1977-78 Annual Report RR-00612 Section 2.3

More elaborate functions for automatically identifying
discriminating features in sets of structures are being
developed. Currently, these experimental routines (contained
within the "PLAN" program) can be used to analyze functionality,
or to identify differences in the ways that superatoms have been
imbedded in structures. These routines will shortly be capable of
exploiting a simplified library of chemical/spectral tests for
particular substructural features; this will allow the program to
identify possible discriminating experiments. The current
capabilities of these functions are described in subsequent
sections.

2.3.1 EXAMINE

The EXAMINE function allows for the identification and
selection of structures characterized by particular combinations
of substructures, ring-systems and Isoprene-patterns. Further, if
relative merits can be associated with the substructural
features, then these merit values can be used to rank the
structures. In addition to providing information on the frequency
of different structural features, the EXAMINE function allows
structures with unacceptable combinations of features to be
pruned away.

EXAMINE thus extends both the earlier SURVEY function
(which EXAMINE has now totally subsumed) and the PRUNE function
in CONGEN. (PRUNE remains in CONGEN because of its greater
efficiency in simply rejecting undesired structures.) EXAMINE
allows structures to be segregated on the basis of combinations
of (desired or undesired) structural features. For example,
EXAMINE can be used to segregate structures which possess feature
A or 8B, or generally, any arbitrary Boolean expression of
relationships among structural features.

The EXAMINE function involves the following steps:
1) the definition of relevant substructural features.

2) {EXAMINE matches the features to the structures produced
by an earlier GENERATE or IMBED step, and summarizes their

frequency. ]

3) [if some form of merit rating is being used, then
details of the ranking process are provided.]

4) then, in “EXAMINE sub-command" mode, subsets of
structures possessing different combinations of features may be
selected. Features may be combined using standard AND/OR/XOR/NOT
operators. These subset selection procedures are basically non-
destructive; however, it is possible to use them to prune the
structure list.

26
1977-78 Annual Report RR-00612 Section 2.3

5) if examination of the structures has suggested
additional selection features, then the entire process may be
repeated (information on the current selection features being
preserved to allow new selection criteria to be combined with
those already in existence). Previously defined libraries of
selection features can be used, either alone or as a supplement
to selection features specified for a particular problem. It is
also possible to save the current set of selection criteria for
future use.

2.3.1.1 Example - Unknown Metabolite from Human Urine

Use EXAMINE to determine which members of a set of
candidate structures possess naturally occurring, alpha-amino
acid part structures. The compound for which CONGEN provided
structural candidates was an unknown component of human urine.
The empirical formula was C,s5Hj9NO.. There were 78 structural
candidates based on this empirical formula and chemical
constraints. Ten of the 78 formally possess an alpha-amino acid
substructure (-NHCHCOO-). Examination of these structures
proceeded as follows (note that the examination would yield the
same results if the entire 78 were examined) .

EXAMINE
Do you require simply to prune your structure list?:
Do you want to rank your structures?(Y for Yes, ? for
explanation) :
Do you want 'to use a library?Y
FILE NAME: AMINOACID.LIBRARY;8 [Old version]
READING <SMITH>AMINOACID. LIBRARY ;8
Do you want all substructures in the file?:¥Y

(file read OK)
Do you want to enter new selection features?:
ALA-1-? Substructure ALA min/max (1 . ANY) present in 1
structures.
GLY-1-? Substructure GLY min/max (1 . ANY) present in 0

structures.

VAL-1-? Substructure VAL min/max (1 . ANY) present in 0
structures.

LEU-1-? Substructure LEU min/max (1 . ANY) present in 0
structures.

ILEU-1-2? Substructure ILEU min/max (1 . ANY) present in 0
structures.

THRE-1-? Substructure THRE min/max (1 . ANY) present in 0
structures.

PHE-1-? Substructure PHE min/max (1 . ANY) present in 2
structures.

TYR-1-? Substructure TYR min/max (1 . ANY) present in 0
structures.

PRO-1-? Substructure PRO min/max (1 . ANY) present in 0
structures.

OH=-PRO-1-? Substructure OH-PRO min/max (1 . ANY) present in 0
structures.

27
1977-78 Annual Report RR-00612 Section 2.3

ASP-1-? Substructure ASP min/max (1 . ANY) present in 1
structures.

GLU-1-? Substructure GLU min/max (1 . ANY) present in 1
structures.

BETA-ALA-1-? Substructure BETA-ALA min/max (1 . ANY) present
in 0 structures.

SER~1-? Substructure SER min/max (1 . ANY) present in 0
structures.

{note that only four of the amino acids have their part
structures (-NHCHR-COO-) represented in the set of candidates,
alanine (ALA), phenylalanine (PHE), glutamine (GLU) and
asparagine (ASP) ]

Enter commands for selecting subsets of structures with
particular features.

Do you want help?:

10 STRUCTURES

~>SELECT

> (ALA~1-? OR PHE-1-? OR ASP-1-? OR GLU-1-?)

