo1ified 370/155 Connection

W? di scussed with personnel in the Stanford Center for Information
Processing (SUP) various approaches f3r improving 373/158
secvic0.  netailed planninq is still under way within SCTP in
regatr$ t3 real-time support and futllre pricing policies.   Thus the
fnll.0uir.q  conclusions are tentative.  It appears that the
lonq-term cost woul? bc prohibitive to continue real-time data
acquisition by the 370/158.  1nst.e3l, a store-and-forward system
was proposed.  This would entail an augmentation of the existing
PnP-11/23 front-end mini-computer with memory, disk, tape, and a
n?w interface to the 370/15A, totaling about $28,000.   This
approach is workable,  if limited near real-time instrument
perf?rnance evaluations could be made to assure satisfactory
in;tr*xment setup and data acquisition,   It was recommended that
thr! exisrlnq software be converted from PL/AZMF to a more
?ffici.ent lanquaqe (such as FORTRAN)  to rpducc operating costs.
This would require approximately 4-h man-months of effort.   The
resulting decrease in operating costs could not he estimated in
tine for this proposal because the new SCIP pricing policies are
not formulated and inadequate 370/158 system analysis tools are
operational to evalllatc our benchmarks in terms of detailed
CRSOUrza consumption.  We have therefore budqeted an approach
b~se3 on the remaini.r_q two options with the understanding that we
WZll recnn;ider the SCTP option before proceeding nith an
innlea?ntation should this proposal be funded.

Phe recently approved AI,: "-SIJPEX PD?-10 facility will provide
I?z"ss3cy zomputinq support for the development and use of DENDRAL
A r  Droqram;s.  The ??S laboratory produces data which these
proqrams analyze an3 thus has a close relationship to the RI
tcsPar-ch.  The SUMEX computer could help in the off-line reduction
3f instrument data, particularly during the early stayes of the
proie~- -t when the machine load will be relatively light (20-258).
The present programs would require conversion from PLfACME as in
option II),  which would take 4-6 man-months.  Such computing may
use from 35-31, mlnutcs of CPIJ time per day, depending on the
amount of  ;C/MS work,  while this approach saves operating
zninputin~ costs,  the front-end PDP-II/20 would require
auqaentation as in option (I) ($28,000) to allow store-and-forward
3p-Pration wi.th subsequent off-line data reduction on SIIMEX,   This
is neede? because SIJVPX is not confiqnred to allow real-time
acqrlisitior of the volume of ?ata anticipated.   This approach,
vhil$ t4e least costly,  wollld entail a measurable use of the
PI)P-10 resources whi.ch we feel are better reserved for the
i?t?n!le,l AI,Y-SIJVSX applications.  In addition,  because of the
pcinrriti?s anticipated for allocation of S'JYSX to AT research,
Darticularly as loading increases, scheduling may he required
which will constrain the PS laboratory operation.   For these
reasons,  w2 feel a better, even though slightly more expensive,
ripproach is a stand-alone PDP-1 l/23 data reduction system.

3)   ~t3nd-Aloce PT)P-1 l/20

54


Th2 3uam2ntatJons of the existing front -end PnP-11/20 required for
5t3ro-and-f orward operation in conjunction vith the 370/158 or
with  SW! T;'x, come close to meeting the nes3s of a stand-alone data
syste.n,  In addition to the menory, disk and +2pe, an augmented
arithmetic capability is neede3 to allow rapid floating point
zalculstions.  A special device for this purpose costs about
`3 7 , 500.   T!le SJJCEX interface can be less sophisticated in this
case,  however, accoun+ir,q for the much lower data volume after
ce3uction, so that the total cost of the stand-alone system would
bF? 3;34,000.  As with the other ontions,  conversion of the present
proqrams would be required.

vhis  approach, while sliqhtly more expensive, has the advantages
af off-loaling all. data loqqicg an3 reduction functions from SDHEX
an3 affords an adequate capacity for non-real-time, stand-alone
?ata reduction on the PDP-11/2D.   It furthermore allows more
fresqoa an? responsiveness in the operation of *he MS laboratory
since ?ata collection or reriucf ion  can he scheduled without
vorryinq about the impart on AIM-SUKEX users.   we therefore
sropose an3 have budgeted an augmentation of our existtng
mini-computer system as a stana-alone ?ata reduction facility.

IT tie biomedical community (see !Jser Community, Sec. I.D.4 above)
Iziirinq access to our facilities  for strflcture elucidation have a
vac5et.y of problems, some of which can be solved by existing
instrumcltation and computer techniques, as noted ribove.   However,
~anv problems consist of complex mlxturcs of compounds where
inalysis by conventional GC/T,RP? S rloes not lead to unambiguous
;3l?tions,  wi separation of components on  a preparative scale for
other spectroscopic analysis is 3ifficult (e.g., see marine
ster313,  section D, belou),  These problems are amenable to attack
by a system comprised of a GC/Hl?YIS combination, the GC providing
S?P3Ctlti.OiY, coupled with the PYS operating at hiqh resolution to
provi?e el?mcntsl compositions.  Thus,  upgra-linq of our current.
svstem so that SC/HPMS data can be provided on a routine basis is
a -lesirabla,  and we believe necessary,  st2p to solve many of these
problnms.

tl:? propose to continue the development of the ;:/HPYS system while
nairtai cinq existing capabilities of rr>uti.nc BRAS analysis and
?r/Pl: uhera this efficiently responds to local needs.   Many
aembers of the user community will require in addition to GC/HRMS,
YPYS analvsis of relatively pure compounds or mixtures of small
a~mbess of compounds.  me will provide this capability on an
t~terim basis,  usinq St_anfordls TRY 370/1513 system while the PDP
11/23 syst"m is being upqrade?.

We wer? able,  using the ACRE ssmpr~ter facility, to start
zvafultinq the operation of a GC/YS system at high mass
resolutions.  These experiments were hampered somewhat by the
Liaits+lons of the computer system useI to acquire the data (only
occasi 31131, sinqle scans were possible); they were necessarily
liscontinaed (as well as all Hf)wS operation!) upon the termination
of AC"4E.  we do have, however, some benchmark figures for the
oerformanca of the proposed system.  flixturos of fatty acid esters
(e.q., methyl pslmitate and methy, 1 stearatc) qave good quality


naas measlrrements (+-lo ppm) over a dynamic range of 100~1 for
sample sizes of the order of 0.5-l-O micrograms/component during
19 sen/l2c33e in mass scans (resolvinq powers S,OOO-R,qOO).

W? 3~0 haltinqly cont.icu inq our evaluation of the SC/HI?% system
aven without a data system, making measurements on individual ions
>f the 1113s~ standard and known materials in the GC effluent.
fhas~ data can be approximately translated into expectations
7 ur!.nq dyn3tmic scanninq.  We have performed an extensive series of
mnasurements on both methyl stearate and cholesterol (not
?erivatFzed), the fatter compound being more representative of our
7urr?at research problems. These measurements tend to confirm the
2r*limi?arv data described above.  Firmer data will be available
slxhsequnnt to the submission of t-his proposal.

~5 prorose to operate our existinq GC/YS system under high
c?s.~l:~+ion conditions siminq toward optimization of resolving
D-Jd"T?,  sc3n rates and GC and molecular separator operatinq
z9rditions to determine the paxlmum usable sensitivity of the
SYStI?C!l.

            .
i42 r3zoqnlze that the ultimate sensitivity will not approach that
attainable by photographic methods of recorclinq; WP feel that the
3kilit.y for on-line operation and evaluation of the operating
conditions of the ?lS partially offsets the sensitivity
di;advantaTes.   We realize that  some structure elucidation
Droblems will not be amensblc to study because of the sensitivity
limi tstions;; we feel, however, that many problems of interest to
the U;?SC Community can be studied effectively with this
performance? capability.  Rather +h3r. propose a research program to
inzrcase the sensitivity of hiqh resolution mass spectrometers
(P.4.,  McLafferty, et-al., Anal.   Chem., 44,  2282 (1972), dynamic
CP ;:l3inninq of peaks; Jet Propulsion Laboratory - chemical
multipl!er emission/detector arrays,  private  communication to T.
Rindfle'sch), we propose to identify our limitations and, with our
z~LlaSnrat~r5, use discretion In selecting 'and preparing samples.

FlIrther 3 s;3lerstions of technical capability to meet the statp of
+!-i3 3rt jc sensitivity will require  investments in hardware that
233 he better justified at a later staqe of a successful facility
program ,  Yeanwhile, other laboratories can be expected to make
siqrlifi zInt contributions to this important problem.   Practical
r-?arl  for budqet 15mitatiops is the main reason we do not press
t'lis issue ourselves at the present time.

yiqnifizant improvements in sensitivity (with only small decreases
in mass measurement accuracy)  can be achiave3 by operating the MS
?t reduced resolving POWPI--: coupled wit. h intelliqent analysis of
tha cesultinq data to detect and resolve the potentially greater
n!~aFer rf 2verlappinq peak envelopes,  This proposal is not
??kire'lv new (e.q.# see Smith, et-al., Anal. Chem., 43, 1796
(1371) ; nurlinqame, et.. al,, in l*ComputPrs in gnalytical
Chemistry," C.H. Orr and J.A. Morris, Yd,, Progress in Analytical
"hnmistry,  Vol. 4, Plenum Press, N2w York, ??.Y., 1970, Chap. III).
Jr?  can,  however, siqnificantly extend these earlier techniques by
ztiliza+iol of 3ur multiplet resolution alqBrit hm.  This algorithm
?~holie? in a computer proqram,  has been shown to increase the
effective resolvinq power of the ?9S up to a factor of three. It
hnses its ooeration on a dynamic model of peak shape computed
3iccl:tly from the data,  For computational efficiency and to avoid


splri?us  infDrm3tlon,  this algorithm would be best implemented as
3 post-processor, basin9 its search for multiplets on the results
nf pri>r elemental. composition determination.

"he ability to detect and analyze for unresolved peaks is mediated
by consideration 0, f the mass measurement accuracy of an MS system.
r'hese svst?ms are capable of determining peak positions (and thus
nasszs) to a sm3Ll fraction of the peak width. The high accuracy
nf such neasurements (+- 2-10 ppm) can, in fact, be utilized to
I?!. ezt. and "resoLven muftiplets in instances where the unresolved
so,': UPS ar3  known precisely (see Durlinqame, et al., ref. above,
??I?  ::-I vs.  13C doublet fietection and resolution),

