Chlorination Studies II. The Reaction of Aqueous Hypochlorous Acid
with -Amino Acids and Dipeptides.
Biochim. et Biophys. Acta, 313, 170 (1973).
By. W.E. Pereira, Y. Hoyano, R. Summons, V.A. Bacon and A.M. Duffield.

Chlorination Studies IV. The Reaction of Aqueous Hypochlorous Acid
with Pyrimidine and Purine Bases.

Biochem. Biophys. Res. Commun., 53, 1195 (1973).

By Y- Hoyano, V. Bacon, KeE. Summons, W.E. Pereira, 8B. Halpern and

A.Me Duffield.

a /Of
Part C. EXTENSION OF THE THEORY OF MASS SPECTROMETRY BY COMPUTER

OBJECTIVES:

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 judymental rules for use in a computer program is
common to many biomedical computing projects, such as medical diagnosis
and therapy recommendation programs. <See, for example, Shortliffe,
et.al.> In order to give these programs the knowledge that makes then
perform at acceptable levels, a medical expert is often asked to
Summarize his own knowledge of the prohlem area in rules that the
program can use. The Meta-DENDRAL theory formation program is a
paradigm for the kind of assistance that computers can give to the
medical experts in this role. Prograns of this sort can, first of all,
provide the expert with 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.

Thus, the expert is able to assimilate large collections of data in the
rules jyiven to the computer. We believe that the meta-DENDRAL work is a
useful model on which fruitful work in other biomedical problems can be

based.

The over-all strategy of this research is to model the theory
formation activity of scientists. #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
nass 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
refineaents of the rules give more sophisticated restatements of the

theory.

we have designed the programs in such a way 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
programs 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

ay J00
classes of compounds, including estrogens, equilenins and other
estrogenic steroids, androstanes, alkyl pregnanes, vinyl gquinazalones,
amino acids and aromatic acids. An article written for mass
spectroscopists and soon to appear in Tetrahedron {Smith, et.al,
enclosed) describes this program and its usefulness in understanding
the previously unreported mass spectrometry of the equilenins. The
amino acid and aromatic acid results are useful for interpreting the
mass spectra taken from those fractions of urine (see Part 8).

The INISUM program is available to anyone who requests it, as stated
in the article soon to appears Because of the complexity of the
progran, we recommend that mass Spectroscopists use

this program on a network 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 INTSUM program. A simple rule formation program, described
previously (HI7), finds the characteristic mass spectrometry mechanisms
for a class of compounds, assuming that 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 be found by the
progran within the set of given compounds. The procedure was described
in a paper for the Third international Joint Conterence on Artificial
Intelligence, which is enclosed. At the same time that the rule
formation program looks for characteristic mechanisms, tne class
separation procedure retines 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 29f the precise relations of the bonds to the skeletal structure
common to the class. For example, a rule is now stated as "Any

bond that is the second bond from a nitrogen atom is likely to
break", rather than “In the skeleton R1-C2-N3-C4-R5S the bond between
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 be placed on the INTSUM results. It is
possible, tor example, to alter the set of predicates used to describe
bonds without altering the program.

The program can be conceptualized as a search program through the space
of possible combinations of predicates. Some predicates describe the
type of bond (e.g., 'sSingle'), others describe the atoms joined by the
bond {e.ge, ‘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
ts addition reduces the explanatory power of the existing description.
For example, if a high average intensity is associated with breaking the

4 Jos
X-X bond in ¥-X-" and further 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 pursued by Carl Farrell in his dissertation work
directed by Professor Feigenbaum and Dr. Buchanan. He has written

a program which accepts, as input, descriptions of specific molecules
and all the primitive actions that might explain the mass spectra of
those molecules. The output of the program 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
of 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 adda
preprocessor that can select a Nsimnle" set of molecules for the rule
Formation to work with. For example, unbranched (straight-chain)
compounds should be expected to present fewer complications for initial
theory formation than highly branched compounds. The eftects of the
complicating features can be studied after the simple rules have been

found.
2. Rule Clarification.

After simple rules have been found, we want the program to clarify
the conditions under which the rules hold. By studying nore
complicated molecules, the program can find the simple rules that no
longer hold for these cases. For example, we want the program to
discover that terminal alpna carbons (as in CH3-X-N) are special.
Or, the program should discover the effects of double bonds by
examining new cases even though 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
progran 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 cules. 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
should be able to integrate the results into a common set of mechanisms
{if any are common). 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 general.
For example, integrating aliphatic amine and ether results should
combine the separate alpha-cleavage rules (one with nitrogen, one

bo Jo¥
with Oxygen) into a more ganeral rule (specifying ‘N or O*%, oF
theteroatom').