5 STRUCTURES WITH ((ALA-1-? OR PHE-1-? OR ASP-1-? OR GLU-1-?) )

[Only five of the ten (or 78) have any one of the four amino acid
substructures. They are drawn below. The first structure drawn
is the 77th of the 78 original candidates. The second number
refers to its rank based on a comparison of the mass spectrum
predicted for this compound against that observed for the
unknown. This compound was among the three top-ranked structures
(MSRANK) in the original set of 78. It is clearly ranked higher
than the other four candidates under the (biochemical) constraint
that the compound contain the substructure of a naturally
eccurring amino acid. Subsequent synthesis and comparison of GC
and MS confirmed the identity of the unknown as
phenylacetylglutamic acid dimethyl ester .]

28
1977-78 Annual Report RR-00612
—>DRAW
(77 . 84)
Cc
|
0
|
C=O
|
C
|
oc C
COCCNCC—C OC
= | =
0 c 60°C
= /
C
(57 . 57)
Cc
|
0
|
C Cc=0
=\ |
C  C—C-C-N-C-C-C-0-€
| = = |=
c 66°C Ooco
= /
C

29

Section 2.3
1977-78 Annual Report

(55 . 57)
C=C
/ \
Cc Cc
Cc-C
|
|
|
0 Oo Cc
= = |
C-O0-C-C-C-C-N-C
|
O=Cc
|
0
|
Cc
(51 . 57)
Cc
|
0
|
C=C Oo C=O
/ \ = |
C C—C-C-C-N-C
= 4 |
c-c Cc
|
O=C
|
0
|
Cc

RR-00612

30

Section 2.3
1977-78 Annual Report RR-00612 Section 2.3

| = = |=

che Oo co

2.3.2 PLAN

As mentioned previously, the PLAN program represents our
initial efforts toward assembling the heart of an experiment
planning program. The goal of PLAN is to identify all structural
features which distinguish among structural candidates ffor an
unknown. In the next year we will develop the program which will
use this information to suggest experiments. The EXAMINE
function, described above, can only look for structural features
explicitly supplied by the chemist. Although a summary of such
features is quite useful, EXAMINE is insufficient to solve the
more general problem of identifying distinguishing substructures.

PLAN in its current form provides the following
capabilities:

1) Using a starting substructure supplied by the chemist
(for example, one of the superatoms used to construct structural
candidates), PLAN can search the local environment of the
substructure for distinguishing features, continuing the search
until discriminatory characteristics are found.

2) PLAN checks (if requested) for simple differences in the
distribution of carbon and hydrogen atoms which could be detected

by l3cmp or luye.

3) PLAN can begin at existing functional groups and examine
larger substructures by expanding the local environment (as in
(1), above) until distinguishing features are found. The example
below represents PLAN operated in this mode.

31
1977-78 Annual Report RR-00612 Section 2.3

4) PLAN, if requested, performs the operations specified in
(3) beginning with double bond systems in the candidates.

2.3.2.1 Example

In the following example, 88 structural candidates for the
compound palustrol[8], based on spectroscopic information, were
processed by PLAN. The following is a recording of that terminal
session. Bracketed comments ( [ ] ) are inserted to explain the
flow of the program.

@congen [begin CONGEN]
(<SMITH>CONGEN. 722 . <LISP>CARHART. SAV; 70702)

:OK

(LISP)

DO YOU WANT TO SPECIFY AN EMPIRICAL FORMULA? (Y FOR YES):

RE [RESTORE file of structures]

INPUT FILE:PAL.REACT [Old version]
READING <SMITH>PAL. REACT; 2
THIS IS A PILE WRITTEN BY CONGEN
(COMPOSITION RESTORED)
(EMPIRICAL FORMULA RESTORED)
(AROMATICS RESTORED)
(CONSTRAINTS RESTORED)
USERATOMS HEP Al Bl CH3 CH2 CH ETH MET C N O
ALL RESTORED

(88 STRUCTURES) (88 candidates]
LISP

(LISP):

: (PLAN) [Begin PLAN]

Do you want to specify starting superatoms? [No starting

point specified]
Do you want the program to check for simple differences in the
off-resonance decoupled 13c spectrum?Y

These structures show no simple differences in their carbon
distributions.

Do you want the program to check for simple differences in
proton distributions? Y

These structures show no simple differences in their
hydrogen distributions.

Do you want the program to check functional groups?Y (See
mode (3), above)

Only one substructural class was generated

32
1977-78 Annual Report RR-00612 Section 2.3

All compounds have this feature:

OH-C
(All compounds possess a
tertiary hydroxyl group,
so PLAN continues]

OH-C

present in 88 structures

Only one substructural class was generated

All compounds have this feature:

Cc
|
C-C-0 {All compounds have three carbon atoms
| bonded to the tertiary OH, but the
Cc hydrogen distributions on those carbons
differ]

By considering proton distributions, 3 subclasses can be
distinguished. Do you want to see the protonated structures?Y
CH
Cc |
H-C-OH8
2 |
CH

CH
Cc |
H-C-OH
2 |
CH2

CH2
Cc |
H-C-OH
2 |
CH2

{This fact alone is sufficient to consider a dehydration
experiment, which is the experiment performed by the chemist when
the work was originally done.]