For instlnzes where the !keteroatom content of a molecule is known
3r where fhe possibilities are reduced severely by chemical,
SD? ., -tr3sc>oic and mass measurement heuristics, there may be a
r3Pqe of possible overlapping ions resulting from fragmentation of
??? ??o???o? o  These poten%ial overlaps may be computed and then
used (in combination with  the known resolvinq power and mass
~~~qsurn~?nt acctlracy of the MS and the measure1 mass of the peak,
zssuminq it was comprised of only 3ne type of ion)  to direct the
nulti~let resolution proqran.

As an exnmnle, we have computed the possible mass doublets for
various ranqes of compositions (Sederberg, et al.., to be          *
ouhlishod),  A sample t3bl.e for C, N,  D =<4 is appended ITable 1).
7nLv 28 of the 364 possibilities are shown, namely those whose
n <as:  d%fference {e) <*OS mass units.

nf these 25,  13 show e>.03 and would he fairly easy to resolve,
requirirq ?/5OOfi resolution at i"W=150.

9t the other extreme, 5 doublets show e<.Ol   fCY4 vs.  H404; C2H20
VS.  N3; :2Y2 vs H403; C3N vs F203; and E4 vs H2N02) which would
?exan,'l special treatment for resolution.

T!lz?  10 dnllbLets for which -01 =< e =< .03 pose the interesting
~h~lleaq~s for tradeoff of resoluti.on vs. scpsi+.ivit.y in the
coat2xt of given problems,  For example, i.f W is absent, the only
ambiquities are C3 vs.  H402 (e = -.02) and C4 vs 03 (e = ,015).

N!uzh 3s WC would wish always t3 have unambiguous empirical
formulas f9r all ioEs, HRHS remaLns a valuable tool despite these
limitations.  AS shown by these examples,  even modoratp resolution
c educes  the number of candidates tn a manageably small number of
aLternativ?s,  eontextual. ar,d intnrval ?ata (within the spectrum)
c39r-i 03  us21 to trim these fwthec at tjro levels:  (a) pooling of
3231:  statistics to sharper. d ecision probabilities on the presence
>f hztPr9atoms -- the fraqments  are subsets of the mol+?cule and
(h) the assemblaqe of candidate solutions unt?er each of the
alternative formulae.  ?anif?stly,  computer processing can sort
>t),t branchas of decision trees  that would soon exhaust human
p;itienze.

These heuristics arp built i_nt3 the DEWDRFL programs {solutions
b?SE?.l  ?n fraqmentation theory),  but are also applicable to table
look-up approaches.

WC {ref. 29,33), and others (e.g., H.-K, iaipf, et, al., J. Amer.
:il?n. Sot., 95,  3369 (1973)) have illustrated the importance of


neta<t?hle ion determinations in automated structure elucidation
has-? on MS data.  Data on metastahle ions must be judiciously
;LTstecl because of the time and sample normally required to
nerform the  measurements.  Our programs ar? now capable of precise
snecification of those experiments necessary and sufficient to
?istinquish among a se+. of candidate structures.   We seek more
efficient vays of acquirinq these selected instrumental dat.a.
T3i.s can be accomplished with minimal cost by Developing the
hTri7,irare and software necessary to perform (defocussed) metastable
scan-;  and  zafculate the data.  Zuch of the hardware, except an
accurate sensor for accelerstinq voltage, alre3dy exists.   We have
ha? consideraS3.e experience in peak detection on the software
3i.12;  Fhn calculations to determine transitions are simple.   rt is
assumed that the operator would manually adjust the instrument to
the 'i3sirel "!auqh teP pass prior to initiation of the scan of
met astable oriqins ("parents*') of this daughter.

Th? recent availability of reversed-geometry instruments has
ncovided n?w methods of metastable defocnssinq (e.q., Beynon,
-3+.al., Anal.  Zhem.,  45 (721,  1023A (1973)).   We have illustrated
the power of these techniques in mixture analysis {ref. 69).   NO
"n-,tmal" qnometry instrument is equipped to perform these
aeasutaments to determine al.1 the dauqhters of a given parent,
information which is frequently more useful than the converse.
This infornation can be obtained, in principle, hy synchronous
v3ri3tion 9f twr, of the three fields (maqnati.c/ accelerating/
electrostatic deflection) in a very accurate way.   We would like
to explore this possibility because we feel that this technique,
if f?asi.hf?, would represent a significant upgrading of the many
z;tan.Iard qeometry, double-fncussinq instruments in existence.

n.  :omputer Assisted Structure Elucidation

A; m3ntionz3 above, some existinq proqrams c3n be used immediately
For structsre elucidation problems using IIS data.  The programs
have been Iascribed in detail elsewhere and are mentioned in the
se:tion on existing capabilities (Sec. I.B.E, above).   The
?13nnor's performance,  for example,  is excellent precisely in the
areas where ES, by itself, is capable of definitive structure
Tnllysis.  The general intellectual flexibility of the human
chemist is beyond the reach of plausible programs.   On the other
ban 3,  whnva
          \> - (._ the history of a sample is known, so as to restrict
th potential classes of compounds and for classes where the rules
3f FAC; fraqaentation are well nnderstood,  the program's performance
matches +hlt of trained mass spectroscopists, the program also
3ffets SO@l? advantaqes !.n its exhaustive and r3tpi.d analysis of the
! a+ a.  r?any structure elucidation problems of the user community
fit into this cateqory ana existing resources can fulfill these
nee?s,

#h,lthec man- or computer-implemented. PlS cannot solve all
5 tcuct IIre elucidation Problems, however,  In such cases, recourse
i- to other spectroscopic techniques if sample size permits. As
&z-l
?ns:ribed in the introductory section, diverse information is
ni2c33. Coyether to achieve a solution.  Interactive computer
nroqrsms can assist in seqments of this procedure, with the
advantaqes of exhaustive eva luayion of the data and the molecular
structures suqqested by these data.


rn 311r own and in planned col!abocative work, we rdill call upon
t h I  extensive facilities of the chemistry department for
3cJuisition of additions1 spec+roscopic data.   These services are
filsnce3 by fees,  paid from existing research qrants of the user
zoaEuci.ty.  There are sufficient documente? examples of structure
?t,rcidati?? problems to obviate the requirement for extensive use
3f +!-%ns:p a43itional facilities in 3evelopment. of the programs. On
the other hand, the intensive pursuit of mechan iced "intelligenceW
in the domain of MS requires more than availability of public ES
'lata .  Tt requires the collaboration of skilled chemists actively
enqa?erl in practical ES research and, Rt the same time, committed
to the nxploration of innovations in the application of AI to the
solution of the problems

As il +!lc? 3ast, we will deoelon the computer programs throuqh
:los3 collaboration among Drs. Duffiefd an3 Smith (and other
nembers of their qroups) and the pcnqram aesiqners and
proqtammecs.  for us, this means daily consultation for discussion
3f str?iteqy, extensions to the proqram, an3 solutions to new
prDblelFS,  Tn particular, we propose to continue software
devclooment (on the AIM-SUMEX facility) as folllows:

1)   The ret zntly completed structure generating alqorithm will be
t he core OF our efforts to assist in structure elucidation.   The
strnztura generator can quarantee that the correct solution is
somewhere in the list of possibilities.  A?dit ional programs, such
as th? Planner allow us to avoid exhaustive generation in
dractice.  Some parts of the cyclic  structure generator program
h2ve nDt. baen extensively tested yet, 2nd these tests will be the
first task to complete.

2)   The structure elucidation task is stronqly directed toward
rejection of whole cateqories (e.g., compound classes) of
--,lSrtlons as quickly as possible by usinq as mlrch knowledge about
z-3.
t he chemical history or characteristics of a sample as is
avail3hle.  Details of spectroscopic data then define the
nDlezil!ar framework more precisely.  Each st.ep in this procedure
ronroser.ts the application of constraints on the set of possible
3313tions.  Computational effici_ency demands that these
constra5nt.s be applied early in the qeneration process when the
structure qenerslor is utillze.1.

`.J? have ma3e some effort to examine th3 kinds of constraints used
by scientists engaged in structure elucidation.  We have begun
Iesiqninq strateqies so that these constraints can be brought to
be3.r 3n th2 structure generator.  Some of these strategies involve
nilor changes to tt?e existinq program; others require significant
extensions of existinq qenerating functions. One approach which
sc3!ns particularly attractive to us is presently under
3evel3pm?n%.  This approach will utilize the existing s%ructure
qeaer3tor, with some modifications, to generate a dictionary of
cyclic skeletons up to those containinq a maximum of twelve
tertiary vertices.  The dictionary will be a complete, irredundant
list of rinq systems which contain no multiple bon3s and no
cut-edges (acyclic parts),  This .lictionary will be organized and
<F?V?l 5;n t'lat many constraints can be implemented easily.   The
3iztior?ary will allow exhaustivr specification of ring systems
with 1o?1bl~ bonds and/or aromaticity.  The rings themselves can he
13be11ed with heteroatoms to qenerate heterocyclic ring systems,


3r with acyclic radicals t.0 qenerate substituted ring systems.
rh? existence of the dictionary will lea? to greater computational
3ffj.ciency as it nepds to be generated only once, ana specific
__
cocfi.qarations of rinqs (numbers, sizes, fusions) can be pulled
immediatelv from the dictionary.

CI'? or-~posc to continue these investiqations so that a reasonable
ilariatv of constraints can be recognized and utilized effectively
hv a com!nuter proqram.  This rapresents the first step touard
increaisfnq the chemical knowledqe of a proqram which views
molecular structures and their manipulation as mathematical
entities and transforms.

3)   Prssent,  effective use of the structure qenerator or its
subroutine: for special problems requires a detailed knowledge of
t*e proqram.  We propose to develop an interface between users and
the proqraa to remove tkis requirement.   The interface would
contain claments of s+ruct:lre input and display routines and a
simple lanquaqe for application of constraints.   Portions of these
el333nts are available from other workers (e-q., Richard Feldman,
R!TV) ar.3 we would draw on these sources whenever possible,

4)  Ue pr333se that initial efforts will be directed toward a
system where the scientist examines his own data and inputs his
fildinns (in terms of allowed and disallowed structural features)
t.9 the prosram as constraints.  The generator would then provide a
list of possible solutions to he evaluated, followed by iteration
on this procedure.

5)  ,A?any structure elucidation problems can be characterized as
assembly nf sub-structures inferred from spectroscopic data into
23trplE?tn  molecular structures.  Although there are two instances
in the literature dsscriblnq programs with the capability to solve
this problem (see S, Sasaki, "Determination of Organic Structures
ay Physical. nethods, Vol, 5," P.C. Nachod and J.J. Zuchermanl Ed.,
Aca.dem<.c Press,  Neu York an;! London.  1973, p. 285; M.E. Munk, C.S.
Sanijano, R.L. P?cLean, and T.H. waskel, J. Rmer. Ehern. Sot., 89,
415s flQ6-q). we do not feel these approaches flrlfill the
requirements for qenerating complete lists of structures and
avoi?inq duplicate structures.  We have some strategies to solve
this problem, thus extending the scope of the qenerator while
tying iC more closely to the methods used by chemists engaged in
structure elucidation,  Our existing structure generator has this
capabilitv;  as long as the sub-structures are connected only by a
si.r!qle I-ol-zrl,  PO new rinqs are formed.

61  IJe wish to implement general routines for finding molecular
Lens from spectroscopic data in order to improve the general power
of the Planninq proqram,   Th2 current Planning program depends on
7arinq som3 metastable ion information with HF!lS data, together
with knnwladqe of the structural class with special rules for the
class.  k'e will incorporate strateqies sugqosted by Biemann (K.
Fiemann and W.J. "IcPurray, Tet. Lett.,  647 (1965)) and Plcbafferty
(1.  Venkataraqhavan, F. U, YcLafferty, and G. ?. Van Lear, Org.
YilSS  Spectrom., 2,  1 {1969)) for finding molecular ions, hut also
q'ae the proqram the flexibility to use class-specific information
wh?r available.  The procedure will be to use these kinds of
Flformstiol within a general ha!lristic search paradigm.

?)   fha section on aims indicat.ed some lonqer-term directions


v5i.c~ miqhk he Dursuea.  CJf th2Re,  we feel that the incorporation
3f three-iii mensional information into the proqram is perhaps most
i npnrtant-. {e.g., representation of three dimensional information,
molecular modellinq includinq stcric factors).  Lederberg has
pcevigusly discussed ways {Ref.  1) in which three dimersional
information can be considered in the generation and representation
of m3lecular structures.  ?!orc recently,  the work of aipke (J.
4mer. Chin, Snc, in press; personal communication) in connection
with computnr assisted orqanic synthesis has provided important
re5ul.t.s which we wouli! attempt to utilize to avoid unnecessary
luplica+ion of effort.  We plan to collaborate uith Dr. G. Loew
[Stanford Genetics Uept.)  to utilize her available programs on
nolezn!ar orbital methods to determine  local minima for
conformations.

&nD+her lonqer term qoal which we feel is both interesting and
important is the use of an extended Predictor (which we have
previously described in the context of MS) to assist in
ji;ti~qnishinq amonu potential solutions to a structure
3Lacida+ion problem,  We have recently carried out some extensions
tn the  2xist inq Predictor by IncDcporatinq the ability to suggest
n?ka?taible defozussing experiments.   Further extensions to include
Cnovlcdqe about other spectroscopic techniques and the information
which can be elicited from these techniques are clearly feasible
3n8 could be a powerful extension to our computer assistance
?ff>rts,

m
-*  Theory Formation

322 inoortant aim of this project is to improve the existing
theory formation capabilities  and thus provide more assistance to
scientists investigating reqularities within classes of compounds.
This is a theory formation task at a very praqmatic level.   The Il.5
Fheorv that the proqram attempts to find is of the same form as
the one practicinq mass spectroscopists use for structure
?llci33+i3rl*  Thus, resultinq pieces of theory are extensions to
both thn szlentis+s' theory ani the computer's theory of the
?iscipl,ine.  To improve this program  we need to complete the
Plqn- Generate-Test program that has been started (as described in
th? 3ppendsd annual report) an3 tune it over many test cases. We
also w;sh to make the proqrams interactive and easy to use so that
thay are more readily accessible.  This can he done when the
prorTrams are transferred +o the AIR-SUMEX facility.

We plan to apply the theory formation proqram to two different
kinds of data:  (a) the data collected in the interest of
gnderstandinq the mass spectronetrg of a particular class of
conpnlin<s,  as was done for estroqenic steroids, and (b)
collections of diverse data that may provide some insight into
note aener31 fraqmentation mechanisms.  For example, we hope to
fi73 qeneral rules analogous to the alpha-cleavage rule or the
stability of aromatic rinqs.

Th? INTSrJP proqram mentioned in Section (I) is the planning phase
3f the theory formation proqram,  It currently runs in batch mode
32 StarforI*s 360/67 computer,  We wish to add an interactive
moni.tor to IYTSUY to qive an investigator the ability to set up
his ?,wn conditions for interpreting tha mass spectra and to
control tha type of summary he wis?-es to see.  For example, if he


is interested in the allowable hydroqcn transfers associated with
329 spec ific process the proqram could be instructed to produce a
very specific summary.  Also,  we wish to add an interactive
proTram for answering questions about the results.  For example,
3~ investiqator should be able to find out easily how many
nroc?ssns involve cleavaqe of a specific bond and how strong their
r~sultinq P?S peaks are.

?'he ZNTSUY proqram is now used routinely by mass spectroscopists
at Stanfor enqaqed in investiqations of the mass spectrometric
fr3qmPrtation of various classc?s of organic compounds, primarily
;t.eroids.  A manuscript is now in preparation (Ref. 54) describing
the fraameltation of proqesterone and related compounds.   The
nrnqram was  used extensively in this work.  We are now heginninq a
jetailed examination of the fragmentation of steroids related to
tb3 an3rostane skeleton,  particularly the biologically important
t:>stosterones,  We propose t:, continue to use the INTSUP! program
in  its present form and as i+ is improved in support of these
st.udic?s.

I'ha qsnerator of rules that we POW have, 9ULEGEN, does a credible
iob of explaininq the reqularities summarized by XNTSUE1.   It has
foun3,  for example,  the well-known alpha -cl?avaqe fragmentation
PlY3,?2SS an1 beta cleavaqe followed by rearrangement in the low
r?sol,lti2n data for  fifteen aliphatic amines.  The program will be
extended in two important ways to increase its utility:   (i) the
proaram needs to be able to work with an increase3 number of
'=szrintive predicates in the qeneration of rules, and (ii) it
1 ._
neeas to h? qiver? a more flexible representation of complex
fraqmFntati.on mechanisms so that it can m3ce easily find rules
involving note than two bonds.

We will continua workinq with low resolution P!S data of the
753-200 nonofunctional aliphatic compounds studied previously in
t-he  context of the performance proqram.   These compounds are
dell-under-stood and thus provide a go03 test of the program*s
-Ffectiven2ss.
_          Tn order to insure generality in the theory
Formation nroqrams,  we will also test the system aqainst the high
rnsoluti.on mass spectra of the 68 estrogrnic steroids.  Since they
3re also w211-understood,  these compounds will show how well the
nroqram can deal with complex ring systems, multifunctional
Z3ntP5;1Il?3,  cleawaqes involvinq more than two bonds, and high
resolution data.

"he existinq proqrar?s arc in qsnd working order - within definite
Linits - s3 we expect to apply them to new sets of data from the
SS 13Sosatory as interest arises.  For example,  as the high and
10~ resolution KS from marine sterols are collected we expect to
use INTSUP! and F.ULEGE?J (at least) to assist in the interpretation
au3 generalization of these data,  Since these problems will
a?vanze th? state of knowledqc of PS,  it is not correct to look on
then as test problems,   However,  in the past  the programs
l?velone? most rapidly when they were applied to unsolved problems
3f interest to our colleagues in the chemistry department.

?or ??velopment of the interactive programs, we will rely heavily
nn the criteria of acceptability by Stlnfor3 users.   The   programs
themselves will be written in TNTZ:RLISP on the SUMEX computer,
Tyitialtv,  we will provide intaractive access to the control
nararaetors of the proqrams in order to allow users to tailor their


runt;  C.o their immediate interests.  Zater we hope to expand these
to 2lLow intcrroqatlon of the programs with respect to both
Tontents of the results and the program's reasoning steps,

3.  Ipplic3 tions to Biomedical Problems

v " c*n immediately offer +.c the user community the Planner,   for
analysis of HF/f!S in terms of molecular structure.  The program is
icsensi+ivs to the source of the 11s data, and we foresee
significant use of the proqram for analysis of spectra of mixtures
~itbont prior separation and spectra from the GC/HRFS facility
witb3ut adjitional proqr3mminq effort.  Examples of applications
3~~3s are  summarized below.

ii? wis-, h to exploit our existinq capabilities of the analysis of
hioloqical mixtures without prior separation (ref. 33).   This
approach will prove particularly useful in studies of mixtures
which 3c1 difficult to separate and analyze by GC.  Phytoecdysones
r~lsted to ecdysone,  an insect molting hormone, present swh a
problem.  EC of these compounds is very ?iFficult, although
high-pressure li_quid chromatnqraphy has recently been used to
zarry out separations.  This class of compounds represents an
lPt?resCI. nq and valuable test case for our combined MS and
computer techniques, particularly the specification and subsequent
acquisition of motastahlP defocussing data for precise linking of
nStrent and  frnqmenf ions in tFe spectrum of a complex mixture
(r3fs. 28, 33).  mad compourIs,  mixtures and current structure
eI.ucidatioo problems are available (Nakanishi, Columbia; Takemoto,
fohoku University, Senc?ai, Japan).  Althouqh most users cannot be
s2mnle+ely specific as to the n2itur?  of their future structure
?lX~cidation problems,  we feel that several of these problems can
b? handled by such an approach.

4s the str!lcture qenerater and its extensions are developed
further. we foresee con+inuing use of an interactive version
3p~lFe:l to specific problems of the user community.   As an
PX3ZlPler the work in collaboration uith the GFC project will
involve sttllies cf several classes of compounds extracted from
hunan body fluids (c. q.,  aromatic and aliphatic acids, various
classes of bases, amino acids and carbohydrates)  which contain
cenresentatives varyinq by substitutions about a small number of
~~L?cl~lar skeletons.  The generator can define all isomers uhich
aust be considered as possible solutions.

FDr th>se prnblems which are amenable to attack by library search
pfoce?!ures, e-q.,  screeninq of SC/LRRS runs  of marine sterols to
-#P?? out known COInpOUndS, ue pcop~se to use these procedures and
to investiqate extensions to them.  usinq a procedure related to
that described by 3cLaffcrt.y [K-S. IZwok, et al., J  Amer. Chem.
sot.,  95,  b185 (1973),  we seek to 3ltermino from nDdified library
search techriques the known structures which yield similisr
spectr;l. Utilizing the DEND%L structural manipulation routines,
me would then seek to determlnp those related structures (#hose
spectra are not in the library)  which are possible solutions. R
Librlrv,  incl.udinq Wiswesser Line E;otation names, Pxists (F, W.
YcLaEferty, private communication)  and would be of some utility in
ttTi.5  w3rk.

The YS facility in conjunction with our proqrams will be used in
studies of Ihe f 0llovir.q nature:


1)   Prof.  Djerassi - we plan use of the t?S facilities and computer
Droqrams in onqoinq research  connected with existing NIH-supported
stu lies on  steroids an:!  marine ster3fs and continued collaboration
with Prof.  Adlcrcreutz on estrogen mixtures isolated from body
flUidS.  Further collaboration with Praf. Adlercreatz will be on
structural studies of new estroqen mefaholitcs whose presence in
n!xt*lres has been inferred through our previous collaborative
?ffC.JlT?LS.

fhe work on marine sterols presently utilizes ?:/LE?r(rS and
frequently laborious separation procedures to isolate individual
fractions for HRMS analysis.  GC/HRMS will be a siqcificant
assistince in this effort.  We plan MS stildies of known marine
sterols (u+.ilizinq TNTS!JPI) to derive fraqmentation rules, which
than will be used in the Planner to aid structure elucidation of

rl?'W z ?mp2unds.

:s~e also plan further work on extensions of IqS theory in the
::tcr3i3 field, initially focussed on additional hiomedicafly
iapoctact classes of steroids related to the preqnane
fnroqesterones) and androstane (testosterones) skeletons.   This