PART C REPERENCES (Published or subnitted during this year)

D-H. Smith, B.G. Buchanan, W.C. White, E.A. Feigenbaum, C. Djerassi
and J. Lederberg, "Applications of Artificial Intelligence for
Chemical Inference Xe INTSUM. A Data Interpretation Program as
Applied to the Collected Mass Spectra of Estrogenic Steroids".
Tetrahedron. In press.

B.G. Buchanan and N.S. Sridharan, "Analysis of Behavior of Chemical
Molecules: Rule Formation on Non-Homogeneous Classes of Objects".

In proceedings of the Third International Joint Conference on Artificial
Intelligence, Stanford University (August, 1973). (Also Stanford
Artificial Intelligence Project Meno No. 215.)

Related Publications

D. Michie and B.G. Buchanan, "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. puchanan, T.C. Merrigan and
S.N. Cohen, "An Artificial Intelligence Program to Advise Physicians
Regarding Antimicrobial Therapy". Computers & Biomedical Research.

In Press.
HUMAN SUBJECTS

JOb
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.

poi.
L tun

Car ¥Djerassi/

Principal Investigator

 

(ok
APPENDIX A

FIGURES 1-3

{07
 

Ta.ce 4. c a = wus ~
vloh mass diffecerces (a) S50 mma (9.08 am,.
E
c t N 0 rmu
Oo 0 0 0 6.0-
0 ‘ L -L Cathe os
0 l 2 —2 LG.
0 -5 4 -3 ~35.h
1 m2 -3 2 = .6
au G =o i who. os
1 2 ~ 3 Woe? ae
i ‘ o -1 35.6
1 -t 2 -2 -uh.7
1 -€ 3 -5 ~21.3
1 -) 4 <4 2.1 --
2 c a4 2 22.5 o.
o e -3 L O09 -—
. L, -e 0 Oa oe
2 é -—_ mi. LT
2 <é - -2 =e
2 a 2 -3 “Jew --
o =: 3 - Lu 3 os
. - 4 x 13.4 oe
Ss é - 3 3 36.5
z ~= -_ -1 -43.7
= me C -2 -20.3 oe
mr L -3 3.4 -~
. 2 —4 26.5 _
c -4 Cc 48.7 —
ai ~2 -.. -31.6
“ 2 ~o -2 -~9,2 ~“-
4 C o -3 15. oe

ct

* iisned tables will consider various ra.iges of
cons.der isotopic nuclides, and will be sortec by e as wel. ¢

fin Se LET.

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ee a eam
FITE DPHO1594

UNCEFINED

THRESHOLD= 4,

SAMP RATE=10800 ,
(15.6 SECS,
NUMBER CF PEAKS POUND=170
38 CALIE MASSES WITH LAST CNE=455.0,

FIGURE 2

USING REP FILE MPRO15S64
SCURCE IS urine-fractionzaminoacids (Gehrke technique
CEE ID IS GVP-17H
MASS OF MCL ICN
RUN CN MAT 711 EXP DOWN SCAR
PEAKS REJECTED

MISSED CALIBRATION MASSES:

281.0
HESEEKRAEE ATER FERS
28.0 9.5 31.0
81.0 0.3 93.0
131.0 -0.2 143.0
181.0 2-4 193.0
231.0 4.10 0 243.0
305.0 0.4 319.0
367.0 0.6 381.0
431.0 2.9 443.0