[If desired, each of the three subclasses can be expanded

33
1977-78 Annual Report RR-00612 Section 2.3

in turn. Only the expansion of the first class is shown (this
class contains the correct structure) .]

Do you want this feature to be further enlarged?Y (each
subclass will be enlarged separately)

CH
Cc |
H-C-O8
2 |
CH [PLAN can continue expansion of each
subclass to search for further
discriminatory features if requested.
The results are omitted for brevity.]

present in 24 structures

CH
Cc |
H-C-O8
2 |
CH2

present in 49 structures

(end of report)

CH2
Cc |
H-C-OH
2 |
CH2

present in 15 structures

(end of report)

(continuing now with earlier report stage)

(end of report)

(continuing now with earlier report stage)

(end of report)

Do you want the program to check double bond systems?N

34
1977-78 Annual Report RR-00612 Section 2.4

2.4 The Reaction Chemistry Program

During the past year we have made good progress in
developing the reaction chemistry program, REACT, into a working
tool for laboratory chemists. Two main areas of application are
discussed in the subsequent sections. These areas and the
examples included are currently in the process of appearing in
the literature. Additional details can be obtained by referring
to those papers when they appear. The first area of application
(subsequent section) is the subject of a paper to appear soon in
Tetrahedron. The second area is being written up for publication
in the Journal of Chemical Information and Computer Science.

2.4.1 Studies in the Biosynthesis of Natural Products

Manual elucidation of structures arising from chemical
reactions which may yield a large number of products via a number
of complex, interrelated pathways is a difficult problem. Such
reactions are, however, natural candidates for computer-assisted
studies because the computer can easily record all intermediates
and products as well as interrelationships among them. [22]
Examples of these reactions include carbonium ion rearrangements,
reactions of free radicals and biochemical processes.

REACT is designed to carry out representations of chemical
reactions on representations of chemical structures. Reactions,
defined by the chemist using the program, are carried out in the
synthetic direction as opposed to the retro-syqthetic direction
of programs for computer-aided synthesis. In structure
elucidation problems, the set of structures undergoing reaction
is the current set of candidate structures for an unknown. It is
clear, however, that the program can also be used effectively in
following reactions of a single, known compound participating in
a complex sequence of reactions. For example, we showed [22]
that CONGEN together with REACT provides a convenient method for
studying acid catalyzed rearrangements such as the conversion of
tetrahydrodicyclopentadiene to adamantane. In that example, the
complete set of isomers was generated by CONGEN. Subsequently, a
one-step reaction carried out on each isomer afforded the
complete rearrangement graph. An alternative method, similar to
that discussed in subsequent sections, is to use a single isomer
aS a precursor. In the examples given in this work, a single
precursor was subjected to repetitive application of a set of
reactions.

 

1p, om. Gund, P. v. R. Schleyer, P. H. Gund and W. T.
Wipke, J.Am.Chem.Soc. 97, 743 (1975).

25. A. Godleski, P. v. R. Schleyer, E. Osawa, Y. Inamoto
and Y. Fujikura, J.Org.Chem. 41, 2596 (1976).

3
E.J. Corey and W.T. Wipke, Selence 166, 178 (1969) .

35
1977-78 Annual Report RR-00612 Section 2.4

To demonstrate the utility of REACT we present two examples
where a given precursor of known structure is subjected to an
extended sequence of reactions. At each step in the sequence one
or more reactions may apply to the products from the previous
step. As will be shown in the sequel such an approach is
especially well suited to problems involving the biosynthesis of
natural products. A complete description of this work will
appear shortly [22].

2.4.1.1 Generation of Biosynthetically Plausible Sterol
Side Chains

Sterols are naturally occurring steroidal alcohols (usually
3-ols) which differ in the number and the position of methyl
groups and the degree of unsaturation (present as a double bond
or cyclopropyl ring). New sterols are frequently isolated in
minute quantities from natural sources. Because of their
structural similarities and the large number of different sterols
present as amixture in the same source (a recent paper
documents the isolation of ca. 50 sterols from one marine source)
it is often difficult to separate them and to obtain pure
compounds in quantities large enough for structure determination
by conventional methods. Some structural assignments are based on
biogenetic considerations, assuming that compounds from the same
origin are related to each other through formation along the same
biochemical pathway. This pathway can be a series of complicated
chemical reactions which yield a large number of intermediates
and products. It is difficult to follow manually such a series of
reactions in order to explore all possible structural
alternatives. To date, over 100 different 3-hydroxy sterols have
been isolated, the majority of them based on the seven nuclear
skeletons ~.