work is currently being Carrie1 out by Dr. Smith ir. collaboration
with two visitinq senior scientists [Dr. Roy Gritter, Dr. Geoff
3?T3"l>V)  currently on saFha+ical l.Pave fellowships.

3)  Chemistry Department Collaborators - as indicated by the
responses summarized in the letters of interest (Appendix A),
*here is significant interest in use of the HS facility by other
NIS-supported members of the chemistry department.   All those
listed are famjliar with the technique of MS as applied to
structure elucidation problems.  East have 11sed MS frequently,
aacticalarly Prof. Van Tamelen in his studies of the cyclization
3f squalene  and related studies in the terpenoid and steroid
F;=ll,
L ir.    The interests of these collaborators are generally in HBES
and  7C/HRrnS,  with occasional use of other capabilities of the
97;t.arn.  The tvpos of compourds studied hv this group and an
ildiz2tinn of the amount of USC expected are summarized in the
letters of interest.

31   "J-anet+cs Pesearch Center  (Gr?C) :  (Profs. J. Lederberg, H. Cann;
Dr. fi.  Duffield)

?he body fluids analyzed by GC/LRMS
+D ?ate include urine, blood,  amniotic fluid and cerebrospinal
FLli.3.  'Sach body fluid is fractionated into the following
z9mpounr'l classes:
   9)  Drqanic acids and neutral compounds
   b)   amin? acids
    cl   carbohydrates
which ?ftcc appropriate derivatization are analyzed by
;C/T,BYS/coraputer system.  A library of known LRHS will serve as
!-!I? nrimary means of identifyinq metabolites from their
experimentally recorded LBYS.

T i.n t\oss instances where the LP?lS is insufficient for metabolite
!!3n+Ffication GC/Hl?PS data will be  necessary to determine the
cnmposi?ion of al1 ions in its mass spectrum.   These data will
qr?atlv enhance the prospects of identifying the metabolite in
susstinn.


It. is known [on past performance)  that if a compound is present in
303~ flnicl; at the level of 1 microqram per GC peak then gO0a
qliality HRlMS will be recorded (ion amplitude dynamic range of
1: lc?D, mass accuracy of +-5pnm) using the Varian !?AT 711 mass
sD?ctrnneter.  Tf the GC peak 3f interest contains insufficient
nateria?. f or a HRMS scan then preparative "JC could be used to
cDncen+rate that portion of the chromatograph effluent prior to
s C/HR ?!s *

2ri.or to the demise of the ACME comnutcr system {July 31, 1973) we
developed a GC/HRM!S system 3na applied it to the analysis of
3x+r3c+s from body fluids.  The followlnq example represents
r?slllts obtained with this system durinq its development.   The
example used was a routine analysis and was run to determine the
can;lhility of the overall system dqrinq its development and not as
3:: unknown. sample of extreme interest.

The total ion plot recorded durinq the lifetime of the GC/HRMS
analysis of an amniotic fluid is reproducea as Figure 1. A
complete hiqh resolution scan was recorded on each of the peaks
shoun in Fiqure 1.  Filinq timz of the time-shared ACME computer
s.yst?m did not allow the system to operate in a repetitive scan
nole.  For the sake of brevity only th2 GC/HRMS scan (# 1594,
"irlara 3)  correspondinq to qlutamic acid N-TFA O-n-Butyl ester
deriv3tivr  Fs produced.   (The corresponding GC/T,PYS scan is Figllre
2). The szan time per aeca?e of mass was 10.5 seconds, the
resolution 6,500 and the matchinq tolerance for the assignment of
emairic31 composition  set to 4 mmu.  The results shou that the
systam was capable of accurate mass measurement with a dynamic
ra!- qe in ion amplitude of about 33: 1 in this instance.

"h?  cessation of computer suppart  for the GC/HRMS system did not
allow a HFYS analysis to be made which was crucial to the
Lde?tification of a metaholite present in a body fluid.   Since
th3i time however, several instances have arisen where GC/HRMS
l3ta would have been collected in an effort to identify
net abolLtes not previously seen.

Th? cxp?cted sample throuqbput in the SRU project vith existing
personnel is expected to approach 5 to 7 body fluir's per week (15-
21 G:/I,?MS fractions to be run in the Genetics Department per
week) a  On avernqe GC/RFMS would be required on 1 - 2 samples per
wnck.

The research interests of the Medical School collaborators
relative ta the proposed F'S resource  are summarized in the letters
of interest {Appendix A).  The #S services required hy this
cormunity will include GC/LRMS (Forrest, Sera, Kalman for drug and
flruq metaholite identification,  Rabinowitz and Wilkinson for
nrostaqlandin identification,  Pobin for identification of
oxidized/r?duced rerlox pairs, Hollister foe Farihuana metabolites,
E! archas, naarotransmitters, Pair, polynmines and the prostatic
anttbacterial factor in urine); SC/HRRS (Trude11, drug metabolite
i?entification,  Kvenvolden,  structure of amino acids and related
,3mpoucc?s plus samples as required from interests described under
;C/'T,RMS) * In thnse instances where the biological extract contains
insufficient material for a GC/HRMS sc.an preparative GC, using
3xistFnq Instrumentation within the chemistry department, can be


usr3 +.n c?ncent.rate the materill prior to tha GC/!IRMS ar.alysis.
rf t.he m3t?rial of interest is obtained relatively pure by this
technique then ARMS analysis usinq direct snmpfe insertion into
f-h
. ..3 ion sotlrce would! be utilized.

ts m?ntion2d above, several of the computer proqrams have
immediate utilitv for assistinq with structure elucidation
3c~hlems*  Car example,  the Structure Generator proqram can answer
itciiCtllral isomerism questions Independently of mass spectrometry,
14-q. I to prov%de lists of isomers in conjunction with isomer
:ntercocversion problems such as carbonium ion rearranqements).
alec311se the program will he able to qenerate complete lists of
isomers with (or uithout)  some specified structural features, a
rn-,e3rchpr caE have confidence that no possibilities have been
,v?rlnoked.  Some interest in the structure qenerator has been
exprassed by representatives of the phacmacelltical industry.   The
n??.erafior could be use? to sugqest complete sets of structural
3 ltern3tivas for possible synthesis,  once a physiologically active
cnn qer~?r hss ken idectified.

In more qzneral terms, the structure generator cap he
cizhly suqqestive of new,  unexplored areas of synthetic
oraanir chemistry.  for example, the qenerator has been used
t7y 3 qr33u3t.e student in chemistry, Mr. Jan Simek, to
i?nntify the space of possible Diels-Alder condensation
products consisting of six atoms of any combination of
C3Cb3R,  nitroqen, oxyqer., and 5ulfrr in a
six-aamhered rirq with one double bond. A literature
se3r.. -h thronqh the Finq Index revealed that many of the
rinq sys*ens have never hcen r~port.33.

66


Ttruzture eluci?!ation is an important and difficult problem for
biomedical scientists.  Rany of them lack  +.he detailed chemical
backqroond necessary to be effici.ent in this endeavor.  Generally
speakinq, they also lack the freguently complex and expensive
?zfuipm?nt (e.q.,  high resolution mass spectrometers) to provide
spectroscooic data to assist them in solving problems of molecular
3truzt:1rs.  We plan to provide the chemical and analytical
exoertise to facilitate t?ie solution of their struct.ural problems.
This research aims at providing more powerful techniques for
Ietcrmininq molecular structures than are now routinely available.
r p ~319 icular, we have proposed (a) providinq extended MS services
3.c a means of collecting povarful analytic data for scientists;
(h) -lenelosinq {and extendinq) sophisticated computer programs to
issi_st wits the interpretation of the 3ata from mass spectrometry
3~3 alseuhare  (c) developing (and extending) novel. compilter
programs to agsist with formulation of the rules of
interpretation, and (d) applying these state of the art techniques
to problem;  of biomedical relevance.  3ur research group is thus
dedicated to a broad-based attack on the apD1ication.s of structure
?luci?Ia+ion to bioloqical and biomedical problems.

YhP Droposet'l research not only holds promise for significant
L>nq-term advances, it can 4av2  immediate benefits as well,   Many
nemberr of the biomedical community at Stanford have called upon
the "1s laboratory for assistance in the past and will continue to
?o so ir:  the future.  Thv proposed resource will provide the
non?uit for a substantial increase in the ntilization of P!S within
+h?  Stanforl! biomedical community.  The ability of the proposed            9
c=.33urze to interpret the experimental data it generates (enhanced
by the close proximity of the resource and biomedical community)
sh3llll rnsalt in a successfIll program of interdisciplinary
rQseesrc!l.

3RYS i; 3n important source of data for these problems, and
;C/!+R*S is still more important.  Previous investment by the NIH
in the !rarian FAT-711 HRES system at Stanford can he utilized now
an< built lpon for the future.  Continued operation of the K/MS
;vstam will qiva the Ttanford common ity access to state-of-the-art
spectroscopic techniques and to professional mass spectroscopists
w h 3 sari heln with onqoing problems.

?he computer programs themselves constitute a unique resource for
assistinq with the structure determination.  The previous NIH
Jrlnt sunported development of the programs.  Tn part, we are
reguastinq funds to exploit these programs.

OR? 3f the most siqnifican? aspects of this work is its
icterAiscipli.nary view of solving molecular structure problems by
in;elliqently directed search of the space of chemical graph
structur=s.  As a result of nosing the structure determination
probqem in this framework,  WC have been able to further the
knowledge about struct-urc elucidation jn at least three ways.
First, s3rn? of the knowledge used by analytlcaf chemists has been
made more precise for use in a computer program.  Second,
coIlifyi??q such knowledqe for the computer has led to the discovery


3f ICW rese;lrch areas to cxten3. our existirg knowledge of MS.
liC?VPra'l.  publications listed ir, the bihlioqraphy (RPfs. 42 and
fnIlDwinq) are reports of exactly this kind of research.  Finally,
t he zomp~t3r~s systematic search through th? space of possible
structures qives the prac+.icinq  scien+ist the confidence that no
3 tr :1 ctures were merely overlooked.  the efficiency of the program
?epenl!.s on the exclusions of many whola classes of compounds, but
th3 co~nnnfer will have reject el those classes using precise,
?xnlicitly stated criteria,

C!ur recent work on finding "IS interpretation rules (theory
fDrm3+ion) can provide additional unique capabilities for
assi.st!.nq with the problem solving.   We wish to continue this
research hacause it offers h3pe fnr a solution to the problem of
Fucnisbinq real-world knowledq? to computer programs -- in
natticillar t:, the computer proqrams  that assist with structure
eluciIatlon.  This is a pressjng problem in current AI research.
9igh norfocmance proqrams, of which DEIDRAL is most often cited,
ierlve their power from l;irqe stores of knowledqe.   Yet there are
no routine methods for infusing such systems with knowledge of the
task domail.  We believe our research in theory formation holds a
rev to the solution of this problem,


v.   FhCILIPfES F, EO?lIP!!ENT

fh? Stanford gass Spectrometry Laboratory will provide YS services
OR the Varian MAT-711 mass spe ztrometer couple:! with a
".?wl?tt-P3ekard qas chromatoqraph (Model 76108).   As service
ilstrunents for more routine mass spectral analyses, the
??kora+orY has a MS-? and CH-11 mass sp?ctrometcrs.

nata red11ction is currently provided on Stanford's IBM 3701158
z?,inpltcr il conjunction with a front-end PDP-l1/20 data
ac7:lisi.t ion computer.  (The PDP-11/20 presently has only the
zauahilitv for buffering peak profile data between the mass
spectrometer and the IEM 370/158 zoBputer at the Stanford Computer
3entar.j An alternative to buying time on thi? 3?0/158 is proposed
an3 discussed in the budget justification.

T!ha 8:
h,\    programs will he run on the NIH-sponsored AIM-SJJMEX
zoapzter facility (a PDP-10 computer with the TRNEX operating
syst3n,  192~ words of memory,  and adequate peripherals for our
purposes;) .  Running these programs on SUPIFX rill incur no charge.


A.   D3?1'D?F.L PURLICATIONS

(1) J.  Lel:?rb?lTCJ,  "DPWDRAL-64 -  A system for computer
2 0 !-! s t IT 1.1 r t. i ? n ,  Fnumeration and Vcltation of CIrqanic Polecules as
l'ree Structures and Cyclic Graphs",  (technical reports to NASA,
31s~ 3v3il3ble from the author and summarized in (12)).
   (la) ?3rt I.  Notational algorithm for tree
      structures (1964) CT.57029
   (15, Part II. Topoloqy of cyclic graphs 1196s) CR.68898
   {fz) P3rt III.  Complete ckemical graphs;
      enheddinq rings ir! trees 11969)

(2) J.  LeYerberq, "Zomputation of ~ol~cu13r Formulas for Mass
SD?ctrometry**, Holdc~-Day, Inc.  (1964).

{3) J.  ?,23prherg,  JJT~poloqical Yappinq of nrganic Molecule~*~,
Proc.  Nat. Acal. Sci., 53:1, Januasy 1565, pp.   134-139.

(`J) J.  Lp'f?rberq,  "Systematics of orq;zlnic molecules, qrsph
topnlony 3nd Hamilton circuits.  A general outline of the DENDRAL
Sy;ten.'J  NASA CR-YRR99 fl965)

(5) J,  L312rberq,  JiHamilton Circuits 3f Convex Trivalent
Folyhcdra [up to 18 vertices), Aa, Math. Monthly, !!ay 7967,

16) s;. L.  Sutherf3nd, '*DENDRAL - A Computer Program for
;enerati?q and Filtering Chemical Structurnsll, Stanford Artificial
rntelfiqenco Project Ycmo No. $9, February 1967.

17) 3.  Lei?rberq and E. A. Feigecbaum, JJMechanizatior! of
T?3u:tiv~ Inference in r)rqanic Chemistry", in P. Kleinmuntz (ed)
Form31 F?presentations for Hum3.r: Ju3qm?nt, [Wiley, 1968) (also
3t?nford Artificial Tntelliqec:e Project FlemD Yo, S4, August
1767).

(8) J*  Le32rk?rq,  "Online computation of molecular formulas from
llas5 IlllmbPr. '*  NASA CR-94977 (i968)

(9) I?. A.  Feiqenhaum and B. G. Duchansn,  "Heuristic DENDRAL: A Program
F-Jr Zeneratinq Explanatory Hypnthesss in Drqanic Chemistry", in
Pr3ceedinqs,  Hawaii International Conference on System Sciences,
3. K.  Yin3riwal3 and F. F. Ku3 (eds),  Yn iversit y of Hanaii Press,
1968,

(10) E. G. Ruchanar?, G. L. Sutherland, and E. A,  Peigenbaum,
`JHeuri?ti,c DRNDRAL: A Program  for Generatinq Explanatory
Pypothas~s in organic Chemistry".  In Machine Intelliger?ce 4 (B.
Se!tzer an?! D. Michie, eds)  Edinburqh University Press (1969).
(31s~ St3rlford Artificial Intelligence Project llemo Ho, 62, July

(11) F. A.  Peiqenhaum,  "Artificial Intelligence: Themes in the
Secnn4 ?eca3c1$.  Tn Fir,al Supplement t3 Pr3ceedinqs of the IFIP68
In?~cn?iti3z?l Conqress, Edinhurqh,  August 1968 (also Stanford
Artificial Tntelligence Project Memo No, 67,  August 1968).


{12) J.  I,e;lPfhPrg, f*Topolo,;ry of fl31ecul~s~~, in The !!nthematical
Tci f?rlC?F: -  h C3llection of Essays,  (ed. )  "9mmittee on Support of
?ssesrch i? the ?+athematical Szienzcls (COSRIMS), National Academy
-tf- Sciences - National Research Council, "J1.I.T. Press,  (1969) 8 PP.
3 7-51.

(13) 2.  Sutherland,  **Neuristic DENDRAL: A Family of LISP
'r9qraws",  Stagford ArtifScial Intellinence Project Fen0 No. 80,
Yarch 1969.

{lcr) J.  Lrsdecberq, G. L. Sloth-rland, R, 5. Puchanan, E. A.
Fziqsnbaum, A, V. Robertson, A. M. Duffielil, and C.  Djerassi,
't?np!ications of ArtifiC:al Intelliqence for Chemical Inference I.
The Yumker of Possible orqaci:: Compounds: Acyclic Structures
33nt3ininq C, H, 0 and Y'?.  Journal of the American Chemical
CO,i?ty I  31211 (Ray 21, 1969).

(15) A, H. Duffield, A. V. Robertson, C. Djerassi, B. G.
3uch3n3n, 2. 'L. Sutherland, E. A, Feiqenbaum, 2nd J.  Lederberq,
"Appllc2ti3n of Artificial Intzlligencs for Chemic31 Inference II.
Lqterpretation of Low Resolution Hess Spectra of Ketones".
Inurnal of the American Chemical Society, 91: 11 (t?ay 21, 1969).

(16) IT!.  S. Buchanan, G, T,. Sutherland, E. A.  Feigenhaum, "Toward
an Vndsrst3nding cf Information Processes  of Scientific Inference
i.n ths Context. of Orqartic Chemistry", in flachine Intelligence 5,
f3*  ?eltz?r and D, Ficbie,  eds) Edinburqh rJniversity Press
[1373),  (also Stanford Artificial fntelliqence Project Hem0 No.
"9,  SPDtemher 1969).

(17) J.  I.?derberq, G. L, Sutherland, B. G. Buchanan, and E. A.
"eiqenhaum,  1,A Heuristic Proqram for S3lvinq a Scientific
f?ferecce Problem: Summary of Motivation an4 Implementation", in
".  Bln3rji 6 FI.D, Hssarovic [eds,) Thenvtical Approaches to
Xon-NumPrizal Problem Solving, New York:  Springer-Verlag, 1970.
(4150,  S+23ford Artificial Intelligence Project Remo Yo. 104,
Vovemher 1963.)

(19, c. w.  Churchman rind B, G. Buchanan, "On the Design of
Indnztive Tystems:  Some Philosophical Problems".   Fritish Journal
f3r the Philosophy of Science, 20 (1969), up.   311-323.

/19t  S. Szhrnll, Al M. Duffield, ::. Djerassi, B. G.  Buchanan, G.
T ?? o  Su?her!3nd, E. A. Pei7enbaum, and 3.  Sederberg,  "Application
of atrtificlal Tntelliqence for Chemical Inference TII.  Aliphatlc
Fthsr; Dinqnosed by Their Low Resolution '?ass Spectra and Nt!R
Dita".  Journal of the American Chemical Society, 91:26 [December
17,  1969).

(231 A. !3achs, A. Y. Duffield, G. Schroll, C. njerazsi, A. 3.
Dplfino, R. S, Buchanan, G, L. Sutherfsnd, 9. h.  feiqenhaum, and
3.  Lcdcrberq, "Applicatior.5 of Artificial Intelligence For
:hemicsl  Inference. IV.  Saturated Amines Diagnose<1 by Their Low
Qesofut i0" Mass Spectra and Nuclsar Fagnetic Resonance Spectra",
3ournal of the American Chemical Society, 92, 6831 (1970).

(91) Y.M. Sheikh, A. Bucks, A.B. Delfin3, G. Schroll, A.!.
Dnffield, Z. Djerassi, F.G. Buchanan, G.L. Sutherland, E-A.
"?iq?~baum and J, Lederberq, ?qhpplications of Artificial
In!el?iqence for Chemical ?nference V.  An Approach to the


J>mput?r 53neration of Cycl?c Structures.   Differentiation Retween
911 the Possible Isomeric Ketones of Composition ChHlOO'*, Orqanic
@ass Spec+rometry, 4, 493 (1970).

(2?) A.  Ruchs, A.B. Delfino, A.PI.  Duffield, C. D jcrassi,
B .`;.  Yuchaaan, E.A.  Feigenhaum and J, Lederherq, "Applications of
&,rtiFiy-i
  . *L   al Tntelliqence for  Chemicsl Inference VI.  Approach to a
Ger.eral Method of Tr?terpretlnq Low !?osolution Mass Spectra with a
Znm ou+erfr , Helvetica Chemica A:ta, 53, 1394 (1970).

(23) ??.A. Feiqenbaum, E.G. Buchanan, and ,'I. Lederherg, "On Generality
3x3 Prs5lcn Solvinq:  A Case Study Using the DT;:NDBAL Program*'. In
nac+i.na Intelliqence 6 (B. ?leltzer and D. Yichis, eds.) Edinburgh
7ziv2csity Press (19711.  [Also Stanford Artificial Intelligence
?r-eject. YF?mo No.  137.)

(2Ul A. 9:lchs, A-B. Delfino, C. Djerassi, A.H. Duffield, B.G. Buchanan,
s.1.  Feiqsnhaum, J. Ledcrherq, G. Schroll, and G.L. Sutherland,
"The 4pplication of Artificial Tntelliqencc in the Interpretation
2E Low- Resolution mass Spectra", Advances in Mass Spectrometry,
5,  314.

(25, B.C.  Buchanan. and J. Lederherq,  "The Heuristic DENDRAL Program
for Explaininq Empirical Dataf4.  Tn proceedings of the IFIP
Cnnqre~s 71, Liubljana, Yugoslavia 11971).   (Also Stanford
Artificial Tntelliqence Project Memo No.  141.)

(25) P.G, Buchanan, E,A. Feiqenbaum, and J. Lcderberg, '*A Heuristic
Proqramminq Study of Theory formation in Science."  In proceedings
II? tbs SecoPrl Internationa 1 Joint Conference on Artificial
rntelli~qanse, Imperial Cofleqe, London (September, 1971).   (Also
Stanford Artificial 'Intelligence Project tierno No. 145.)

(27) Buchanan, B, G., Duffiel3, A.M.* Robertson, A.V., "An Application
of 9r%ificial Tntelligencc to the Interpretation of Mass Spectra",
Yass Spectrometry Techniques and Appliances, Edited by G. W. A.
"iIn?,  John Wiley F; Sons, Inc., 7971, p. 121-77.

(29) D.H. Smith, B.G. Fuchanan, B.S. Enqelmore, A.M. Duffield, A. Yeo,
p.n. Peiqenbaum, J. Lederberq, and C, Djerassi, "Applications of
?rt ificial Intelliqence for Chemical Inference VIII.  An approach
f:, the Computer Interpretation of the Aiqh Resolution 13ass Spectra
nf Complex Rolecules.  Structure Elucidation of Estrogenic
St7r3i3sn, Journal of the American Chemical Society, 94, 5952-5973
(lq73).

129) B-G. Buchanan, E,A, Peigenhaum, and N.S. Sridharan, *'Heuristic
ThPnry Formation:  Data InterpcetatiDn and Yule Formation". In
*actline Intelfiqence 7,  E'dinburqh University Press (1972).

(30)  Ledsrberq, J.,  "Rapid Calculation of  Molecular Formulas from
"as3 Value3,".   3nl.  of Chemical Education, 49, 613 (1972).

131) Drown, Al, qasinter L., Hjalweland, L., l*Constructive Graph
tahe1i.n q t7s inq Double C~set.s'~,  Discrete YathematFcs (in press).
( AlFO Computer Science nemo 319, 1972).

(32) F. G, Buchanan,  Review of Hubert Dreyfus'  "What Computers Canlt
n 0:  A Critique of Artificial. Reason",  Computing f)evietls (January,
1773).  (Also Stanford Artificial Intelliqence Project Remo No.


(31) D, 9. Sm;J.th, B. G. Buchanan, R. S. Enqelmorc?!, H. Aldercreutz and
- Djerassi,
UI       *'Applications of Artificial Intelliqence for Chemical
Tnferercc IX.  Analysis of Mixtures Wikhout Prior Separation as
Illustrated for EstroqensrV.  Journal of the American Chemical
solzz?ty 95, 6075 11973).

(34) ?. U, Smith, B. G. Puchanan, W. C, Yhitc, E. A. Feigenbaum,
_
. . .  Djerassi and J, Lederherg, "Applications of Artificial
Tntelliqence for Chemical Inference X.   Intsum.   A Data
Tnterpretati.qn Proqram as Applie d to the Collected Mass Spectra of
Pstroqenic Steroids",  Tetrahedron, 29, 3117 (1973).

(35) F. G.  Euchanan and N. S. Sridharan, "Rule Pormation on
Van-Yomoqeneous Classes of f3hjectstq.  In proceedinqs of the Third
Tctarnatfonal Joint Conference on Artificial Intelligence
(St?ilf?rfi, California, Auqust, 1973).  {Also Stanford Artificial
Intelliqence Project nemo No. 215.)

(6) n.  flichic and E.G. BUCh3nan,  "Current Status of the Heuristic
DENDRAL. Proqram for Applyinq Artificial Intelliqence to the
Interpretation of Mass Spec+ran.  August, 1973.

(371 P.  Scown and L. Masinter,  "An Algorithm for the Construction
of the Graphs of Organic Molec~les~~, Discrete rsthematics (in
press).  Also Stanford Computer Science Department Memo
;~Av-cs-73-367,  May,  1973)

(39) 9.H. Smith, L.M. Masinter and M.S. Sridhiran, **Heuristic
Q!?:E!DRAL:  Analysis of Molecular Structure," Proceedinqs of the
N4TO/CNA Alvanced Study Institute on Computer Representation and
Yanioufati3n of Chemical Information, in press.

(39) P.  Clrhart and C. Djerassi, wApplications of Artificial
Intslliqenze for Chemical Ir:ference XI:  The Analysis of Cl3 NMR
Data for Structure Elucidation Df Acyclic Aminesgl, J. Chem. Sot.
I??rkin II),  1753 (1973).

(40) L. Yasinter, N, Sridharan, and D.H. Smith, VtApplications
of Artificial Intelfiqence for  Chemical Inference XIX:   Exhaustive
??neration of cyclic and Acyclic Isomers.tl, submitted to Journal
of the American Chemical Society.

(41) I.. Yssinter, N. Sridharan, R. Carhart and D.H. Smith, HApplicatLons
of Artificial fnte.lliaenc~ for Chemical Inference XIII: An
AlqorFthm for tabellinq Chemical Graphs", submitted to Journal of
th? !m?rican Chemical Society.

(47) ThcJ Determination of Phenylalanine in Ser!lm by Mass
"raqm?n+oqraphy.  Clinical 9iochem.. 6 (1973).   By W.E*  Pereira,
T'.A.  F13con, Y. Hoya!?o, 9. Summons snd A.P. Duffield.

(43) The Simultaneous Quantitation of Ten Amino Acids in Soil
Yxtracts by Mass Fraqmentoqraphy.   Anal.  Si ochew.,  55, 236 (1973).
"y H.E.  Pereira, Y. Hoyaro, !J.F, Reynolds, 9-E. Summons and A-M.
Duffie1-i.

144) An Analysis of Twelve Amino Acids in Rioloqical Fluids by Bass
Fraqmsntoqraphv.  Anal. Chea., in press.   Ry F.P.  Summons, #.E.

73


P-Zf?FC3, W.?.  Beyn3lds, 1`.C.  Pindfleisch and A.tr. Duffield,

(45) l'hz :!u3ntitat_ion 3f R-AmLnoizDbukyric Reid in Urine by Mass
Fraqmentoqcaphy.  Cfin. Chim. Acta, in press.   ey W.E.  Pereira,