TABLE OF

83.6
0.3
0.1

-2.3

-4.4

-2.0
1.1

-&.0

DATE RUN 730313

107 FOR AREA,

44.0
100.0
155.0
205.0
255.0
331.0
393.0
455.0

MIN WIDTH= 2, MIN AREA= 1
1.49 DECS), TOEC=10.5
ARRAY SPACE USED( 1750/ 80C00)= 21.9%
6 MASSES ABCVE NOT FOUND

2.8

UID os ws OO

aw ON OD OU at AD

6.6

MASS PILE MPHO1594 HAS BEEN CREATED WITH 84
3.7 VOLTS AND REF BASE PEAK IS 174.5 YCLTS

SAMPLE BASE PEAK IS
MATCHING TCLERANCE= 4.COQ0O MMU FRCF MASS

MATCHING C 15 H 20 0 5 N

FASS
41.03931
§3.05388
55.01820

55.05510
56.06277
57.07068
64.01433
68.96245
68.97505
69.03101

70.03085

75.00519

83.01006

84.04382
85.02852

AREA
58.0
5.4
3.5

12.0

33.9
72.8

6.0

35.3

4 F
EMU ERR
0. 183
-0.899
1.153
~0. 189
0. 321
0. 180
0.248
-1.721
1. 863

-1.698
2.907
1.564

-1. 682

-3.025
0.560

~0.585

-1.911

~3.253

-0.814
0.230

-1. 113
0.914

-0. 429

L 6
CR

8

Q FOR WIDTH

51.0
112.0
162.0
212.0
269.0
343.0
405.0

24.9
-0.5

0.1
-0.2
-0.9
“2.2

4.4

MASSES FOUND

40 TO 700
CCMHPCSITICN
c 3H 5
c 3H 7
c tH 128
c 38 390
c 48 7
c AH 8
c 4H 9
c 14th 4cC
C 2H 2 FL
c 2H 3B
c 4H SC
c tH 245
c 38 46
H 4 N 2 FL
H 10 248
c 25H 360
c 38 1 FL
fH 20 1 FL
c 248 %1dCc
c YH 3 O
C 2H 2 FL
c 2H 4B
c 48 6C
c 24H 3 C
c 4H 560

Ww le

Nw ot & N a

a Sew Ww

mW

te oe

69.0
119.0
169.0
219.0
293.0
355.0
417.0

PROJECTICN ERRCRS 4948994454 944400 042

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APPENDIX B

LETTERS OF INTEREST

JRO
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 anticipated use of mass spectral
facilities by my research group in the fornseeable 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,

Gin

E. E. van Tamelen
Professor of Chemistry

EEvT/j1b

/Ry
OFFICE MEMORANDUM e STANFORD UNIVERSITY «© OFFICE MEMORANDUM

Date! December 3, 73

T :
° Carl Djerassi

From : .
Keith Hodgson

SuBsecT: 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.

    
  

3. No, no research is cur
for the next 6 mon

(at least
Cd by NIH.

WNGNVYOW3W 3Dd1ddO © ALISUZAINN GUOINVLS = WNONVYOWIW 3914d0 © ALISUZAINN GYOINVIS

PRR
OFFICE MEMORANDUM e@ STANFORD UNIVERSITY #© OFFICE MEMORANDUM e STANFORD UNIVERSITY # OFFICE MEMORANDUM

Date: December 3, 1973

To: Professor Carl Djerassi

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 P45, based monooxygenases currently
supported by the NIH.

secs Fine CAL

WNGONVYOWIW 3D1ddO © ALISUZAINN GYOSNVIS © WNONVYOWIW Jd1ddO © ALISUTAINN GYOSNVIS © WNONVYOWSW 43IdgO © ALISHZAINN GYOSNVLS ©

SRS
OFFICE MEMORANDUM e STANFORD UNIVERSITY @ OFFICE MEMORANDUM ¢ STANFORD UNIVERSITY @ OFFICE MEMORANDUM

To

From

SuBJEct:

Date: December 13, 1973

Professor Djerassi
Professor Harry S. Mosher

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 (nudibranchs) as
well as some new chaline 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.

Mae So Snnl—

/2Y

WNGNVYOWEW 351ddO © ALISUZTAINA QYOINVLS «© WNONVEOW IW JFdIddO © ALISUZAINN GYOINVIS *¢ WNONVYOW3W F5HdO © ALISHFAINN QYOINVIS ©
OFFICE MEMORANDUM e STANFORD UNIVERSITY © OFFICE MEMORANDUM ¢ STANFORD UNIVERSITY @ OFFICE MEMORANDUM

To

From

SuBJect:

Date: 14 December 1973

C. Djerassi

W. S. Johnson

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.

Ww. & ohne

JAS

WNONVYOWRW 3D1ddO © ALISYTAINM GYOANVIS © WNONVYOWIW JDIdJO © ALISYUJAINN AYOANVWIS ©®© WNAONVYOWSW 3D1ddO © ALISYZAINN GYOINVIS ©