We use a method of combined gas chromatography/mass
spectrometry (GC/MS) to analyze complex mixtures of sterols ina
search for new compounds which may represent important
biosynthetic intermediates. Part of this method involves
research in interpretation and prediction of mass spectra. [23]
We have used the REACT program as an additional tool to predict
Plausible structural candidates to guide both our manual and
computer-based interpretations.

The set of reactions used in REACT to carry out possible
transformations of sterol side chains have been suggested

 

4s. Popov, R. M. K. Carlson, A. Wegmann and C. Djerassi,
Steroids 28, 699 (1976).

5c, Djerassi, R. M. K. Carlson, S. Popov and T. d.
Varkony, in "Marine Natural Products Chemistry" by D. J. Faulkner
and W. H. Fenical (ed.), Plenum : New York, N.Y., 1977, p 111.

36
1977-78 Annual Report RR-00612 Section 2.4

previously 6 the precursor, (a 24,25 unsaturated side chain
numbered 8 at the top of the following chart) the order of
application of the various reactions and the classes of products
which result are shown in the following chart. The sequence of
reactions consists of repetitive application of the following
steps:

 

 

 

town
N a

 

 

3 R
. a n
oe ee ¢
o . -H*
fg

 

 

 

 

 

 

 

Oxidationic ' Cyclopropyl
at 22,23 | %satucated | Reduction|g te Rearvangement containing |
\———— side chains | <——— OLEFINS; __

 

 

 

 

 

 

a
89.
99
3 & y
Sn

; Re
rs “ 6 re \
ee + ~Nig6
q ~H aS me
—? t
“.

Methylation

 

6 E. Lederer, Quart.Rev.,Chem.Soc. 23, 453 (1969).

37
1977-78 Annual Report RR-00612 Section 2.4

1) Methylation. C-methylation of a double bond. In nature
this reaction occurs via the ylide of S-adenosylmethionine. This
reaction is constrained for general application later in the
sequence to forbid the sterically unfavorable methylation of
tetra-substituted double bonds.

2) The carbonium ion obtained by the alkylation can undergo
several reactions:

a) proton elimination and formation of a double bond; b)
cyclization to form a cyclopropyl system with subsequent
elimination of a proton; c) quenching to form saturated side
chains.

3) The olefin is allowed to undergo several additional
reactions:

a) reduction to form a _ saturated side chain; b)
rearrangement to a cyclopropyl system; c) degradation to shorter
side chains via loss of allylic methyl groups; d) methylation to
produce longer side chains.

Constraints on reactions of the olefin included

a) subsequent migration of the double bond is not allowed;
b) olefins obtained by degradation are allowed to umdergo only
one step of methylation.

4) Subsequent oxidation of saturated side chains proceeds
to form a new double bond at C-22,23, a mechanism proposed by
Knapp, et al. This set of reactions was applied sequentially a
total of three times. Thus, side chains possessing from seven to
eleven carbon atoms are accessible by this sequence.

Results. A numerical summary of results is presented in our
Table below. The table is organized by summarizing the side
chains produced by the different biochemical pathways. The only
known, naturally occurring C7 saturated side chain was correctly
predicted by REACT. Three C7 unsaturated side chains were
predicted. Two of these three exist in nature. In the C8 series
five unsaturated side chains out of 12 predicted are observed in
nature. For the longer side chains, more are possible but fewer
are observed. For example, only one out of the 76 predicted Cll
side chains has so far been found in nature.

 

Tel oF, Knapp, J. B. Greig, L. J. Gad and T. W.
Goodwin, J.Chem.Soc.,Chem. Comm. 707 (1971).

38
1977-78 Annual Report RR-00612 Section 2.4

 

 

Number of SATURATED OLEFINS CYCLOPROPANES
C in side .
chains A B E Nature A BE F Nature Cc oD Nature
7 - 1 - 1 - 2 - 1 2 -  - -
8 1 - 1 1 6 3 2 5 - - -
9 1 7 - 1 4 13 6 4 4 2 4 -
10 3°12 - 4 13 17 #19 8 6 8 il 1
ll 8 - 8 1 31 - 37 8 1 17°) (21
A methylation only.
B methylation followed by degradation only.
C rearrangement of carbonium ion.
D rearrangement of olefin.
E degradation followed by methylation only.
F oxidation of saturated side chains at 22,23 position.

Table I. Number of Side Chains Produced by Different
Pathways.

The total number of sterols which obey our biosynthetic
constraints is 1778. This number is manageable by techniques of
computer-assisted structure elucidation. Separating the
structures by molecular weight reduces considerably the number of
candidate structures which must be considered in a given problem.
Thus, in a GC/MS experiment the maximum number of structures we
have to consider is not larger than 264 (the number of isomers

with empirical formula C29 H4,0. Any additional spectroscopic or
chemical data reduce this number still further. For other
molecular weights the number of possibilities is considerably
fewer. Structural information from the mass spectral
fragmentation pattern of the molecule may leave only a small
number of possibilities from which to choose.