n .F.  Summons, B.E. Reynolds, T.C. Rindffeisch 3nd 1.Y. Duffield.

('IF;) ?hz npternination of Wh3nol in Plood an3 Urine by Pass
~r3~me~t.oqra phy*  Clin. Chim. 9ct3, in press.   ?y W.E.  Pereira,
c u
..-.  qnmmon s, T, C.  Pi~dfleiscE 31-11 R.N. I7uffielk3.


~ub?icsti.ons Descrihinq  DENDRAL-Relate d  Fesesrch But lJot Funded By
?tiiS  nrant

(47) An Automated Gas Chromatoqraphic Analysis of Phenylalanine In
ser,1m.  Clinical Biochem., 5, 166 [1972).   Ry E.  Steed, 8.
??C"if3* R.  Falpern, pl. D.  SoLomnn and A.!?. Duffield.

(48)  PVrrolizi3ine Alkaloids. XIX.   Structure of the Alkaloid
PracifDline.   Cofl.  Czech. Chen. Commur!., 37, 4112 (1g72).   By P.
Sedmora, 4.  Klasek, A.??.  Duffield and F. Santavy.

(`14)  chlorination Studies T.  The Reaction of Aqueous Hypochlorous
Aci? with Cvtosine.  Riochem. Riophys. Res. Commun., 48, 880
(1172).   Tzy w.  Patton, V. Sacon, A.M. Dufficld, F. Halpern, Y.
'loya?l>, k'.  Pereira and J, LedcrSetq.

(571 A Stl!.ly of thy Flectran Impact Fragmentation of Promazine
Sulphoxide and Promazine usinq Specifically Deuterated Analoques.
Austral .  J.  Chem., 25, 325 (19731.   !3y 4l.D.  Solomon, R. Summons,
2.  Pzreira and A.?!. Duffield.

(511 Snectrometrie de Masse VIII.  Eliminat.ion d'can Induite par
fmpat=t "lectronique dans Lr

Totrahydro-1,2,3,4-Napthtal-1,2.   Qrg.  Rass Spectre., 7,
'357 (1973).   By P. Petros, J.P. rYlorizur, J. Kossanyi and A.M.
Duffield.

1521  Chlorination StlIdies II.  The Reaction of Aq'leous Hypochlornus
Aci? with a-Amino Acids and Dipeptiqes.   Bio-him.  et Biophys.
Acta, 313, 170 [1373).  By W.??.  Pereira, Y. Hoyano, Ii. Summons,
V.A.  Dacon and A.!l. Duffield.

(43) Spectromstrie de wasse.   IX.  Fraqmentations Induites par
rmpazt Flez+ rorique de GLycoLs-   En Serie Tetraline.   Eull. Chim.
sot, Trance,  2105 (1973).  By P.  Perros, J.?. Morizur, J, rtossanyi
3n;l A.+!*  Doff ield.

(54) The Use of I?ass Spectrometry for the Identification of
!?~+.abolitss  of Phenothiazines.  Pr3ceedinqs Df the Third
Tn+ornationaf Svmposium or Phenothiazlnes, Raven Press, New York,
lQ73.   Ry A.M.  Duffield.

[SS)  chlorination Studies TV.  The Reaction of Aqueous HypochLorous
fizzi3 wi+b Pyriaidine and Purine Eases.  Biochem. Biophys. Res.
Zommun., 53,  1195 {1373) *   R-7 Y.  FJoyan3, V. fincon, R.E. Summons,
K.F.  ?erPira, R,  Halpern and A.!?. Duffinld.

(56)  F;as;s Spectrometry in Structural and stereochemical Problems.
ZCXXYVIT.   Electron Impact Tnduced Rydrogen Losses and gigrations
in S9,mc Aromatic Amides.   prq.  Hass Spectry., in press.   Ry A.M.
nrlfLfiClI1, ';.  deWartino and C. Djerassi.

(57)  StabLe Ts3tope Mass Fraqmentoqravhy:  Quantitation and
Tvdrctqen-Deuteri.um Fxchanqe Studies of Eight F"urchison Meteorite
Amino Rcizls.   Geochem.  it CosmQchim.  Acta,  suhaitted for
nublicati3n.   Eiy W.E.  Pereira, F.E. Summons, T.t. Rindfleisch,


4 . Pi *  Pllffi?ld, Es.  Zeitmari ani! J.G. Lawless.   (58) Pass
Yp-cfrometry in Structural and Stereochemical Problems CLXXXIII.
A Ctadv of the Electron fmpact Induced fragmentation of Aliphatic
911ehy3's.  J, Amer. Chem. Sot,, 91, 6814 (1969).  Ry R.3.  Liedtke
I?? c.  Pjecassi.

(`;?I Y3ss Speztrometry in Structural and Stereochemical Problems -
CYZVTI.  Flectron-Impact Tcducad Functional Group Interaction in
s-~~nzyloxycyclohexy1 Trimnthglsilyl Ether.   Orq.  ??ass Spectrom.
3,  257 (7970).  By Paul D. Woodqate, R3bi.n T. Gray and Carl
ni3r3::si.

IS'))  y3ss Spectrometry in S+ru,, -t.?lral and Stereochemical Problems -
CYzvIIr.  A study of the fraqmentation Processes of Some
a,B-Unsaturated Aliphatic Ketones.  Orq.  ?lass Spectrom., 4, 273
(1971)) .  By Younus M. Sheikh, h.?!.  Duffield and Carl Djerassi,

(c;lr "ass Snectrometry in Structural and Stereochemical Problems
err T,  Int?ractioc of Remote Functional Groups in Acyclic Systems
anan Efcctron Impact, J. Drg, Chem,, 36, 1796 (1471).   By ?I.
Sheehan, R.J. Spangler, M. Tkeda and C.  Djerassi.

(62) mass Spectrometry in Structural and Stereochemical Problems
TJVTT.  Praqmentation of Unsaturated Ethers.   Orq.  Yass Spectrom.,
5,  895 (1971) s   By 3. P.  Morizur and C. Djerassi.

(63) "4ass Spectromotry in Structural and Stereochemical Problems
"CVITT.  The Effect of Double Bonds Upon the McLafferty
Rsarranqemant of Carbonyl Compounds. J. Amcr. Chem, Sot., 94, 473
(1712).  Fv J.R, Dias, Y.Y. Sheikh and C. Djerassi.

(64) P3s;s Spectrometry in Structxtral and Stereochemical Problems
C~XV.  Rehsvior of Phenyl-Substituted a,B-Unsaturated Ketones Upon
Electron Tmpact.  Promotion of f!ydrogen 9e3rranqement Processes.
7, orq.  Them.,  37, 776 (1972).  By R.J.  Liedtke, A.F. Gerrard, 3.
Diekman  32:! c.  Pjerassi.

(55) Mass Spectrometry in  Structural and Stereochemical Problems
??????? o
?????    Delineation of C0mpetir.g Fraqment3tion Pathways of
Zompfex Yolecules from a Study of Yetastable Ion Transitions of
neuterate Derivatives,  Orq. Mass Spectrom., 7, 367 (1973),  UY
D.A.  Smith, A.M.  Duffield and C, Djnrassi.

(55) T!h= Clrhon-13 maqnetic Resonance Spectra of Acyclic Aliphatic
3 mi23s.  J. Amer. Chem. SOC., 95, 3710 (1973).  By Ii.  Fqgert and
- Djec3ssi.
. .

(57) The Carbon-13 Ruelear Plaqnetic Resonance Spectra of Keto
Steroids.  J, Orq. Chem,, 33, 3788 (1973).   Ry H.  Fqgert and C.
Dj?rzssi.

(68)  nlsss Spectrometry in Structural and  Stereochemical Problems
"CYUXVIIT.  The Effect of Heteroatoms [Jpon the Mass Spectrometric
?raqmen+ntion of Cyclohexanonps, J, 3rq. Them., in press, By
7.5.  Rlock, P.H, Smith, and C. Djarassi.

(63) M3ss Spectrometry in Structural and Stereochemical Problems
ZCY LI7:.  Applications of DADI, a Technique for Study of fletastable
T3"3, to F?ixt,lre Analysis, J. Amer. Chem. SOC., submitted for
aubfication.  By D.F. Smith, C. Djerassi, P.R. ilaurer, and U.


[70) Y2.s~ Spectrom~try in Sfruct.xral and Stereochemical Problems.
The PraqRentation of Proqesteror.e an:3 Alkyl-Substituted
Przq?secrnnes, in prenaration, by S, Hammerun and C. Djerassi.

f711  Ar?pLications of Molecular Orbital Theory +o the
Tctcroretation of Yass Spectra: Prediction of Primary
Frs7nentatioR Sites in Clrqanic Folecu13~,~~ Org. Mass Spectrom,, 7,
1241 (19731, by G. Loew, 8.  Chadwick, and 3.P. Smith.


PROGRESS REPORT


  TEXT OF 1973 ANNUAL REPORT FOR RESEARCH
PROJECT:  RESOURCE-RELATED RESEARCH -- COMPUTERS
            AND CHEMISTRY


Progress Report

Part A.   APPLICATIONS OF ARTIFICIAL INTELLIGENCE TO MASS SPECTROMETRY

3BJECTEVES:

Research activities carried out under Pact A of this project have been
directed toward extending the reasoning power of heuristic DENDRAL.
Heuristic DENDRAL reresents a paradigm for attacking problems in one of
the major areas of importance to any scientific discipline dealing vith
nnlecules,  the area of structure elucidation.   We have focused our
attenti.Dn on the use of heuristic programming techniques for analysis of
mass spectra and ancillary analytical data which can be obtained
utilizing a mass spectrometer.   It is convenient to discuss objectives,
progress and plans by examining three broad areas of activity in
research connected with ?art A.   He wish to note that these areas
conform to our overall strategy of PLAN-GENERATE-TEST.  We have shovn,
earlier, how pouerful this strategy is when applied to the task of
structure elucidation utilizing mass spectral data,   The areas and their
objectives are the following:

(I) PLSNNER:
  (a] Extend th e programs used for structure elucidation to
     structural analysis of complex molecules.
  (b) Assess the capabilities an3 limitations of the PLANNER.
  (c) Generalize the programming techniques to reduce compound
     class dependence.
  (4) Explor e the utility of ancillary data available from the mass
     spectrometer.

(11)   STRUCTURE GENERATOR:
  (a) Complete the exhaustive,  irredundant generator of molecular
       structures.

(b) Develop efficient constraints an the generator to exploit its
  potential utility.

(c) Exploit the concepts developed for the structure generator in
  solving various structure-problems (related to m.s. and
  others) and isomer-problems,

(III) PREDICTOR

(a) Extend the Predictor to still more complex molecular structures.
(b) Explore the design of experimental strategies, utilizing
  Predictor functions, t3 differentiate among candidate solutions.

We point out that the PLAN-GENERATE-TEST strategy, although applied to
structure elucidation, has potential utility as a strategy for solving
other chemical problems.  Similarly,  although we utilize mass spectral
data aln3st exclusively, the same heuristic programming techniques
allow facile extension to analysis of jata from other types of
analytical instrumentation.  These vere not objectives of the
original research proposal but seem logical extensions for future
work.  We have illustrated the potential of these techniques for
analysis of 13C NMR data (Carhart and Djerassi, 1973).   This is
discussed briefly under the PLANS section, below.

PRC)GRESS:

(I) PLANNER


The function of the Planner is to analyze mass spectral data acquired
in a compound.  The Planner StttiI1pt.S to derive structural information
from these data using the rules of behavior of compounds in the mass
spectrometer.

3bjective (a):  Extend Programs.

The Planner is presently embodied in a program which also contains a
set of functions to assemble this structural information into complete
molecules {a primitive Structure Generator) and to test these
molecular structures with other,  not necessarily mass spectral, rules
(a prinitive Predictor).  This performance program was written in
this way to provide a useful tool for chemical studies while more
qeneral versions of the Structure Generator and Predictor were being
~oveLor>ed.  This program and its performance have been described in
some detail in a publication and ia previous progress reports. A
manuscript (Smith, et-al.,  1973) has now appeared describing the
application of this program to the analysis of mixtures of compounds
without prior separation.

Dbjective (b):   Assess Capabilities.

We have extended the capabilities of the Planner so that we can
snalyza both lou and high resolution mass spectral data.   A low
resolution mass spectrum is regarded by the program as a pseudo high
resolution spectrum wherein possible elemental compositions of each
peak are limited only by the inferred molecular formula of the
compound.  This results in more ambiguity with a commensurate increase
in number of candidate solutions as would be expected considering the lower
specificity of low resolution data as compared to high resolution
data.

We have extended our capabilities for molecular ion determination
utilizing a heuristic search technique through the space of plausible
molecular ions.  This technique has had significant success even when
dealing with the low resolution mass spectra of compounds which
display no molecular ion,  for example the class of derivatized amino
acids  [trifluoracetyl, n-butyL esters) important to studies carried
out under Part B, below.

We have segmented the performance program to decrease the amount of
memory required for its operation.   This should increase the chances
for other groups to make use of the program.

llhe limitations of the present performance program are primarily the
requirement that some information about the class of compounds be
ic3own,  and that, for each class,  relatively detailed rules about the
Bass spectral fragmentation of this class be available.   The former
limitation results primarily from the nature of the program in that a
complete structure generator is not incorporated.  The primitive
structure generator available to the program can only place substituents
about an assumed skeleton.   This limitation will be alleviated uhen
a structure generator vith  GOODLIST and BADLIST constraints is
available (see Structure Generator, below).   The latter limitation
is mora fundamental,  but is characteristic of every spectroscopic
technique to one degree or another.  It Dust be assumed that analysis
3f a mass spectrum.!, alona,  m3y not lead to sufticicntly unambiguous
information about the structure of the compound yielding the spectrum.
It is Eor this reason that extensions of the programming techniques
to encompass data from other spectroscopic techniques are attractive.

3


3bjective (c):  Generalize Techniques.

Pe have carried out several successful experiments to ensure that the
performance program, used originally for analysis of estrogenic
steroids, retains only procedures which are compound-class independent.
By supplying fragmentation rules for other classes of compounds, we nave
successfully carried out structure elucidation of molecules in several
diverse classes including other steroijal hormones and related compounds
(progesterones, testosteroaes, androsterones), steroidal sapogenins and
derivatized amino acids.

Objective (d):  Explore Utility
Previous progress reports have summarized in some detail the ways in
which data from ancillary techniques in mass spectrometry (metastable
ion an3 low ionizing voltage data, labile hydrogen exchange) can be
used bg the program.  The utility of metastable ions for aid in
stucture elucidation continues as an active area of interest.
Experience with the program has inspired studies on metastable ions,
first,  to help delineate the course of fragmentation of molecules with
the purpose of extending and refining fragmentation rules used by the
program (Smith,  Duffield and Djerassi, 1973).  Experience with the
increased specificity of structural information with concomitant
reduction in analysis time when metastable ion information is
nvailaole (Smith, et-al., 1973) has led to a study of a new technique
for detection and analysis of netastable ions (Direct Analysis of
Daughter Ions, or DADI) and has illustrated the utility of this
technique in mixture analysis (Smith, Djerassi, Haurer and Rapp,
1973) o

Experience with the PLANNER has led to several research activities
relate3 to, but not supported by, this grant.  Our studies of estrogen
aixtures isolated from pregnancy urine have suggested new compounds
likely to be important in the human metabolism of estrogens.   Some of
these compounds are hitherto unreported structures and a synthesis
prograa is underway in Professor Djerassl "s laboratory to produce some
of these compounds.  The Planner will be used as one method of
comparison of the synthesized, authentic standards with those isolated
from pregnancy urine.

Hark is also being carried out to explore the fragmentation of model
systems possessing two heteroatoms in close proximity.   It is clear
from the first of these studies (Block, Smith, and Djcrassi, 1973)
that the fragmentation of these difunctional systems does not reflect
that of monofunctional analogs.  More groundwork is required in this
area to obtain better fragmentation rules for these systems,

II.   STRUCTURE GENERATOR

Objective (a):  Complete the Generator

l!he last progress report discu ssed the completion of both the basic
structure generator algorithm and program, which provide the capability     6'
for exhaustive generation of graph isomers of a given empirical formula,
uith prospective avoidance of duplicate structures.  Since the time of
the submission of that report, manuscripts describing the structure
generator, directed specifically to an audience of chemists, have been
submitted (Masinter, Sridharan, Lederberg, and Smith, 1973; Masintcr and
Sridharan, 1973).  Some effort over the past year has been devoted to


verification of the completeness and irredundancy of the method. He
have extended existing combinatorial counting algorithms to check that
'the numbers of isomers generated are correct,   iie have used an
interactive version of the generator to verify that variations (allowed
by the algorithm) of the mechanism of generation yield the same set of
.
Lsoiner3.  Ir, this nay we are n3w increasingly confident that the
program's performance accurately reflects the mathematically proven
algorithm on which it is based.

The Structure Generator has been briefly described, and placed in its
context within Heuristic DENDRAL,  in an invited paper presented at a
NATO/CBS sponsored conference on Computer Representation and
Manipulation of Chemical IaforaatiDn, held in Amsterdam in June, 1973
(Smith,  nasinter and Sridhlrsn, 1973).

We have also begun 60 develop techniques to expand the scope of the
3eneratBr.  One example,  which has been completed, is adding extensions
to the CATALOG.  The CATALOG contains the set of vertex-graphs from
uhich structures are assembled.  The original CATALOG was not sufficient
to generate all isomers of some potentially interesting compositions,
2. .,
Q   those involving graphs possessing nodes of degree >3.   He now have
a program which constructs complete sets of vertex-graphs containing
nodes sf degree >3 from the set of trivalent graphs in the original
EATAL3;.  IJ~ have thus extended the capabilities of the generator,
Other such extensions are discussed in the PLANS section, below.

Objective (b) :  Develop Constraints

It is absolutely essential that we provide the mechanism for
constraining the Structure Generator:   without constraints it is
merely a legal move generator,  as in a chess-playing program-   For
structure elucidation problems,  the Planner can determine many
features of the molecular structure from various types of experimental
Data such as presence of functional groups, and the numbers of double
bonds and rings.   Partial information of this sort can be used to
constrain the Structure Generator to the space of plausible candidate
structures.  From a graph-theoretic point of view, however,
constraining the graph generating algorithm is a difficult unsolved
problem.

14e are presently formulating sever31 types of constraints to apply to
the structure Generatoi,  Some types of constraint await the development
of neu mathematical tools (see PLANS),  vhile others can be immediately
implemented with relatively minor alterations to the algorithm.   The
class of constraints presently receiving attention deals with types
of unsaturation (rings or double bonds) desired in the final
structures.   Related to this constraint is the constraint of number of
quaternary carbons present.  The former information (number and
nature of multiple bonds) is readily available from several spectroscopic
techniques,  uhile the latter may be obtained from 13C NtiR.   The
implementation of this class of  constraints will be used as the model
for future implementation 3f a GOODLIST (structural features known to
be present) and a BADLIST {structural features known to be absent).

It is possible that some types of constraints may not be easily
implemented within the algorithm.  Thus,  retrospective tests of
isomer;  may be required to search fDr desired or unwanted features.
We have developed some new approaches to graph matching which seem to
be significantly more efficient than previous methods.   Should
prospective implementation of a constraint prove difficult, we vi11

5

83


have at our disposal some powerful graph matching tools to exercise
the constraint.

3bjective (c) :  Exploit th? Generator for Structure Elucidation

ble have demonstrated the utility of some subsystems of the structure
generator, e.g., the LABELLER,  by exploring some problems of isomerism
noted in the chemical literature.   We have corrected the member and
presented the identities of isomers formed by different substitutions of
slkyl chains about a porphyrin nucleus.  tie are presently exploring some
problens of isomerism of carbocyclic ring systems, specifically C10~10
snd (CH) 10 and CJOHZn-4 tricyclic ring systems, n = 8 - 12, related to
the mechanistics of isomeric interconversion.

We hsve the complete list of all topologically possible 1176 &membered
Diels- Rider ring systems,  using any combination of C,N,O and S.   This
list was generated using the PARTITIONER and an extended version of
the LABELLER.  These are all the 6-membered ring systems that can be
embedded in structures resulting from the well-known Diels-Alder
reaction.  Of the 1176 possible ring systems, approximately 80% are
unreported in the Ring Indax.  tiany of these are chemically unstable -
underscoring the need for a BADLISF implementation for the Cyclic
Structilre Generator.   However,  many of these unreported ring systems
are certainly chemically plausible.  Awareness of such gaps in
relatively simple synthetic categories might lead to discovery of new
categories of compounds with important biological effects,

{III) PREDICTOR

The function of the Predictor in the PLAN-GENERATE-TEST strategy is
to perform a detailed evaluation of candidate solutions (structures)
to a structure elucidation problem.  It may use a more detailed model
of spectroscopic behavior than that embodied in a Planner to attempt
to differentiate among possible solutions.

3bjective (a):  Extend the Program

We have extended and generalized the Predictor used previously for
saturated, aliphatic,  monofunctional compounds.   Given a list of
structures and rules of fragmentation processes, it will predict a mass
spectrum for each structure.   Prediction of relative ion abundances is
crude,  but previous work has shown that even crude measures of ion
abundance are usually satisfactory.  The predicted spectrum can be
matched then with the observed and candidates ranked according to the
quality of the match.   The  program works with structures and rules of
any complexity,  An interesting philosophical question is how much
klouledge should be brought to bear on interpretation of the data at the
Planning vs.  Predicting stages of analysis.  It is our feeling that if
more can be accomplished during Planning to constrain the Structure
Generator, the analysis vi11 be more efficient.  On the other hand, some
knowledge can be utilized only if a complete structure is specified, so
that its use is restricted to a predictive role.   boreover,  Predic,tor
Functions have a greater utility,  as indicated in the subsequent-
section.

Objective (b):   Differentiate Structures

The Predictor has a more obvious application in the design of


experiaental strategies to differentiate among candidate structures.
Rules of spectroscopic hehrlvior utilized during Planning demand the
presence of some data to evaluate.   The Predictor can then be used to
request additional data from any  source to aid in differentiation. We
have explored this approach by analyzing the spectrum of a compound
nith the performance program.   The Predictor uas  used to evaluate the
the set of candidate structures to define the minimum number of
metastsble defocussing experiments necessary to achieve a unique
solution.  Thus,  no time need be spent acquiring unnecessary or
redundant data.  Clearly,  this has important implications for future
work in that many different types of data (e-g,, NMR, IR) might be
requested by the Predictor to facilitate identification.

PLANS

For tha remaining period of this grant we Propose to carry out the
following extensions of the research outlined above.

(I) PLANNER

The major area of activity related to the present version of the Planner
will be to focus our attention on using the program in support of
chemical studies outlined under Part i3 (see below).   The chemical
extraction and derivitization procedures used in the analysis of body
fluids restricts the types of compounds present in each separated
fraction.  Such simplifications make this a problem more amenaDLe to
attack.  Only certain class'es of compounds are present in each fraction,
and we have some knowledge of the mass spectral fragmentation of these