2.4.1.2 Elucidation of Biosynthetic Pathways
Elucidation of biosynthetic pathways can be accomplished in

several ways, including for example co-occurrence of structurally
related compounds or use of mutant organisms which accumulate

39
1977-78 Annual Report RR-00612 Section 2.4

intermediates. 8 These methods usually leave the structures of
intermediates and/or the details of the biochemical pathways open
to question. More detailed experiments are required to establish
rigorously reaction pathways from precursor to product.

Isotopic labelling experiments are capable of providing
additional detail through synthesis of labelled precursors
followed by incorporation of labelled substrate and determination
of the labelling pattern of the products of biochemical
transformation. The incorporation of labelled precursors into
desired products is generally low and elucidation of the
labelling pattern in minute amounts of product is difficult.
Thus, these experiments are generally time consuming and costly.
They can be complicated by the existence of different biochemical
pathways, some of which yield products with the same distribution
of isotopic labels. Therefore, care must be used in designing
such experiments. It is important to select a labelled precursor
that will allow one to distinguish among most of the possible
pathways, and that will lead to a product with labels distributed
in easily detectable positions. Manual methods are often
insufficient to determine all the theoretically possible pathways
when the number of possible pathways and the number of
intermediate structures is very large. However, this type of
problem is easily managed by REACT, which can accurately and
systematically monitor transformations of the precursor into
products, follow the isotopic labels throughout a_ reaction
sequence and detect the formation of equivalent structures and
labelling patterns. We stress that this is not an exercise in
"paper chemistry", but a systematic way to investigate all the
possible aspects of a proposed experiment before devoting
valuable time and resources to an exper iment which leads to
ambiguous results.

An example which illustrates our method is the exploration
of biosynthetic pathyays leading to formation of a family of
fungal metabolites The complete paper (22] describes our
results in detail. Briefly, use of REACT enabled us to: 1)
verify proposed pathways and suggest alternatives; 2)
demonstrate how different patterns of isotopic labelling lead to
unambiguous assignment of pathways for certain molecules; and 3)
demonstrate that several pathways are possible for certain other
fungal metabolites, pathways which would not be differentiated by
proposed labelling schemes.

2.4.2 Applications to Structure Elucidation

The first version of REACT and its applications were

 

8 3. D. Bu'Lock, "The Biosynthesis of Natural Products",
McGraw Hill, New York, N.Y., 1965, p.94.

9 G. A. Cordell, Chem. Rev. 76, 425 (1976) .

40
1977-78 Annual Report RR-00612 Section 2.4

described previously [22]. Subsequently, the structure of the
program was revised significantly to include commands and
internal operations which more closely parallel laboratory
procedures. The new version has been described briefly and some
applications of REACT to mechanistic problems have been discussed
[24]. In subsequent sections we describe the REACT program in
detail, together with an example of the application of the
program to a structural problem.

To demonstrate the application of REACT we choose an
example which illustrates some (but not all) aspects of the use
of REACT in a structure elucidation problem. A contrived example
might illustrate many of the other features and subtleties of the
program, but would not be as meaningful chemically. The example
involves a dehydration reaction (see reaction definition) applied
during the course of elucidation of the structure of palustrol
(1) [8]. Structural features of the products were powerful
constraints on the identity of the compound. This problem was
solved prior to the existence of the REACT program,

OH , OH

We pick up the example at the point at which the reaction
waS applied in the laboratory. This example is of interest
because it represents a case where direct translation of
observations on products back to structural constraints on the
starting materials is difficult. Using REACT, expression of
Structural information is straightforward and logical. The
laboratory reaction, separation and key structural information
are summarized below. The starting materials, in a flask called
STRUCS, are the candidate structures for palustrol (1).

41
=

DEHYDRATION

SEPARATE

r=}

1 vinyl H O vinyl H O vinyl H

0 vinyl CH, 1 vinyl CH 0 vinyl CH

Figure 1. Diagram of REACT's
Separation of CONGEN Structures with
Respect to Dehydration.
1977-78 Annual Report RR-00612 Section 2.4

Consideration of all available spectroscopic data had
reduced the problem to a_ set of 88 candidates prior to carrying
out the dehydration reaction. The contents of the flask STRUCS
were dehydrated and the products placed in a flask called DEHYD.
Separation of the reaction mixture yielded three products, placed
in flasks Dl, D2 and D3. The numbers of vinyl protons and vinyl
methyl groups detected by H NMR for each product are summarized
in Figure 1.

2.4.2.1 The Reaction Tree

The reaction tree is a representation of the sequence of
laboratory procedures (reactions and separations) to which
precursors and their products have been subjected. Formally, it
consists of named flasks and their interrelationships in the form
of reaction names and separation steps. If there are multiple
precursors (i.e. more then one structure in a flask), as in the
example, each is allowed to react, independently, resulting ina
data structure internal to REACT which records the reactions of
each structure separately. The chemical meaning of multiple
Structures in the starting material flask STRUCS is that the
exact identity of the compound is not known; its structure is
represented by one of all the possible structures in the flask.
If the flask was created via a reaction(s), the structures
represent the collection of all products from all precursors
where, again, the identity of each of the products in the
laboratory application of the reaction is not necessarily known.
In our representation, an example of which is shown in Figure l,
flasks which could possess multiple structures, such as multiple
candidates for an unknown, are depicted as containing all
structures, and all possible products appear lumped together in a
product flask. The dehydration reaction applied above (see Table
III) is summarized in Fig. 2.

STRUCS=88
|
*DEHYDRATION->DEHYD=241

Figure 2. Result of Dehydration in REACT

This figure is interpreted to mean that the 88 candidate
structures, any one of which could be the true unknown in the
flask STRUCS, yield a total of 241 possible products, all
associated with the flask DEHYD. Confusion related to this
presentation can be avoided by remembering that the internal
representation is effectively n copies of the reaction tree where
n is the number of precursors in the flask STRUCS, or 88 for the

42
1977-78 Annual Report RR-00612 Section 2.4

example of Fig. 1 For example, one such copy encodes the
information about the conversion of 3 to 4a - 4c.

In our example we discuss only a single reaction. In
general, however, the reaction tree can be of arbitrary
complexity. Several different reactions can be applied to
aliquots of a precursor (whether it be an original starting
material or a product of a previous reaction). In addition, an
extended sequence of reactions can be carried out. Thus, the
reaction tree can grow arbitrarily in width and depth.

2.4.2.2 Separation

A flask obtained by reaction can contain a mixture of
products. A single precursor can yield multiple products in three
ways in a reaction: 1) presence of multiple reaction sites, each
yielding a different product; 2) multiple reactions; and 3)
cleavage reactions where all fragments are isolable. The usual
laboratory step subsequent to reaction is separation of the
products. Thus, REACT has a SEPARATE command which allows the
chemist to express to the program his laboratory observations on
performing the separation. The number of products obtained on
separation is a constraint on the identity of the starting-
material, and is information useful in applications of REACT to
structural problems. The separation requires placement of each
separated product into a designated or named, flask (Table IT).

Table II. The Dialog with REACT on Separation of Contents

of Product Flask
DEHYD into Flasks Dl, D2 and D3

Command Comment

SEPARATE Enter separation mode

NAME OF FLASK TO BE SEPARATED:DEHYD Select product flask

NEW FLASK NAME:D1 Select names for flasks

NEW FLASK NAME:D2 for three separated products
NEW FLASK NAME:D3

NEW FLASK NAME: No other flasks

MAXIMUM NUMBER OF ADDITIONAL PRODUCTS:0 No additional products
in the tar flask

210 STRUCTURES SURVIVED SEPARATION Results
BEGINNING RAMIFICATION. ..DONE Implications of separation

Return to REACT

43
1977-78 Annual Report RR-00612 Section 2.4

It is characteristic of many laboratory reactions that an
unspecified, perhaps large, number of additional products are
obtained, some legitimate, but at low concentration, others from
side reactions which may not be incorporated in the definition of
the reaction used in REACT. The chemist using REACT must base
his use of the SEPARATE command on his own evaluation of the
reaction applied in the laboratory. Selection of a named flask
in which to place a separated product implies that the product so
separated arose from the named reaction, and not from some other
unspecified reaction. However, to accommodate the fact that the
reaction may have been incomplete or side reactions may have
occurred, additional products can be specified to be in a "tar"
flask associated with each set of separated products. On
separation, the new flasks each contain one unique product, whose
identity is not known. The structure of the product must be one
of the structural possibilities associated with the flask.
However, the structures in the "tar" flask, (or in any flask
prior to separation) can be a mixture of products, where each
product in the mixture may be represented by several structural
possibilities.

The dialog to establish separated products and a tar flask
with REACT is summarized in Table II. In _ the laboratory,
separation yielded three products (Fig. 1). In this example we
choose to specify exactly three products by selection of three
flasks to receive the products, Dl, D2 and D3, and no other.

The fact that three products, all assumed to arise from the
dehydration, were observed is a constraint on the identity of the
Starting material in the flask STRUCS. Those structural
possibilities (according to CONGEN) for palustrol which would
yield only two products (e.g., 8, to yield 8a and 8b) can be
rejected independently of the identity of the products, while
those structures which yield three products on dehydration remain
under consideration (e.g., 1 and 2) until additional data on the
identities of the products are gathered and specified to REACT
(see subsequent section).

OH |
8a. 8b

44

8

—
1977-78 Annual Report RR-00612 Section 2.4

 

 

 

 

 

 

 

 

iA City”
NN

 

 

 

 

The reaction tree which results from the separation (Table
II) is shown in Figure 3.

STRUCS=72

DEHYDRATION >DeHTD=210-e-|b3=210
{p2=210
ip1=210

Figure 3. Results of Separation in REACT.