classes.  He wish to couple the program to the results of library
matching procedures so that we direct our efforts to structure
elucidation of those components which have not been previously
identified.  This is particularly important in the context of analysis
problems such as those discussed under Part B,

He propose increasing the utility of the program by removing two
present constraints:  (a) allow unspecified "dummy" atoms in the
skeleton instead of requiring a rigidly fixed structural skeleton,
and (b) allow fragmentation processes to be specified more flexibly -
in particular,  allow fragmentations in substituents on the skeleton
instead of requiring all fragmentations to cut through the skeleton.

Although we are presently uncomfortable with immediate coupling of
the structure Generator to the Planner, we propose continued
exploration of the problems of controlling the generator automatically.
Actual implementation auaits a more comprehensive treatment of the
problem of constraints.

II.   STRUCTURE GENERATOR

The inclusion of a reasonable set of constraints is obviously required
and will be the subject of most of our future development work. We
plan to develop an interface to the present interactive version of
the Structure Generator that speaks a more chemical language.   This
intecfsce will be designed to avoid the present requirement that the         b..
user knov something about the program before he can use it.   As the
Dptinum method for implementation of a constraint is determined, the
interface will be extended to translate the usual specification of the
constraint in chemical terms into rules acting at the level of the
program.  As we stressed ir, development of the PLANNER, there are
considerable advantages to building a powerful program in an


incremental fashion.  These steps are loyically directed to our longer
term goal of developing a useful structure elucidation tool for the
chemist, based on the structure generator,

There are several other areas of interest which are l,eripherally related
to the problem of constraints and which will occupy our attention.   The
Structure Generator knows no chemistry other than atom names and their
associated valence.  There are several important areas where this is an
immediate problem.  For example,  the program has no explicit awareness
of the aromatic resonances,  leading to a remediable redundancy in the
list of isomers.  An aromaticity-predictor is also indispensable for
anticipating chemical behavior of a structure.

We wish to deal with types of isomers besides simple connectivity
isomers.  We need to have the facility for assembly of molecular
sub-structures (the usual type of information inferred from
spectroscopic data) uhen such an assembly yields new rings or multiple
bonds,  All the above questions need a reexamination of the fundamental
mathematical considerations.  The present algorithm has been proven to
yield complete and irredundant solutions.  In devising new algorithms or
variants of the present one, the burden of proof can be reduced to (the
usually easier) equivalence to the previous algorithm,   Professor Harold
Brown,  who was the mathematician instrumental in initial development of
the labelling algorithms for structure generation, will be with us again
for several months to help attack the problems outlined above.

XII.   PREDICTOR

Although the Predictor has been essentially finished for our own
internal use,  we propose to spend a modest amount of time in the
coming months making it more usable by others.  In particular, we
uish to extend the initial work on predicting the new experiments
necessary for distinguishing among candi.-iate structures (e.g.,
predicting that a metastable peak at mass 70.1 vould confirm one
structure and disconfirm another).  In addition,  we plan to vork on
cataloging some existing sets of mass spectrometry rules in such a
way that the program can be easily used for different classes of
problems.

d

.


Part A references (Published or submitted during year)

R. Carhart and C. Djerassi,  "Applications of Artificial Intelligence
for  Chemical Inference XI....

D.H. Smith, B.G. Buchanan, R.S. Engelmore, H. Adlercreutz, and
C. Djerassi,  **Applications of Artificial Intelligence for Chemical
Inference, IX.  Analysis of Mixtures Without Prior Separation as
Illustrated for Estrogens,t' J. Amer. Chem. Sot., September 5, 1973.

D.H.  Smith, A.M. Duffield, and C. Djerassi, '*Mass Spectrometry in
Structural snd Stereochemical Problems, CCXXII.   Delineation of
Competing Fragmentation Pathways of Complex Molecules from a Study
of Metastable Ion Transitions of Deuterated Derivatives," Org. Mass
Spectrom.,  7, 367 (1973).

D.H.  Smith, C. Djerassi, K.H. Maurer, and U. Rapp, "flass Spectrometry
in Structural and Stereochemical Problems, CCXXXIV.  Applications of
DADI, A Technique for Study of Metastable Ions, to Mixture Analysis,"
J.  Amer. Chem, Sot., submitted (1973).

J.H.  Block, D.H. Smith, and C. Djerassi, "Mass Spectrometry in
Structural and Stereochemical Problems, CCXXXVIII.   The Effect of
Heteroatoms upon the Zass Spectrometric fragmentation of Cyclohexanones,"
J.  Org.  Chem.,  submitted (1973).

L.M.  tiasinter, N.S. Sridharan, J. Lederberg, and D.H. Smith,
"Applications of krtificial Intelligence for Chemical Inference XII.
Exhaustive Generation of Cyclic and Acyclic Isomers,l' J. Amer. Chem.
sot.,  submitted (1973).

L.H.  Hasinter and N.S. Sridharan, 18Applications of Artificial
Intelligence  for Chemical Inference, XIII.  Labelling Objects Having
Symmetry, J. Amer. Chem. Sot., submitted (1973).

D.H.  Smith, L.M. Hasinter, and N.S. Sridharan, tlHeuristic DENDRAL:
Analysis of Molecular Structures," to be published in the proceedings
of the NATO/CRS Advanced Study Institute on Computer Representation
and Hanipulation of Chemical Information.

N.S.  Sridharan, nComputer Generation of Vertex Graphs", Stanford
Computer Science Memo CS-73-381, Stanford University, July, 1973.


Part B-i  Gas Chromatograph -  Mass Spectrometer Data System Development

3BJECTIVES AND RATIONALE

The objectives of this part 3f the research project are the
improvement of GC/MS data system capabilities and the coupling of
extracted data to the Heuristic  DENDRAL programs for analysis-   ide
ultimately seek a substantial degree of interaction between the
instruaentation and the analysis programs including computer
specification and control of the data to be collected.   In addition
to the development goals,  this portion of the project provides for the
day-to-day operation of the GC/MS systems in support of mass spectrum
interpretation computer program development (Parts A and C) and
applications 0 f GC and MS to biomedical and natural product sample
analysis nith collaborators.

Our rationale for this approach is that the overall system should be
designed for problem solving rather than just for data acquisition.
This implies that analytical computer programs, after review of
available experimental data,  could be able to specify additional
information needed to confirm a solution or distinguish between
alternative solutions.  Such reguests could be passed back to an
instrument management program to set up proper instrument parameters
and collect the additional information.  Our initial objectives to
implement an on-line, closed-loop system using the ACME computer
facility have met with a number of difficulties.  These grow
principally out of ACME's limited computing capacity and commitments
as a general time-sharing service.  In addition,  the scanning high
resolution mass spectrometer has inherent sensitivity limitations,
which do not preclude a demonstration but rather limit the practical
sample volume which could be analyzed.   Until such limitations can
be overcome,  particularly in terms of computing support, ue have
focussed our efforts on an open-loop demonstration of such an
approach.

PROGRESS

Progress has been made in demonstrating a GC/fiigh Resolution Mass
Spectrometry capability,  in further developing automated data analysis
algorithms,  and in planning for the implementation of a data system
for the collection of metastable ion information,  Progress in these
and other areas directed toward the main research goals has been
impacted by a transition in computing support uhich is still underway.
This transition,  discussed in more detail below, vas occasioned by
the phase-over of the ACME computing facility, which we had been
using,  from NIH grant subsidy to a fully fee-for-service operation
under Stanford University auspices.

Summaries of the results and problems encountered in each of the areas
follow.

Gas Chromatography/High Resolution Mass Spectrometry (GC/HRMS)

iie have verified the feasibility of combined gas chromatography/high
resolution mass spectrometry (GC/HRMS).  Using programs described in
previoils reports,  we can acquire selected scans and reduce them
automatically.  The procedures are slow compared to Vtreal-timeN1
because of the limitations of the time-shared ACME facility.   Ue have
recorded sufficient spectra of standard compounds to show that the


system is performing well.  A typical experiment which illustrates
some of the parameters involved was the following.   A mixture
(approximately 1  aicrogran/c3mponent) of methyl palmitate and
methyl stearate was analyzed by GC under conditions such that the GC
peaks were well separated and of approximately 25 sec. duration-   The
mass spectrometer was scanned at a rate of 10.5 set/decade, and a
resolving power of 5000.  The resulting mass spectra displayed peaks
over a dynamic range of 103 to 1 and were automatically reduced to
masses and elemental compositions without difficulty.   Mass
measurement accuracy appears to be 10 ppm over this dynamic range.

14e have begun to exercise the GC/HRMS system on urine fractions
containing significant components whose structures have not been
elucidated on the basis of low resolution spectra alone.   k'hereas
more work is required to establish system performance capabilities,
two things have become clear: 1)  GC/HR:fS can be a useful analytical
adjunct to our low resolution  SC/MS clinical studies (Part B-ii),
and 2)  the sensitivity of the present system limits analysis to
relatively intense GC peaks.  This sensitivity limitation is inherent
in scanning instruments where one gives up a factor of 20-50 in
sensitivity over photographic image plane systems in return for
on-line data read-out.  This limitation may be relieved by using
television read-out systems in coniunction with extended channeltron
detector arrays as has been proposed by researchers at the Jet
Propulsion Laboratory.  iJe can nevertheless make progress in applying
SC/HRMS techniques to accessible effluent peaks and can adapt the
i.mprDved sensor capability when available.

These experiments have also shown that the ACHE computer facility
cannot reliably provide the rapid service required to acquire and
file repetitive spectometer scans.  This problem is to be expected
in a heavily used time-shared facility without special configuration
for high rate,  real time support.  Excepting possible requirements for
real time data analysis [such as in a closed-loop system), this
problem could be solved by implementing a large local buffer (e.g.,
disk) at the front-end data acquisition mini-computer.   We are
exploring this possibility in conjunction with the overall planning
for computer support discussed below,

Data Analysis Algorithms

Al   Peak Resolution

One of the significant trade-offs to be made in GC/HRMS is that Of
sensitivity versus resolution.  In maintaining high instrument
resolution (in the range of 5,000-10,000) while scanning fast enough
to analyze a Gc effluent peak (approximately 10 secldecade), system
sensitivity is constrained as discussed above.   We have worked on a
method for reducing instrument resolution requirements through more
sophisticated computer analysis of a lover resolution output, In
effect this transters the burden of overlapping peak detection and
mass determination to the computer instead of reguiring inherently
well resolved data out of the instrument.  The advantage comes in
better system sensitivity,

Unresolved peaks are separated by an analytical algorithm, the
operation of which is based on a model peak derived from known
singlet peaks in the data.  Actual tabulated peak models are used
rather than the assumption of a particular parametric shape (e.g.,
triangular, Gaussian, etc.).  This algorithm provides an effective


increase in system resolution by approximately a factor ot tnree
thereby effectively increasing system sensitivity.  By measuring
and comparing successive moments of the sample and model peaks, a
series of hypotheses are tested to establish the multiplicity of the
peak, minimizing computing requirements for the usually encountered
simple peaks.  Analytic expressions for the amplitudes and positions
of component peaks have been derived in the doublet case in terms of
the first four moments of the peak complex,   This eliminates time
consuming iteration procedures for this important multiplet case.
Iteration is still required for more complex multiplets.

B.  GC Analysis

The application of GC/MS techniques to clinical problems as described
in Part B(ii) of this proposal has indicated the desirability of
automating the analysis of the results of a GC/MS experiment.   The
SC/tiS output involves extracting from the approximately 700 spectra
collected during a GC run,  the 50 or so representing components of
the body fluid sample. The raw spectra are in part contaminated with
background "column bleedI and in part composited with adjacent
constituent spectra unresolved by the CC.

We have begun to develop a solution to this problem with promising
results.  By using a disk-oriented aAtrix transposition algorithm, the
array of 700 spectra by 500 mass samples per spectrum can be rotated
to gain convenient access to the "mass fragmentogram" form of the
rlata.  The transposition alqorithm avoids many successive passes over
the input data file as would be required in a straightforuard approach.
By yenzrating a reorganize3 intermediate file, time savings by
factors of 5-10 are achieved.

The fraqmentogram form of the data displayed at a few selected mass
values,  has been used at Stanford, MIT, and elseuhere for some time
I to evaluate the GC effluent profile as seen from these masses.   Mass
fragmentograms have the important property of displaying higher
resolution in localizing CC effluent constituents.  Thus by
transposing the raw data to the mass fragmentogram domain, ye can
systematically analyze these data for baselines, peak positions, and
amplitudes, and thus derive better mass spectra for the relatively
few constituent materials.  These are free from background
contamination and influences of adjacent GC peaks unresolved in the
overall gas chromatogram.  These spectra can then be analyzed by
library search techniques or first principles as necessary.

We have applied a preliminary version of this algorithm to several
urine samples.  These contain several apparently simple peaks which
in fact consist of multiple components.  The algorithm performs well
in separating out these constituents although further testing is
required.

Closed-loop Instrument Control

In the long term, it could be possible for the data interpretation
software to direct the acquisition of data in order to minimize
ambiguities in problem solutions and to optimize system efficiency.
The task of deciding among and collecting various types of mass
spectral information (e-g-, high resolution spectra, low ionizing
voltage spectra,  or sel?ctzd metastable ion information) under
closed-loop control during a GC experiment is difficult.   Problems
arise because of the large requirements placed on computer resources


and present limitations in instrument sensitivity or data read-out
imposed by the time constraints ot GC effluent peak widths.
solutions to these problems may not bc economically feasible within
currently existing tecc!irioloqy but  seem achievable in the future- We
are studying this problem in a manner which would entail a multi (two'
DC three)  - pass system.  This permits the collectioc of one type of
data (e.g., high resolution mass spectra) during the first GC/MS
analysis.  Processing of these data by DENDRAL will reveal what
additional data are necessary on specific GC peaks during a subsequent
GC/MS run.  Such additional data could help to uniquely solve a
structure or at least to reduce the number of candidate structures.
This simulated closed-loop procedure could demonstrate the utility of
DENDRAL type programs to examine data, determine solutions and
propose additional strategies,  but will not have the requirement of
operating in real-time,  Some parameters in the acquisition of
particular types of information, such as metastable data, will
require computer control,  even in the open-loop mode.

We have considered plans to implement tuo aspects of instrument
control,  in addition to the magnetic scan control implemented for GC
operation and reported previously.  These include system resolution
control,  such as would be required t3 change from normal spectrum
scanning mode to metastable scanning mode,  and high voltage control
necessary to selectively measure metastable ion fragmentation data.
In addition to these we have considered the discrete switching of
various electronic mode controls which are straightforward and not
discussed in detail.

Implementation plans for computer control of these instrument
functions have been delayed because of the ACHE computing facility
transition which diverted the necessary hardware and software
manpover.

Resolution control involves changing the widths of the slits at the
exit of the ion source and the entrance to the ion multiplier
detector.  Additional source and electrostatic analyzer voltages must
be controlled to optimize performance, as discussed later.   Mechanical
slit adjustment is accomplished on the MAT-717 instrument by heating
wires uhich support the slit jaws.  The resulting expansion or
contraction of those wires move the spring-loaded jaws. As
implemented by the manufacturer,  the time constants involved in
heating the control wires are 5-10 seconds.  It is possible to speed
this up to approximately 0.5 seconds by application of a controlled
over voltage decreasing to the appropriate equilibrium value for the
desired slit width.  This was demonstrated by a series of experiments
on an extra slit assembly mounted in a vacuum jar in our laboratory.
Cooling of the wires is relatively fast in the way they are mounted
so no problem exists in that direction.

It is desirable to have feedback to indicate the actual slit width
achieved rather than relying on a slit assembly calibration.
Stretching of the support wires or changes in the spring tension
under temperature cycling would change this calibration- An
optical scheme to measure slit width in situ is possible.   We do not
contemplate implementing this feedback immediately because it requires
major chanqes to the instrument flight tube.

Two types of metastable ion relationships are obtainable by
suitable control of the double-focussing instrument,  First, for a
given daughter ion,  one can trace the parent ions which give rise to


it.   second, for a given parent ion one can trace the various
daughters to which it gives rise,  The first measurement ("metastable
defocussing") is the more straightforward for this instrument since
parent ions can hC? enumerated by a si:aple scan of the accelerating
voltage, holding the electrostatic analyzer (ESA) voltage and
magnetic  field constant.  The second type of scan requires the
coordinated scan to two of the three fields.  Ye feel that joint
computer control of the accelerating voltage and ESA voltage is the
simpler approach since the magnetic field is more difficult to set
and monitor because of hysteresis effects.  For a resolution of 1000
in the metastable ion mass measurement,  the voltages must be set to
approximately .O 1-.02X accuracy.  This requires a 14-16 bit digital-
to-analog (D/A) converter to control the input (10 volts) to the
operational amplifier which generates the high voltage.   Similar
D/A controls of ion source voltages for ion current and focus
optimization can be implemented using optical isolators to allou
vernier control of the various high voltages around the nominal
8KV values.

Computing Transition

As mentioned earlier, the transition of the ACME computing facility
from NI1-i subsidy to Stanford -sponsored fee-for-service operation has
impacted our development efforts this past year.   Both the low
resolution instrument used for routine body fluid analysis research
nnd the high resolution instrument are affected.  All computing
support was previously obtained from the ACME facility, much of it
as core research without explicit transfer of funds.   The transition
has required consideration of both technical and economic factors.
The neu facility represents a combination of the previous ACME
interactive and real time computing load with various administrative
and batch computiag loads on a new IBM 370/158 computer.   This new
environment will have even more difficulty in supporting real time
computing needs than ACNE did.  No real time support has been
available since the 360/50 service was discontinued on July 31,
although terminal service was reestablished in mid-August,   Data
acquisition service via the IBM 1800 is expected to be operational
by early Novemher-

For the high resolution instrument, this transition, as a minimum,
necessitates an i.nterfacc modification (we previously sent data
through the IBM 2701 interface no longer to be supported).   It also
amplifies the problems we ancountered in sending and filing high
rate mass spectrometer data (particularly during GC/MS runs).   These
problems mould be present to s3me extent in any general time-sharing
service machine without specific hardware and software configuration
provision for these needs (such provisions for real time support had
been proposed in our SUMEX computer application).

After examiLing a variety of alternatives, we conclude that a
d.?dicated mini-computer solution (built around a machine uith the
arithmetic capability of a PDP-11/45) would be highly attractive
technically and relatively inexpensive.  A stand-alone mini-computer
system would cost in the range of RSO,OOO-$60,000, augmenting
existing equipment, plus approximately $9,000 per year maintenance
and $2,000 per year for supplies.  Estimates for 370/158 support,
based on current chargin,; ril~jc)r`i'ih~:s and previous utilization
experience,  run from 535,000-550,000 per year.  This spread is caused
by uncertainties in the effects of planned measures to increase
operating efficiency and possible changes to the rate structure- In


any case,  the mini-computer approach pays for itself in 7 to 2 years
of operation and provides the responsiveness or` a dedicated machine
for real time support.  Unfortunately our existing budget does not
provida for this solution.  The budget is very marginal for purchase
of computing support from the 370/158 as well.  This later approach
is the only currently available one, however, since it can be
implemented with relatively low start-up cost.  The effect of budget
limitations appears in terms of a reduced number of samples which
can be run.  We have attempted to minimize the other budget costs
(manpower principally) to increase the computing funds available.
This will necessarily impact our development goals.  We hope, in the