The reduction in the numbers of structures in flasks STRUCS
and DEHYD (compare Fig. 3 to Fig. 2) results from _ the
implications, or ramifications, of the statement on separation.
REACT has a record of how many products are obtained from each
structure and the identities of each precursor and product. It
can eliminate automatically from further consideration precursors
which yield an undesired number of products. If three products
are observed, as in the example, only 72 0f the original 88
structures remain as candidates. Sixteen of the structures
yielded, by the computer program, other than exactly three
products and were therefore removed from consideration as
candidates. The products of these sixteen structures are also
removed from the product flasks, resulting in a decrease in the
number of structures in DEHYD from 241 to 210. The remaining 210
structures are not exactly three times 72 because several
candidates yield equivalent products. For example, the
dehydration of both 9 and 10 yields, among other products, 11.

45
1977-78 Annual Report RR-00612 Section 2.4

As mentioned previously, duplicate structures are detected
and removed for efficiency, except in mechanistic reactions.

What of the contents of the flasks Dl, D2 and D3? Up to
this point, no statements about the structural identity of any
product have been made, paralleling the laboratory events of,
first, separation, and, later, gathering of data on the products.
Thus, any of the 210 products in DEHYD might be in any of the
flasks Dl - D3 (see Fig. 3, where all 210 products remain
allocated to Dl - D3). Stated at the level of internal
representation in REACT (see also above discussion), where the
original structures are represented individually, each structure
(in STRUCS) yielded three products, any of which might be in any
flask. Subsequent operations will perform the appropriate
allocations of structures to flasks.

Details of the internal representation and the algorithm
which performs ramification after SEPARATE and PRUNE (see below)
are given in a separate publication. This algorithm is
responsible for determining legal allocations for structures to
flasks throughout the reaction tree whenever the tree is modified
in any way.

2.4.2.3 PRUNE - Expression of Constraints on Products

In laboratory procedures, the next step would be to collect
data on the product in each flask. Structural information gained
represents constraints not only on the identity of the products,
but also on the identity of the precursor and its precursor and
so forth throughout an entire reaction Sequence. REACT allows
structural statements to be made as constraints on the contents
of any flask in a reaction tree. The command to express
constraints is PRUNE (a word which is jargon but does carry with
it the concept of trimming the reaction tree and also corresponds
to the same command in CONGEN.

Substructural constraints can be obtained froma file or
defined by the chemist as required, using EDITSTRUC. In our
example, the product in one of the flasks (Dl) was observed
according to H NMR analysis to possess one vinyl proton and no
vinyl methyl groups. These substructures, PT] (12) and VINM
(13), respectively, were defined and the substructures supplied
to PRUNE.

H-c=C
12(PT1)

OH
CH, - C=C
—3 14 5
13(VINM) — i

46
1977-78 Annual Report RR-00612 Section 2.4

STRUCS=72

ACEH URAITON->PeEYD=210-=- [3187
{D2=18)
{1-129

Figure 4, Application of PRUNE in REACT.

The reaction tree which results on application of PRUNE is
shown in Figure 4. There remain 129 structures which could be in
the flask Dl. The number of structural candidates (72) has not
been reduced, implying that all 72 can yield at least one
structure possessing one vinyl proton and no vinyl methyls. Some
candidate structures can yield more than one product which obeys
these constraints and might therefore be in Dl, resulting in 129
rather than only 72 structures in that flask. For example, 2
yields two products obeying the constraints; either could be the
product observed in Dl. However, for structure 1, only one of
the products (14) is a legal structure under the constraints;
that structure must be in flask Dl.

If one product is forced to be in a certain flask it can be
in no other flask. Thus, the number of dehydration products which
could be in D2 and D3 decreases from 210 to 187 (compare Figs.
3,4). Opoviously, with a more complex reaction tree, such logical
decisions become complicated. REACT determines allowable
allocations automatically.

Flask D2 contains a product which possesses no vinyl
protons and one vinyl methyl group (Fig. 1). Constraining the
contents of D2 with this structural information results in the
allocation summarized in Figure 5.

STRUCS=45

|

*DEHYDRATION->DEHYD=135-s- |D3=76
|
|D2=52
|
|D1=69

Figure 5. Results of Constraining Contents of Flasks in REACT.

Now the number of candidate structures in STRUCS is reduced
to 45, implying that there are 72-45=27 structures which cannot

47
1977-78 Annual Report RR-00612 Section 2.4

yield a product distribution which satisfies the structural
constraints placed on both flasks Dl and D2. An example is 12,
which, although it yields at least one (two) products satisfying
the constraints on flask Dl, yields no products satisfying the
constraints on flask D2. It is therefore discarded as a
candidate structure. At the same time, any products of discarded
structures (and precursors in a more complex tree) are removed
from DEHYD and flasks Dl - D3.

Application of the constraints on flask D3 (Fig. 1), that
the product contained therein possess neither a vinyl methyl nor
a vinyl proton results in the reaction tree shown in Figure 6.
Now only fourteen structural candidates remain, and from the
allocation of products to flasks (Fig. 6a) each yields three
unique products. Each of the structural candidates was tested
for the presence of exactly two secondary methyl groups; the
reaction tree of Figure 7 results.

Previously, translation of the results of the dehydration
into a substructure used to test the 88 candidates reduced the
number of candidates to 22, rather than 14 (Fig. 6a).

STRUCS=14

SoEHYDRATION->DEHyD=42-2~[p3-14
{D2e14
IDeA

Figure 6. Further Application of Constraints.

STRUCS=12

*DEHYDRATION-»DEHYD=36-s-|D3#12
tp2=12
(Di=12

Figure 7. Constraining Contents of Flasks Still Further.

The substructure used was correct, but incomplete in that
eight structures which obeyed the substructural constraint could
not yield the observed products. Through use of REACT,
structural information can be applied directly to the structures
of potential products without the necessity of translating
observations back to the precursors.

48
1977-78 Annual Report RR-00612 Section 2.4

2.4.3 Utilities
We discuss the utilities briefly here not because they are
critical to understanding the method but because they are an
essential part of the interactive nature of REACT.

1) Displaying Reaction Tree. Examples of reaction trees in
Figures 2-6 illustrate the format in which the reaction sequence
can be observed. The DISPLAY] command allows the chemist to view
selected portions of the tree, i.e., one named flask together
with any separations or reactions performed on that flask.

2) Drawing Structures. The structures (or any subset) in
any selected flask can be drawn. To check numbering of atoms,
particularly in the use of MREACT, structures can also be drawn
with structure numbers (NDRAW) .

3) Determining Structural Relationships. Relationships
between precursors and products can be obtained using the PARENTS
and PRODUCTS commands. A report can be obtained for all or
selected structures ina flask, either to summarize precursors
which led to a structure (PARENTS reports flask and structure
number of every parent of every structure) or products of all or
selected structures (PRODUCTS reports flask and structure number
of every product of every structure) . These commands were used
to examine the reaction tree in the example to determine
relationships among structures presented in the text.

4) File Manipulation and Other Commands. These utility
commands allow a chemist to save and restore problems or portions
thereof at will, thereby maintaining a computer-based "lab
notebook" of his operations. Other commands simplify the
reporting of problems and subsequent improvement of REACT and
correction of errors. CHECKPOINT and UNDO are useful when the
chemist wants to explore the consequences of a separation or
pruning and still return to his previous reaction tree if
desired.

2.5 Mass Spectral Prediction and Ranking

2.5.1 Predicting Spectra Using MSRANK and the Half-Order
Theory

The MSRANK program has been incorporated as part of CONGEN,
but is not yet available for general use by outside per sons
accessing CONGEN. We have during the past year been giving the
program some extensive tests to determine its scope and
limitations. We have studied the following classes of compounds
{all closely related to current research problems): 1) marine
sterols; 2) substituted pregnanes; 3) aliphatic and aromatic
esters; and 4) macrolide antibiotics.

49
1977-78 Annual Report RR-00612 Section 2.5

We conclude that MSRANK is a powerful filter for
eliminating from further consideration structures which cannot
yield the observed mass spectrum for an unknown by “reasonable”
fragmentation pathways. The greater the structural diversity of
isomeric candidates for an unknown, the better the performance of
MSRANK in focussing inon the correct structure. When the
structures are quite similar, for example when they have been
constructed from the same set of superatoms and few remaining
atoms, the ranking by MSRANK is quite similar (as one might
expect). When this situation occurs, the chemist must still
consider the top 10 - 50 percent of the structures as
possibilities, depending on the distribution of scores.

We have added an explanation feature to MSRANK. Upon
request the program prints a list of peaks in the observed
Spectrum which have different "reasonable" explanations. for
different candidate structures. Based on this information the
chemist can accept the ranking or change the parameters which
define his theory of fragmentation to obtain a different ranking.
This procedure helps detect and reduce the plausibility of
"nonsense" fragmentation processes.

2.5.2 Prediction Using Fragmentation Rules Supplied by
Chemists

When the candidate structure is known to belong toa
previously investigated class of compounds, then we can use
additional information to predict a more precise mass spectrum.
This information is in the form of specific fragmentation rules.
These rules are described by a subgraph, a break (or cleavage)
and related hydrogen or neutral transfers, intensity ranges
associated with rules and a parameter describing the confidence
in a rule. We are working on a program which allows the user to
enter rules defining his theory of mass spectral fragmentation.
A computer session for entering rules which describe
fragmentation of ring D in 17-substituted steroids is presented
below to convey the nature of a fragmentation rule and associated
parameters.

 

@<wew>dendr1 <begin program>
using <LISP>CARHART.SAV; 70702
<WCW>DENDRL.SAV;8 created 26-JAN-78 06:06:39
what do you want to do? : CRF

create user rule file.

new rule set.

=? <query for options>
one of the following:

RESTORE ENTER DELETE SHOW SAVE QUIT ??

= ENTER RL <enter rule named "RI">
enter rule:
:= SHOW <query rule>

50