renewal application for DEUDRAL support,  to be able to implement the
more effective mini-computer approach for the high resolution
spectrometer as a longer term solution.

We have undertaken an interim mini-computer solution for the low
resolution spectrometer (Finnigan 1015 quadrupole) which is primarily
used for our body fluid analysis studies.   For the same reasons outlined
above,  a mini-computer solution is attractive.   In the case of the low
resolution quadrupole instrument,  a lesser capacity machine will suffice
for immediate data acquisition and display functions.   We have
implemented such an interim system on a PDP-11/20 machine available from
other funding sources.  This system,  which is now operational, allows
the acq?lisition of GC/YS data,  limited by the capacity of the DEC tape
storaqe medium to approximately 600 spectra, per experiment.   Por
certain types of GC analyses,  up to 1030 spectra per experiment are
required so this limits. to some extent,  the utility of this interim
system.  A calcomp plotter is supported for display purposes.   A fixed
head disk 'provides for library search procedures which are still being
converted from the ACME system.   We have applied to the NIH-GMS for
funds to augment this system  in order to relieve current limitations as
part of a Genetics Center research proposal.

FUTURE PLANS

Our future plans are basically to continue development along the lines
outlined above.  We vi.11 complete the computing support transition steps
described.   These include primarily establishing a connection to the new
370/158 facility to provide interim support for the high resolution
system.  We will pursue additional software and hardware development
goals as far as possible within the limited budget available,   These
efforts  will concentrate for the most part on bringing up a metastable
ion analysis data system.  It should be reemphasized that the manpower
levels proposed in the follow-on budget have been minimized to allow for
purchasing computing time on the 370/158.  The allocated manpower is
required primarily for instrument operation and maintenance with minimal
provision for development efforts.


Part B(ii).  Analysis of Body Fluids by Gas Chromatography/Mass
Spectronetry.

The chemical separation of urine into the following fractions prior
to GC/MS analysis has been described in previous DENDRAL Reports:

free acids  (analyzed by gc/ms as their methyl esters)
amino acids (analyzed by gc/ms as their N-trifluoroacetate
   n-butyl ester)

carbohydrates (analyzed by gc/ms as their trimethyl silyl ether
   derivatives)

hydrolyzed acids (analyzed by gc/ms as their methyl esters)
hydrolyzed amino acids (analyzed by gc/ms as their
   N-trifluoroacetate n-butyl esters)

During the past year we have extended these methods of fractionation
to the follouing body fluids:  bload [after an initial precipitation
of proteins by the addition of ethanol) and amniotic and
cerebrospinal fluids.  The following summarizes the results obtained
from an analysis of these fluids during the past year by gas
chromatography-mass spectrometry-

URINE ANALYSIS:

A.  The Development of a  flMetaboliclt Profile Characteristic of
  NeDnatal Tyroscnemia Using Combined Gas Chromatography-Mass
   Spectrometry.

This work vas carried out in collaboration with clinical colleagues
from the Department of Pediatrics at Stanford University and a joint
publication describing this research is in preparation.

The study was based on a total of one hundred and four 24-hour urine
samples from sixteen premature or small birthweight infants receiving
treatment in the Stanford nursery.   After exclusion of infants who
became ill, died,  or left the nursery,  we were able to follow nine
infants closely for periods of between 4 and 6 weeks from day 3 of
life.  All nine infants had birthweights of below 15OOg and three
of these uere below 1OOOg.

Of the nine infants studied,  five showed transient tyrosinemia as
shown by a marked elevation in the urinary excretion of the tyrosine
metabolites,  p-hydroxyphecyllactic acid,  p-hydroxyphenylpyruvic acid
and p-hydroxyphenylacetic acid.   There was also a less marked but
distinct elevation in the urinary tyrosine output.  Figures 1 and 2
show the metabolic profiles of the same infant (J.L.) in the
normal (a) and tyrosinemic[b) states,  Figure 1 shows the free acid
z)utputs, chromatographed as the methyl ester-methyl ether derivatives
and Figure 2 is an expression of the free amino acids of the same
urines,  chromatographed as the N-trifluoroacetyl n-butyl ester
derivatives.  In each case the concentration of each metabolite is a
function of the peak heiqht as compared to the height of the internal
standard.  Table 1 is a summary of the ranges of urinary output of
tyrosine and metabolites observed for all the infants in the study.

ThBLE 1   Dally Excretion in mg/kg

Tyrosine    p-HPLactic   p-HPPyruvic   p-RP Acetic


Normal

0.2 - 3     o-5          0 - 0.5     0.2 - 2

Tyrosinemic 3   - '15    5 - 50       0.5 - 5       0.5 - 5

As shown by Table 1 and Figure 1 neonatal tyrosinemia is characterized
by a very large increase in the output of p-hydroxyphcnyllactic acid
and by a lo-50 fold excess of the latter over p-hydroxyphenylpyruvic
acid.  Studies of the hereditary defects in tyrosine metabolism
initially indicated that p-hydroxyphenylpyruvic acid uas the major
metabolite althouqh more recently cases have been reported where
p-hydrDxyphcnyllactic is in 3 2-5 excess over p-hydroxyphenylpyrnvic.
These latter determinations  were made using GC and GC/MS
methods and therefore probably reflect the improved specificity of
the analytical procedure (previously colormetric methods Here used)
rather than a difference of actual metabolic profile.  Apart from the
very large excess of p-hydroxyphenyllactic acid over its keto analog
we could detect no significant differences between the profiles shoun
in neonatal tyrosinemia and those published for hereditary disease.
Other metabolites such as p-hydroxymandelic acid, DOPA
N-acetyltyrosine,  which have previously been reported in tyrosinemic
urine were not seen to be elevated,

B.  SC/MS Analysis of Urine from Children Suffering from Leukemia.

This research was carried out with twenty 24-hour urine samples
supplied by Drs.  Jordan iiilbur and Tom Long of the Stanford Children's
Hospital.

The acidic fraction of all urines studied in this project showed no
abnormal metaholites nor were gross  amounts of knoun acids detected.
I'he amino acid fraction, however,  of six of the urine samples showed
the presence of an non-protein amino acid, beta-aminoisobutyric acid
(BAIB).  In several of these instances the patients were excreting
in excess of 1 gram of DAIB per day.  The literature contains many
references to increased BAX:B excretion (genetic excretors, lead
poisoning, pulmonary tuberculosis, march hemoglobinuria, thalassaemia
and Down's Syndrome).  The reported excretion of BAIB by leukemic
patients was not substantiated by another investigator.   There are
several criticisms in the literature of the methods used for the
quantitation of BAIB in biological fluids and in order to fill this
void a sensitive, specific and rapid method for the quantitation of
BAIB has been developed.   (SEE:  The Quantitation of bAIB in Urine
by Mass Fragmentography; Ii-E. Pereira, 8, Summons, W.E. Reynolds,
TIC.  Rindfleisch and A.M. Duffield, in press).

C.  GC/MS Analysis of Urine from Patients Suffering from Hodgkin's
   Disease.

During this study 20 urine samples from patients with diagnosed
Hodqkin's Disease (Department of Oncology, Stanford University
Medical Center) were analyzed and in general, no abnormal metabolic
profile could be found in any urine.  There vas one exception in
which an individual was noted to excrete massive quantities of
adipic acid (of the order of 1 gram per day).

I).  Detection of Metabolic Errors by CC/MS Analysis of Body Fluids.

This project results from a collaborative effort between the Departmeuts
of Genetics and Pediatrics of the Stanford University Medical Center.
l!o date over 50 samples have been analyzed; the majority i35) being

`7

%!r


urine,  while amniotic fluid (lo), blood (6) , and cerebrospinal fluid (6)
were also analyzed.   `It has been and will continue to be our practice to
analyze aliquots of fluid samples in  collaboration with clinical
investigators obtained for valid diagnostic purposes completely divorced
from this research on GC/XS analysis techniques.  This investigation is
not iatended to serve as a screening program for a large population hut
rather to focus on those individuals who exhibit suggestive clinical
manifestations such as psychomotor retardation and progressive
neurologic disease as well as suggestive pedigrees.

In the case of amniotic fluid the hope is to be able to monitor the
condition of the fetus in those pregnancies which might be considered
at risk.  To date we have investigated specimens from normal
pregnancies in order to establish the catalog of compounds to be
observed in amniotic fluid.  From this base it could prove possible
to identify materials which might identify the health of the fetus.

We have been able to confirm the presence of erotic acid in a urine
from a person found to have erotic aciduria uhile another urine
sample was used to domonstrate our ability to identify the
characteristic metabolites present in isovaleric acidemia.   The
following description refers to a urine from a child with
hypophosphatasia.

A child died 33 hours after birth in Fresno, California, with the
classical signs of hypophosphatasia.  This genetic defect is marked
by high phosphoethanolamine (PEA) concentrations in urine of
affected honozygotes and unaffected heterozygotes.   Atter
derivatization (in this instance the TNS ethers of the water
soluble carbohydrate fraction were prepared) we were able to detect
by GC/MS large concentrations of ethanolamine and phosphoric acid
but not PEA itself.  The derivatization procedure we used most likely
hydrolyzed PEA.   We were able to quantitate for this compound in the
infant's urine using an amino acid analyzer, and PEA excretion was
extremely high (over 200 times normal values for infants) confirming
the diagnosis.  Next we examined urine samples from the child's
parents,  presumed heterozygotes,  by GC/MS and by the amino acid
analyzer,  Again,  no PEA was detected by the former method although
the presence of ethanolamine and phosphoric acid was demonstrated.
We determined the follouing excretion levels of PZA by amino acid
analyzer:

Newborn infant:   94 micromoles per 100 ml. (Normal 0.21-O-33)
Father:       269 micromoles per 24 hours (normal 17-99)
Hother:        32 micromoles per 24 hours (normal 17-99)

It is of interest that in this family the affected infant and his
unaffected father both show subnormal serum alkaline phosphatase
activity.  The mother,  who did not excrete increased amounts of PEA,
uas found to have normal activity of this enzyme in her serum.   The
following table summarizes the serum phosphatase activity measurements:

Neuborn infant:   0.2 units*  (normal 2.8-6.7)
Father:         0.7 units   (normal 0.8-2-3)
@other:         3.4 units   (normal 0.8-2.3)

f* - 1 unit is that phosphatase activity which vi11 liberate 1
millimole of p-nitrophenol per hour per liter of serum)


El  Drug Analysis Service Using GC/ES

Ide were recently contacted by physicians to rapidly identify a drug
self-administered by a patient  in the Stanford University Hospital.
From the mass spectrum the drug was identified as pentazocaine
within the hour.  Although not part of the formal DENDRAL proposal
we expect that similar cases may arise in the t`uture and we intend
to respond positively to such requests.

Development of Library Search Routines for Mass Spectrum Identification

The analysis of a single body fluid fraction produces between 600 and
750 mass spectra.  In order to cope with the interpretation of the
daily production of mass specta (about 8 body fluid fractions for a
total >f between 4,800 and 6,000 mass spectra) ue have begun the
implementation of library search routines.  Concurrent with the
analysis of body fluids for metabolic content we have been recording
the mass spectra of many reference compounds.   This collection
represents the beginning of the construction of a library of
reference spectra.  Late in 1973 ue expect to receive from Dr. S. Markey,
University of Colorado Medical Center,  a more comprehensive library
which he has collated from contribrltors (including our own laboratory)
in the field of biological applications of gas chromatography/mass
spectrometry.

Prior to the demise of the ACME computer faciliity at Stanford
University,  we ran library search routines on data collected from
urine fractions.  Because of the ACHE system being heavily loaded,
our programs took about one minute per compound identification-
However,  the experience gained will be used to implement library
search routines on our current PDF 11 GC/MS data system.   In addition
ye have sent mass spectra from several urine analyses to Dr. S. Gsotch,
Jet Propulsion Laboratory, Pasadena, California, in order that he
.could use his library search routines on real data.   In this instance
the limiting factor for efficient compound identification was the
library content vhich was limited to a few compounds of biological
significance,  In addition those compounds of interest that were
present in the library were often in a derivatized form different
from that used in our analytical methodology.

Application of GC/HRMS to Body Fluid Analysis

We reported in the last annual report of the DENDHAL project that
the Varian MAT 711 mass spectrometer uas interfaced with a gas
chromatograph for the recording of low resolution mass spectra. We
have n3w used this system tar the recording of HRflS of gas
chromatographic rractions f con urine analyses.   We were able to
record HRMS scans over several gas chromatographic peaks of interest
in a number or urine fractions.  The high resolution results were
found to be of a high quality in mass measurement accuracy.   Uhen
using the MAT 711 instrument for GC/HRMS the sensitivity of the ion
source was a limiting factor in that less intense gas chromatographic
peaks often lacked sufficient material to generate acceptable high
resolution mass spectra.  Notwithstanding this limitation the HRMS
data recorded on different urine fractions uas used to confirm the
identification of several metabolites.  If by chance the metabolite
of concern was available only in quantities insufficient for direct
GC/HRMS, preparative GC would be used to concentrate the component
of interest for subsequent HRMS,

`7

97


RESOURZE OPERATXON

Over the term of this cjrant our mass spectrometry laboratories have
provided support to cuu'.~rous research projects in addition to the
DENDRAL computer program development project funded under this
grant.  These cover a variety of applications at Stanford, in the
United States, and abroad.  Included are problems in the study of
human netabolites, biochemistry, and natural product chemistry.
Samples have been run in wllaboration biith outside people both on
the EAT-711 GC/High Resolution Xass Spectrometer system and the
Pinnigan 1015 GC/Low Resolution Quadrupole Plass Spectrometer system.
The low resolution system has also been supported by a NASA research
grant.

The following tables summarize the support rendered in terms of
numbers of samples run through various types of analysis:

I.  EAT-711 High Resolution System (Period covered 11/'71 - 6/73).

Batch       Batch     GC/High GC/Low
High Resol. Low Resol, Resol.   Hesol.
HS           MS          MS       MS

DENDRAL program devel.           317         3

Stanford Genetics (Body
fluid analysis)

39        17

Stanford Chemistry (non-
DENDRAL - Dr. Djerassi's
qroue 1

91        112

Stanford Chemistry (non-
DBUDRAL - Drs. Vantamelen,
Johnson, Masher, Collman,
Altman,  Goldstein)

29

23                  4

Stanford Surgery {Dr. Fair)         8

Dr.  Adlerkreutz (Finland)         10

DC.  Venien (France)               26

Dr. Gilbert, Hors, Baker (Brazil) 40

Dr.  Orazi (Argentina)              19

Dr.  Subramanian (India)           10

Dr.  Khastqir (India)

Dr.  O'Sullivan (Ireland)

Dr.  Badr (Libya)

Dr.  Hital (India)

           44

            1

           5

            5

            5

  30

   5

-e-e         ----

T3
G-LA

13

50


624        215          13      54
s~lllplcs   samples    samplc5 sa mp 1~:;

11)  FINNIGAN 1015 Low Resolution System (period covered 8/72-a/73)

Note the samples run are specified by fluid type.   Each fluid is
extracted and dzrivatized as described in Part B (ii) and therefore
may represent several GC/LK?iS analyses.  Specific discussions of the
results of various of the analyses run are discussed earlier in
Part B(ii).

Stanford Pediatrics (Drs. Cann, Sunshine
and Johnson)

Stanford Oncology (Dr. Rosenberg)

Stanford Psychiatry - Genetics
(Drs.  Brodie and Cavalli-Sforza)

Stanford Respiratory Medicine (Dr. Robin)

Stanford Pharmacology (Dr. Kalman)

Stanford Biochemistry (Dr. Stark)

Stanford Children's Hospital
(Drs.  Wilbur and Long)

UC San Francisco Eedical School -
Dermatoloqy (Dr. Banda)

Hen10 Park V-A. Hospital (Dr. Forrest)

Palo Alto V. A. E!ospital {Drs. Hollister
and Green)

University of Puerto Rico School of
Medicine (Dr.  Garcia-Castro)

GC/Low
Resolution MS

---I_ -----

141  urines

  7  Amniotic Fluids

  6  bloods

  2 cerebrospinal fluids

20  urines

  4 cerebrospinal fluids

  2  urines

  2  bloods

  2  extracts

  4  extracts

24  urines

  2  urines

13  extracts

7  extracts

7  urines

----I---------

243 samples

? /'
i-I-

??@


PART B PUBLICATIONS

Ilhe following sjlmmarizes the publications resulting from research in
the low resolution mass spectrometry laboratory over the past year,
including body fluid analysis,   This laboratory has been jointly
supported by NItl (DE:NDi(AL) and NASA.  'L'he listed publications include
rosearch relevant to hoth sponsors.

The Determination of Phenylalanine in Serum by Hass Fragmentography.
  Clinical Biochem., 6 (1973)
  By W.E. Pereira, V.A. Bacon, Y. Hoyano, R. Summons and A.M. Duffield.

The Simultaneous Quantitation of Ten Amino Acids in Soil Extracts by
Mass Fragmentography
  Anal.  Biochem-,  55, 236 (7973)
By. W.E- Pcreira, Y. Hoyano, W.E. Reynolds, R-E. Summons and
   A.M.  Duffield.

An Analysis of Twelve Amino Acids in Biological Fluids by Hass
Fragmentography.
  Anal. Chem.,
  By R.E. Summons, W.E. Pereira, W.E. Reynolds, T.C. Rindfleisch and
   A.Fi.  Duffield.

The Quantitation of B-Amino isobutyric Acid in Urine by Mass
Pragmentography.

clin. Chim. Acta, in press
By d,E, Percira, 6.E. Summons, il.E. Reynolds, T.C. Rindfleisch and
A. M.  Duffield.

The DetermFnation of Ethanol in Blood and Urine by Mass Pragmentography.
  clin. Chim. kcta
  By W.E. Pereira, H.E. Summons, TIC. Rindfleisch and A.M. Duffield.

A Study of the Electron Impact Fragmentation of Promazine Sulphoxide
and Promazine using Specifically Deuterated Analogues.
  Austral. J. Chem,, 26, 325 (1373)
  By M.D. Solomon, R. Summons, W. Pereira and A.M. Duffield.

Mass Spectrometry in Structural and Stereochemical Problems. CCXXXVII.
Electron Impact Induced Hydrogen Losses and Migrations in Some
Aromatic Amides

Org. Mass Spectry-, in press.
By A.M.  Duffield, G.  DeMartino and C. Djerassi.

Spectrometrie de Masse. IX.  Fragmentations Xnduites par Impact
Electrorique de Glycols-  -En Serie Tetraline
  Bull Sot. Chim. France, 2105 (1973).

Spectrometric de passe VIII.  Elimination d*can Induite par Impact
Electronique dans Le Tetrahydro-1,2,3,4-Napthtal-ene-diol-1,2-
  Or9  Mass Spectrc., 7, 357 (1973).
  By P.  Perros, J.P, Morizur, J, Kossanyi and A.M. Duffield.

Chlorination Studies I.  The Reaction of Aqueous Hypochlorous Acid
with Cytosirie.

Biochem, Biophys, Hes. Commun., 48, 8RO (Y972)
By w.  Patton, V. Bacon, A.M. Duffield, B. Halpern, Y. Hoyano,
ii.  Pereira and J. Lederberg.


Chlorination S%udies II.  The Xeaction of Aqueous Hypochlorous Acid
with   -Amir~o Acids and Dipeptidcs.
  Biochim.  et Biophys. Acta, 313, 170 (1973).
  BY-  w. E. Pereira, Y. Hoyano, R. Summons, V,A, Bacon and A.IY, Duffield.

Chlorination Studies IV-  The Reaction of Aqueous Hypochlorous Acid
with Pyrimidine and Purine Bases.
  Biochem. Biophys. Res. Cornmun., 53, 1195 (1973).
  By Y. Hoyano, V. Bacon, H-E. Summons, W.E, Pereira, B. Halpern and
  A.H. Duffield.


Part C.   EXTENSION OF THE THEORY OF MASS SPECTROflETRY BY COiClPUTER

DBJECTIVES:

Part C of the DENDRAL effort,  termed Meta-DENDRAL, aims at providing

theory formation help for chemists interested in the mass spectrometric
behavior of new classes of compounds.  Our goals are necessarily
long-range because theory formation by computer is itself an exciting,
unsolved problem in computer science.  We have chosen to explore this
problem in the context of mass spectrometry in order to make
frontier computer research results available to working scientists.

The problem of finding judgmental rules for use in a computer program is
common to many biomedical computing projects,  such as medical diagnosis
and therapy recommendation programs.  <SC?@,  for example, Shortliffe,
et-al.>  In oriier to give these programs the knowledge that makes them
perform at acceptable levels,  a medical expert is often asked to
summarize his own knowledge of the problem area in rules that the
proqram can use.  The neta-DENDRAL theory formation program is a
paradigm for the kind of assistance that computers can give to the
medical experts in this role.  Programs of this sort can, first of all,
provide the expert uith an interpreted summary of a large collection of
"hard" empirical data.  Second,  the program can suggest to the expert
plausible rules that appear to explain major features of the data.
rhus,  the expert is abl e to assimilate large collections of data in the
rules given to the computer.   iue believe that the meta-DENDRAL work is a
useful model on which fruitful uork in other biomedical problems can be
based.

The over-all strategy of this research is to model the theory
formation activity of scientists.  i/e start with a set of empirical
data which are known molecular  structures and their associated mass
spectra.  By exploring the possible  mechanistic explanations of each
sass spectrum, the program  is able to find a set of mechanisms that
appear to be characteristic for the class of molecules.   These
characteristic mechanisms constitute the general mass spectrometry
rules for the class,  or a first-level theory for the class.   Further
refinements of the rules give more sophisticated restatements of the
theory.

k'e have designed the programs in such a uay as to provide useful
results  from the intermediate steps.  The progress section discusses
several sets of results that have been obtained, even though the
entire program has not yet been completed.

PROGRESS:

In the past ten months (since January,  1973) the theory formation
proqrams have seen significant application and significant new
extensions.   In addition,  the work has been described in publications
for both chemists and computer scientists.

Applications of Existing Programs.

The INTSUM program,  for interpreting and summarizing the mass spectra
of many known compounds of one class,  was described in the previous
annual report as essentially tinished.  In this last period we have
used this program to help understand the mass spectrometry of several


classes of compounds, including estrogens, equilenins and other
estroqenic steroids, androstanes, alkyd pregnanes, vinyl quinazalones,
amino acids  and aromatic acids.   An article written for mass
spectroscopists and soon to appear in Tetrahedron {Smith, et-al,
enclosed) describes this proqram and its usefulness in unbcrstanding
the previously unreported mass spectromctry of the equilenins.   The
amino acid and aromatic dcid results are useful for interpreting the
mass spectra taken from those fractions of urine (see Part b).

The INTSUM program is available to anyone who requests it, as stated
in the article soon to appear.  Because of the complexity of the
proqraa, we recommend that mass spectroscopists use
this program on a netuork computer after they have collected a number
of mass spectra from a class of compounds whose fragmentation
mechanisms they wish to investigate,

Recent Extensions to Meta-DENDRAL.

In this last period significant progress has been made on the theory
formation programs that use the interpreted summary of the data provided
by the INTS'IJH program.  h simple rule formation program, described
previously {HI7),  finds the characteristic mass spectrometry mechanisms
for a class of c0mpounds,  assuming +.hat the compounds exhibit regular
behavior as a class.   Recent work has removed the restriction that the
compounds must behave as a class - important classes can he found by the
proqram within the set of given compounds,  The procedure was described
in a paper for the Third International Joint Conference on Artificial
Intelligence,  which is enclosed.   [it the same time that the rule
formation program looks for characteristic mechanisms, the class
separation procedure refines the class of molecules that appear to
behave uniformly (i.e.,  appear to exhibit most of the characteristic
mechanisms).

Another important extension of the theory formation program makes the
rule descriptions more general and less specific to the class of
compounds studied.   The mechanisms in the rules are now described
generally in terms of the kinds of bonds that break, and not in
terms of the precise relations of the bonds to the skeletal structure
common to the class.  For example,  a rule is now stated as "hny
bond that is the second bond from a nitrogen atom is likely to
break",  rather than "In the skeleton R1-C2-N3-C4-R5 the bond betueen
atoms 1 and 2 and the bond between atoms 4 and 5 are both likely to
break".

These general descriptions will allow much more freedom in the kinds of
interpretations that can bz  placed on the INTSUH results.   It is
possible,  for example, to alter the set of predicates used to describe
bonds without altering the program.

The program can be conceptualized 3s a search program throuqh the space
of possible combinations of predicates.  Some predicates describe the
type of bond (e-q., 'single'),  others describe the atoms joined by the
bond (e.g., 'nitrogen', 'secondary'),  and others describe the bonds and
atoms next away from the bond that breaks.  Some a priori heuristics          .
limit consideration of complex predicates to chemically meaningful
combinations, for example,  by forbidding consideration of a single atom
as both carbon and nitrogen,  Other heuristics guide the process of
expansion by forbidding a new predicate to be added to a description if
its addition reduces the explanatory poner of the existing description.
For example,  if a high average intensity is associated with breaking the


X-X bond in X-X-Y dnd frtrtt:er specification of either of the X's reduces
the average intensity,  then the description is not changed.

In addition to the  work just mentioned,  a generative model of rule
formation has been pursu!:d bj7 Carl Farrell in his dissertation work
directed by Professor I:ei.g?nbaulr; and Dr. Uuchanan.   He has written
a program which accepts, as input, descriptions of specific molecules
and all the primitive actions that miqht explain the mass spectra of
those molecules.  The output of the proqram is a set of general
situation-action rules that describe classes of molecules that seem
to be characteristically show evidence of significant actions.

PLANS

In the following period we plan to increase the performance capabilities
Df the theory formation program in several ways,

1.  Sample Selection.

The program's current strategy is to find the rules exhibited by most
or all of the molecules in the initial sample.   If the molecules are
diverse,  the rules will be diverse,   Thus,  we plan to add a
preprocessor that can select a  lfsim~lel' set of molecules for the rule
formation to work with.  For example,  unbranched {straight-chain)
compounds should be expected to preticnt fewer complications for initial
theory formation than highly branched compounds.   The effects of the
complicating features can be studied after the simple rules have been
found.

2.   Rule Clarification.

After simple rules have been found,  ue want the program to clarify
the conditions under which the rules hold.  By studying more
complicated molecules, the proqram can find the simple rules that no
longer hold for these cases.  For example,  we want the program to
discover that terminal alpoa carbons (as in CH3-X-N) are special-
Or, the program should discover the effects of double bonds by
examining neu cases even thouqh the molecules in the original set
contained no double bonds.

3.  Experimentation.

Because the original set of molecules contains the simpler examples
from which it is easier to find characteristic mechanisms, the
program will need to clarify rules in the way suggested under (2).
For a human scientist,  this means describing new experiments to
perform that will help place limits on the range of applicability of
the rules,  Looking at additional arbitrary molecules may be helpful,
but not as helpful as looking at the specific molecules that will
resolve specific questions about the preliminary rule set.

4.  Integration of Results.

When the program has examined two or more classes of molecules, it            I_
should be able to integrate the results  into a common set of mechanisms
(if any are co,nmon).  The set of predicates used by the integration
program may not have  to be wider than the set used by the rule formation
progran,  but one would expect the rules themselves to be more qeneral.
For example,  intcqrating aliphatic amine and ether results should
combine the separate alpha-cleavage rules {one with nitrogen, one


with oxyqen) lnt0 a more goneral rule (specifying 'N Or O', Or
'hetcroatom').

PART C REPERZMCZS  {Published or submitted during this year)

D.H.  Smith, B.G. Buchanan, h'.C, L'hite, B.A. Feigenbaum, C. Djerassi
and J.  Lederberg,  "Ap;r;lications of Artificial Intelligence for
Chemical Inference X.   INTSUM.  A Data Interpretation Program as
Appliezl to the Collected Mass Spectra of Estrogenic Steroidst@.
Tetrahedron.  In press.

B-G,  Buchanan and N-S. Sridharan,  "Analysis of Behavior of Cheaical
Molecules:  Rule Formation on Non-Homogeneous Classes of Objscts4'.
In proceedinqs of the Third International Joint Conference on Artificial
Intelligence,  Stanford University (August, 1973)-   (Also Stanford
Artificial Intelligence Project Memo No. 215.)

Related Publications

DI Michie and D.G. Buchanan, l'Current Status of the Heuristic DENDRAL
Program for Applying Artificial Intelligence to the Interpretation of
Mass Spectra".  August, 1973.

E.H.  Shortliffe, S.G. Axline, B.G. bUChanan, T-C. Merrigan and
S.N.  Cohen,  "An Artificial Intelligence Program to Advise Physicians
Regarding Antimicrobial Therapy".  Computers & Biomedical Research.
In Press.


HUMAN SUBJECTS


HUMAN SUBJECTS

   As a part of this research project, GC/MS analysis techniques will
be applied to human body fluids in collaboration with clinical investigators,
and blood and urine specimens will be collected from human subjects.
Collection of VOIDED URINE SPECIMENS presents no risk to the patient.
Collection of blood samples will not be taken solely for the purpose of
this research but rather would be collected as part of a diagnostic procedure
deemed necessary for clinical diagnosis.


  The undersigned agrees to accept responsibility
for the scientific and technical conduct of the project
and for provision of required progress reports if a
grant is awarded as the result of this application.

Principal In+stigator


APPEhDIX A

FIGURES l-3



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__C_`-;__-_

FIGURE


FIGURE 2

Ff',E DPH01594   USING REP FILE HP801564
SOURCE IS urine-fraction:aminoacids(Gehrke technique
Ct?P ID IS GVP-17H
UBCEFINED     MASS OF flC!L ICN  DATE RUB 730313
RUN CN KAT 711 E8P DOUN SCAEi
THBESHOLD= 4,  FEAKS REJECTED   107 POE AREA,    0 FOR UIDTB
SAflP RATE=10800 , MIN WIDTfI= 2, !!IP AREA= 18
(15.6 SECS, 1.49 DECS), TDEC=10,5
NUHRER CF PEAKS POUND=170   ARRAY SPACE USED( 1750/ 8000)= 21.9%
38 CALIE MASSES WITH LAST CKE=455,0,   6 BASSES ABCVE NOT POUND
PlISSED CALIBRATION MASSES:
281.0
************t*****   TABLE OF PROJECTICN EBRCBS ***+*+**************

28.0     9.5   31.0    43.6   44.0     2.8   51.0    24.9   69.0    -0.3
81.0     0.3   93.0     O-3   100.0     0.2  112.0    -0.5   119.0    -0.2
131.0    -0-2   143.0     0.1   155.0     0-l   162.0     0.1   169.0    -0.9
18 1.0     2.4  .193.0    -2.3   205.0     1.5  212.0    -0.2  219.0    -0.9
231-O     4.1   243.0    -4.4   255.0     1.8  269.0    -0.9   293.0     1.5
305.0     0.4   319.0    -2.0   331.0     2.6  343.0    -2.2   355.0     2.6
367.0     0.6  381.0     1.1   393.0     5.1  405.0     4.4   417.0    -1.7
431.0     2.0   443.0    -4.0   455.0     6.6
BASS PILE flPHOl594 HAS BPEN CREATED WITH 84 MASSES FOUND
SABPLE EASE PEAK IS   3.7 VCLTS Ah'D REF EASE PEAK IS 174.5 YCLTS
HATCHING TCLERAHCE= 4,COO M!lU FRCE WASS 40 TO 300

WATCHING C
  BASS
41.03931
43,053t?8
55.01820

55.05510
56.06277
57.07068
64.01433

6e.96245
68-97505
69.03101

70.03085

75.00519

83.01006

84.04382

85-02852

15  H 20  0   5  N 4 FL   6
  AREA     Ri!lU ERRCR      CCBPOSITICN
  58.0       0,183        c   3H  5
   5.4      -0,899         C   3H   7
   3.5       1.153        C   1H   1N   3
           -0,189        c   3E   30   1
  12.0       0.321        c   4E   7
  33.9       0,180         C   4H   8
  72.8       0.248        C   4H  9
   4.0      -1,721        C   1H  45:  3
           1.863        C  2H   2PL   2
   3.7
   4.7
   5.4      -1,698        c  2R   31   3
          -3.04 1        c   4H   5c   1
   4.4       2.907        C   1H   21   4
           1.564         c  3E   40   1B   1
          -3,405         H   4 N   2FL   2
   6.2      -1,682         H   10  21J   3
          -3,025        c   2E   30   3
           0,560        C  3H   1PL   2
          -0,585        fI  20   IFL   3
   5.8      -1.911         c  2H   1c   1N   3
          -3.253        c   4H  30  2
          -'O-814       c 2B 2PL 3
   6.0       0,230         c  2R   4bi   4
          -1,113        C   4B   6C   11   1
  35.3       0.914        c  2H  3c   1P  3
          -0.429        c   4H  50  2


                     2.011
96.00504      9.5    -0:83 3
                    -2.170
                    -3.513
                     3.896
                     2.751
                     -1.073
134.96208      3.3
137.00035      4.6       1.639
                     -2,383
                     0,494
                    -2, 185
                    -3,528
                    -3.330
                    2,934
                     -1.088
                     -0,890
                    -2.233
139.02548      5.6       2,567
                     -0.113
                    -1.455
                    -1.258
                    -2.600
                      2.129
                      2.327
                      0,984
                    -0.160
                    -3.985
140.03549      3-2       2.065
                     0.722
                    -1.957
                     0,920
                   -0.423
                    -3.102
                    3.162
                   -1.807
152.03288     66.1       2.141
                    -0,538
                    -1.881
                    -1.683
                    -3.025
                     1,704
                     1.902
                     0,559
                     0,757
                   -0.586
153-03532      4.9       2,620
                    1.278
                   -3,254
                   ' 1,475
                     0.133
                     0.331
                    -3.69 1
                    -1.012

C'2H   4FL   3
C   la 4c 5
c 20   1H   4
C 4H   20  21   1
C 5fl   10   1FL    1
C 2H   20  2FL   2
c 2B  ,l bl   1 FL   3

C 5H   10   3N  2
C 10 H   10   1
C 2B 2C 4N 2EL 1
C 50   1N  3fL   1
C 7H 2C 2PL 1
C 2H   10  2nr  3PL   2
H 10  2ti  2FL   4
c 58   1FL   4
U3PL   5
c 2ti 2c   1FL 5
C 1H 5C 5N 3
c 4R   3c   21   4
C 6B 5C 3N.l
C  1H 4C 3N YPL 1
C 3H 6C 4N   1PL 1
C 78  3N   1PL   2
C 2H 2N 4FL 3
C 4H   40   1N   1PL   3
C 1B 5C 2N   1FL 4
C   1E 4B 2FL 5
C 4FI   4C  21   4
C 6H   60   3N  1
C 9H 4N 2
C 1 H  5 0   31   4FL   1
C 3B 7C 4N   1FL 1
C 6H   50   1N   2PL    1
C 48   5-O   1I   1PL   3
C 1H 5N 2PL 5
C 2H 6C 5N 3
C 5R 4C 2N 4
C 7H   60  3N   1
C 2E! 5C 3bl 4FL 1
C 4B   70   4N   1FL   1
C 8R 4N   1FL 2
C 38   3N   4PL    3
C 5H   SC 1N   1PL 3
H 40   1N  4PL    4
c 2B   60   21   1PL   4
C 9R   31   3
CllR   50   1
C 2B 7C 5N 3
C 6B   40   1N  3PL   1
C 8B 6C 2PL 1
C 3H SG 2N3fL 2
C 8EI 5bl 1FL 2
C 5H 7C 3fL 2


                    -3.494
                    2.573
164.00092      3.5       3.877
                      2.534
                    -0.145
                     2,732
                      1,389
                   -1.290
                    -2,435
                    -3,580
                     3.829
                      1.150
173.98967     6.3 .      1.963
                    -2.059
                     0.818
                    -3,204
                   -3,006
                    3.258
                   -0.764
174.99890     11,4-     -2.896
                    0.491
                      3,368
                    -0,654
                    2.223
                   -0,456
                   -1,799
                   -1.601
                   -2.943
                      0.641
178.98990     3.5      0,599
                   -3.423
                   -0,546
                   -1.691
                      1,894
                   -2,128
                   -3.273
180-02676     87.2      -1,582
                    -2,925
                      1.805
                   -2.727
                     3.339
                     2.003
                     0,660
                   -3,872
                     3.537
                     2.194
181.03192     12.5       2.973
                     3.171
                   -0,851
                    al.828
                     2.026
                    0.683
               -0,653
                    -l-Q96

C 3H  4N 4PL 3
C 6R 5FL 4
C 50 3N   4
C 7H 2G 4N 1
c 10 0 1N  2
C 28 1C  4N  4FL   1
C 4H 3C 5N   1PL 1
C 7H 1C  21  2PL
C 4H 20  3N  2FL  :
C 1Ei 30   4N   2FL   3
C 2R 20  31   1FL    4
c 5at 2FL 4
C 50 3N   2PL   2
c 10 0 1FL   2
C 2H 10  4N  2FL   3
C 7H 10  2PL   3
C 20 2N  3FL    4
C 21 2FL   6
C 5FL 6
C 7H 10  3N  3
C 13 FL 1
c SH 10   3N  2FL   2
ClOH 1C   1 FL   2
C 2B 20   4N  2FL   3
c 50 1N  3PL   3
C 7H 2C 2FL 3
C 2B 10  2N  3FL    4
C 4H  3C 3FL 4
C 5H 1FL 6
c 70 3N  2FL    1
C12C 1FL    1
c 4n 10   4N  2PL   2
C  1H 2C 5N 2FL 3
C 20 2%   2FL   5
c 7FL 5
C 4H 1C   1FL 6
c 6H 40  3N   4
c 8H  6C 4N 1
C12E.I 3N   1FL 1
C 3H 5C  4N 4FL 1
c 6H  6C 4FL 2
c 7B 2N  4PL 2
C 9H 40   1N  1PL   2
Ef 6C 5N 4PL 2
C  1B SC  4N 3FL 3
c 3a 7C 5FL 3
~12~50  2
c 7H  '4C 2N 3FL 1
c12EJ 4N  1PL 1
c 9A 6C 3FL 1
c 4H  SO 3N 3PL 2
c 6H  7C 4FL 2
c 7H  3P 4FL
c 9H 50 1I 1;L 2


190.99983

196.95901
198.03705

199.03798

199.98764

200.99631

217.99628

          0.881
         -01462
7.6       3,119
          1.776
         -0.903
          1.974
         -0,705
         -2.048
        -1,850
        -3.193
          1.537
        -2,995
3.2
1co.o       2.670
         -1.862
        -3.204
          2: 868
         1.525
        -3,006
         3,060
          1,723
         0,380
         3,258
3.7      -0.190
         2,489
        -1,533
        -1.33s
        -2.678
         2.052
         -2,480
        -3,823
        .     2,250
         0,907
7-7       1,838
        -2,184
         0,693
        -3.329
        -3,131
          3.133
         -0,889
          1.988
         -2,039
        -1,836
26.7      2.681
        -1,341
        -1,143
\         1,536
        72,486
        -2,288
        -3.63 1
         1,099
        -3.433
          3.976
4.4      -2.778

C 1E 60 4P 3FL
C 3H 80 5FL 3
c 7H  10 411 3
C 9'8 30 S
C128.10 2B 1
c 4B 20 5B 3PL
C 70 2N 4EL 1
c 9H 20 3bT 1PL
C 4H   10 319 4FL
C 6H 3C 41J   1 FL
C 10 N   1PL 3
C  1H 20 41p 4PL

C15B 4N 1
C 6H 60 419 4
C 8H 80 5s 1
C 10H 3 P 4 FL 1
C12H 5c lB1   1 FL
C 3H 70 SH 4FL
c 6H 8C SFL 2
c 7H   4C lbi'4FL
C 9H 60 21   1 FL
C  1H 7C SI 3FL
ClOB 50 2a 3
c 7H  7c SN 2
c 12 H   70 3
c 7B  6G 38 3FL
C 9H 80.4PL 1
C13H 5FL 2
C 4H 7C 4Ei 3PL
C  6H 90 SPL 2
C 8B 4N 3PL 3
C 10 H  60 1FL 3
c 90   4-B 2
Cl40 2

3

1

1 .
2
2

3

1
1

2
2
3

1

2

c 6H   10  sat  2PL.  1
c 11 ?I   1c   3fL    1
C 60  3N   3FL   2
C 40   31  2FL    4'
c 90   1EL    4
C 1H   lG   48  2PL   5
c 6H   10   TFL   5
C lo   28   3fL   6
C9HlO4a2
c 14 H   1c  2
C 90  20   3PL    1
c 6H   IO   58  2FL    1
CllH 2C 3FL 1
c 6H  1C 3% 3PL 2'
c 8H  3C &FL 2
C12PL   3
c 3B   20   UN  3PL   3   .,
C 4H 1C 3N 2FL 4
Cl20  2li  3


223.98842 -            -3.231
            10,9-      2.617
                     1.472
                   -2.550
                    -2,352
                    -3.695
                    -3.497
                     3.912
                    -0.110
                    2.767
                    -1,255
225.02393      6.2      3.802
                    -2.072
                    3,994
                    2.657
                    1,315
                     1.512
                    0.170
                    -2,510
                     0.368
                   -0;975
226.03322     10.2      -0,603
                     2.784
                     2.982
                    1,639
                   -1.040
                    1.837
                    0,494
                    -2,185
                    0,692
                   -0.651
236.99301      3.3     -0.625
                   'I.770
                   1.815
                   -2.207
                    Ok670
                  -3.352

                   -0.099
                   -3.923
                     3.486
                   -0.536
                     2,341
                    -1.681
                    1,196
                   -1,483
                    -2,826
219.99133      7.9      0.443
                  -0.702
                   -1,846
                     1,738
                   -2.284
                    0,593
                   -3,429
                   -0.551

C 9H 2C 5N 2
c 9n   10  3N 3PL    1
Cl00 2ar 2FL 2
c 15 PL   2
C 7H   1c  31 2EL   3
c 12 n   10  1PL 3
C 4H 2C UN 2FL 4
c 70   1N  3PL 4
C 9H   20  2FL 4
Cl20  3ii  2
C 9H   10  UN 2FL    1
C 6H   20  5N 2PL   2
C 70   2N  2PL 4
c 12 PL    4
C 4H   10  3N 2PL   5
C 9H 1C  1PL 5
C 1H   20  UN 2FL   6
c 40   1P  3PL 6
Cl10   4P   2
c 8H  IC 5N 2FL 1
C 13 H   1c  3PL. 1
C 80   3N  3FL 2
ClOH   20  UPL 2
C 5H  1C 4N 3fL 3
C 60  3N  2PL 4
c 11 0   1FL   4
C 3H  1C 4E 2FL 5
c 8H  1c 2PL 5
C 14 H   1N   4
C 7H   50   51 4
C 10 H   6C   5PL 1
CllH   20   1N 4PL   1
C13H 4C 2N   1FL 1
C 8H   30  2N 4FL   2
c 10 H  5 $7  3N 1PL   2
C13H 3N 2PL 2
C 5H 4C 3N 4PL
C 7H   6C  4N 1 FL  3
C 7H 6C 5B 4
C13H   50  2N 1FL   1
C 8H 4C   2N 4PL
ClOH 6C 3N  1 FL  ;
C13H   48   2FL 2
c 5H   50  31 4PL   3
C 78   7C  4N 1FL   3
C 10 H   50  1N 2FL   3
C 2H 6C 4B 4FL 4
c 4H   80  5N 1PL    4
C 12 H   1c  4B 2
C 9A 2C 5N 2FL 1
c 10 0  2N  2PL 3
c 1s FL 3
C 7H   10  3N 2PL   4
c 12 H   10   1PL 4


243.99310

245-00569

247.98857

248*99155

254.10146

2=-10947

5.8

'5.3

4.2

4.7

39.5

3.3

-0.475
-3.154
-1.620
2,209
1,064
-2.760
-0.081
-3,905
3.504
-0.518
2.359
-1.663
1.214
-1.586
3.144
1.999
0.656
0.854
-0.489
-1.825
-3,168
-0,291
-1,634
2.766
1.621
-2,401
-2.203
-3.546
-3.348
0.039
2.916
-1.106
-0.908
-2.087
-3.232
3.032
0.353
3,230
-0,792
-1,937
-3.082
-01052
-1,395
-1,197
3.533
2.190
2.388
1,545
-at, 634
1,243
-0,100
0.143
-1.200
-3.879

C 4H   20  4N 2 PL
c 7c   1N 3FL 5
C 1H 30 5N2PL
c14c   3E   2
C 11 H   10   4N 2FL
CllC 2ti 3PL 2
C 88   20  5N 2PL
C 8B   1c 3s 3PL
C 9C 2N2FL 4
c 14 PL    4
C 6H 10 3N2PL
C 11 H   1c   1PL 5
C  3H 20  4N2PL
ClOH 3C 5N 3
c 14 0   1N  3PL 1
c 11 tf   1C 2N 3FL
C13H   30  3FL 2
c 8H PO   3N 3PL
ClOH 4C 4PL 3
C 11 N   4PL   3
c13a 2c   1 bl.1 PL
C 5H  30 4N 3PL
c 7B SC 5PL 4
Cl30   4N   2
c 10 B   1C 5N 2PL
c 15 H   1C  3FL 1
Cl00 3N 3FL 2
C12H   20  4Ft 2
C 7H   10 4N 3PL
c 13 0   1PL    4
C 5H   1C 4N 2FL
C 10 H   10  2PL 5
C SC 2E 3PL 6
C 13 H   1C  412
ClOH 2i: 5N 2PL
C 88  2C  5N 1 FL
CtlO 2N 2FL 3
C 3H 10 5N 4FL

5

6

2

3

3
4

1

3

5

1
3

4

c 8H 1C   31   2FLa  4
C 5H 2C 4N 2TL 5
C 2H 30   SN  2PL   6
ClOH140   UN   4
C12H160   5N   1
C 7HlSC 5tI 4FL 1
Cl18120 1N 4FL 2
C13Hl4C 2N   1PL 2

C  8H13C 2H.
/c-rti --i5.c -
L        .-la   1 PL
c 1nTiTrzr 3     N-co,

C 5Hl4C 3N 4PL 4
C 7Hl6C 4N   1PL 4
ClOH150  4N   4               . .
C12H170   5X   1
C15H15C  21  2


i55.99071

4.4

256.94409     19.5
261.98169      7.4

262.99121

19.3

269.98486

4.2

273.98608

10.7

-1,002
3.728
2.385
2.583
1.240
-1.439
1,438
3.833
-0.189
-1.334
-2.479
3,785
1,106
-2,916
3,983
-0,039
-1,184

-0.900
-2.045
  0.395
-3.627
  2,023
  0.878
-2,946
-0.267
  3.3.18
-q*704
  2.173
. .-1,849
  '1.101
  1.299
La2.723
-3.868
  3.541
  2.396
  2.594
  1,251
-1,428
-2.749
-3,894
  3.515
  2.370
-1,652
-1.454
-2,797
-2,599
-3.942
  0.788

c 7H160 SN 4FL
c 11 H 13 0   1N 4PL
Cl3Hl5C 2N   1 FL
C 8H14C 2N 4PL
ClOB160 3N 1PL
C13H14N 2FL 3
c 5H150 3ar 4PL
Cl00 5N 4
Cl50 312
C 12 H   1C 4N 2PL
C 9H 2C 5H 2FL
C 7H 2C 5N   1 PL
Cl00 2N  2FL 4
Cl5PL 4
C 2H   1C 5s 4FL
C 7H 10 3N 2PL
c 4H   2 c  4N 2FL

Cl10 4b( 2PL 2
  C 8R   1C 51 2PL
  C 60 3rm 2PL 6
  c 11 0   1PL 6
  c 14 c   3N 2PL 1
  c 11 H   1C 4N 2PL
Cl10 2N 3PL 3
  c 8H   2C 5N 2PL
C 9C 2N 2FL 5
  C14FL s
  c 6H   1C 3N2PL
  CllH 10   1PL 6
C 13 H   1C 5N   1 FL
C 80 5ti 4PL 2
Cl30 3N 2PL 2
C 10 H   1C 4N 2FL
Cl10 3N   1 FL 4.
  c 8H   1C 4N   1 FL
C 30 4-B 4FL 6
C 5H 2C 5N 1PL
C 80 21 2FL 6
  c 14 0  41J 3
c 11 H  1C 5N 3FL
c 12 0  4N 2PL 2
C 9H  1C 5N 2PL
  C 14 H   1c 3PL 3
  C   9 c  3Ei 3PL 4
c 11 Ef  20 4FL 4
,c 6H  1C 4N3PL
c 8H  30 5FL 5
c 12 0   1FL 6

*$$$**$*****$*t*$ MISSED CALfBRATICN &!~sS=281.O ***lb*****
28 1.08472      4.7      -3,910        CllH13C  SN   4
                    3,499       C12H120 4N 3PL
                    2,157       ClYHlYC  SPL   1
                    0,820       c 15 H 10 c   1N   4FL

4

1
2
4

5"
6

3

2

3

6

1

3

5 '

6

1

3

5

1

1
    ,,


                     2.354
                    -0.325
                   -1.668
                   -1.470
                    -2.812
                    1.917
282.09814     11.1.      1.728
                   -3,439
                     3.970
                     2.825
                     0.146
                     3.023
                     l-681
                    -0.999
                    -2,144
282.99707      5.3       1.422
                     0.277
                    -2.403
                    -3.547
                   -0,868
                     3.861
                     2.717
                    -1.305
285.98682      9.3      -2.073
                    -3.218
                      3.046
                    -0,976
                   -0.778
                    -2,121
                   -1.923
                   -3.266
                    1.464
                   -3.068
290.98560,     4.6       1.376
                     0,231
                    -3,593
                     2.671
                   -1.351
                     1,526
                   -2.496
                     0.548
                   -0.597
                    -1,742
                     i-843
                    -2,179
                    3,794
                   -1.175
                    -2.320
                    -2.122
                   -0,576
                    0.719
                     1,882
                    0.737
                   -3,087

293.98828

4.7

297.97510      3.2

. 311.97388

312.98804

4.9

11.1

c 9R13C 5N   3YL 2
C12H110 2N  4FL 2
C14~13C 3N 1FL 2
C 9H12C  3N 4PL 3
C  11  H 14 C   4 N   1FL   3
C  15  H 11 N   1FL    4
C  11  H14C   5N   4
C13H150 4N 2FL 1

C  14  H 1_4_.-C-~_ 3. He  1 FL
  -n-i!3 C        -2
            4 N   1 FL
c  14  u 13 0   1N   2FL   3

C 6Hl40 4N   4 FL
C 8H16C 5N   1 FL
CllH140 2N 2FL
C 8H15C  31 2FL
C 14 H   1C  31  2FL
c 11 A   SO  41  2FL
c 14 0   1P  3FL   3
C 11 H  1C 2N 3FL
C 8H 3C 5N 2FL
c 12 0   1N   2FL   5
c 9H   1C 21 2FL
c 14 R   1FL    6
c 15 0   41  3
c 12 R   10   5N   3 FL
ClOH 1C 5ti 2PL
c 15 H   10  3PL   3
c 10 c   3u   3PL    4
C 12 H  2 0   4FL    4
C 7H   1C 41 3PL
c 9A   30  5FL   5
C 13 G   1FL   6
C 4H 20 5N 3FL
Cl50   41  2PL    1
c 12 R  1-G 5H 2fL
c 12 0   3Ei   3PL   3
Cl00 3bl 2FL 5
c 15 c   1EL   5
c 7H   10 4N 2FL
c 12 H   1C   2PL    6
C 150   3 N  2 FL    2
c 12 A   1C Ybl 2FL
C 9H 2C SN 2FL
C 10 0  2 El  2 FL    6
c 15 FL   6
c 15 0   5FL   2
Cl20 4N   1FL 4
C 9H   10  5N  1 FL
c 40   5s   4PL   6
c 15 0   51   1PL
ClOC   4N   1 FL  :
c750 3H 2PL 3
c 12 H   1C 4N 2FL
C 12 0  2 N  3 FL   5

4
4

6

:

2

6

3
4

5

4 .,


40   60   80   100   120   140  160

                co<
FIGURE 3.

Gc/LRMs (FINNIGAN 1015 QUADR~POLF: mss SPECTRO~~ETER).

SAMPLE: GLUTAJUC ACID N-TFA O-n-BUTYL ESTER DERIVATIVE.

$+&j+
    

.

180 200 220 240
       w
          L-

260

280 300 320 340 360 380 400 420   440  460   480  500

GUP L7H flMNIOTIC FLUID
D400

. .

FILE 344                                                                           .


   APPENDIX B

LETTERS OF INTEREST


STANFORD UNIVERSITY
STANFORD, CALIFORNIA 94305

DEPARTMENT OF CHEMISTRY

December 17, 1973

Professor Carl Djerassi
Department of Chemistry
Stanford University
Stanford, California 94305

Dear Carl:

  I am writing to indicate the anticipaJed use of mass spectral
facilities by my research group in the for,seeable future. As has
been true in the past, we plan to utilize both GC/HRMS and simple
HRMS for various purposes, especially 1) the determination of structure
of enzymic cyclization products, including members of the lanosterol
class, derived from squalene oxide-like substrates, the purpose being
the elucidation of the mechanism of enzymic steroid synthesis, and 2)
the characterization and confirmation of structures of intermediates
in the synthesis of natural products, including polycyclic terpenoids,
alkaloids of physiological interest, and nucleosides,and 3) identifi-
cation and/or structure determination of organic materials employed
in our organic-inorganic program devoted to nitrogen fixation and
related processes.

Very truly yours,

E. E. van Tamelen
Professor of-chemistry

EEvT/jlb


OFFICE MEMORANDUM o STANFORD UNIVERSITY a OFFICE MEMORANDUM

To :

FROM :

SUBJECT:

DATE: December 3,

Carl Djerassi

Keith Hodgson

Response to inquiry about GC/HRMS
facility

   In response to your three questions
concerning the potential use of upgraded
GC/HRMS facilities:

1. Yes, especially in the study of certain
biological ligands and lower molecular
weight ligand-metal complexes.

2. Potential use of the facility might run
in the range of 8-10 samples per year most
of which probably would be handled most
easily by simple HRMS.

for the next


OFFICE MEMORANDUM o STANFORD UNIVERSITY o OFFICE MEMORANDUM . STANFORD UNIVERSITY . OFFICE MEMORANDUM
                                                                           .

                              DATE: December 3, 1973         lz
                                              2:
To :    Professor Carl Djerassi                           2
                                                                E:
                                                                           =i
                                                                           4

FROM :

James P. Collman
Professor of Chemistry

.

SUBJECT:

  Please excuse our belated response to your inquiry of November 20
concerning a potential upgrading of mass spectrometry facilities.  The
service you mention in your memo of the 20th would be valuable to us.
We would have significant use for the GC/HRMS for a project dealing
with models for cytochrome Pdso based monooxygenases currently
supported by the NIH.

JPC :lb

o


OFFICE MEMORANDUM . STANFORD UNIVERSITY o OFFICE MEMORANDUM  . STANFORD UNIVERSITY . OFFICE MEMORAFtDUM
                                                                           .

DATE: December 13, 1973

To   : Professor Djerassi

FROM :  Professor Harry S. Mosher

SUBJECT:  Your proposal to the NIH

On our NIH Grant on the investigation of animal toxins we have been
studying natural products from the skin of Central American frogs
(atelopidtoxin) and some products from marine animals (n&dibranchs! as
well as some new choline esters isolated from the hypobranchial gland
of various sea snails.  Some, if not all,of these are mixtures.
Obviously the new capabilities of the mass spectrometry laboratory
would be of value to me,  I expect only occasional use of HRMS an.'.
GC/HRMS, but on these occasions these techniques would be very important.

o

0
=:
z
m

3
T
0'
E
0
s


DATE:   14 December 1973

To :

FROM :

SUBJECT:

C. Djerassi

W. S. Johnson

OFFICE MEMORANDUM o STANFORD UNIVERSITY o OFFICE MEMORANDUM o STANFORD UNIVERSITY o OFFICE MEMORANDUM
                                                                           .

The contemplated new facility for high resolution mass spectrometry and
combined gas chromatography/high resolution mass spectrometry would be
of extreme value to our research program concerning the non-enzymic
biogenetic-like cyclization of polyolefines, a project which is presently
supported by NIH Grant AM 3787-14. If this facility were to become
available, we would expect to use it extensively in the analysis of product
mixtures of the aforementioned cyclizations. We estimate that our need for
the gas chromatographic capability would be about 20% of the total need
for the mass spectrometry service.

o

4

o


STANFORD UNIVERSITY MEDICAL CENTER

STANFORD, CALIFORNM 94305 . (415) 321.12C'O EXT. 5785
                                b

STANFORD U~w3t.m~ SCHOOL OF MEO~CINE
De$dmcnt of Ancsthrsia

November 30, 1973

Professor Joshua Lederberg
Department of Genetics
School of Medicine
Sta'nlford University
Stanford, California 94305

Dear Dr. Lederberg:

  Thank you for including my laboratories in the group which
could be served by a GC[ERMS facility.  As you know,.Dr. Cohen
and I have our own GC/FE/ Computer System.  Cur use of the pro-
posed facility would be limited to those times when it is
necessary to use high resolution ot identify a metabolite. I
would estimate a need for three GC/HFW and three HEWS Spectra
per year.  My work is entirely supported by the National Institutes
of Health.

S~~lQ;~&Kq(w
  James R. Trudell, Ph.D.

JRT:rw


VETERANS ADMINISTRATION

        HOSPITAL

      3801 MIRANDA AVENUE
PALO ALTO, CALIFORNIA 94304

IN REPLY
REFER TO:

November 26, 1973

Professor J. Lederberg
Department of Genetics
Stanford University School of
Medicine
Stanford, California 94305

Dear Prof. Lederberg:

  Dr. Allen Duffield of your department has informed me that you
plan to obtain additional apparatus that would provide high resolution
C&/MS as a service to the Stanford community.

  We have in the past used the hospitality of your department in the
identification of metabolites and derivatives of phenothiazine drugs and
cannabinoids by GC/MS.  Originally, we had the collaboration of Dr. B.
Halpern and more recently Dr. A. Duffield, who was instrumental in
helping us with some of our problems.                             *

  Our department would indeed be most interested in availing ourselves
of Gc/Ms analyses in the course of our current NIH projects which again
are concerned essentially with drug metabolism and the isolation and
characterization of unknown drug derivatives.

As a rough estimate, I would think that we may-be interested in
the analyses of about five samples per month, two of which will require
high resolution MS.

  I certainly hope that your project to acquire the sbphisticated
new instrumentation you are.seeking will be successful.

Sincerely yours,

Irene S. Forrest, Ph.D.
Chief, Biochem. Research Lab.
(15W

ISF:jr

Show vcrcrm's jcd~ nam, VA jil c number, and racial security nmhr on al/ rortw~ondcncc.


OFFICE MEMORANDUM .  STANFORD UNIVERSITY o OFFICE MEMORANDUM  . STANFORD UNIVERSITY . OFFICE MEMORANOUM

                                                                           I
                                                      zi
                                          DATE:  November 30; 1973         ci

To :                                             z
     Joshua Lederberg                               z
                                                                E
FROM I                                                                           Z
                                                                           -c
     1. Rabinowitz, Ph.D.                                                                        0
     D.I. Wilkinson, Ph.D.

SUBJECT:   RE: NIH GC/HRMS Proposal
                    *

3

Research carried out in this department has strongly implicated a role    :
for the prostaglandins in the etiology of psoriasis (E. M. Farber, K. Aso,     :
32nd Annual Meeting, American Academy Dermatology, Chicago, Ill., Dec. 1973;        z
E. M. Farber et al, J. Invest. Derm,,in preparation; E. M. Farber et al,      c"
Nature New Biology, in preparation).  The prostaglandins are a class of           3
C2D fatty acids, having molecular weights near 350 and basal tissue con-            e
centrations in the nanogram and picogram per gram range.  The Frostaglandins are 2
presently detected by radioimmunoassay, bioassay and mass spectr,ometric      :
techniques, among others.  There is considerable controversy concerning the     s
method of choice for measurement of absolute amounts of prostaglandin in various Z
tissues.  In particular, it has been suggested that mass spectrometric techniques c
yield more accurate quantitative assays than radioimmunoassay techniques (Adv.
Biosciences, 2, 71-123, 1973, Ed. G. Rasp.6, S. Bernhard, Pergamon Press, f4.Y.).  f
Radioimmunoassay techniques are currently in use in our laboratories, and       ;
                                                                           c"
the addition of mass spectrometry capability would greatly increase the       :
definitiveness of our studies, as well as make available to us a powerful
tool for the study of prostaglandin precursors and metabolites. Work to date
has been supported in part by NIH Grant No. AM 15107.       i
                                                                n
                                                                           In

1

D. I. Wilkinson, Ph.D.
Departmect of Dermatology

                                                                           a
IR:DIW:ss                                                                           0

. .


.

OFFICE MEMORANDUM o STANFORD UNIVERSITY o OFFICE MEMORANDUM o STANFORD UNIVERSITY . OFFICE MEMORANbWh

me 3 1973

DATE: November 30, 1973

To :  Joshua Lederberg, Department of Genetics
    Carl Djerassf, Department of Chemistry

FROM 1 Eugene D. Robfn, M.D., Department of Respd atory Medicine

SUBJECT:  Your memo of November 20, 1973 describing a proposed GC/HRMS facility.

I have applfed to the NIX for a contfnuatfon of my research grant, Adaptations
To 02 Depletion in which I have proposed to measure the redox state of NAD+/NADH
and NADP+/NADPH by measuring the ratio of oxidized to reduced redox pairs usfng g
gas chromatography/mass spectrometry.  These analyses will be conducted with
the assistance of Drs. Alan Duffield and Wilfred Pereira of the Department of
Genetics.  I welcome the opportunity to have a GC/HRMS facility available on
campus to support the GC/LRMS available fn the department of genetics.  The
facility you propose to establish wfll ba of importance to us in those
instances where assignment of molecular composition to ionized fragmerts is
crucial for kass spectral interpretation.  I would anticipate using this service
between one and two times a month.

Sfncerely yours,

Professor of Medicine and Physiology

EDR:ods


VETERANS ADMINISTRATION

         tdOSPITAL

      3801 MIRANDA AVENUE
PALOALTO,CALIFORNIA 94304

November 28, 1973

IN REPLY
REFER TO:

Dr. Joshua Lederberg
Department of Genetics
Stanford University School of Medicine
Palo Alto, California 94305

Dear Dr. Lederberg:

  I should be very pleased if you were able to obtain through
the National Institutes of Health a GC/MS facility which could be
shared jointly by members of the Stanford University faculty.

  At present, I am being funded under grant DA-00424-01 for a
study of the metabolism of ma,ihuana.  We have made significant
progress in our methods of extracting metabolites, in isolating
new ones by thin-layer chromatographic techniques, and by purify-
ing them to some degree as determined by GLC. The big bottleneck
has been the lack of ready access to a GC/MS set-up which would
permit further characterization of the metabolites.

  Our needs would be primarily for GC/low resolution MS, for
which we have extensive need, perhaps the analysis of 15-25 samples
per month.  Depending on the outcome of these analyses, we might
have 1 to 2 samples per month requiring GC/high resolution MS. We
anticipate having little need for high resolution MS without GC
because of the fact that our samples are isolated from complex
mixtures and are nearly impossible to purify.

  If there is any way in which I could assist in helping obtain
such a facility for the University, please let-me know.

LEH:bh

$brw vc~crm's full name, VA f;l c numbtt, and social Jccurity number on aI/ corrtspondtnct.


STANFORD Uh'IVERSITY HOSPITAL
  Pharmacy Department

Date     September 5, 1973

To: Y Dr. J. Lederberg, Director

Department of Genetics

From:    Hiram H. Sera, Director

Su?ject:  Drug Analysis Service with Gas Chromatograph and Mass Spect-
        rometer.                                                  .

1 wish to express our appreciation to your department for assisting us in
identifying a drug -sample submitted to us from the El patient :are area.

                                                  .
BACKG iiODiD :            .

The patient on ElA with G.I. disturbance, joint "pains and occasional
spike temperature was found to possess an unidentified medication in a
plastic vial and was found to have self-administered the drug intramus-
cularly while in the hospital.  The house staff was notified and the drug
sample was submitted to us for immediate identification.

Through my previous association and knowledge of Drs. Summons' and W.
Perieras' (in Dr. Duffield's instrumentation research laboratory) wcrk
with gas chromatograph and mass spectrometer, I had taken the liberty
to request .their assistance in the identification.

In an hour, the determination was made and the drug was found to be
Pentazocaine or Talwin which is a synthetic analgesic used commonlv in
this hospital in tablet and injection forms.
.
Since we do occasionally receive similar requests from physicians, I
wish to call on your staff again in the future.  Thank you.

EEIS:lh
.cc:  Mr. John Williams
   Dr. Roger Summons
   Dr. W. Periera
   Dr. A. Duffield


OFFICE MEMORANDUM o STANFORD UNIVERSITY o OFFICE MEMORANDUM o STANFORD UNIVERSITY o OFFICE MEMORANDUM
                                                                           c
                                                                   2
                                              iii-
                                                        2;
                              DATE: November 26, 1973          z
                                                z
   To :     Joshua Lederberg, Department of Chemistry                             7
         Carl 'Djerassi, Department of Chemistry                             =:
                                                                           E

FROM :

SUBJECT:

Sumner M. Kalman                                        z

                                                                           0

                                                                           0

Mass Spectrometry, Your Memo'of November 20, 1973.                        ,"
                                                              rc

  A central facility for mass spectrometry and GC/MS would be
highly desirable from my point of view. We often need to identify
metabolites of drugs that interfere with our assays, and that represent
research problems as well.  Frequently we need to check the purity of
a reference material which is in short supply.  I have received much
heln from both your laboratories in the past and would welcome the
opportunity to use an expanded facility.  For many of our prcjlems low
resolution MS is satisfactory and I hope you mean to provide this
service too.

With respect to your questions I anticipate that

(1) Yes.

(2) We would probably use GC/MS once a month or more. We
would use MS at about the same rate.

(3) Yes.

Sincerely yours,

Sumner M. Kalman, M.D.
Professor of Pharmacology
Director, Drug Assay Lab


OFFICE MEMORANDUM o STANFORD UNIVERSITY o OFFICE MEMORANDUM o STANFORD UNIVERSITY o OFFICE MEMORANDUM
                                       OEC 3 l9?J            .
                                                                  r=
                                             ii
                                                      s
                                    DATE: 3 December 1973           0"

                                                                           c
   To :    Joshua Lederberg                                                                       z
                                                                 2
                                                               z
                                                                           VI

FROM :'                                                                         z
       Jack Barchas                                                                           4

                                                                           b

                                                                           0
SUBJECT:    GC/HRMS                                                      =:
                                                                   ii
                                                                           In

bur thanks to you and Alan Duffield for inquiring of our interest
in the proposed GC/HRMS.  We would find it quite useful, as we
are currently applying for funding for a quadrupole mass spectro-
`meter for mass fragmentography studies.  With such a unit, there
would be many times when the capability of the HRMS instrumentation
w0ul.o be valuable in structural elucidation.  We would expect very
heavy utilization of our instrument if we were to.obtain the funding,
ad, therefore, would expect to make considerable use of the proposed
GC/HRMS, which is an essential ancillary tool.

The GC aspects of the instrument would be valuable, since we would

expect to be studying a number of unknowns and the GC separation

would be an integral part of that process.                                              b

Our work is supported by NIMH, ONR, NASA, and the Alcohol Abuse
division of HEW.

JDB/rs


REPLY TO
M-TNOF: LPE: 239-q

NATIONAL AERONAIJTICS AND SPACE ADMINISTRATION
            AMES RESEARCH CENTER
          MOFFETT FIELD. CALIFORNIA 94035

November 28, 1973

Professor Joshua Lederberg
Department of Genetics
School of Medicine
Stanford University
Stanford, CA 94305

Dear Professor Lederberg:

I was delighted to learn of your proposed plans to upgrade your
mass spectrometry capabilities by providing routine high retolution
mass spectrometry (HRMS) and combined gas chromatography/
high resolution mass spectrometry (GC/HRMS). Such a service
could be of inestimable value to our program.  As you know we are
developing gas chromatography/high resolution mass spectrometry
facilities for NASA's interests.  In particular we are modifying
our equipment in order to determine carbon and nitrogen isotopic
compositions of organic molecules.  If available we would use your
proposed facilities for our routine GC/HRMS analysis of biologically
significant molecules which are sought in our program.  Most of
our work requires GC/HRMS as opposed to HR*MS. In addition, we
are al+o most interested in computer programs which aid in mass
spectral interpretations.  Although we have a few of our own
programs, we would be most eager to upgrade our own interpretation
capabilities through use of programs from your facility.

Our work thus far has been supported solely by NASA;.we are not
supported at present by NIH.

I hope that our expression of interest will be of use to you in obtaining
funding for a potentially most useful analytical facility.

Sincerely yours,

Keith A. Kvenvolden
Chief, Chemical Evolution
Branch


OFFICE MEMORANDUM o  STANFORD UNIVERSITY . OFFICE MEMORANDUM o STANFORD UNIVERSITY o OFFICE MEMORANDUM

DATE:  November 28,%1973

To :  Joshua Lederberg, Ph.D.
     s331

FROM :  William R. Fair, M.D.
     S287

SUBJECT:  Use of facilities for high resolution mass spectral analysis with gas
    chromatography.

As your memo of November 1973 requested, we have answered the questions concerning
our interest in GC/HRMS.

1.

This service would be of definite value to us in two projects currently being
investigated in our laboratories.  a) The identification, distribution, and
biological significance of the prostatic antibacterial factor (PAF). Our
preliminary experiments indicate that this is a basic polypeptide, perhaps
attached to a divalent metal such as zinc.  b) This service would also be of
valCe in the determination of the urinary polyamine levels in patients with
various genitourinary tract malignancies.  Our initial experiments along this
line indicate that there is significant elevation of polyamines in patients with
pros tatic carcinoma.  The use of GC/HRMS would enable a more precise quantita-
tion of these differences and enable us to expand our research into other areas
concerning the biochemical significance of the polyamines.

2.  I would estimate that on the PAF project we would, use approximately 2-4 samples
per month and perhaps lo-12 samples per month on the polyamine projects. Both
of these projects would require the use of GC/HRMS.

3. A portion of our research on the PAF is currently supported by a grant from the
  NIH.  The amount of this grant is $36,698, and this grant will terminate on
  December 31, 1974.